Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4
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College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of Dundee, James Black Centre, Dow Street, Dundee, DD1 5EH, United Kingdom
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Abstract
The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.
The Bromo- and Extra-terminal (BET) family of proteins, including the ubiquitously expressed BRD2, BRD3, and BRD4 and the testis-specific BRDT, recruit transcriptional regulatory complexes to acetylated chromatin thereby controlling specific networks of genes involved in cellular proliferation and cell cycle progression. (1) Deregulation of BET protein activity, in particular BRD4, has been strongly linked to cancer and inflammatory diseases, making BET proteins attractive drug targets. (2) For example, RNAi screens have identified BRD4 as a therapeutic target in acute myeloid leukemia, (3) ovarian carcinoma, (4) and siRNA knock down of BRD4, but not of BRD2 or BRD3, induced upregulation of apolipoprotein A1 (ApoA1), which protects from atherosclerosis progression and other inflammatory processes. (5) The silencing of BRD4 furthermore identified BRD4 as a target to treat chronic obstructive pulmonary disease (COPD). (6) These results underscore the potential of targeting BRD4 as a therapeutic strategy and motivate further research in validating BRD4 as a drug target.
Crucial to the function of BET proteins are two highly homologous bromodomains that are present in their amino-terminal regions and direct recruitment to nucleosomes by binding to specific acetylated lysines (KAc) within histone tails. (7) Small molecule BET inhibitors, including the triazolodiazepine-based JQ1 (8) and I-BET762 (9) (Figure 1a) among others, (10-13) bind to the KAc-binding pocket of the bromodomains and disrupt interaction with histones, thereby displacing BET proteins and their associated transcriptional regulatory complexes from chromatin. BET inhibitors are highly potent (Kd ∼100 nM), cell-penetrant, and active in vivo against a range of solid, hematological, and other tumors, which has prompted compounds entering phase I clinical trials for cancer. (14-16) However, BET inhibitors show no selectivity for individual BET family members, thereby limiting their scope as chemical probes for validating the roles of individual BET targets in physiology and disease. To this end, chemical genetic strategies have been recently developed to engineer orthogonal selective BET bromodomain-ligand pairs. (17) While this approach has the advantage of enabling disruption at will of a single or more bromodomains, it requires a mutation to be introduced into the target protein. Therapeutically, the effects of BET inhibitors on different transcriptional pathways have raised concerns about the safety and tolerability of BET inhibitors in humans. Crucially, none of the inhibitors described to date is selective for binding BRD4 bromodomains over those of its paralogs BRD2 and BRD3.
Figure 1
Figure 1. Design, synthesis, and biophysical and biological evaluation of BET bromodomain PROTACs. (a) Chemical structures of BET-bromodomain inhibitors JQ1 and I-BET762 and binders of von Hippel-Lindau protein VHL-1 and VHL-2. (b) Scheme of the synthesis of PROTAC compounds MZ1–3 and cisMZ1; for detailed synthetic procedures see the Supporting Information. (c) Isothermal titration calorimetry data for titration of MZ1 into the individual members of the BET-bromodomain subfamily. Titrations were performed at 30 °C with a protein concentration of 15 μM and ligand concentration of 150 μM (entry 1–6). Titration of MZ1 and cisMZ1 into VBC at 25 °C with identical concentrations (entry 9, 12) and reverse titration of VBC protein (150 μM) into MZ3 (15 μM) at 25 °C (entry 10) were conducted. For ΔS and ΔG values, see the Supporting Information. (d) HeLa cells were treated with either siRNA targeting individual BET proteins or negative control siRNA 24 h prior to treatment with the compounds MZ1–3, cisMZ1, and JQ1 or vehicle control (0.01% DMSO) for an additional 24 h. Abundance of individual BET protein was analyzed by Western blotting using corresponding specific antibodies accordingly after SDS-PAGE. i, data from ref 8; ii, data from ref 26.
Small molecule chemical probes or inhibitors acting at the post-translational level hold several advantages for target validation over genetic techniques such as dominant-negative mutants or knockouts and RNAi knockdowns, including affording spatial and temporal control in a reversible fashion. A general limitation associated with conventional occupancy-driven target inhibition is that it often demands full target engagement, requiring sustained high concentration of a potent small molecule inhibitor over a prolonged time. This in turn enhances off-target effects and can lead to unwanted side effects or toxicity in a therapeutic setting. To provide an alternative small molecule approach that could address these issues, we hypothesized that it would be possible to design a molecule that can remove BET proteins entirely from the cell as opposed to just inhibit them, yielding new tools for studying BET bromodomain proteins and validating them as drug targets. In order to achieve intracellular BET-protein degradation, we applied a small molecule PROTAC (Proteolysis Targeting Chimera) approach. (18, 19) A PROTAC is a heterobifunctional compound that contains two ligands connected by a linker unit. One ligand binds an E3 ubiquitin ligase protein, while the other ligand binds to the target protein of interest, thereby bringing the ligase and the target in close proximity. This in turn triggers the polyubiquitination and subsequent proteasome-dependent degradation of the target. Proof-of-concept examples have been described where PROTACs were used to degrade the estrogen (20)- and androgen-receptor, (21) methionine aminopeptidase-2, (22) as well as the aryl hydrocarbon receptor. (23) However, all first-generation PROTACs included a peptidic moiety as the E3 ligase ligand. For example, a hydroxyproline-containing heptapeptide sequence ALA-Hyp-YIP from the transcription factor Hypoxia-Inducible Factor 1 alpha subunit (HIF-1α) has been widely used, (24) as this represents the minimal epitope for HIF-1α binding to the ubiquitously expressed E3 ligase von Hippel-Lindau protein (VHL). (25) The high peptidic nature of the first-generation PROTACs resulted in poor physicochemical properties such as low intracellular stability and poor cell permeability, which limited their applicability as chemical probes and their potential therapeutic development. To overcome these limitations here, we develop a nonpeptidic PROTAC approach that exploits our recently discovered and optimized drug-like VHL ligands (26) and show that it can be applied to target BET bromodomains and potently induce effective and selective degradation of BRD4.
We began by designing a series of PROTACs that would link together specific VHL ligands and BET bromodomain ligands. Recent work has established compounds VHL-1 and VHL-2 as strong binders with Kd values below 300 nM to VHL (Figure 1a). (26) Inspection of the protein–ligand crystal structures show that the methyl group of the terminal acetyl groups in compounds VHL-1 and VHL-2 is solvent exposed, and we therefore reasoned that it could provide a suitable connecting point for a linker (Figure S1). The BET inhibitor JQ1 (8) was chosen as the bromodomains-recruiting scaffold, and its t-butyl ester group was selected as a connecting point for a linker because it is solvent-exposed and not involved in key interaction with the BET bromodomains as revealed by cocrystal structures (Figure S1). Linkers with different lengths comprised of polyethylene glycol chains with either three or four ethylene glycol units were chosen to connect JQ1 with the VHL ligand. To achieve the desired ligands, a generally applicable two-step synthetic strategy was devised. First, the linker bearing a carboxylic acid at one end and an azide group at the other end was connected with the terminal free amine of the VHL ligand by a HATU-mediated amide bond formation. In the second step, reduction of the azide group to an amine and subsequent amide bond formation with the carboxylic acid of the ester-hydrolyzed JQ1 analogue afforded the desired PROTAC compounds MZ1, MZ2, MZ3, and cisMZ1 (Figure 1b).
To assess whether PROTAC molecules retained their binding to the target proteins VHL and BET bromodomains in a similar fashion as the parental ligands, isothermal titration calorimetry (ITC) experiments were performed (Figure 1; all ITC titrations are shown in the Supporting Information). MZ1, as a representative of all PROTAC molecules that share the same JQ1 moiety for binding bromodomains, was titrated into individual first and second bromodomains of BRD2, BRD3, and BRD4 (Figure 1c, entries 1−6). The measured binding affinities (Kd of 115–382 nM) and ΔH (−6.1 to −10.0 kcal/mol) compared well with those reported for unmodified JQ1 (8) (literature values for BRD4 bromodomains shown in Figure 1c, entries 7, 8), suggesting that JQ1 binding mode is conserved within the context of our PROTACs. Similarly, as binding to the VHL protein is crucial for the recruitment of target proteins to the E3 ligase, the binding of MZ1 and MZ3 to the VHL-ElonginB-ElonginC complex (VBC) was also quantified using ITC (Figure 1c, entries 9, 10). The measured affinities (Kd of 150 and 310 nM for MZ1 and MZ3, respectively) and ΔH (−6.9 and −4.9 kcal/mol, respectively) compared very closely to those of the parental unmodified ligands VHL-1 (Kd = 185 nM, ΔH = −5.5 kcal/mol, entry 11) and VHL-2 (Kd = 290 nM, ΔH = −5.3 kcal/mol). (26) As the stereochemistry of the hydroxyl group of the central hydroxyproline moiety is crucial for ligand binding to VHL, compound cisMZ1 was synthesized that is structurally identical to MZ1 except for a reversed stereocenter at the C-4 position bearing the hydroxyl group. As expected, cisMZ1 did not exhibit any measurable binding affinity for VHL in the ITC experiment (Figure 1c, entry 12) and thus was elected as a negative control compound in cellular assays.
To demonstrate that PROTACs are able to induce degradation of BET proteins, HeLa cells transfected with control siRNA were treated with 1 μM of compounds MZ1–3 alongside negative controls JQ1 and cisMZ1 for 24 h (Figure 1d). In parallel, HeLa cells with BRD2, BRD3, and BRD4 individually and separately silenced by transfection with the respective siRNA were treated with vehicle DMSO to compare the protein depletion effect of RNAi knockdown and PROTACs. BET protein abundance was evaluated by SDS-PAGE followed by Western blot using corresponding specific antibodies to probe for BRD2, BRD3 or BRD4, respectively. All three PROTAC compounds demonstrated complete removal of BRD4 with no detectable protein observed after 24 h of treatment. In contrast, removal of BRD2 and BRD3 was not complete after 24 h. MZ1 exhibited the highest efficacy among the three compounds. MZ2, which is structurally analogous to MZ1 except for a longer linker containing four PEG units, showed a weaker removal effect compared to MZ1. MZ3, containing an additional phenylalanine moiety between the linker and the VHL ligand, showed to be the least effective at removing BRD2 and BRD3. Together, the data demonstrate potent and effective degradation of BET proteins and suggested a preferential degradation effect on BRD4 over BRD2 and BRD3. The latter observation was unexpected given the parental compound JQ1 is a pan-BET inhibitor and our PROTACs bind with similar affinities to BET bromodomains. Nevertheless, the attractive opportunity to achieve single target selectivity prompted us to conduct further characterization.
To assess the compound dose- and time-dependent intracellular activities, HeLa cells were first treated with various concentrations of MZ1, MZ2, and MZ3 (Figure 2a). All three compounds showed concentration dependent BET removal activity with higher activity at higher concentrations. As in the initial experiment, MZ1 proved the most active compound, with more than 90% of all BET proteins being removed at compound concentration down to 1 μM. Remarkably, preferential removal of BRD4 over BRD2 and BRD3 was confirmed with all three compounds. Such preference is more prominent with treatment at lower concentrations, e.g., 0.1–0.5 μM. To study the activities of our PROTACs over time, HeLa cells were treated with 1 μM or 0.1 μM MZ1, and cellular BET protein levels were monitored in a time course experiment (Figure 2b for representative data with MZ1, see Figure S2 for additional data with other compounds and concentrations). Progressive removal of BET proteins over time was observed in all experimental setups, and BRD4 consistently exhibited the strongest and fastest reduction in protein level. Reassuringly, no BET protein degradation was observed in the presence of either DMSO or JQ1 (Figure S3) or cisMZ1 (Figure 3a). To verify whether the observation of preferential removal for BRD4 by our PROTACs can be observed in another cell line, the same study was carried out in U2OS osteosarcoma cells, and the same activity profile was observed (Figure S4). To visualize the BET protein degradation process, U2OS cells were transfected with a plasmid coding for a green fluorescent protein (GFP) tagged BRD4 protein, allowing fluorescence readout of BRD4 within the cell nuclei. Cells were induced to express GFP-BRD4 for 24 h and then were treated with either 5 μM MZ1 or 5 μM cisMZ1, and the fluorescence was observed over time. In the presence of the active compound MZ1, a complete depletion of the fluorescence signal was observed after just 3 h, whereas cisMZ1 caused no change in the fluorescence signal over the course of the experiment (Figure 2c, Figure S5 and Supporting Information videos a and b). These data confirmed that BRD4 is removed from the cell nuclei in a time dependent manner due to the presence of MZ1. Taken together, time and dose–response activity profiles revealed rapid and effective PROTAC-induced preferential degradation of BRD4 over BRD2 and BRD3.
Figure 2
Figure 2. PROTACs induce concentration- and time-dependent selective degradation of BRD4. (a) HeLa cells treated for 24 h with different concentrations of MZ1 (panel I), MZ2 (panel II), and MZ3 (panel III). The bands observed in the BRD4 short isoform lane at a high concentration of each compound are correlated to nonspecific binding. (b) Time dependent treatment over 36 h of HeLa cells with 1 μM (panel I) and 100 nM (panel II) of MZ1. (c) U2OS cells transfected with GFP-BRD4 were treated with either 5 μM of MZ1 or cisMZ1 over a time course of 4 h. BRD4 degradation over time was followed by live fluorescence imaging.
Figure 3
Figure 3. Mechanistic studies on PROTAC biological activity. (a) Time dependent treatment over 36 h of HeLa cells with 1 μM inactive compound cisMZ1. (b) HeLa cells treated with JQ1 or MZ1 at 1 μM in the absence or presence of the proteasome inhibitor MG132. (c) Time dependent treatment over 36 h of HeLa cells with 1 μM MZ1 observing the levels of the von Hippel-Lindau (VHL) protein. (d) HeLa cells treated with 100 μM CoCl2 as a hypoxia control or 0.1, 1, and 10 μM MZ1. (e) BRD4 protein levels were observed (panel I) with single treatment of MZ1 at t = 0 for 4 h and then exchange of media, (panel II) single treatment with MZ1 at t = 0 but no exchange of media, and (panel III) single treatment with 0.01% DMSO for 4 h and then exchange of media.
To gain mechanistic insights, the VHL and proteasome dependency of PROTAC-mediated protein degradation was first examined. cisMZ1 was unable to induce degradation of any of the BET proteins over time (Figure 3a), demonstrating that PROTAC efficacy is dependent on productive recruitment of VHL. Next, the reliance of the PROTAC-induced protein degradation on proteasome activity was assessed using proteasome inhibitor MG132. Treatment with MG132 completely abrogated MZ1-induced degradation of all BET proteins (compare lanes 3 and 6 in Figure 3b), establishing the expected proteasome-dependence of the approach. Interestingly, MG132 treatment in the absence of PROTAC showed no significant accumulation in BET protein levels, either alone or in combination with JQ1 (compare lanes 1 and 2 with 4 and 5 in Figure 3b, respectively), suggesting that basal proteasome activity level against BET proteins is negligible under those conditions and only becomes significant as a result of PROTAC treatment.
To further evaluate the biological activity of our compounds, we asked whether PROTAC treatment had any effect on the levels of its target E3 ligase (VHL) and on the level of HIF-1α, the natural substrate of VHL. VHL levels in the presence of MZ1 (1 μM) remained unaffected over the course of up to 36 h, thus indicating that the amount of E3 ligase is not influenced by MZ1 binding (Figure 3c). On the other hand, as the VHL ligand portions of our PROTACs occupy the same binding site on VHL that is used to recruit HIF-1α, PROTACs could block HIF-1α binding to VHL to an extent that it may lead to potential stabilization of HIF-1α within cells. For the approach herein described, this effect would not be desirable as up-regulation of HIF-1α transcriptional activity would confound the effects resulting from degradation of BET proteins and would be expected to result in induction of the hypoxic response, potentially giving rise to unwanted side effects. To assess whether any HIF-1α stabilization could be observed, HeLa cells were treated with MZ1 and with cobalt(II) chloride as a hypoxia mimicking positive control. Reassuringly, we could not observe any evidence of HIF-1α stabilization even at concentrations of MZ1 up to 10 μM, while clear HIF-1α stabilization is observed in the presence of CoCl2 (Figure 3d).
