Understanding the thermodynamic and binding characteristics of DNA minor groove binders (MGBs) is important for the rational design and development of novel MGBs; however, there are contradicting results in the literature regarding the thermodynamic signature of MGBs. The expansion of the thermodynamic database for MGBs in the literature was encouraging to evaluate and critically test the previously reported hypothesis that MGB binding is mainly entropically driven. In this review, the thermodynamic data of a group of MGBs published in the literature were analyzed to better understand the factors that drive minor groove recognition. Analysis of the enthalpic and entropic contributions to the free energy of binding for 20 interactions from a total of 14 different compounds reveals that MGB binding can be driven by enthalpy, entropy, or by both and that is mainly dictated by ligand structural heterogeneity. These findings could be useful in the design of MGBs for therapeutic purposes.

Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission within the mammalian central nervous system. iGluRs exist as three main groups: N-methyl-d-aspartate receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), and kainate receptors. The past decades have witnessed a remarkable development of PET tracers targeting different iGluRs including NMDARs and AMPARs, and several of the tracers have advanced to clinical imaging studies. Here, we assess the recent development of iGluR PET probes, focusing on tracer design, brain kinetics, and performance in PET imaging studies. Furthermore, this review will not only present challenges in the tracer development but also provide novel approaches in conjunction with most recent drug discovery efforts on these iGluRs, including subtype-selective NMDAR and transmembrane AMPAR regulatory protein modulators and positive allosteric modulators (PAMs) of AMPARs. These approaches, if successful as PET tracers, may provide fundamental knowledge to understand the roles of iGluR receptors under physiological and pathological conditions.

Multitargeting compounds comprising activity on more than a single biological target have gained remarkable relevance in drug discovery owing to the complexity of multifactorial diseases such as cancer, inflammation, or the metabolic syndrome. Polypharmacological drug profiles can produce additive or synergistic effects while reducing side effects and significantly contribute to the high therapeutic success of indispensable drugs such as aspirin. While their identification has long been the result of serendipity, medicinal chemistry now tends to design polypharmacology. Modern in vitro pharmacological methods and chemical probes allow a systematic search for rational target combinations and recent innovations in computational technologies, crystallography, or fragment-based design equip multitarget compound development with valuable tools. In this Perspective, we analyze the relevance of multiple ligands in drug discovery and the versatile toolbox to design polypharmacology. We conclude that despite some characteristic challenges remaining unresolved, designed polypharmacology holds enormous potential to secure future therapeutic innovation.

MDM2 is a key oncogenic protein that serves as a negative regulator of the tumor suppressor p53. While a number of inhibitors of the MDM2-p53 interaction have progressed to clinical testing as treatments for a variety of hematologic and solid tumor cancers, the results thus far have been mixed, with perhaps the strongest responses observed in relapsed/refractory acute myeloid leukemia (AML). In an effort to improve the efficacy for this class of compounds, researchers have turned to targeted degradation of MDM2. IMiD-based MDM2 PROTAC 8, which potently reduces MDM2 protein levels through targeted degradation, exhibits enhanced efficacy in the RS4;11 xenograft model relative to a nondegrading MDM2-p53 inhibitor MI-1061.

Human murine double minute 2 (MDM2) protein is a primary endogenous cellular inhibitor of the tumor suppressor p53 and has been pursued as an attractive cancer therapeutic target. Several potent, nonpeptide, small-molecule inhibitors of MDM2 are currently in clinical development. In this paper, we report our design, synthesis, and evaluation of small-molecule MDM2 degraders based on the proteolysis targeting chimera (PROTAC) concept. The most promising compound (MD-224) effectively induces rapid degradation of MDM2 at concentrations <1 nM in human leukemia cells. It achieves an IC₅₀ value of 1.5 nM in inhibition of growth of RS4;11 cells and also low nanomolar IC₅₀ values in a panel of leukemia cell lines. MD-224 achieves complete and durable tumor regression in vivo in the RS4;11 xenograft tumor model in mice at well-tolerated dose schedules. MD-224 is thus a highly potent and efficacious MDM2 degrader and warrants extensive evaluations as a new class of anticancer agent.

The F9/Yde/Fml pilus, tipped with the FmlH adhesin, has been shown to provide uropathogenic Escherichia coli (UPEC) a fitness advantage in urinary tract infections (UTIs). Here, we used X-ray structure guided design to optimize our previously described ortho-biphenyl Gal and GalNAc FmlH antagonists such as compound 1 by replacing the carboxylate with a sulfonamide as in 50. Other groups which can accept H-bonds were also tolerated. We pursued further modifications to the biphenyl aglycone resulting in significantly improved activity. Two of the most potent compounds, 86 (IC₅₀ = 0.051 μM) and 90 (IC₅₀ = 0.034 μM), exhibited excellent metabolic stability in mouse plasma and liver microsomes but showed only limited oral bioavailability (<1%) in rats. Compound 84 also showed a good pharmacokinetic (PK) profile in mice after IP dosing with compound exposure above the IC₅₀ for 6 h. These new FmlH antagonists represent new antivirulence drugs for UTIs.