A number of non-BET potential off-targets of JQ1 have been recently reported, among which proteins DDB1 and RAD23B (hHR23b) were validated by proteome labeling and Western blotting. (27) To assess whether MZ1 causes degradation of these off-targets, protein levels were examined in HeLa cells treated with MZ1 at 1 μM and 100 nM over a time course of 36 h, and no degradation was observed (Figure S6). Next, to determine whether the removal of BET proteins by PROTAC treatment is reversible, and to establish how long it would take for cells to reverse the effect, we treated HeLa cells for 4 h with 1 μM of MZ1, removed the compound from the media, and then monitored protein levels over a period of 48 h. The washed cells showed detectable recovery of intracellular BRD4 only by 20 h after washout, while in the absence of the wash step, no protein could be detected even after 48 h (Figure 3e, see Figure S7 for the same experiment monitoring time-dependent levels of BRD2 and BRD3). Taken together, these results demonstrate that PROTAC-induced protein degradation is strictly dependent on binding to VHL, on proteasome activity, and does not interfere with the normal endogenous levels of both VHL and HIF-1α. Furthermore, the degradation effect is not only rapid but also sustained and long lasting even upon removal of the compound.
BET inhibitors such as JQ1 influence the expression of an assortment of genes. (28) Selective targeting of individual BET family members would be predicted to elicit distinct and more limited transcriptional responses, because the genome occupancy patterns of BET proteins are not identical. (29) To evaluate the functional consequences of removing BET proteins using PROTACs, and in particular of inducing selective degradation of BRD4 over BRD2 and BRD3, we next monitored the mRNA expression profiles of a selection of cancer-related genes which respond to JQ1 treatment and BET protein inhibition: MYC, P21, AREG, FAS, TYRO3, and FGFR1. The dependence of MYC and P21 expression on BRD4 activity is well characterized. MYC stimulates cell cycle progression and is constantly expressed upon misregulation in cancer, thus leading to continuous overexpression of downstream MYC-dependent genes. (30) In bone associated tumors (31) as well as leukemia and lymphoma cell lines, (32) JQ1 treatment or silencing of BRD4 resulted in downregulation of MYC. MYC represses transcription of the cell cycle CDK inhibitor P21, a tumor suppressor. (33) Downregulation of MYC and consequent derepression of P21 promotes cell cycle arrest. In contrast to the well characterized BRD4 dependency of MYC and P21, FAS, which encodes a proapoptotic protein belonging to the tumor necrosis factor receptor family, (34) is downregulated by depletion of BRD2, (35) while for the growth factors AREG and FGFR1 as well as the protein tyrosine kinase TYRO3 little is known about any BET protein specific regulation. However, these four genes are known to strongly respond to treatment with JQ1 (36) and therefore were included as a representative set of genes to compare between the pan-BET inhibitory effect caused by JQ1 and a selective BRD4 degradation caused by MZ1. Treatment with MZ1 at 100 nM for 24 h was chosen as this provided an optimal condition and the lowest effective concentration for achieving selective degradation of BRD4 over BRD2 and BRD3 and at the same time minimizing potential interference due to BET bromodomain inhibition (Figure 2a panel I and Figure 2b panel II). In addition, treatments with negative control compound VHL-1′ (Figure S8) lacking the JQ1 moiety, as well as with JQ1 itself, were also conducted to provide comparisons. Treatment of MZ1 resulted in down regulation of MYC, similar to JQ1, after 12 h (Figure S9), although MYC levels recovered after 24 h. Treatment with MZ1 and JQ1 resulted in similar upregulation of P21 and AREG both after 12 h (Figure S9) and 24 h (Figure 4a). Interestingly, in contrast to JQ1, which resulted in significant changes on FAS, TYRO3, and FGFR1, MZ1 showed more subtle and less significant effects on these genes relative to VHL-1′ (Figure 4a and Figure S9). We hypothesize that such differences observed in gene modulation may be the result of preferential degradation of BRD4 over the other two BET proteins caused by MZ1. To test this hypothesis, we suppressed individual BRD2, BRD3, or BRD4 genes using siRNA to mimic the protein removal effect (Figure S10) and analyzed the gene expression level of the target genes of interest (Figure 4b). While MYC, P21, and AREG levels were confirmed to be affected by suppression of BRD4, we found that FAS was downregulated upon suppression of BRD2 only but not BRD4 (Figure 4b), while FGFR1 is upregulated upon suppression of either BRD3 or BRD4. These results are consistent with preferential degradation of BRD4 over BRD2 and BRD3 by MZ1 and point to a more BRD4-selective pharmacological profile of MZ1 compared with pan-selective inhibitor JQ1.
Figure 4
Figure 4. Selective degradation of BRD4 leads to a differential response between JQ1 and MZ1 on selected genes. mRNA expression profiles of MYC, P21, AREG, FAS, FGFR1, and TYRO3 upon treatment with PROTAC MZ1 and JQ1 were compared. (a) HeLa cells were treated with 100 nM MZ1, VHL-1′, or JQ1 or 0.01% DMSO vehicle control (Veh.) for 24 h. (b) To mimic the protein removal effect, HeLa cells were transfected with siRNA targeting individual BRD2, BRD3, or BRD4 or negative control siRNA and were harvested after 48 h. Quantitative PCR was performed to analyze relative gene expression level of treated HeLa cells using target specific primers. Gene expression levels relative to GAPDH were normalized to control treatment. The data shown represent the mean ± SEM (n = 3 technical replicates) of one experiment. Statistical significance compared to the control was determined with two-tailed t tests: *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. = not significant.
In summary, we report a small molecule PROTAC approach achieving rapid, effective, and prolonged intracellular degradation of BET bromodomain proteins. The PROTAC-induced protein degradation is dependent on binding to VHL, is reversed upon blocking proteasome activity, and does not interfere with the endogenous, physiological levels of VHL and of its natural substrate HIF-1α. All investigated compounds showed preferential degradation of BRD4 over BRD2 and BRD3 at low concentrations. The downstream gene expression pattern resulting from treatment with our potent and selective PROTAC MZ1 is similar to JQ1 inhibition for BRD4-dependent genes MYC, P21, and AREG but not for FAS, FGFR1, and TYRO3. Our results suggest a different pharmacological response resulting from selectively depleting BRD4 with MZ1 compared to inhibiting the whole BET protein subfamily with JQ1. Given that no preference for binding the bromodomains of BRD4 over the highly homologous bromodomains of BRD2 and BRD3 was observed by ITC within the context of the purified proteins, we speculate that the observed selectivity could arise from preferential and more efficient polyubiquitination of lysine residues on the surface of BRD4 compared to those of BRD2 and BRD3. Alternatively or in addition, preferential direct interaction or reduced steric constraints between VHL and BRD4 compared to BRD2/3 may occur as a result of PROTAC binding, triggering a more productive formation of a VHL:PROTAC:BRD4 ternary complex. Elucidation of the molecular basis for the BRD4-selective activity of PROTACs will warrant further mechanistic investigation in the future. Our findings demonstrating effective and selective degradation of BRD4 with a PROTAC approach open up unprecedented opportunities to study the downstream physiological and pathological consequences of BRD4 modulation. It will allow determination of whether more selective pharmacological perturbations of BET protein function will have improved therapeutic efficacy, potentially leading to more efficient and specific new drugs in the future. Finally, potent chemical probes that bind to human bromodomains outside the BET subfamily are beginning to emerge, (37) which could be similarly conjugated to a VHL ligand to induce selective intracellular degradation of their respective target bromodomain-containing proteins.
Methods
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For detailed descriptions of synthetic and biological methods, see the Supporting Information.
Supporting Information
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Additional figures, tables, materials and methods, and .avi videos. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00216.
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Acknowledgment
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This work was supported by awards to A.C. from the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant BB/J001201/2 and David Phillips Fellowship BB/G023123/2) and the European Research Council (ERC, Starting Grant ERC-2012-StG-311460 DrugE3CRLs). Light microscopy is supported by a Wellcome Trust strategic award to the University of Dundee (097945/Z/11/Z). We thank P. Soares and S. Scaffidi for providing VHL ligand precursors, M. Ferguson, L. Guther and S. Damerow for providing access and assistance with the LI-COR Odyssey system, S. Swift and C. Thomson of the Dundee Imaging Facility for assistance with the light microscopy, and K. Airey of the Dundee MRC tissue culture facility for training and assistance. The University of Dundee and the authors have filed a patent application (GB1504314.4) related to the use of BET bromodomain targeting PROTACs to induce degradation of BET proteins.
Added in Proof
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Around the time the "Just Accepted" version of this paper was published online, two independent reports have come out online describing pan-BET selective PROTAC compounds dBET1 (DOI: 10.1126/science.aab1433) and ARV-825 (DOI: 10.1016/j.chembiol.2015.05.009) that conjugate the same BET bromodomain recruiting ligand JQ1 to a ligand for a different E3 ligase, cereblon.
References
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This article references 37 other publications.
- 1Gallenkamp, D., Gelato, K. A., Haendler, B., and Weinmann, H. (2014) Bromodomains and their pharmacological inhibitors ChemMedChem. 9, 438– 464[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsl2gtL4%253D&md5=2ce7c6b5c37001fb8fb901fe04aeb606Bromodomains and Their Pharmacological InhibitorsGallenkamp, Daniel; Gelato, Kathy A.; Haendler, Bernard; Weinmann, HilmarChemMedChem (2014), 9 (3), 438-464CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Over 60 bromodomains belonging to proteins with very different functions have been identified in humans. Several of them interact with acetylated lysine residues, leading to the recruitment and stabilization of protein complexes. The bromodomain and extra-terminal domain (BET) proteins contain tandem bromodomains which bind to acetylated histones and are thereby implicated in a no. of DNA-centered processes, including the regulation of gene expression. The recent identification of inhibitors of BET and non-BET bromodomains is one of the few examples in which effective blockade of a protein-protein interaction can be achieved with a small mol. This has led to major strides in the understanding of the function of bromodomain-contg. proteins and their involvement in diseases such as cancer and inflammation. Indeed, BET bromodomain inhibitors are now being clin. evaluated for the treatment of hematol. tumors and have also been tested in clin. trials for the relatively rare BRD-NUT midline carcinoma. This review gives an overview of the newest developments in the field, with a focus on the biol. of selected bromodomain proteins on the one hand, and on reported pharmacol. inhibitors on the other, including recent examples from the patent literature.
- 2Belkina, A. C. and Denis, G. V. (2012) BET domain co-regulators in obesity, inflammation and cancer Nat. Rev. Cancer 12, 465– 477[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovFyks7s%253D&md5=d06d88ef0d5d4d71115f40a2f901f53dBET domain co-regulators in obesity, inflammation and cancerBelkina, Anna C.; Denis, Gerald V.Nature Reviews Cancer (2012), 12 (7), 465-477CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. The bromodomain is a highly conserved motif of 110 amino acids that is bundled into four anti-parallel α-helixes and found in proteins that interact with chromatin, such as transcription factors, histone acetylases and nucleosome remodelling complexes. Bromodomain proteins are chromatin 'readers'; they recruit chromatin-regulating enzymes, including 'writers' and 'erasers' of histone modification, to target promoters and to regulate gene expression. Conventional wisdom held that complexes involved in chromatin dynamics are not 'druggable' targets. However, small mols. that inhibit bromodomain and extraterminal (BET) proteins have been described. We examine these developments and discuss the implications for small mol. epigenetic targeting of chromatin networks in cancer.
- 3Zuber, J., Shi, J., Wang, E., Rappaport, A. R., Herrmann, H., Sison, E. A., Magoon, D., Qi, J., Blatt, K., Wunderlich, M., Taylor, M. J., Johns, C., Chicas, A., Mulloy, J. C., Kogan, S. C., Brown, P., Valent, P., Bradner, J. E., Lowe, S. W., and Vakoc, C. R. (2011) RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia Nature 478, 524– 528[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFers7%252FP&md5=618793a766f037bff5909524743fedb7RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemiaZuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R.; Herrmann, Harald; Sison, Edward A.; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J.; Johns, Christopher; Chicas, Agustin; Mulloy, James C.; Kogan, Scott C.; Brown, Patrick; Valent, Peter; Bradner, James E.; Lowe, Scott W.; Vakoc, Christopher R.Nature (London, United Kingdom) (2011), 478 (7370), 524-528CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here the authors describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukemia (AML), an aggressive hematopoietic malignancy that is often assocd. with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, the authors identify the protein bromodomain-contg. 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-mol. inhibitor JQ1 led to robust antileukemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukemia stem cells. Similar sensitivities were obsd. in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacol. means to suppress MYC in cancer. The authors' results establish small-mol. inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacol. intervention.
- 4Baratta, M. G., Schinzel, A. C., Zwang, Y., Bandopadhayay, P., Bowman-Colin, C., Kutt, J., Curtis, J., Piao, H., Wong, L. C., Kung, A. L., Beroukhim, R., Bradner, J. E., Drapkin, R., Hahn, W. C., Liu, J. F., and Livingston, D. M. (2015) An in-tumor genetic screen reveals that the BET bromodomain protein, BRD4, is a potential therapeutic target in ovarian carcinoma Proc. Natl. Acad. Sci. U. S. A. 112, 232– 237
- 5Chung, C.-W., Coste, H., White, J. H., Mirguet, O., Wilde, J., Gosmini, R. L., Delves, C., Magny, S. M., Woodward, R., Hughes, S. A., Boursier, E. V., Flynn, H., Bouillot, A. M., Bamborough, P., Brusq, J.-M. G., Gellibert, F. J., Jones, E. J., Riou, A. M., Homes, P., Martin, S. L., Uings, I. J., Toum, J., Clément, C. A., Boullay, A.-B., Grimley, R. L., Blandel, F. M., Prinjha, R. K., Lee, K., Kirilovsky, J., and Nicodeme, E. (2011) Discovery and characterization of small molecule inhibitors of the BET family bromodomains J. Med. Chem. 54, 3827– 3838[ACS Full Text
], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtVWnurs%253D&md5=ebc0f6062321dece877259d775f1c19bDiscovery and characterization of small molecule inhibitors of the BET family bromodomainsChung, Chun-wa; Coste, Herve; White, Julia H.; Mirguet, Olivier; Wilde, Jonathan; Gosmini, Romain L.; Delves, Chris; Magny, Sylvie M.; Woodward, Robert; Hughes, Stephen A.; Boursier, Eric V.; Flynn, Helen; Bouillot, Anne M.; Bamborough, Paul; Brusq, Jean-Marie G.; Gellibert, Francoise J.; Jones, Emma J.; Riou, Alizon M.; Homes, Paul; Martin, Sandrine L.; Uings, Iain J.; Toum, Jerome; Clement, Catherine A.; Boullay, Anne-Benedicte; Grimley, Rachel L.; Blandel, Florence M.; Prinjha, Rab K.; Lee, Kevin; Kirilovsky, Jorge; Nicodeme, EdwigeJournal of Medicinal Chemistry (2011), 54 (11), 3827-3838CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biol. and structural characterization of many bromodomain contg. proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and mol. characterization of potent (nM) small mol. inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophys. studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small mols. that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compds. bound into bromodomains of Brd2 and Brd4 elucidate the mol. interactions of binding and explain the precisely defined stereochem. required for activity. - 6Khan, Y. M., Kirkham, P., Barnes, P. J., and Adcock, I. M. (2014) Brd4 is essential for IL-1β-induced inflammation in human airway epithelial cells PLoS One 9, e95051
- 7Filippakopoulos, P. and Knapp, S. (2012) The bromodomain interaction module FEBS Lett. 586, 2692– 2704[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntFahsbg%253D&md5=c02dee22c330b1d5c1ba39579360ebc5The bromodomain interaction moduleFilippakopoulos, Panagis; Knapp, StefanFEBS Letters (2012), 586 (17), 2692-2704CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. The ε-N-acetylation of protein Lys residues (Kac) is one of the most abundant post-translation modifications (PTMs) in the human proteome. In the nucleus, the acetylation of histones has been linked to transcriptional activation of genes but the functional consequences of most acetylation events and proteins recruited to these sites remains largely unknown. Bromodomains (BRDs) are small helical interaction modules that specifically recognize acetylation sites in proteins. BRDs have recently emerged as interesting targets for the development of specific protein interaction inhibitors, enabling a novel exiting strategy for the development of new therapies. Here, the author provides an overview over sequence requirements of BRDs, known substrates, and the structural mechanisms of specific Kac recognition.
- 8Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O., Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET bromodomains Nature 468, 1067– 1073[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXotlGmtA%253D%253D&md5=bd9cadac86124d2c6c3cd5b2a92d68c2Selective inhibition of BET bromodomainsFilippakopoulos, Panagis; Qi, Jun; Picaud, Sarah; Shen, Yao; Smith, William B.; Fedorov, Oleg; Morse, Elizabeth M.; Keates, Tracey; Hickman, Tyler T.; Felletar, Ildiko; Philpott, Martin; Munro, Shongah; McKeown, Michael R.; Wang, Yuchuan; Christie, Amanda L.; West, Nathan; Cameron, Michael J.; Schwartz, Brian; Heightman, Tom D.; La Thangue, Nicholas; French, Christopher; Wiest, Olaf; Kung, Andrew L.; Knapp, Stefan; Bradner, James E.Nature (London, United Kingdom) (2010), 468 (7327), 1067-1073CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here the authors report a cell-permeable small mol. (I,JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is obsd. in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chem. scaffold for the development of chem. probes more broadly throughout the bromodomain family.
- 9Nicodeme, E., Jeffrey, K. L., Schaefer, U., Beinke, S., Dewell, S., Chung, C.-W., Chandwani, R., Marazzi, I., Wilson, P., Coste, H., White, J., Kirilovsky, J., Rice, C. M., Lora, J. M., Prinjha, R. K., Lee, K., and Tarakhovsky, A. (2010) Suppression of inflammation by a synthetic histone mimic Nature 468, 1119– 1123[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtl2rtb%252FP&md5=0dfccd9d01b87859512bf591e1c5a93cSuppression of inflammation by a synthetic histone mimicNicodeme, Edwige; Jeffrey, Kate L.; Schaefer, Uwe; Beinke, Soren; Dewell, Scott; Chung, Chun-wa; Chandwani, Rohit; Marazzi, Ivan; Wilson, Paul; Coste, Herve; White, Julia; Kirilovsky, Jorge; Rice, Charles M.; Lora, Jose M.; Prinjha, Rab K.; Lee, Kevin; Tarakhovsky, AlexanderNature (London, United Kingdom) (2010), 468 (7327), 1119-1123CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defense, is frequently deleterious to the host due to the exaggerated prodn. of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signaling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a crit. step in the regulation of gene expression. Here we present a novel pharmacol. approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compd. (I-BET) that by mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compds. specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.
- 10Boi, M., Gaudio, E., Bonetti, P., Kwee, I., Bernasconi, E., Tarantelli, C., Rinaldi, A., Testoni, M., Cascione, L., Ponzoni, M., Mensah, A. A., Stathis, A., Stussi, G., Riveiro, M. E., Herait, P., Inghirami, G., Cvitkovic, E., Zucca, E., and Bertoni, F. (2015) The BET Bromodomain inhibitor OTX015 affects pathogenetic pathways in pre-clinical B-cell tumor models and synergizes with targeted drugs Clin. Cancer Res. 21, 1628– 1638[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtVahsbc%253D&md5=5742b4d5884261d546588a339f3c2ea0The BET Bromodomain Inhibitor OTX015 Affects Pathogenetic Pathways in Preclinical B-cell Tumor Models and Synergizes with Targeted DrugsBoi, Michela; Gaudio, Eugenio; Bonetti, Paola; Kwee, Ivo; Bernasconi, Elena; Tarantelli, Chiara; Rinaldi, Andrea; Testoni, Monica; Cascione, Luciano; Ponzoni, Maurilio; Mensah, Afua Adjeiwaa; Stathis, Anastasios; Stussi, Georg; Riveiro, Maria Eugenia; Herait, Patrice; Inghirami, Giorgio; Cvitkovic, Esteban; Zucca, Emanuele; Bertoni, FrancescoClinical Cancer Research (2015), 21 (7), 1628-1638CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-contg. proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015. We assessed the preclin. activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo expts. to identify the mechanism of action and the genetic features assocd. with sensitivity to the compd. OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo expts. showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, esp. with mTOR and BTK inhibitors. Gene expression signatures assocd. with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compd. induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell-like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11. Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clin. investigation of OTX015 as single agent and in combination therapies. Clin Cancer Res; 21(7); 1628-38. ©2015 AACR.
- 11Gosmini, R., Nguyen, V. L., Toum, J., Simon, C., Brusq, J.-M. G., Krysa, G., Mirguet, O., Riou-Eymard, A. M., Boursier, E. V., Trottet, L., Bamborough, P., Clark, H., Chung, C.-W., Cutler, L., Demont, E. H., Kaur, R., Lewis, A. J., Schilling, M. B., Soden, P. E., Taylor, S., Walker, A. L., Walker, M. D., Prinjha, R. K., and Nicodeme, E. (2014) The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor J. Med. Chem. 57, 8111– 8131[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Wlur7J&md5=b1c9c9cae73c41252cf73908d561bd9bThe Discovery of I-BET726 (GSK1324726A), a Potent Tetrahydroquinoline ApoA1 Up-Regulator and Selective BET Bromodomain InhibitorGosmini, Romain; Nguyen, Van Loc; Toum, Jerome; Simon, Christophe; Brusq, Jean-Marie G.; Krysa, Gael; Mirguet, Olivier; Riou-Eymard, Alizon M.; Boursier, Eric V.; Trottet, Lionel; Bamborough, Paul; Clark, Hugh; Chung, Chun-wa; Cutler, Leanne; Demont, Emmanuel H.; Kaur, Rejbinder; Lewis, Antonia J.; Schilling, Mark B.; Soden, Peter E.; Taylor, Simon; Walker, Ann L.; Walker, Matthew D.; Prinjha, Rab K.; Nicodeme, EdwigeJournal of Medicinal Chemistry (2014), 57 (19), 8111-8131CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Through their function as epigenetic readers of the histone code, the BET family of bromodomain-contg. proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncol. and inflammation models, and the first compds. have now progressed into clin. trials. The exciting biol. of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compds. active in murine models of septic shock and neuroblastoma. At the mol. level, these effects are produced by inhibition of BET bromodomains. X-ray crystallog. reveals the interactions explaining the structure-activity relationships of binding. The resulting lead mol., I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.
- 12Mirguet, O., Lamotte, Y., Donche, F., Toum, J., Gellibert, F., Bouillot, A., Gosmini, R., Nguyen, V. L., Delannée, D., Seal, J., Blandel, F., Boullay, A.-B., Boursier, E., Martin, S., Brusq, J.-M., Krysa, G., Riou, A., Tellier, R., Costaz, A., Huet, P., Dudit, Y., Trottet, L., Kirilovsky, J., and Nicodeme, E. (2012) From ApoA1 upregulation to BET family bromodomain inhibition: Discovery of I-BET151 Bioorg. Med. Chem. Lett. 22, 2963– 2967[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjt1elsbY%253D&md5=48c9a8dc4b70bea4a86807f7a1cec5f1From ApoA1 upregulation to BET family bromodomain inhibition: Discovery of I-BET151Mirguet, Olivier; Lamotte, Yann; Donche, Frederic; Toum, Jerome; Gellibert, Francoise; Bouillot, Anne; Gosmini, Romain; Nguyen, Van-Loc; Delannee, Delphine; Seal, Jonathan; Blandel, Florence; Boullay, Anne-Benedicte; Boursier, Eric; Martin, Sandrine; Brusq, Jean-Marie; Krysa, Gael; Riou, Alizon; Tellier, Remi; Costaz, Agnes; Huet, Pascal; Dudit, Yann; Trottet, Lionel; Kirilovsky, Jorge; Nicodeme, EdwigeBioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2963-2967CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The discovery, synthesis, and biol. evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.
- 13McLure, K. G., Gesner, E. M., Tsujikawa, L., Kharenko, O. A., Attwell, S., Campeau, E., Wasiak, S., Stein, A., White, A., Fontano, E., Suto, R. K., Wong, N. C. W., Wagner, G. S., Hansen, H. C., and Young, P. R. (2013) RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain antagonist PLoS One 8, e83190
- 14Cheng, Z., Gong, Y., Ma, Y., Lu, K., Lu, X., Pierce, L. A., Thompson, R. C., Müller, S., Knapp, S., and Wang, J. (2013) Inhibition of BET bromodomain targets genetically diverse glioblastoma Clin. Cancer Res. 19, 1748– 1759[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsVCmsLo%253D&md5=1722189e5d950da233b785558f2c9f58Inhibition of BET Bromodomain Targets Genetically Diverse GlioblastomaCheng, Zhixiang; Gong, Yuanying; Ma, Yufang; Lu, Kaihua; Lu, Xiang; Pierce, Larry A.; Thompson, Reid C.; Muller, Susanne; Knapp, Stefan; Wang, JialiangClinical Cancer Research (2013), 19 (7), 1748-1759CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Purpose: Glioblastoma is refractory to conventional therapies. The bromodomain and extraterminal domain (BET) proteins are epigenetic readers that selectively bind to acetylated lysine residues on histone tails. These proteins recently emerged as important therapeutic targets in NUT midline carcinoma and several types of hematopoietic cancers. In this study, the therapeutic potential of a novel BET bromodomain inhibitor, JQ1, was assessed in a panel of genetically heterogeneous glioblastoma samples. Exptl. Design: The antineoplastic effects of JQ1 were shown using ex vivo cultures derived from primary glioblastoma xenograft lines and surgical specimens of different genetic background. The in vivo efficacy was assessed in orthotopic glioblastoma tumors. Results: We showed that JQ1 induced marked G1 cell-cycle arrest and apoptosis, which was phenocopied by knockdown of individual BET family members. JQ1 treatment resulted in significant changes in expression of genes that play important roles in glioblastoma such as c-Myc, p21CIP1/WAF1, hTERT, Bcl-2, and Bcl-xL. Unlike the observations in some hematopoietic cancer cell lines, exogenous c-Myc did not significantly protect glioblastoma cells against JQ1. In contrast, ectopically expressed Bcl-xL partially rescued cells from JQ1-induced apoptosis, and knockdown of p21CIP1/WAF1 attenuated JQ1-induced cell-cycle arrest. Cells genetically engineered for Akt hyperactivation or p53/Rb inactivation did not compromise JQ1 efficacy, suggesting that these frequently mutated signaling pathways may not confer resistance to JQ1. Furthermore, JQ1 significantly repressed growth of orthotopic glioblastoma tumors. Conclusion: Our results suggest potentially broad therapeutic use of BET bromodomain inhibitors for treating genetically diverse glioblastoma tumors. Clin Cancer Res; 19(7); 1748-59. ©2013 AACR.
- 15Prinjha, R. K., Witherington, J., and Lee, K. (2012) Place your BETs: The therapeutic potential of bromodomains Trends Pharmacol. Sci. 33, 146– 153[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtlOlt74%253D&md5=ea1e9e5740b1131239aa0407748d549bPlace your BETs: the therapeutic potential of bromodomainsPrinjha, R. K.; Witherington, J.; Lee, K.Trends in Pharmacological Sciences (2012), 33 (3), 146-153CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Ltd.)A review. Therapeutic targeting of the processes that regulate histone modification is a growing area of scientific exploration. Although most interest has concd. on the various families of enzymes that contribute to these processes, this review focuses on emerging data demonstrating the chem. tractability and therapeutic potential of a hitherto underexplored family of proteins, namely the bromodomain (BRD) family of reader proteins. These proteins perform a crucial role in translating histone modifications with powerful transcriptional consequences. We review current knowledge of the biol. of this emergent target class and highlight recent breakthroughs that now make the BRD family of reader proteins attractive for drug discovery.
- 16Filippakopoulos, P. and Knapp, S. (2014) Targeting bromodomains: Epigenetic readers of lysine acetylation Nat. Rev. Drug Discovery 13, 337– 356[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmsFalsr4%253D&md5=77049c82ce6a19b4685e3b7950399a07Targeting bromodomains: epigenetic readers of lysine acetylationFilippakopoulos, Panagis; Knapp, StefanNature Reviews Drug Discovery (2014), 13 (5), 337-356CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Lysine acetylation is a key mechanism that regulates chromatin structure; aberrant acetylation levels have been linked to the development of several diseases. Acetyl-lysine modifications create docking sites for bromodomains, which are small interaction modules found on diverse proteins, some of which have a key role in the acetylation-dependent assembly of transcriptional regulator complexes. These complexes can then initiate transcriptional programs that result in phenotypic changes. The recent discovery of potent and highly specific inhibitors for the BET (bromodomain and extra-terminal) family of bromodomains has stimulated intensive research activity in diverse therapeutic areas, particularly in oncol., where BET proteins regulate the expression of key oncogenes and anti-apoptotic proteins. In addn., targeting BET bromodomains could hold potential for the treatment of inflammation and viral infection. Here, we highlight recent progress in the development of bromodomain inhibitors, and their potential applications in drug discovery.
- 17Baud, M. G. J., Lin-Shiao, E., Cardote, T., Tallant, C., Pschibul, A., Chan, K.-H., Zengerle, M., Garcia, J. R., Kwan, T. T. L., Ferguson, F. M., and Ciulli, A. (2014) A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes Science 346, 638– 641[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVWlt7nL&md5=04fe967e4681fb4b2b3168f4d5f8ff81A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probesBaud, Matthias G. J.; Lin-Shiao, Enrique; Cardote, Teresa; Tallant, Cynthia; Pschibul, Annica; Chan, Kwok-Ho; Zengerle, Michael; Garcia, Jordi R.; Kwan, Terence T.-L.; Ferguson, Fleur M.; Ciulli, AlessioScience (Washington, DC, United States) (2014), 346 (6209), 638-641CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Small mols. are useful tools for probing the biol. function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The bromodomain and extra-terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-mol. inhibitors, but these inhibitors lack selectivity for individual family members. We developed an Et deriv. of an existing small-mol. inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BET protein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BET proteins in human physiol. and disease.
- 18Raina, K. and Crews, C. M. (2010) Chemical inducers of targeted protein degradation J. Biol. Chem. 285, 11057– 11060[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktFGlsrs%253D&md5=f5142a890c2e1c859d58ddb25c077052Chemical Inducers of Targeted Protein DegradationRaina, Kanak; Crews, Craig M.Journal of Biological Chemistry (2010), 285 (15), 11057-11060CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. The functions of many cellular proteins have been elucidated by selective gene inactivation and subsequent phenotypic anal. For example, genetic mutations, gene knock-out generation, and the use of RNA interference to target mRNA for degrdn. can all result in decreased prodn. of a specific protein, yielding informative cellular phenotypes. However, these techniques each have certain inherent limitations. This minireview focuses on the recent development of new approaches to study protein function at the post-translational level, namely chem. induction of targeted protein degrdn.
- 19Corson, T. W., Aberle, N., and Crews, C. M. (2008) Design and applications of bifunctional small molecules: Why two heads are better than one ACS Chem. Biol. 3, 677– 692[ACS Full Text], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1yiurzF&md5=2edac15286d3a739dd66214a332a545bDesign and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than OneCorson, Timothy W.; Aberle, Nicholas; Crews, Craig M.ACS Chemical Biology (2008), 3 (11), 677-692CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Induction of protein-protein interactions is a daunting challenge, but recent studies show promise for small mols. that specifically bring two or more protein mols. together for enhanced or novel biol. effect. The first such bifunctional mols. were the rapamycin- and FK506-based "chem. inducers of dimerization", but the field has since expanded with new mols. and new applications in chem. genetics and cell biol. Examples include coumermycin-mediated gyrase B dimerization, proteolysis targeting chimeric mols. (PROTACs), drug hybrids, and strategies for exploiting multivalency in toxin binding and antibody recruitment. This Review discusses these and other advances in the design and use of bifunctional small mols. and potential strategies for future systems.
- 20Cyrus, K., Wehenkel, M., Choi, E. Y., Swanson, H., and Kim, K. B. (2010) Two-headed PROTAC: An effective new tool for targeted protein degradation ChemBioChem. 11, 1531– 1534
- 21Sakamoto, K. M., Kim, K. B., Verma, R., Ransick, A., Stein, B., Crews, C. M., and Deshaies, R. J. (2003) Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation Mol. Cell. Proteomics 2, 1350– 1358[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtVShu7jE&md5=5a9f4cf5d5cc1d990bf59ac824902111Development of Protacs to target cancer-promoting proteins for ubiquitination and degradationSakamoto, Kathleen M.; Kim, Kyung B.; Verma, Rati; Ransick, Andy; Stein, Bernd; Crews, Craig M.; Deshaies, Raymond J.Molecular and Cellular Proteomics (2003), 2 (12), 1350-1358CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small mol. inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. The authors previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degrdn. through a chimeric bridging mol. or Protac (proteolysis targeting chimeric mol.). This Protac consisted of an SCFβ-TRCP-binding phosphopeptide derived from IκBα linked to ovalicin, which covalently binds MetAP-2. In this study, the authors employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, resp. The authors show here that an estradiol-based Protac can enforce the ubiquitination and degrdn. of the α isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technol. may yield a general approach to treat a no. of human diseases, including cancer.