Matriptase and hepsin belong to the family of type II transmembrane serine proteases (TTSPs). Increased activity of these and the plasma protease, hepatocyte growth factor activator (HGFA), is associated with unregulated cell signaling and tumor progression through increased MET and RON kinase signaling pathways. These proteases are highly expressed in multiple solid tumors and hematological malignancies. Herein, we detail the synthesis and structure–activity relationships (SAR) of a dipeptide library bearing Arg α-ketobenozothiazole (kbt) warheads as novel inhibitors of HGFA, matriptase, and hepsin. We elucidated the substrate specificity for HGFA using positional scanning of substrate combinatorial libraries (PS-SCL), which was used to discover selective inhibitors of matriptase and hepsin. Using these selective inhibitors, we have clarified the specific role of hepsin in maintaining epithelial cell membrane integrity, known to be lost in breast cancer progression. These selective compounds are useful as chemical biology tools and for future drug discovery efforts.

In our effort to find small-molecule treatments of advanced prostate cancers (PCs), a novel series of indolyl and indolinyl propanamides (series II and III) were discovered as selective androgen receptor degraders (SARDs). Initial studies of androgen receptor (AR) antagonist (1) and agonist (2) propanamides yielded a tertiary aniline (3) with novel SARD activity but poor metabolic stability. Cyclization to II and III produced submicromolar AR antagonism and protein degradation selective to AR and AR splice variant (AR SV). II and III maintained potency against enzalutamide-resistant (Enz-R) mutant ARs and PC cells and were efficacious in Enz-R xenografts, suggesting their potential to treat advanced PCs. Design, synthesis, and biological activity of novel SARDs that could potentially be used for the treatment of a wide spectrum of PCs including castration-resistant, Enz-R, and/or AR SV-dependent advanced PCs that are often untreatable with known hormone therapies are discussed.

Six morpholine-(iso)thiosemicarbazone hybrids HL¹–HL⁶ and their Cu(II) complexes with good-to-moderate solubility and stability in water were synthesized and characterized. Cu(II) complexes [Cu(L^1–6)Cl] (1–6) formed weak dimeric associates in the solid state, which did not remain intact in solution as evidenced by ESI-MS. The lead proligands and Cu(II) complexes displayed higher antiproliferative activity in cancer cells than triapine. In addition, complexes 2–5 were found to specifically inhibit the growth of Gram-positive bacteria Staphylococcus aureus with MIC₅₀ values at 2–5 μg/mL. Insights into the processes controlling intracellular accumulation and mechanism of action were investigated for 2 and 5, including the role of ribonucleotide reductase (RNR) inhibition, endoplasmic reticulum stress induction, and regulation of other cancer signaling pathways. Their ability to moderately inhibit R2 RNR protein in the presence of dithiothreitol is likely related to Fe chelating properties of the proligands liberated upon reduction.

The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth, and survival. A novel series of HSP90 inhibitors were discovered by structure–activity relationship (SAR)-based optimization of an initial hit compound 11a having a 4-(4-(quinolin-3-yl)-1H-indol-1-yl)benzamide structure. The pyrazolo[3,4-b]pyridine derivative, 16e (TAS-116), is a selective inhibitor of HSP90α and HSP90β among the HSP90 family proteins and exhibits oral availability in mice. The X-ray cocrystal structure of the 16e analogue 16d demonstrated a unique binding mode at the N-terminal ATP binding site. Oral administration of 16e demonstrated potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body weight loss.

Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses, and nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of highly potent and selective fibrinolysis inhibitors. Here, we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent (K_i = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin, and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects.

μ opioid receptor (MOR) agonists have been widely applied for treating moderate to severe pain. However, numerous adverse effects have been associated with their application, including opioid-induced constipation (OIC), respiratory depression, and addiction. On the basis of previous work in our laboratory, NAP, a 6β-N-4′-pyridyl substituted naltrexamine derivative, was identified as a peripheral MOR antagonist that may be used to treat OIC. To further explore its structure–activity relationship, a new series of NAP derivatives were designed, synthesized, and biologically evaluated. Among these derivatives, NFP and NYP significantly antagonized the antinociception effect of morphine. Whereas NAP acted mainly peripherally, its derivatives NFP and NYP actually can act centrally. Furthermore, NFP produced significantly lesser withdrawal symptoms than naloxone at similar doses. These results suggest that NFP has the potential to be a lead compound to treat opioid abuse and addiction.