- 22Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., and Deshaies, R. J. (2001) Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation Proc. Natl. Acad. Sci. U. S. A. 98, 8554– 8559[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXls1Wisbk%253D&md5=540358035222f745f7b6367b38781a21Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradationSakamoto, Kathleen M.; Kim, Kyung B.; Kumagai, Akiko; Mercurio, Frank; Crews, Craig M.; Deshaies, Raymond J.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (15), 8554-8559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex contg. Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degrdn. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compd., protein-targeting chimeric mol. 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IκBα phosphopeptide that is recognized by the F-box protein β-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCFβ-TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.
- 23Lee, H., Puppala, D., Choi, E. Y., Swanson, H., and Kim, K. B. (2007) Targeted degradation of the aryl hydrocarbon receptor by the PROTAC approach: A useful chemical genetic tool ChemBioChem. 8, 2058– 2062
- 24Schneekloth, J. S., Fonseca, F. N., Koldobskiy, M., Mandal, A., Deshaies, R., Sakamoto, K., and Crews, C. M. (2004) Chemical genetic control of protein levels: Selective in vivo targeted degradation J. Am. Chem. Soc. 126, 3748– 3754[ACS Full Text], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvVegur0%253D&md5=f0855157e4daf32080a23e1728c4b9ccChemical genetic control of protein levels: selective in vivo targeted degradationSchneekloth, John S., Jr.; Fonseca, Fabiana N.; Koldobskiy, Michael; Mandal, Amit; Deshaies, Raymond; Sakamoto, Kathleen; Crews, Craig M.Journal of the American Chemical Society (2004), 126 (12), 3748-3754CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Genetic loss of function anal. is a powerful method for the study of protein function. However, some cell biol. questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of mols. capable of inducing the degrdn. of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric mols. (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, resp. These are the first in vivo examples of direct small mol.-induced recruitment of target proteins to the proteasome for degrdn. upon addn. to cultured cells. Moreover, PROTAC-mediated protein degrdn. offers a general strategy to create "chem. knockouts," thus opening new possibilities for the control of protein function.
- 25Hon, W.-C., Wilson, M. I., Harlos, K., Claridge, T. D. W., Schofield, C. J., Pugh, C. W., Maxwell, P. H., Ratcliffe, P. J., Stuart, D. I., and Jones, E. Y. (2002) Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL Nature 417, 975– 978
- 26Galdeano, C., Gadd, M. S., Soares, P., Scaffidi, S., Van Molle, I., Birced, I., Hewitt, S., Dias, D. M., and Ciulli, A. (2014) Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities J. Med. Chem. 57, 8657– 8663[ACS Full Text], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVGhtb%252FP&md5=831aa2387d94cb03d735a4be7bdd2cddStructure-Guided Design and Optimization of Small Molecules Targeting the Protein-Protein Interaction between the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase and the Hypoxia Inducible Factor (HIF) Alpha Subunit with in Vitro Nanomolar AffinitiesGaldeano, Carles; Gadd, Morgan S.; Soares, Pedro; Scaffidi, Salvatore; Van Molle, Inge; Birced, Ipek; Hewitt, Sarah; Dias, David M.; Ciulli, AlessioJournal of Medicinal Chemistry (2014), 57 (20), 8657-8663CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-mol. ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degrdn. The authors recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, the authors report the design and optimization, guided by x-ray crystal structures, of a ligand series with nanomolar binding affinities.
- 27Li, Z., Wang, D., Li, L., Pan, S., Na, Z., Tan, C. Y. J., and Yao, S. Q. (2014) “Minimalist” cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling J. Am. Chem. Soc. 136, 9990– 9998
- 28Shi, J. and Vakoc, C. R. (2014) The mechanisms behind the therapeutic activity of BET bromodomain inhibition Mol. Cell 54, 728– 736[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpslCrtrk%253D&md5=f49bf3524ec378576fb1a25c64a464aeThe Mechanisms behind the Therapeutic Activity of BET Bromodomain InhibitionShi, Junwei; Vakoc, Christopher R.Molecular Cell (2014), 54 (5), 728-736CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)A review. The bromodomain and extraterminal (BET) protein Brd4 recruits transcriptional regulatory complexes to acetylated chromatin. While Brd4 is considered to be a general transcriptional regulator, pharmacol. inhibition of BET proteins shows therapeutic activity in a variety of different pathologies, particularly in models of cancer and inflammation. Such effects have been attributed to a specific set of downstream target genes whose expression is disproportionately sensitive to pharmacol. targeting of BET proteins. Emerging evidence links the transcriptional consequences of BET inhibition to the assocn. of Brd4 with enhancer elements, which tend to be involved in lineage-specific gene regulation. Furthermore, Brd4 engages in direct regulatory interactions with several DNA-binding transcription factors to influence their disease-relevant functions. Here we review the current understanding of mol. mechanisms that underlie the promising therapeutic effects of BET bromodomain inhibition.
- 29Anders, L., Guenther, M. G., Qi, J., Fan, Z. P., Marineau, J. J., Rahl, P. B., Lovén, J., Sigova, A. A., Smith, W. B., Lee, T. I., Bradner, J. E., and Young, R. A. (2014) Genome-wide localization of small molecules Nat. Biotechnol. 32, 92– 96[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFentbrI&md5=6703796ae735da5279eb6a1f4e4d7d4dGenome-wide localization of small moleculesAnders, Lars; Guenther, Matthew G.; Qi, Jun; Fan, Zi Peng; Marineau, Jason J.; Rahl, Peter B.; Loven, Jakob; Sigova, Alla A.; Smith, William B.; Lee, Tong Ihn; Bradner, James E.; Young, Richard A.Nature Biotechnology (2014), 32 (1), 92-96CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)A vast no. of small-mol. ligands, including therapeutic drugs under development and in clin. use, elicit their effects by binding specific proteins assocd. with the genome. An ability to map the direct interactions of a chem. entity with chromatin genome-wide could provide important insights into chem. perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chem. mols. throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chem. entities that interact with DNA or genome-assocd. proteins.
- 30Bretones, G., Delgado, M. D., and León, J. (2014) Myc and cell cycle control Biochim. Biophys. Acta 1849, 506– 516
- 31Lamoureux, F., Baud’huin, M., Rodriguez Calleja, L., Jacques, C., Berreur, M., Rédini, F., Lecanda, F., Bradner, J. E., Heymann, D., and Ory, B. (2014) Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle Nat. Commun. 5, 3511[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2crltlentg%253D%253D&md5=a07f9e31ee509a1958d6c81071c6ea3bSelective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycleLamoureux Francois; Baud'huin Marc; Rodriguez Calleja Lidia; Jacques Camille; Berreur Martine; Redini Francoise; Ory Benjamin; Lecanda Fernando; Bradner James E; Heymann DominiqueNature communications (2014), 5 (), 3511 ISSN:.The vicious cycle established between bone-associated tumours and bone resorption is the central problem with therapeutic strategies against primary bone tumours and bone metastasis. Here we report data to support inhibition of BET bromodomain proteins as a promising therapeutic strategy that target simultaneously the three partners of the vicious cycle. Treatment with JQ1, a BET bromodomain inhibitor, reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation both in vitro and in vivo. These effects are associated with transcriptional silencing of MYC and RUNX2, resulting from the depletion of BRD4 from their respective loci. Moreover, JQ1 also inhibits osteoclast differentiation by interfering with BRD4-dependent RANKL activation of NFATC1 transcription. Collectively, our data indicate that JQ1 is a potent inhibitor of osteoblast and osteoclast differentiation as well as bone tumour development.
- 32Mertz, J. A., Conery, A. R., Bryant, B. M., Sandy, P., Balasubramanian, S., Mele, D. A., Bergeron, L., and Sims, R. J. (2011) Targeting MYC dependence in cancer by inhibiting BET bromodomains Proc. Natl. Acad. Sci. U. S. A. 108, 16669– 16674[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlWksrnL&md5=b1f0fd41fa56bbfcbb1ad6c8d48f7f80Targeting MYC dependence in cancer by inhibiting BET bromodomainsMertz, Jennifer A.; Conery, Andrew R.; Bryant, Barbara M.; Sandy, Peter; Balasubramanian, Srividya; Mele, Deanna A.; Bergeron, Louise; Sims, Robert J., IIIProceedings of the National Academy of Sciences of the United States of America (2011), 108 (40), 16669-16674, S16669/1-S16669/14CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacol. inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small mol. inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was obsd. in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent redn. of MYC transcript and protein levels resulted in G1 arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacol. inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clin. utility given the widespread pathogenetic role of MYC in cancer.
- 33Seoane, J., Le, H.-V., and Massagué, J. (2002) Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage Nature 419, 729– 734
- 34Strasser, A., Jost, P. J., and Nagata, S. (2009) The many roles of FAS receptor signaling in the immune system Immunity 30, 180– 192
- 35Hnilicová, J., Hozeifi, S., Stejskalová, E., Dušková, E., Poser, I., Humpolíčková, J., Hof, M., and Staněk, D. (2013) The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing Mol. Biol. Cell 24, 3557– 3568[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVygsL7O&md5=52ed5c841e333c58248b23febde32844The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicingHnilicova, Jarmila; Hozeifi, Samira; Stejskalova, Eva; Duskova, Eva; Poser, Ina; Humpolickova, Jana; Hof, Martin; Stanek, DavidMolecular Biology of the Cell (2013), 24 (22), 3557-3568CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addn., almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunopptn. and live-cell imaging, we show that the C-terminal part is crucial for Brd2 assocn. with chromatin. Live-cell microscopy also allows us to map the av. binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.
- 36Delmore, J. E., Issa, G. C., Lemieux, M. E., Rahl, P. B., Shi, J., Jacobs, H. M., Kastritis, E., Gilpatrick, T., Paranal, R. M., Qi, J., Chesi, M., Schinzel, A. C., McKeown, M. R., Heffernan, T. P., Vakoc, C. R., Bergsagel, P. L., Ghobrial, I. M., Richardson, P. G., Young, R. A., Hahn, W. C., Anderson, K. C., Kung, A. L., Bradner, J. E., and Mitsiades, C. S. (2011) BET bromodomain inhibition as a therapeutic strategy to target c-Myc Cell 146, 904– 917[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtF2ht7fF&md5=013674b8224214a9a1c8f0b75583a5a6BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-MycDelmore, Jake E.; Issa, Ghayas C.; Lemieux, Madeleine E.; Rahl, Peter B.; Shi, Jun-Wei; Jacobs, Hannah M.; Kastritis, Efstathios; Gilpatrick, Timothy; Paranal, Ronald M.; Qi, Jun; Chesi, Marta; Schinzel, Anna C.; McKeown, Michael R.; Heffernan, Timothy P.; Vakoc, Christopher R.; Bergsagel, P. Leif; Ghobrial, Irene M.; Richardson, Paul G.; Young, Richard A.; Hahn, William C.; Anderson, Kenneth C.; Kung, Andrew L.; Bradner, James E.; Mitsiades, Constantine S.Cell (Cambridge, MA, United States) (2011), 146 (6), 904-917CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Summary: MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-mol. bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In exptl. models of multiple myeloma, a Myc-dependent hematol. malignancy, JQ1 produces a potent antiproliferative effect assocd. with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathol. activation of c-Myc.
- 37Brand, M., Measures, A. M., Wilson, B. G., Cortopassi, W. A., Alexander, R., Höss, M., Hewings, D. S., Rooney, T. P. C., Paton, R. S., and Conway, S. J. (2015) Small molecule inhibitors of bromodomain–acetyl-lysine interactions ACS Chem. Biol. 10, 22– 39
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- Bowen Zhang, Chang Liu, Zhenqian Yang, Sai Zhang, Xiaolin Hu, Baohu Li, Mei Mao, Xiao Wang, Zhuoyue Li, Shumin Ma, Siqi Zhang, Chong Qin. Discovery of BWA-522, a First-in-Class and Orally Bioavailable PROTAC Degrader of the Androgen Receptor Targeting N-Terminal Domain for the Treatment of Prostate Cancer. Journal of Medicinal Chemistry 2023, 66 (16) , 11158-11186. https://doi.org/10.1021/acs.jmedchem.3c00585
- Diego Garcia Jimenez, Maura Vallaro, Matteo Rossi Sebastiano, Giulia Apprato, Giulia D’Agostini, Paolo Rossetti, Giuseppe Ermondi, Giulia Caron. Chamelogk: A Chromatographic Chameleonicity Quantifier to Design Orally Bioavailable Beyond-Rule-of-5 Drugs. Journal of Medicinal Chemistry 2023, 66 (15) , 10681-10693. https://doi.org/10.1021/acs.jmedchem.3c00823
- Can Zhao, Henian Wang, Wenhu Zhan, Xiaoqing Lv, Xiaodong Ma. Exploitation of Proximity-Mediated Effects in Drug Discovery: An Update of Recent Research Highlights in Perturbing Pathogenic Proteins and Correlated Issues. Journal of Medicinal Chemistry 2023, 66 (15) , 10122-10149. https://doi.org/10.1021/acs.jmedchem.3c00079
- Jieyu Jin, Dong Wang, Guqin Shi, Jingxiao Bao, Jike Wang, Haotian Zhang, Peichen Pan, Dan Li, Xiaojun Yao, Huanxiang Liu, Tingjun Hou, Yu Kang. FFLOM: A Flow-Based Autoregressive Model for Fragment-to-Lead Optimization. Journal of Medicinal Chemistry 2023, 66 (15) , 10808-10823. https://doi.org/10.1021/acs.jmedchem.3c01009
- Ingo V. Hartung, Joachim Rudolph, Mary M. Mader, Monique P. C. Mulder, Paul Workman. Expanding Chemical Probe Space: Quality Criteria for Covalent and Degrader Probes. Journal of Medicinal Chemistry 2023, 66 (14) , 9297-9312. https://doi.org/10.1021/acs.jmedchem.3c00550
- Andres Valdivia, Purav P. Vagadia, Guangxu Guo, Eilidh O’Brien, Daniela Matei, Gary E. Schiltz. Discovery and Characterization of PROTACs Targeting Tissue Transglutaminase (TG2). Journal of Medicinal Chemistry 2023, 66 (14) , 9445-9465. https://doi.org/10.1021/acs.jmedchem.2c01859
- Oluwatosin R. Ayinde, Chia Sharpe, Emily Stahl, Robert J. Tokarski II, James R. Lerma, Natarajan Muthusamy, John C. Byrd, James R. Fuchs. Examination of the Impact of Triazole Position within Linkers on Solubility and Lipophilicity of a CDK9 Degrader Series. ACS Medicinal Chemistry Letters 2023, 14 (7) , 936-942. https://doi.org/10.1021/acsmedchemlett.3c00082
- Rongfan Tang, Zhe Wang, Sutong Xiang, Lingling Wang, Yang Yu, Qinghua Wang, Qirui Deng, Tingjun Hou, Huiyong Sun. Uncovering the Kinetic Characteristics and Degradation Preference of PROTAC Systems with Advanced Theoretical Analyses. JACS Au 2023, 3 (6) , 1775-1789. https://doi.org/10.1021/jacsau.3c00195
- Jiantao Hu, Biao Hu, Fuming Xu, Mi Wang, Chong Qin, Donna McEachern, Jeanne Stuckey, Shaomeng Wang. Precise Conformational Control Yielding Highly Potent and Exceptionally Selective BRD4 Degraders with Strong Antitumor Activity. Journal of Medicinal Chemistry 2023, 66 (12) , 8222-8237. https://doi.org/10.1021/acs.jmedchem.3c00520
- Christopher M. Stevens, Yaxian Zhou, Peng Teng, Lauren N. Rault, Yaxian Liao, Weiping Tang. Development of Oligomeric Mannose-6-phosphonate Conjugates for Targeted Protein Degradation. ACS Medicinal Chemistry Letters 2023, 14 (6) , 719-726. https://doi.org/10.1021/acsmedchemlett.2c00479
- Ethan S. Toriki, James W. Papatzimas, Kaila Nishikawa, Dustin Dovala, Andreas O. Frank, Matthew J. Hesse, Daniela Dankova, Jae-Geun Song, Megan Bruce-Smythe, Heidi Struble, Francisco J. Garcia, Scott M. Brittain, Andrew C. Kile, Lynn M. McGregor, Jeffrey M. McKenna, John A. Tallarico, Markus Schirle, Daniel K. Nomura. Rational Chemical Design of Molecular Glue Degraders. ACS Central Science 2023, 9 (5) , 915-926. https://doi.org/10.1021/acscentsci.2c01317
- Zhen Wang, Jing Liu, He Chen, Xing Qiu, Ling Xie, H. Ümit Kaniskan, Xian Chen, Jian Jin, Wenyi Wei. Telomere Targeting Chimera Enables Targeted Destruction of Telomeric Repeat-Binding Factor Proteins. Journal of the American Chemical Society 2023, 145 (19) , 10872-10879. https://doi.org/10.1021/jacs.3c02783
- Nafsika Forte, Dustin Dovala, Matthew J. Hesse, Jeffrey M. McKenna, John A. Tallarico, Markus Schirle, Daniel K. Nomura. Targeted Protein Degradation through E2 Recruitment. ACS Chemical Biology 2023, 18 (4) , 897-904. https://doi.org/10.1021/acschembio.3c00040
- Yuen Lam Dora Ng, Aleša Bricelj, Jacqueline A. Jansen, Arunima Murgai, Kirsten Peter, Katherine A. Donovan, Michael Gütschow, Jan Krönke, Christian Steinebach, Izidor Sosič. Heterobifunctional Ligase Recruiters Enable pan-Degradation of Inhibitor of Apoptosis Proteins. Journal of Medicinal Chemistry 2023, 66 (7) , 4703-4733. https://doi.org/10.1021/acs.jmedchem.2c01817
- Andrea G. Shergalis, Violeta L. Marin, David Y. Rhee, Sameera Senaweera, Rebecca L. McCloud, Judith A. Ronau, Charles W. Hutchins, Shaun McLoughlin, Kevin R. Woller, Scott E. Warder, Anil Vasudevan, Justin M. Reitsma. CRISPR Screen Reveals BRD2/4 Molecular Glue-like Degrader via Recruitment of DCAF16. ACS Chemical Biology 2023, 18 (2) , 331-339. https://doi.org/10.1021/acschembio.2c00747
- Miquel Duran-Frigola, Marko Cigler, Georg E. Winter. Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence. Journal of the American Chemical Society 2023, 145 (5) , 2711-2732. https://doi.org/10.1021/jacs.2c11098
- Janet M. Sasso, Rumiana Tenchov, DaSheng Wang, Linda S. Johnson, Xinmei Wang, Qiongqiong Angela Zhou. Molecular Glues: The Adhesive Connecting Targeted Protein Degradation to the Clinic. Biochemistry 2023, 62 (3) , 601-623. https://doi.org/10.1021/acs.biochem.2c00245
- Huan Sun, Ka Yang, Xue Zhang, Yingxue Fu, Jay Yarbro, Zhiping Wu, Ping-Chung Chen, Taosheng Chen, Junmin Peng. Evaluation of a Pooling Chemoproteomics Strategy with an FDA-Approved Drug Library. Biochemistry 2023, 62 (3) , 624-632. https://doi.org/10.1021/acs.biochem.2c00256
- Bridget P. Belcher, Carl C. Ward, Daniel K. Nomura. Ligandability of E3 Ligases for Targeted Protein Degradation Applications. Biochemistry 2023, 62 (3) , 588-600. https://doi.org/10.1021/acs.biochem.1c00464
- Miao Chen, Ping Zhou, Yun Kong, Jingrui Li, Yan Li, Yao Zhang, Jie Ran, Jun Zhou, Yan Chen, Songbo Xie. Inducible Degradation of Oncogenic Nucleolin Using an Aptamer-Based PROTAC. Journal of Medicinal Chemistry 2023, 66 (2) , 1339-1348. https://doi.org/10.1021/acs.jmedchem.2c01557
- Dacheng Ma, Qichen Yuan, Fei Peng, Victor Paredes, Hongzhi Zeng, Emmanuel C. Osikpa, Qiaochu Yang, Advaith Peddi, Anika Patel, Megan S. Liu, Zheng Sun, Xue Gao. Engineered PROTAC-CID Systems for Mammalian Inducible Gene Regulation. Journal of the American Chemical Society 2023, 145 (3) , 1593-1606. https://doi.org/10.1021/jacs.2c09129
- Alexander Hanzl, Eleonora Barone, Sophie Bauer, Hong Yue, Radosław P. Nowak, Elisa Hahn, Eugenia V. Pankevich, Anna Koren, Stefan Kubicek, Eric S. Fischer, Georg E. Winter. E3-Specific Degrader Discovery by Dynamic Tracing of Substrate Receptor Abundance. Journal of the American Chemical Society 2023, 145 (2) , 1176-1184. https://doi.org/10.1021/jacs.2c10784
- Ki Bum Hong, Hongchan An. Degrader–Antibody Conjugates: Emerging New Modality. Journal of Medicinal Chemistry 2023, 66 (1) , 140-148. https://doi.org/10.1021/acs.jmedchem.2c01791
- Anand Divakaran, Cole R. Scholtz, Huda Zahid, Wenwei Lin, Elizabeth C. Griffith, Richard E. Lee, Taosheng Chen, Daniel A. Harki, William C. K. Pomerantz. Development of an N-Terminal BRD4 Bromodomain-Targeted Degrader. ACS Medicinal Chemistry Letters 2022, 13 (10) , 1621-1627. https://doi.org/10.1021/acsmedchemlett.2c00300
- Diego García Jiménez, Matteo Rossi Sebastiano, Maura Vallaro, Valentina Mileo, Daniela Pizzirani, Elisa Moretti, Giuseppe Ermondi, Giulia Caron. Designing Soluble PROTACs: Strategies and Preliminary Guidelines. Journal of Medicinal Chemistry 2022, 65 (19) , 12639-12649. https://doi.org/10.1021/acs.jmedchem.2c00201
- Chetan K. Chana, Pierre Maisonneuve, Ganna Posternak, Nicolas G.A. Grinberg, Juline Poirson, Samara M. Ona, Derek F. Ceccarelli, Pavel Mader, Daniel J. St-Cyr, Victor Pau, Igor Kurinov, Xiaojing Tang, Dongjing Deng, Weiren Cui, Wenji Su, Letian Kuai, Richard Soll, Mike Tyers, Hannes L. Röst, Robert A. Batey, Mikko Taipale, Anne-Claude Gingras, Frank Sicheri. Discovery and Structural Characterization of Small Molecule Binders of the Human CTLH E3 Ligase Subunit GID4. Journal of Medicinal Chemistry 2022, 65 (19) , 12725-12746. https://doi.org/10.1021/acs.jmedchem.2c00509
- Natsuko Macabuag, William Esmieu, Perla Breccia, Rebecca Jarvis, Wesley Blackaby, Ovadia Lazari, Liudvikas Urbonas, Maria Eznarriaga, Rachel Williams, Annelieke Strijbosch, Rhea Van de Bospoort, Kim Matthews, Cole Clissold, Tammy Ladduwahetty, Huw Vater, Patrick Heaphy, Douglas G. Stafford, Hong-Jun Wang, John E. Mangette, George McAllister, Vahri Beaumont, Thomas F. Vogt, Hilary A. Wilkinson, Elizabeth M. Doherty, Celia Dominguez. Developing HDAC4-Selective Protein Degraders To Investigate the Role of HDAC4 in Huntington’s Disease Pathology. Journal of Medicinal Chemistry 2022, 65 (18) , 12445-12459. https://doi.org/10.1021/acs.jmedchem.2c01149
- Wei Yan, Bo-Syong Pan, Jingwei Shao, Hui-kuan Lin, Hong-yu Li. Feasible Column Chromatography-Free, Multi-Gram Scale Synthetic Process of VH032 Amine, Which Could Enable Rapid PROTAC Library Construction. ACS Omega 2022, 7 (30) , 26015-26021. https://doi.org/10.1021/acsomega.2c00245
- Alessandra Salerno, Francesca Seghetti, Jessica Caciolla, Elisa Uliassi, Eleonora Testi, Melissa Guardigni, Marinella Roberti, Andrea Milelli, Maria Laura Bolognesi. Enriching Proteolysis Targeting Chimeras with a Second Modality: When Two Are Better Than One. Journal of Medicinal Chemistry 2022, 65 (14) , 9507-9530. https://doi.org/10.1021/acs.jmedchem.2c00302
- Jianyu Yan, Tengfei Li, Zhenyuan Miao, Pei Wang, Chunquan Sheng, Chunlin Zhuang. Homobivalent, Trivalent, and Covalent PROTACs: Emerging Strategies for Protein Degradation. Journal of Medicinal Chemistry 2022, 65 (13) , 8798-8827. https://doi.org/10.1021/acs.jmedchem.2c00728
- Brandon Dale, Chris Anderson, Kwang-Su Park, H. Ümit Kaniskan, Anqi Ma, Yudao Shen, Chengwei Zhang, Ling Xie, Xian Chen, Xufen Yu, Jian Jin. Targeting Triple-Negative Breast Cancer by a Novel Proteolysis Targeting Chimera Degrader of Enhancer of Zeste Homolog 2. ACS Pharmacology & Translational Science 2022, 5 (7) , 491-507. https://doi.org/10.1021/acsptsci.2c00100
- Ariamala Gopalsamy. Selectivity through Targeted Protein Degradation (TPD). Journal of Medicinal Chemistry 2022, 65 (12) , 8113-8126. https://doi.org/10.1021/acs.jmedchem.2c00397
- Jin Liu, Lin Yuan, Yong Ruan, Bulian Deng, Zicao Yang, Yichang Ren, Ling Li, Ting Liu, Huiting Zhao, Ruiyao Mai, Jianjun Chen. Novel CRBN-Recruiting Proteolysis-Targeting Chimeras as Degraders of Stimulator of Interferon Genes with In Vivo Anti-Inflammatory Efficacy. Journal of Medicinal Chemistry 2022, 65 (9) , 6593-6611. https://doi.org/10.1021/acs.jmedchem.1c01948
- Dongbo Lu, Caroline A. Foley, Shama V. Birla, Austin J. Hepperla, Jeremy M. Simon, Lindsey I. James, Nathaniel A. Hathaway. Bioorthogonal Chemical Epigenetic Modifiers Enable Dose-Dependent CRISPR Targeted Gene Activation in Mammalian Cells. ACS Synthetic Biology 2022, 11 (4) , 1397-1407. https://doi.org/10.1021/acssynbio.1c00606
- Dhanusha A. Nalawansha, Ke Li, John Hines, Craig M. Crews. Hijacking Methyl Reader Proteins for Nuclear-Specific Protein Degradation. Journal of the American Chemical Society 2022, 144 (12) , 5594-5605. https://doi.org/10.1021/jacs.2c00874
- Shi Shi, Yu Du, Yi Zou, Jing Niu, Zeyu Cai, Xiaonan Wang, Feihuang Qiu, Yi Ding, Gengchen Yang, Yunze Wu, Yungen Xu, Qihua Zhu. Rational Design for Nitroreductase (NTR)-Responsive Proteolysis Targeting Chimeras (PROTACs) Selectively Targeting Tumor Tissues. Journal of Medicinal Chemistry 2022, 65 (6) , 5057-5071. https://doi.org/10.1021/acs.jmedchem.1c02221
- Zhiqing Liu, Yi Li, Haiying Chen, Hsien-Tsung Lai, Pingyuan Wang, Shwu-Yuan Wu, Eric A. Wold, Paul G. Leonard, Sarah Joseph, Haitao Hu, Cheng-Ming Chiang, Allan R. Brasier, Bing Tian, Jia Zhou. Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site. Journal of Medicinal Chemistry 2022, 65 (3) , 2388-2408. https://doi.org/10.1021/acs.jmedchem.1c01851
- Dhanushka Weerakoon, Rodrigo J. Carbajo, Leonardo De Maria, Christian Tyrchan, Hongtao Zhao. Impact of PROTAC Linker Plasticity on the Solution Conformations and Dissociation of the Ternary Complex. Journal of Chemical Information and Modeling 2022, 62 (2) , 340-349. https://doi.org/10.1021/acs.jcim.1c01036
- Yifan Huang, Hiromasa Yokoe, Ai Kaiho-Soma, Kazunori Takahashi, Yusuke Hirasawa, Hiroshi Morita, Fumiaki Ohtake, Naoki Kanoh. Design, Synthesis, and Evaluation of Trivalent PROTACs Having a Functionalization Site with Controlled Orientation. Bioconjugate Chemistry 2022, 33 (1) , 142-151. https://doi.org/10.1021/acs.bioconjchem.1c00490
- Miyako Naganuma, Nobumichi Ohoka, Genichiro Tsuji, Haruna Tsujimura, Kenji Matsuno, Takao Inoue, Mikihiko Naito, Yosuke Demizu. Development of Chimeric Molecules That Degrade the Estrogen Receptor Using Decoy Oligonucleotide Ligands. ACS Medicinal Chemistry Letters 2022, 13 (1) , 134-139. https://doi.org/10.1021/acsmedchemlett.1c00629
- Victoria G. Klein, Adam G. Bond, Conner Craigon, R. Scott Lokey, Alessio Ciulli. Amide-to-Ester Substitution as a Strategy for Optimizing PROTAC Permeability and Cellular Activity. Journal of Medicinal Chemistry 2021, 64 (24) , 18082-18101. https://doi.org/10.1021/acs.jmedchem.1c01496
- Gaoqi Weng, Dan Li, Yu Kang, Tingjun Hou. Integrative Modeling of PROTAC-Mediated Ternary Complexes. Journal of Medicinal Chemistry 2021, 64 (21) , 16271-16281. https://doi.org/10.1021/acs.jmedchem.1c01576
- Adam G. Bond, Conner Craigon, Kwok-Ho Chan, Andrea Testa, Athanasios Karapetsas, Rotimi Fasimoye, Thomas Macartney, J. Julian Blow, Dario R. Alessi, Alessio Ciulli. Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins. Journal of Medicinal Chemistry 2021, 64 (20) , 15477-15502. https://doi.org/10.1021/acs.jmedchem.1c01532
- Yalin Tu, Yameng Sun, Shuang Qiao, Yao Luo, Panpan Liu, Zhong-Xing Jiang, Yumin Hu, Zifeng Wang, Peng Huang, Shijun Wen. Design, Synthesis, and Evaluation of VHL-Based EZH2 Degraders to Enhance Therapeutic Activity against Lymphoma. Journal of Medicinal Chemistry 2021, 64 (14) , 10167-10184. https://doi.org/10.1021/acs.jmedchem.1c00460
- Troy A. Bemis, James J. La Clair, Michael D. Burkart. Unraveling the Role of Linker Design in Proteolysis Targeting Chimeras. Journal of Medicinal Chemistry 2021, 64 (12) , 8042-8052. https://doi.org/10.1021/acs.jmedchem.1c00482
- Nan Bai, Sven A. Miller, Grigorii V. Andrianov, Max Yates, Palani Kirubakaran, John Karanicolas. Rationalizing PROTAC-Mediated Ternary Complex Formation Using Rosetta. Journal of Chemical Information and Modeling 2021, 61 (3) , 1368-1382. https://doi.org/10.1021/acs.jcim.0c01451
- Pan Tang, Jifa Zhang, Jie Liu, Cheng-Ming Chiang, Liang Ouyang. Targeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological Development. Journal of Medicinal Chemistry 2021, 64 (5) , 2419-2435. https://doi.org/10.1021/acs.jmedchem.0c01487
- Peter S. Dragovich, Thomas H. Pillow, Robert A. Blake, Jack D. Sadowsky, Emel Adaligil, Pragya Adhikari, Sunil Bhakta, Nicole Blaquiere, Jinhua Chen, Josefa dela Cruz-Chuh, Karen E. Gascoigne, Steven J. Hartman, Mingtao He, Susan Kaufman, Tracy Kleinheinz, Katherine R. Kozak, Liang Liu, Liling Liu, Qi Liu, Ying Lu, Fanwei Meng, Melinda M. Mulvihill, Aimee O’Donohue, Rebecca K. Rowntree, Leanna R. Staben, Steven T. Staben, John Wai, Jian Wang, BinQing Wei, Catherine Wilson, Jianfeng Xin, Zijin Xu, Hui Yao, Donglu Zhang, Hongyan Zhang, Hao Zhou, Xiaoyu Zhu. Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 1: Exploration of Antibody Linker, Payload Loading, and Payload Molecular Properties. Journal of Medicinal Chemistry 2021, 64 (5) , 2534-2575. https://doi.org/10.1021/acs.jmedchem.0c01845
- Peter S. Dragovich, Thomas H. Pillow, Robert A. Blake, Jack D. Sadowsky, Emel Adaligil, Pragya Adhikari, Jinhua Chen, Nicholas Corr, Josefa dela Cruz-Chuh, Geoffrey Del Rosario, Aaron Fullerton, Steven J. Hartman, Fan Jiang, Susan Kaufman, Tracy Kleinheinz, Katherine R. Kozak, Liling Liu, Ying Lu, Melinda M. Mulvihill, Jeremy M. Murray, Aimee O’Donohue, Rebecca K. Rowntree, William S. Sawyer, Leanna R. Staben, John Wai, Jian Wang, BinQing Wei, Wentao Wei, Zijin Xu, Hui Yao, Shang-Fan Yu, Donglu Zhang, Hongyan Zhang, Shenhua Zhang, Yongxin Zhao, Hao Zhou, Xiaoyu Zhu. Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy. Journal of Medicinal Chemistry 2021, 64 (5) , 2576-2607. https://doi.org/10.1021/acs.jmedchem.0c01846
- Cyrille S. Kounde, Edward W. Tate. Photoactive Bifunctional Degraders: Precision Tools To Regulate Protein Stability. Journal of Medicinal Chemistry 2020, 63 (24) , 15483-15493. https://doi.org/10.1021/acs.jmedchem.0c01542
- Wenwei Lin, Yongtao Li, Jaeki Min, Jiuyu Liu, Lei Yang, Richard E. Lee, Taosheng Chen. Development of BODIPY FL Thalidomide As a High-Affinity Fluorescent Probe for Cereblon in a Time-Resolved Fluorescence Resonance Energy Transfer Assay. Bioconjugate Chemistry 2020, 31 (11) , 2564-2575. https://doi.org/10.1021/acs.bioconjchem.0c00507
- Michael L. Drummond, Andrew Henry, Huifang Li, Christopher I. Williams. Improved Accuracy for Modeling PROTAC-Mediated Ternary Complex Formation and Targeted Protein Degradation via New In Silico Methodologies. Journal of Chemical Information and Modeling 2020, 60 (10) , 5234-5254. https://doi.org/10.1021/acs.jcim.0c00897
- Laura Goracci, Jenny Desantis, Aurora Valeri, Beatrice Castellani, Michela Eleuteri, Gabriele Cruciani. Understanding the Metabolism of Proteolysis Targeting Chimeras (PROTACs): The Next Step toward Pharmaceutical Applications. Journal of Medicinal Chemistry 2020, 63 (20) , 11615-11638. https://doi.org/10.1021/acs.jmedchem.0c00793
- Chaoguo Cao, Jie Yang, Yong Chen, Peiting Zhou, Yingwei Wang, Wu Du, Lifeng Zhao, Yuanwei Chen. Discovery of SK-575 as a Highly Potent and Efficacious Proteolysis-Targeting Chimera Degrader of PARP1 for Treating Cancers. Journal of Medicinal Chemistry 2020, 63 (19) , 11012-11033. https://doi.org/10.1021/acs.jmedchem.0c00821
- Victoria G. Klein, Chad E. Townsend, Andrea Testa, Michael Zengerle, Chiara Maniaci, Scott J. Hughes, Kwok-Ho Chan, Alessio Ciulli, R. Scott Lokey. Understanding and Improving the Membrane Permeability of VH032-Based PROTACs. ACS Medicinal Chemistry Letters 2020, 11 (9) , 1732-1738. https://doi.org/10.1021/acsmedchemlett.0c00265
- Duncan E. Scott, Timothy P. C. Rooney, Elliott D. Bayle, Tashfina Mirza, Henriette M. G. Willems, Jonathan H. Clarke, Stephen P. Andrews, John Skidmore. Systematic Investigation of the Permeability of Androgen Receptor PROTACs. ACS Medicinal Chemistry Letters 2020, 11 (8) , 1539-1547. https://doi.org/10.1021/acsmedchemlett.0c00194
- Mingliang Wang, Jianfeng Lu, Mi Wang, Chao-Yie Yang, Shaomeng Wang. Discovery of SHP2-D26 as a First, Potent, and Effective PROTAC Degrader of SHP2 Protein. Journal of Medicinal Chemistry 2020, 63 (14) , 7510-7528. https://doi.org/10.1021/acs.jmedchem.0c00471
- Rebecca Beveridge, Dirk Kessler, Klaus Rumpel, Peter Ettmayer, Anton Meinhart, Tim Clausen. Native Mass Spectrometry Can Effectively Predict PROTAC Efficacy. ACS Central Science 2020, 6 (7) , 1223-1230. https://doi.org/10.1021/acscentsci.0c00049
- Bingqi Tong, Jessica N. Spradlin, Luiz F. T. Novaes, Erika Zhang, Xirui Hu, Malte Moeller, Scott M. Brittain, Lynn M. McGregor, Jeffrey M. McKenna, John A. Tallarico, Markus Schirle, Thomas J. Maimone, Daniel K. Nomura. A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL. ACS Chemical Biology 2020, 15 (7) , 1788-1794. https://doi.org/10.1021/acschembio.0c00348
- Ronen Gabizon, Amit Shraga, Paul Gehrtz, Ella Livnah, Yamit Shorer, Neta Gurwicz, Liat Avram, Tamar Unger, Hila Aharoni, Shira Albeck, Alexander Brandis, Ziv Shulman, Ben-Zion Katz, Yair Herishanu, Nir London. Efficient Targeted Degradation via Reversible and Irreversible Covalent PROTACs. Journal of the American Chemical Society 2020, 142 (27) , 11734-11742. https://doi.org/10.1021/jacs.9b13907
- Marı́a Maneiro, Nafsika Forte, Maria M. Shchepinova, Cyrille S. Kounde, Vijay Chudasama, James Richard Baker, Edward W. Tate. Antibody–PROTAC Conjugates Enable HER2-Dependent Targeted Protein Degradation of BRD4. ACS Chemical Biology 2020, 15 (6) , 1306-1312. https://doi.org/10.1021/acschembio.0c00285
- Joshua N. Asiaban, Natalia Milosevich, Emily Chen, Timothy R. Bishop, Justin Wang, Yuxiang Zhang, Christopher J. Ackerman, Eric N. Hampton, Travis S. Young, Mitchell V. Hull, Benjamin F. Cravatt, Michael A. Erb. Cell-Based Ligand Discovery for the ENL YEATS Domain. ACS Chemical Biology 2020, 15 (4) , 895-903. https://doi.org/10.1021/acschembio.0c00124
- Sayumi Yamazoe, Jeffrey Tom, Yue Fu, Wenqiong Wu, Liang Zeng, Changlei Sun, Qi Liu, Jie Lin, Kui Lin, Wayne J. Fairbrother, Steven T. Staben. Heterobifunctional Molecules Induce Dephosphorylation of Kinases–A Proof of Concept Study. Journal of Medicinal Chemistry 2020, 63 (6) , 2807-2813. https://doi.org/10.1021/acs.jmedchem.9b01167
- Yuta Naro, Kristie Darrah, Alexander Deiters. Optical Control of Small Molecule-Induced Protein Degradation. Journal of the American Chemical Society 2020, 142 (5) , 2193-2197. https://doi.org/10.1021/jacs.9b12718
- Christopher R. Wellaway, Dominique Amans, Paul Bamborough, Heather Barnett, Rino A. Bit, Jack A. Brown, Neil R. Carlson, Chun-wa Chung, Anthony W. J. Cooper, Peter D. Craggs, Robert P. Davis, Tony W. Dean, John P. Evans, Laurie Gordon, Isobel L. Harada, David J. Hirst, Philip G. Humphreys, Katherine L. Jones, Antonia J. Lewis, Matthew J. Lindon, Dave Lugo, Mahnoor Mahmood, Scott McCleary, Patricia Medeiros, Darren J. Mitchell, Michael O’Sullivan, Armelle Le Gall, Vipulkumar K. Patel, Chris Patten, Darren L. Poole, Rishi R. Shah, Jane E. Smith, Kayleigh A. J. Stafford, Pamela J. Thomas, Mythily Vimal, Ian D. Wall, Robert J. Watson, Natalie Wellaway, Gang Yao, Rab K. Prinjha. Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening. Journal of Medicinal Chemistry 2020, 63 (2) , 714-746. https://doi.org/10.1021/acs.jmedchem.9b01670
- Caroline A. Foley, Frances Potjewyd, Kelsey N. Lamb, Lindsey I. James, Stephen V. Frye. Assessing the Cell Permeability of Bivalent Chemical Degraders Using the Chloroalkane Penetration Assay. ACS Chemical Biology 2020, 15 (1) , 290-295. https://doi.org/10.1021/acschembio.9b00972
- Xin Han, Lijie Zhao, Weiguo Xiang, Chong Qin, Bukeyan Miao, Tianfeng Xu, Mi Wang, Chao-Yie Yang, Krishnapriya Chinnaswamy, Jeanne Stuckey, Shaomeng Wang. Discovery of Highly Potent and Efficient PROTAC Degraders of Androgen Receptor (AR) by Employing Weak Binding Affinity VHL E3 Ligase Ligands. Journal of Medicinal Chemistry 2019, 62 (24) , 11218-11231. https://doi.org/10.1021/acs.jmedchem.9b01393
- Gang Xue, Kun Wang, Danli Zhou, Hanbing Zhong, Zhengying Pan. Light-Induced Protein Degradation with Photocaged PROTACs. Journal of the American Chemical Society 2019, 141 (46) , 18370-18374. https://doi.org/10.1021/jacs.9b06422
- Carl C. Ward, Jordan I. Kleinman, Scott M. Brittain, Patrick S. Lee, Clive Yik Sham Chung, Kenneth Kim, Yana Petri, Jason R. Thomas, John A. Tallarico, Jeffrey M. McKenna, Markus Schirle, Daniel K. Nomura. Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications. ACS Chemical Biology 2019, 14 (11) , 2430-2440. https://doi.org/10.1021/acschembio.8b01083
- Jiuling Yang, Yangbing Li, Angelo Aguilar, Zhaomin Liu, Chao-Yie Yang, Shaomeng Wang. Simple Structural Modifications Converting a Bona fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders. Journal of Medicinal Chemistry 2019, 62 (21) , 9471-9487. https://doi.org/10.1021/acs.jmedchem.9b00846
- Quanju Zhao, Chaowei Ren, Linyi Liu, Jinju Chen, Yubao Shao, Ning Sun, Renhong Sun, Ying Kong, Xinyu Ding, Xianfang Zhang, Youwei Xu, Bei Yang, Qianqian Yin, Xiaobao Yang, Biao Jiang. Discovery of SIAIS178 as an Effective BCR-ABL Degrader by Recruiting Von Hippel–Lindau (VHL) E3 Ubiquitin Ligase. Journal of Medicinal Chemistry 2019, 62 (20) , 9281-9298. https://doi.org/10.1021/acs.jmedchem.9b01264
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- Philipp Ottis, Chiara Palladino, Phillip Thienger, Adrian Britschgi, Christian Heichinger, Marco Berrera, Alice Julien-Laferriere, Filip Roudnicky, Tony Kam-Thong, James R. Bischoff, Bruno Martoglio, Piergiorgio Pettazzoni. Cellular Resistance Mechanisms to Targeted Protein Degradation Converge Toward Impairment of the Engaged Ubiquitin Transfer Pathway. ACS Chemical Biology 2019, 14 (10) , 2215-2223. https://doi.org/10.1021/acschembio.9b00525
- Hannah Tovell, Andrea Testa, Houjiang Zhou, Natalia Shpiro, Claire Crafter, Alessio Ciulli, Dario R. Alessi. Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader. ACS Chemical Biology 2019, 14 (9) , 2024-2034. https://doi.org/10.1021/acschembio.9b00505
- Ziqian Wang, Nianzhe He, Zongwei Guo, Cuili Niu, Ting Song, Yafei Guo, Keke Cao, Anhui Wang, Junjie Zhu, Xiaodong Zhang, Zhichao Zhang. Proteolysis Targeting Chimeras for the Selective Degradation of Mcl-1/Bcl-2 Derived from Nonselective Target Binding Ligands. Journal of Medicinal Chemistry 2019, 62 (17) , 8152-8163. https://doi.org/10.1021/acs.jmedchem.9b00919
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- Xing Qiu, Ning Sun, Ying Kong, Yan Li, Xiaobao Yang, Biao Jiang. Chemoselective Synthesis of Lenalidomide-Based PROTAC Library Using Alkylation Reaction. Organic Letters 2019, 21 (10) , 3838-3841. https://doi.org/10.1021/acs.orglett.9b01326
- Hannah Tovell, Andrea Testa, Chiara Maniaci, Houjiang Zhou, Alan R Prescott, Thomas Macartney, Alessio Ciulli, Dario R Alessi. Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader. ACS Chemical Biology 2019, 14 (5) , 882-892. https://doi.org/10.1021/acschembio.8b01016
- Michael L. Drummond, Christopher I. Williams. In Silico Modeling of PROTAC-Mediated Ternary Complexes: Validation and Application. Journal of Chemical Information and Modeling 2019, 59 (4) , 1634-1644. https://doi.org/10.1021/acs.jcim.8b00872
- Michael J. Roy, Sandra Winkler, Scott J. Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio Ciulli. SPR-Measured Dissociation Kinetics of PROTAC Ternary Complexes Influence Target Degradation Rate. ACS Chemical Biology 2019, 14 (3) , 361-368. https://doi.org/10.1021/acschembio.9b00092
- Johannes Popow, Heribert Arnhof, Gerd Bader, Helmut Berger, Alessio Ciulli, David Covini, Christian Dank, Teresa Gmaschitz, Peter Greb, Jale Karolyi-Özguer, Manfred Koegl, Darryl B. McConnell, Mark Pearson, Maria Rieger, Joerg Rinnenthal, Vanessa Roessler, Andreas Schrenk, Markus Spina, Steffen Steurer, Nicole Trainor, Elisabeth Traxler, Corinna Wieshofer, Andreas Zoephel, Peter Ettmayer. Highly Selective PTK2 Proteolysis Targeting Chimeras to Probe Focal Adhesion Kinase Scaffolding Functions. Journal of Medicinal Chemistry 2019, 62 (5) , 2508-2520. https://doi.org/10.1021/acs.jmedchem.8b01826
- Yangbing Li, Jiuling Yang, Angelo Aguilar, Donna McEachern, Sally Przybranowski, Liu Liu, Chao-Yie Yang, Mi Wang, Xin Han, Shaomeng Wang. Discovery of MD-224 as a First-in-Class, Highly Potent, and Efficacious Proteolysis Targeting Chimera Murine Double Minute 2 Degrader Capable of Achieving Complete and Durable Tumor Regression. Journal of Medicinal Chemistry 2019, 62 (2) , 448-466. https://doi.org/10.1021/acs.jmedchem.8b00909
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- Philipp M. Cromm, Kusal T. G. Samarasinghe, John Hines, Craig M. Crews. Addressing Kinase-Independent Functions of Fak via PROTAC-Mediated Degradation. Journal of the American Chemical Society 2018, 140 (49) , 17019-17026. https://doi.org/10.1021/jacs.8b08008
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- Olesya A. Kharenko, Reena G. Patel, S. David Brown, Cyrus Calosing, Andre White, Damodharan Lakshminarasimhan, Robert K. Suto, Bryan C. Duffy, Douglas B. Kitchen, Kevin G. McLure, Henrik C. Hansen, Edward H. van der Horst, Peter R. Young. Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine. Journal of Medicinal Chemistry 2018, 61 (18) , 8202-8211. https://doi.org/10.1021/acs.jmedchem.8b00666
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- Chong Qin, Yang Hu, Bing Zhou, Ester Fernandez-Salas, Chao-Yie Yang, Liu Liu, Donna McEachern, Sally Przybranowski, Mi Wang, Jeanne Stuckey, Jennifer Meagher, Longchuan Bai, Zhuo Chen, Mei Lin, Jiuling Yang, Danya N. Ziazadeh, Fuming Xu, Jiantao Hu, Weiguo Xiang, Liyue Huang, Siwei Li, Bo Wen, Duxin Sun, Shaomeng Wang. Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression. Journal of Medicinal Chemistry 2018, 61 (15) , 6685-6704. https://doi.org/10.1021/acs.jmedchem.8b00506
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- Anastasia Amato, Xavier Lucas, Alessio Bortoluzzi, David Wright, Alessio Ciulli. Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach. ACS Chemical Biology 2018, 13 (4) , 915-921. https://doi.org/10.1021/acschembio.7b01093
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Abstract
Figure 1
Figure 1. Design, synthesis, and biophysical and biological evaluation of BET bromodomain PROTACs. (a) Chemical structures of BET-bromodomain inhibitors JQ1 and I-BET762 and binders of von Hippel-Lindau protein VHL-1 and VHL-2. (b) Scheme of the synthesis of PROTAC compounds MZ1–3 and cisMZ1; for detailed synthetic procedures see the Supporting Information. (c) Isothermal titration calorimetry data for titration of MZ1 into the individual members of the BET-bromodomain subfamily. Titrations were performed at 30 °C with a protein concentration of 15 μM and ligand concentration of 150 μM (entry 1–6). Titration of MZ1 and cisMZ1 into VBC at 25 °C with identical concentrations (entry 9, 12) and reverse titration of VBC protein (150 μM) into MZ3 (15 μM) at 25 °C (entry 10) were conducted. For ΔS and ΔG values, see the Supporting Information. (d) HeLa cells were treated with either siRNA targeting individual BET proteins or negative control siRNA 24 h prior to treatment with the compounds MZ1–3, cisMZ1, and JQ1 or vehicle control (0.01% DMSO) for an additional 24 h. Abundance of individual BET protein was analyzed by Western blotting using corresponding specific antibodies accordingly after SDS-PAGE. i, data from ref 8; ii, data from ref 26.
Figure 2
Figure 2. PROTACs induce concentration- and time-dependent selective degradation of BRD4. (a) HeLa cells treated for 24 h with different concentrations of MZ1 (panel I), MZ2 (panel II), and MZ3 (panel III). The bands observed in the BRD4 short isoform lane at a high concentration of each compound are correlated to nonspecific binding. (b) Time dependent treatment over 36 h of HeLa cells with 1 μM (panel I) and 100 nM (panel II) of MZ1. (c) U2OS cells transfected with GFP-BRD4 were treated with either 5 μM of MZ1 or cisMZ1 over a time course of 4 h. BRD4 degradation over time was followed by live fluorescence imaging.
Figure 3
Figure 3. Mechanistic studies on PROTAC biological activity. (a) Time dependent treatment over 36 h of HeLa cells with 1 μM inactive compound cisMZ1. (b) HeLa cells treated with JQ1 or MZ1 at 1 μM in the absence or presence of the proteasome inhibitor MG132. (c) Time dependent treatment over 36 h of HeLa cells with 1 μM MZ1 observing the levels of the von Hippel-Lindau (VHL) protein. (d) HeLa cells treated with 100 μM CoCl2 as a hypoxia control or 0.1, 1, and 10 μM MZ1. (e) BRD4 protein levels were observed (panel I) with single treatment of MZ1 at t = 0 for 4 h and then exchange of media, (panel II) single treatment with MZ1 at t = 0 but no exchange of media, and (panel III) single treatment with 0.01% DMSO for 4 h and then exchange of media.
Figure 4
Figure 4. Selective degradation of BRD4 leads to a differential response between JQ1 and MZ1 on selected genes. mRNA expression profiles of MYC, P21, AREG, FAS, FGFR1, and TYRO3 upon treatment with PROTAC MZ1 and JQ1 were compared. (a) HeLa cells were treated with 100 nM MZ1, VHL-1′, or JQ1 or 0.01% DMSO vehicle control (Veh.) for 24 h. (b) To mimic the protein removal effect, HeLa cells were transfected with siRNA targeting individual BRD2, BRD3, or BRD4 or negative control siRNA and were harvested after 48 h. Quantitative PCR was performed to analyze relative gene expression level of treated HeLa cells using target specific primers. Gene expression levels relative to GAPDH were normalized to control treatment. The data shown represent the mean ± SEM (n = 3 technical replicates) of one experiment. Statistical significance compared to the control was determined with two-tailed t tests: *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. = not significant.
References
ARTICLE SECTIONSThis article references 37 other publications.
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- 6Khan, Y. M., Kirkham, P., Barnes, P. J., and Adcock, I. M. (2014) Brd4 is essential for IL-1β-induced inflammation in human airway epithelial cells PLoS One 9, e95051
- 7Filippakopoulos, P. and Knapp, S. (2012) The bromodomain interaction module FEBS Lett. 586, 2692– 2704[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntFahsbg%253D&md5=c02dee22c330b1d5c1ba39579360ebc5The bromodomain interaction moduleFilippakopoulos, Panagis; Knapp, StefanFEBS Letters (2012), 586 (17), 2692-2704CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. The ε-N-acetylation of protein Lys residues (Kac) is one of the most abundant post-translation modifications (PTMs) in the human proteome. In the nucleus, the acetylation of histones has been linked to transcriptional activation of genes but the functional consequences of most acetylation events and proteins recruited to these sites remains largely unknown. Bromodomains (BRDs) are small helical interaction modules that specifically recognize acetylation sites in proteins. BRDs have recently emerged as interesting targets for the development of specific protein interaction inhibitors, enabling a novel exiting strategy for the development of new therapies. Here, the author provides an overview over sequence requirements of BRDs, known substrates, and the structural mechanisms of specific Kac recognition.
- 8Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O., Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET bromodomains Nature 468, 1067– 1073[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXotlGmtA%253D%253D&md5=bd9cadac86124d2c6c3cd5b2a92d68c2Selective inhibition of BET bromodomainsFilippakopoulos, Panagis; Qi, Jun; Picaud, Sarah; Shen, Yao; Smith, William B.; Fedorov, Oleg; Morse, Elizabeth M.; Keates, Tracey; Hickman, Tyler T.; Felletar, Ildiko; Philpott, Martin; Munro, Shongah; McKeown, Michael R.; Wang, Yuchuan; Christie, Amanda L.; West, Nathan; Cameron, Michael J.; Schwartz, Brian; Heightman, Tom D.; La Thangue, Nicholas; French, Christopher; Wiest, Olaf; Kung, Andrew L.; Knapp, Stefan; Bradner, James E.Nature (London, United Kingdom) (2010), 468 (7327), 1067-1073CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here the authors report a cell-permeable small mol. (I,JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is obsd. in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chem. scaffold for the development of chem. probes more broadly throughout the bromodomain family.
- 9Nicodeme, E., Jeffrey, K. L., Schaefer, U., Beinke, S., Dewell, S., Chung, C.-W., Chandwani, R., Marazzi, I., Wilson, P., Coste, H., White, J., Kirilovsky, J., Rice, C. M., Lora, J. M., Prinjha, R. K., Lee, K., and Tarakhovsky, A. (2010) Suppression of inflammation by a synthetic histone mimic Nature 468, 1119– 1123[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtl2rtb%252FP&md5=0dfccd9d01b87859512bf591e1c5a93cSuppression of inflammation by a synthetic histone mimicNicodeme, Edwige; Jeffrey, Kate L.; Schaefer, Uwe; Beinke, Soren; Dewell, Scott; Chung, Chun-wa; Chandwani, Rohit; Marazzi, Ivan; Wilson, Paul; Coste, Herve; White, Julia; Kirilovsky, Jorge; Rice, Charles M.; Lora, Jose M.; Prinjha, Rab K.; Lee, Kevin; Tarakhovsky, AlexanderNature (London, United Kingdom) (2010), 468 (7327), 1119-1123CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defense, is frequently deleterious to the host due to the exaggerated prodn. of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signaling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a crit. step in the regulation of gene expression. Here we present a novel pharmacol. approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compd. (I-BET) that by mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compds. specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.
- 10Boi, M., Gaudio, E., Bonetti, P., Kwee, I., Bernasconi, E., Tarantelli, C., Rinaldi, A., Testoni, M., Cascione, L., Ponzoni, M., Mensah, A. A., Stathis, A., Stussi, G., Riveiro, M. E., Herait, P., Inghirami, G., Cvitkovic, E., Zucca, E., and Bertoni, F. (2015) The BET Bromodomain inhibitor OTX015 affects pathogenetic pathways in pre-clinical B-cell tumor models and synergizes with targeted drugs Clin. Cancer Res. 21, 1628– 1638[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtVahsbc%253D&md5=5742b4d5884261d546588a339f3c2ea0The BET Bromodomain Inhibitor OTX015 Affects Pathogenetic Pathways in Preclinical B-cell Tumor Models and Synergizes with Targeted DrugsBoi, Michela; Gaudio, Eugenio; Bonetti, Paola; Kwee, Ivo; Bernasconi, Elena; Tarantelli, Chiara; Rinaldi, Andrea; Testoni, Monica; Cascione, Luciano; Ponzoni, Maurilio; Mensah, Afua Adjeiwaa; Stathis, Anastasios; Stussi, Georg; Riveiro, Maria Eugenia; Herait, Patrice; Inghirami, Giorgio; Cvitkovic, Esteban; Zucca, Emanuele; Bertoni, FrancescoClinical Cancer Research (2015), 21 (7), 1628-1638CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-contg. proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015. We assessed the preclin. activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo expts. to identify the mechanism of action and the genetic features assocd. with sensitivity to the compd. OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo expts. showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, esp. with mTOR and BTK inhibitors. Gene expression signatures assocd. with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compd. induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell-like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11. Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clin. investigation of OTX015 as single agent and in combination therapies. Clin Cancer Res; 21(7); 1628-38. ©2015 AACR.
- 11Gosmini, R., Nguyen, V. L., Toum, J., Simon, C., Brusq, J.-M. G., Krysa, G., Mirguet, O., Riou-Eymard, A. M., Boursier, E. V., Trottet, L., Bamborough, P., Clark, H., Chung, C.-W., Cutler, L., Demont, E. H., Kaur, R., Lewis, A. J., Schilling, M. B., Soden, P. E., Taylor, S., Walker, A. L., Walker, M. D., Prinjha, R. K., and Nicodeme, E. (2014) The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor J. Med. Chem. 57, 8111– 8131[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Wlur7J&md5=b1c9c9cae73c41252cf73908d561bd9bThe Discovery of I-BET726 (GSK1324726A), a Potent Tetrahydroquinoline ApoA1 Up-Regulator and Selective BET Bromodomain InhibitorGosmini, Romain; Nguyen, Van Loc; Toum, Jerome; Simon, Christophe; Brusq, Jean-Marie G.; Krysa, Gael; Mirguet, Olivier; Riou-Eymard, Alizon M.; Boursier, Eric V.; Trottet, Lionel; Bamborough, Paul; Clark, Hugh; Chung, Chun-wa; Cutler, Leanne; Demont, Emmanuel H.; Kaur, Rejbinder; Lewis, Antonia J.; Schilling, Mark B.; Soden, Peter E.; Taylor, Simon; Walker, Ann L.; Walker, Matthew D.; Prinjha, Rab K.; Nicodeme, EdwigeJournal of Medicinal Chemistry (2014), 57 (19), 8111-8131CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Through their function as epigenetic readers of the histone code, the BET family of bromodomain-contg. proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncol. and inflammation models, and the first compds. have now progressed into clin. trials. The exciting biol. of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compds. active in murine models of septic shock and neuroblastoma. At the mol. level, these effects are produced by inhibition of BET bromodomains. X-ray crystallog. reveals the interactions explaining the structure-activity relationships of binding. The resulting lead mol., I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.
- 12Mirguet, O., Lamotte, Y., Donche, F., Toum, J., Gellibert, F., Bouillot, A., Gosmini, R., Nguyen, V. L., Delannée, D., Seal, J., Blandel, F., Boullay, A.-B., Boursier, E., Martin, S., Brusq, J.-M., Krysa, G., Riou, A., Tellier, R., Costaz, A., Huet, P., Dudit, Y., Trottet, L., Kirilovsky, J., and Nicodeme, E. (2012) From ApoA1 upregulation to BET family bromodomain inhibition: Discovery of I-BET151 Bioorg. Med. Chem. Lett. 22, 2963– 2967[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjt1elsbY%253D&md5=48c9a8dc4b70bea4a86807f7a1cec5f1From ApoA1 upregulation to BET family bromodomain inhibition: Discovery of I-BET151Mirguet, Olivier; Lamotte, Yann; Donche, Frederic; Toum, Jerome; Gellibert, Francoise; Bouillot, Anne; Gosmini, Romain; Nguyen, Van-Loc; Delannee, Delphine; Seal, Jonathan; Blandel, Florence; Boullay, Anne-Benedicte; Boursier, Eric; Martin, Sandrine; Brusq, Jean-Marie; Krysa, Gael; Riou, Alizon; Tellier, Remi; Costaz, Agnes; Huet, Pascal; Dudit, Yann; Trottet, Lionel; Kirilovsky, Jorge; Nicodeme, EdwigeBioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2963-2967CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The discovery, synthesis, and biol. evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.
- 13McLure, K. G., Gesner, E. M., Tsujikawa, L., Kharenko, O. A., Attwell, S., Campeau, E., Wasiak, S., Stein, A., White, A., Fontano, E., Suto, R. K., Wong, N. C. W., Wagner, G. S., Hansen, H. C., and Young, P. R. (2013) RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain antagonist PLoS One 8, e83190
- 14Cheng, Z., Gong, Y., Ma, Y., Lu, K., Lu, X., Pierce, L. A., Thompson, R. C., Müller, S., Knapp, S., and Wang, J. (2013) Inhibition of BET bromodomain targets genetically diverse glioblastoma Clin. Cancer Res. 19, 1748– 1759[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsVCmsLo%253D&md5=1722189e5d950da233b785558f2c9f58Inhibition of BET Bromodomain Targets Genetically Diverse GlioblastomaCheng, Zhixiang; Gong, Yuanying; Ma, Yufang; Lu, Kaihua; Lu, Xiang; Pierce, Larry A.; Thompson, Reid C.; Muller, Susanne; Knapp, Stefan; Wang, JialiangClinical Cancer Research (2013), 19 (7), 1748-1759CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Purpose: Glioblastoma is refractory to conventional therapies. The bromodomain and extraterminal domain (BET) proteins are epigenetic readers that selectively bind to acetylated lysine residues on histone tails. These proteins recently emerged as important therapeutic targets in NUT midline carcinoma and several types of hematopoietic cancers. In this study, the therapeutic potential of a novel BET bromodomain inhibitor, JQ1, was assessed in a panel of genetically heterogeneous glioblastoma samples. Exptl. Design: The antineoplastic effects of JQ1 were shown using ex vivo cultures derived from primary glioblastoma xenograft lines and surgical specimens of different genetic background. The in vivo efficacy was assessed in orthotopic glioblastoma tumors. Results: We showed that JQ1 induced marked G1 cell-cycle arrest and apoptosis, which was phenocopied by knockdown of individual BET family members. JQ1 treatment resulted in significant changes in expression of genes that play important roles in glioblastoma such as c-Myc, p21CIP1/WAF1, hTERT, Bcl-2, and Bcl-xL. Unlike the observations in some hematopoietic cancer cell lines, exogenous c-Myc did not significantly protect glioblastoma cells against JQ1. In contrast, ectopically expressed Bcl-xL partially rescued cells from JQ1-induced apoptosis, and knockdown of p21CIP1/WAF1 attenuated JQ1-induced cell-cycle arrest. Cells genetically engineered for Akt hyperactivation or p53/Rb inactivation did not compromise JQ1 efficacy, suggesting that these frequently mutated signaling pathways may not confer resistance to JQ1. Furthermore, JQ1 significantly repressed growth of orthotopic glioblastoma tumors. Conclusion: Our results suggest potentially broad therapeutic use of BET bromodomain inhibitors for treating genetically diverse glioblastoma tumors. Clin Cancer Res; 19(7); 1748-59. ©2013 AACR.
- 15Prinjha, R. K., Witherington, J., and Lee, K. (2012) Place your BETs: The therapeutic potential of bromodomains Trends Pharmacol. Sci. 33, 146– 153[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtlOlt74%253D&md5=ea1e9e5740b1131239aa0407748d549bPlace your BETs: the therapeutic potential of bromodomainsPrinjha, R. K.; Witherington, J.; Lee, K.Trends in Pharmacological Sciences (2012), 33 (3), 146-153CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Ltd.)A review. Therapeutic targeting of the processes that regulate histone modification is a growing area of scientific exploration. Although most interest has concd. on the various families of enzymes that contribute to these processes, this review focuses on emerging data demonstrating the chem. tractability and therapeutic potential of a hitherto underexplored family of proteins, namely the bromodomain (BRD) family of reader proteins. These proteins perform a crucial role in translating histone modifications with powerful transcriptional consequences. We review current knowledge of the biol. of this emergent target class and highlight recent breakthroughs that now make the BRD family of reader proteins attractive for drug discovery.
- 16Filippakopoulos, P. and Knapp, S. (2014) Targeting bromodomains: Epigenetic readers of lysine acetylation Nat. Rev. Drug Discovery 13, 337– 356[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmsFalsr4%253D&md5=77049c82ce6a19b4685e3b7950399a07Targeting bromodomains: epigenetic readers of lysine acetylationFilippakopoulos, Panagis; Knapp, StefanNature Reviews Drug Discovery (2014), 13 (5), 337-356CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Lysine acetylation is a key mechanism that regulates chromatin structure; aberrant acetylation levels have been linked to the development of several diseases. Acetyl-lysine modifications create docking sites for bromodomains, which are small interaction modules found on diverse proteins, some of which have a key role in the acetylation-dependent assembly of transcriptional regulator complexes. These complexes can then initiate transcriptional programs that result in phenotypic changes. The recent discovery of potent and highly specific inhibitors for the BET (bromodomain and extra-terminal) family of bromodomains has stimulated intensive research activity in diverse therapeutic areas, particularly in oncol., where BET proteins regulate the expression of key oncogenes and anti-apoptotic proteins. In addn., targeting BET bromodomains could hold potential for the treatment of inflammation and viral infection. Here, we highlight recent progress in the development of bromodomain inhibitors, and their potential applications in drug discovery.
- 17Baud, M. G. J., Lin-Shiao, E., Cardote, T., Tallant, C., Pschibul, A., Chan, K.-H., Zengerle, M., Garcia, J. R., Kwan, T. T. L., Ferguson, F. M., and Ciulli, A. (2014) A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes Science 346, 638– 641[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVWlt7nL&md5=04fe967e4681fb4b2b3168f4d5f8ff81A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probesBaud, Matthias G. J.; Lin-Shiao, Enrique; Cardote, Teresa; Tallant, Cynthia; Pschibul, Annica; Chan, Kwok-Ho; Zengerle, Michael; Garcia, Jordi R.; Kwan, Terence T.-L.; Ferguson, Fleur M.; Ciulli, AlessioScience (Washington, DC, United States) (2014), 346 (6209), 638-641CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Small mols. are useful tools for probing the biol. function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The bromodomain and extra-terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-mol. inhibitors, but these inhibitors lack selectivity for individual family members. We developed an Et deriv. of an existing small-mol. inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BET protein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BET proteins in human physiol. and disease.
- 18Raina, K. and Crews, C. M. (2010) Chemical inducers of targeted protein degradation J. Biol. Chem. 285, 11057– 11060[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktFGlsrs%253D&md5=f5142a890c2e1c859d58ddb25c077052Chemical Inducers of Targeted Protein DegradationRaina, Kanak; Crews, Craig M.Journal of Biological Chemistry (2010), 285 (15), 11057-11060CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. The functions of many cellular proteins have been elucidated by selective gene inactivation and subsequent phenotypic anal. For example, genetic mutations, gene knock-out generation, and the use of RNA interference to target mRNA for degrdn. can all result in decreased prodn. of a specific protein, yielding informative cellular phenotypes. However, these techniques each have certain inherent limitations. This minireview focuses on the recent development of new approaches to study protein function at the post-translational level, namely chem. induction of targeted protein degrdn.
- 19Corson, T. W., Aberle, N., and Crews, C. M. (2008) Design and applications of bifunctional small molecules: Why two heads are better than one ACS Chem. Biol. 3, 677– 692[ACS Full Text], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1yiurzF&md5=2edac15286d3a739dd66214a332a545bDesign and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than OneCorson, Timothy W.; Aberle, Nicholas; Crews, Craig M.ACS Chemical Biology (2008), 3 (11), 677-692CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Induction of protein-protein interactions is a daunting challenge, but recent studies show promise for small mols. that specifically bring two or more protein mols. together for enhanced or novel biol. effect. The first such bifunctional mols. were the rapamycin- and FK506-based "chem. inducers of dimerization", but the field has since expanded with new mols. and new applications in chem. genetics and cell biol. Examples include coumermycin-mediated gyrase B dimerization, proteolysis targeting chimeric mols. (PROTACs), drug hybrids, and strategies for exploiting multivalency in toxin binding and antibody recruitment. This Review discusses these and other advances in the design and use of bifunctional small mols. and potential strategies for future systems.
- 20Cyrus, K., Wehenkel, M., Choi, E. Y., Swanson, H., and Kim, K. B. (2010) Two-headed PROTAC: An effective new tool for targeted protein degradation ChemBioChem. 11, 1531– 1534
- 21Sakamoto, K. M., Kim, K. B., Verma, R., Ransick, A., Stein, B., Crews, C. M., and Deshaies, R. J. (2003) Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation Mol. Cell. Proteomics 2, 1350– 1358[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtVShu7jE&md5=5a9f4cf5d5cc1d990bf59ac824902111Development of Protacs to target cancer-promoting proteins for ubiquitination and degradationSakamoto, Kathleen M.; Kim, Kyung B.; Verma, Rati; Ransick, Andy; Stein, Bernd; Crews, Craig M.; Deshaies, Raymond J.Molecular and Cellular Proteomics (2003), 2 (12), 1350-1358CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small mol. inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. The authors previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degrdn. through a chimeric bridging mol. or Protac (proteolysis targeting chimeric mol.). This Protac consisted of an SCFβ-TRCP-binding phosphopeptide derived from IκBα linked to ovalicin, which covalently binds MetAP-2. In this study, the authors employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, resp. The authors show here that an estradiol-based Protac can enforce the ubiquitination and degrdn. of the α isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technol. may yield a general approach to treat a no. of human diseases, including cancer.
- 22Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., and Deshaies, R. J. (2001) Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation Proc. Natl. Acad. Sci. U. S. A. 98, 8554– 8559[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXls1Wisbk%253D&md5=540358035222f745f7b6367b38781a21Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradationSakamoto, Kathleen M.; Kim, Kyung B.; Kumagai, Akiko; Mercurio, Frank; Crews, Craig M.; Deshaies, Raymond J.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (15), 8554-8559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex contg. Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degrdn. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compd., protein-targeting chimeric mol. 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IκBα phosphopeptide that is recognized by the F-box protein β-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCFβ-TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.
- 23Lee, H., Puppala, D., Choi, E. Y., Swanson, H., and Kim, K. B. (2007) Targeted degradation of the aryl hydrocarbon receptor by the PROTAC approach: A useful chemical genetic tool ChemBioChem. 8, 2058– 2062
- 24Schneekloth, J. S., Fonseca, F. N., Koldobskiy, M., Mandal, A., Deshaies, R., Sakamoto, K., and Crews, C. M. (2004) Chemical genetic control of protein levels: Selective in vivo targeted degradation J. Am. Chem. Soc. 126, 3748– 3754[ACS Full Text], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvVegur0%253D&md5=f0855157e4daf32080a23e1728c4b9ccChemical genetic control of protein levels: selective in vivo targeted degradationSchneekloth, John S., Jr.; Fonseca, Fabiana N.; Koldobskiy, Michael; Mandal, Amit; Deshaies, Raymond; Sakamoto, Kathleen; Crews, Craig M.Journal of the American Chemical Society (2004), 126 (12), 3748-3754CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Genetic loss of function anal. is a powerful method for the study of protein function. However, some cell biol. questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of mols. capable of inducing the degrdn. of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric mols. (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, resp. These are the first in vivo examples of direct small mol.-induced recruitment of target proteins to the proteasome for degrdn. upon addn. to cultured cells. Moreover, PROTAC-mediated protein degrdn. offers a general strategy to create "chem. knockouts," thus opening new possibilities for the control of protein function.
- 25Hon, W.-C., Wilson, M. I., Harlos, K., Claridge, T. D. W., Schofield, C. J., Pugh, C. W., Maxwell, P. H., Ratcliffe, P. J., Stuart, D. I., and Jones, E. Y. (2002) Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL Nature 417, 975– 978
- 26Galdeano, C., Gadd, M. S., Soares, P., Scaffidi, S., Van Molle, I., Birced, I., Hewitt, S., Dias, D. M., and Ciulli, A. (2014) Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities J. Med. Chem. 57, 8657– 8663[ACS Full Text], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVGhtb%252FP&md5=831aa2387d94cb03d735a4be7bdd2cddStructure-Guided Design and Optimization of Small Molecules Targeting the Protein-Protein Interaction between the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase and the Hypoxia Inducible Factor (HIF) Alpha Subunit with in Vitro Nanomolar AffinitiesGaldeano, Carles; Gadd, Morgan S.; Soares, Pedro; Scaffidi, Salvatore; Van Molle, Inge; Birced, Ipek; Hewitt, Sarah; Dias, David M.; Ciulli, AlessioJournal of Medicinal Chemistry (2014), 57 (20), 8657-8663CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-mol. ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degrdn. The authors recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, the authors report the design and optimization, guided by x-ray crystal structures, of a ligand series with nanomolar binding affinities.
- 27Li, Z., Wang, D., Li, L., Pan, S., Na, Z., Tan, C. Y. J., and Yao, S. Q. (2014) “Minimalist” cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling J. Am. Chem. Soc. 136, 9990– 9998
- 28Shi, J. and Vakoc, C. R. (2014) The mechanisms behind the therapeutic activity of BET bromodomain inhibition Mol. Cell 54, 728– 736[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpslCrtrk%253D&md5=f49bf3524ec378576fb1a25c64a464aeThe Mechanisms behind the Therapeutic Activity of BET Bromodomain InhibitionShi, Junwei; Vakoc, Christopher R.Molecular Cell (2014), 54 (5), 728-736CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)A review. The bromodomain and extraterminal (BET) protein Brd4 recruits transcriptional regulatory complexes to acetylated chromatin. While Brd4 is considered to be a general transcriptional regulator, pharmacol. inhibition of BET proteins shows therapeutic activity in a variety of different pathologies, particularly in models of cancer and inflammation. Such effects have been attributed to a specific set of downstream target genes whose expression is disproportionately sensitive to pharmacol. targeting of BET proteins. Emerging evidence links the transcriptional consequences of BET inhibition to the assocn. of Brd4 with enhancer elements, which tend to be involved in lineage-specific gene regulation. Furthermore, Brd4 engages in direct regulatory interactions with several DNA-binding transcription factors to influence their disease-relevant functions. Here we review the current understanding of mol. mechanisms that underlie the promising therapeutic effects of BET bromodomain inhibition.
- 29Anders, L., Guenther, M. G., Qi, J., Fan, Z. P., Marineau, J. J., Rahl, P. B., Lovén, J., Sigova, A. A., Smith, W. B., Lee, T. I., Bradner, J. E., and Young, R. A. (2014) Genome-wide localization of small molecules Nat. Biotechnol. 32, 92– 96[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFentbrI&md5=6703796ae735da5279eb6a1f4e4d7d4dGenome-wide localization of small moleculesAnders, Lars; Guenther, Matthew G.; Qi, Jun; Fan, Zi Peng; Marineau, Jason J.; Rahl, Peter B.; Loven, Jakob; Sigova, Alla A.; Smith, William B.; Lee, Tong Ihn; Bradner, James E.; Young, Richard A.Nature Biotechnology (2014), 32 (1), 92-96CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)A vast no. of small-mol. ligands, including therapeutic drugs under development and in clin. use, elicit their effects by binding specific proteins assocd. with the genome. An ability to map the direct interactions of a chem. entity with chromatin genome-wide could provide important insights into chem. perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chem. mols. throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chem. entities that interact with DNA or genome-assocd. proteins.
- 30Bretones, G., Delgado, M. D., and León, J. (2014) Myc and cell cycle control Biochim. Biophys. Acta 1849, 506– 516
- 31Lamoureux, F., Baud’huin, M., Rodriguez Calleja, L., Jacques, C., Berreur, M., Rédini, F., Lecanda, F., Bradner, J. E., Heymann, D., and Ory, B. (2014) Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle Nat. Commun. 5, 3511[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2crltlentg%253D%253D&md5=a07f9e31ee509a1958d6c81071c6ea3bSelective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycleLamoureux Francois; Baud'huin Marc; Rodriguez Calleja Lidia; Jacques Camille; Berreur Martine; Redini Francoise; Ory Benjamin; Lecanda Fernando; Bradner James E; Heymann DominiqueNature communications (2014), 5 (), 3511 ISSN:.The vicious cycle established between bone-associated tumours and bone resorption is the central problem with therapeutic strategies against primary bone tumours and bone metastasis. Here we report data to support inhibition of BET bromodomain proteins as a promising therapeutic strategy that target simultaneously the three partners of the vicious cycle. Treatment with JQ1, a BET bromodomain inhibitor, reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation both in vitro and in vivo. These effects are associated with transcriptional silencing of MYC and RUNX2, resulting from the depletion of BRD4 from their respective loci. Moreover, JQ1 also inhibits osteoclast differentiation by interfering with BRD4-dependent RANKL activation of NFATC1 transcription. Collectively, our data indicate that JQ1 is a potent inhibitor of osteoblast and osteoclast differentiation as well as bone tumour development.
- 32Mertz, J. A., Conery, A. R., Bryant, B. M., Sandy, P., Balasubramanian, S., Mele, D. A., Bergeron, L., and Sims, R. J. (2011) Targeting MYC dependence in cancer by inhibiting BET bromodomains Proc. Natl. Acad. Sci. U. S. A. 108, 16669– 16674[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlWksrnL&md5=b1f0fd41fa56bbfcbb1ad6c8d48f7f80Targeting MYC dependence in cancer by inhibiting BET bromodomainsMertz, Jennifer A.; Conery, Andrew R.; Bryant, Barbara M.; Sandy, Peter; Balasubramanian, Srividya; Mele, Deanna A.; Bergeron, Louise; Sims, Robert J., IIIProceedings of the National Academy of Sciences of the United States of America (2011), 108 (40), 16669-16674, S16669/1-S16669/14CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacol. inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small mol. inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was obsd. in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent redn. of MYC transcript and protein levels resulted in G1 arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacol. inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clin. utility given the widespread pathogenetic role of MYC in cancer.
- 33Seoane, J., Le, H.-V., and Massagué, J. (2002) Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage Nature 419, 729– 734
- 34Strasser, A., Jost, P. J., and Nagata, S. (2009) The many roles of FAS receptor signaling in the immune system Immunity 30, 180– 192
- 35Hnilicová, J., Hozeifi, S., Stejskalová, E., Dušková, E., Poser, I., Humpolíčková, J., Hof, M., and Staněk, D. (2013) The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing Mol. Biol. Cell 24, 3557– 3568[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVygsL7O&md5=52ed5c841e333c58248b23febde32844The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicingHnilicova, Jarmila; Hozeifi, Samira; Stejskalova, Eva; Duskova, Eva; Poser, Ina; Humpolickova, Jana; Hof, Martin; Stanek, DavidMolecular Biology of the Cell (2013), 24 (22), 3557-3568CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addn., almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunopptn. and live-cell imaging, we show that the C-terminal part is crucial for Brd2 assocn. with chromatin. Live-cell microscopy also allows us to map the av. binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.
- 36Delmore, J. E., Issa, G. C., Lemieux, M. E., Rahl, P. B., Shi, J., Jacobs, H. M., Kastritis, E., Gilpatrick, T., Paranal, R. M., Qi, J., Chesi, M., Schinzel, A. C., McKeown, M. R., Heffernan, T. P., Vakoc, C. R., Bergsagel, P. L., Ghobrial, I. M., Richardson, P. G., Young, R. A., Hahn, W. C., Anderson, K. C., Kung, A. L., Bradner, J. E., and Mitsiades, C. S. (2011) BET bromodomain inhibition as a therapeutic strategy to target c-Myc Cell 146, 904– 917[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtF2ht7fF&md5=013674b8224214a9a1c8f0b75583a5a6BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-MycDelmore, Jake E.; Issa, Ghayas C.; Lemieux, Madeleine E.; Rahl, Peter B.; Shi, Jun-Wei; Jacobs, Hannah M.; Kastritis, Efstathios; Gilpatrick, Timothy; Paranal, Ronald M.; Qi, Jun; Chesi, Marta; Schinzel, Anna C.; McKeown, Michael R.; Heffernan, Timothy P.; Vakoc, Christopher R.; Bergsagel, P. Leif; Ghobrial, Irene M.; Richardson, Paul G.; Young, Richard A.; Hahn, William C.; Anderson, Kenneth C.; Kung, Andrew L.; Bradner, James E.; Mitsiades, Constantine S.Cell (Cambridge, MA, United States) (2011), 146 (6), 904-917CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Summary: MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-mol. bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In exptl. models of multiple myeloma, a Myc-dependent hematol. malignancy, JQ1 produces a potent antiproliferative effect assocd. with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathol. activation of c-Myc.
- 37Brand, M., Measures, A. M., Wilson, B. G., Cortopassi, W. A., Alexander, R., Höss, M., Hewings, D. S., Rooney, T. P. C., Paton, R. S., and Conway, S. J. (2015) Small molecule inhibitors of bromodomain–acetyl-lysine interactions ACS Chem. Biol. 10, 22– 39
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