Pyruvate dehydrogenase kinase 4 (PDK4) activation is associated with metabolic diseases including hyperglycemia, insulin resistance, allergies, and cancer. Structural modifications of hit anthraquinone led to the identification of a new series of allosteric PDK4 inhibitors. Among this series, compound 8c showed promising in vitro activity with an IC₅₀ value of 84 nM. Good metabolic stability, pharmacokinetic profiles, and possible metabolites were suggested. Compound 8c improved glucose tolerance in diet-induced obese mice and ameliorated allergic reactions in a passive cutaneous anaphylaxis mouse model. Additionally, compound 8c exhibited anticancer activity by controlling cell proliferation, transformation, and apoptosis. From the molecular docking studies, compound 8c displayed optimal fitting in the lipoamide binding site (allosteric) with a full fitness, providing a new scaffold for drug development toward PDK4 inhibitors.

Kidney-type glutaminase [KGA/isoenzyme glutaminase C (GAC)] is becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839 analogues with selenium using a novel synthesis of the selenadiazole moiety from carboxylic acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition, more potent induction of reactive oxygen species, improved inhibition of cancer cells, and higher cellular and tumor accumulation than the corresponding sulfur-containing molecules. However, both CB839 and its selenium analogues show incomplete inhibition of the tested cancer cells, and a partial reduction in tumor size was observed in both the glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this, tumor tissue damage and prolonged survival were observed in animals treated with the selenium analogue of CB839.

Conformational restriction applied to dihydrobenzylpyrimidin-4-(3H)-ones (DABOs) by the intoduction of a methyl group at the α-benzylic position is known to massively improve the anti-HIV-1 activity of these compounds. Here, we report the effects of methoxy substitution at the α-benzylic position in S-, NH-, and N,N-DABOs carrying 2,6-difluoro, 2-chloro-6-fluoro, or 2,6-dichloro substituted benzyl moieties. The various α-methoxy DABO series (12–14) present different SAR at the dihalo benzyl substitution, with the most potent compounds (12d,e and 13c) showing similar (picomolar/nanomolar) anti-HIV-1 potency as the corresponding α-methyl analogues against wt HIV-1, and 10–100-fold increased potency (up to low nanomolar) against clinically relevant K103N, Y181C, Y188L, IRLL98, and K103N+Y181C HIV-1 mutant strains, highlighting the importance of the α-methoxy substitution to provide highly efficient DABOs as “second generation” NNRTIs. HPLC enantioseparation of three of the most potent derivatives (12d, 13c, and 14c) provided single enantiomers with significant enantioselectivity in HIV-1 inhibition. Computational studies allowed to correlate the best antiviral activity with the (R) absolute configuration at the α-methoxy stereogenic center.

There is an urgent clinical need for antimalarial compounds that target malaria caused by both Plasmodium falciparum and Plasmodium vivax. The M1 and M17 metalloexopeptidases play key roles in Plasmodium hemoglobin digestion and are validated drug targets. We used a multitarget strategy to rationally design inhibitors capable of potent inhibition of the M1 and M17 aminopeptidases from both P. falciparum (Pf-M1 and Pf-M17) and P. vivax (Pv-M1 and Pv-M17). The novel chemical series contains a hydroxamic acid zinc binding group to coordinate catalytic zinc ion/s, and a variety of hydrophobic groups to probe the S1′ pockets of the four target enzymes. Structural characterization by cocrystallization showed that selected compounds utilize new and unexpected binding modes; most notably, compounds substituted with bulky hydrophobic substituents displace the Pf-M17 catalytic zinc ion. Excitingly, key compounds of the series potently inhibit all four molecular targets and show antimalarial activity comparable to current clinical candidates.

Herein, the synthesis and pharmacological characterization of an extended library of differently substituted N-methyl-14-O-methylmorphinans with natural and unnatural amino acids and three dipeptides at position 6 that emerged as potent μ/δ opioid receptor (MOR/DOR) agonists with peripheral antinociceptive efficacy is reported. The current study adds significant value to our initial structure–activity relationships on a series of zwitterionic analogues of 1 (14-O-methyloxymorphone) by targeting additional amino acid residues. The new derivatives showed high binding and potent agonism at MOR and DOR in vitro. In vivo, the new 6-amino acid- and 6-dipeptide-substituted derivatives of 1 were highly effective in inducing antinociception in the writhing test in mice after subcutaneous administration, which was antagonized by naloxone methiodide demonstrating activation of peripheral opioid receptors. Such peripheral opioid analgesics may represent alternatives to presently available drugs for a safer pain therapy.

The interaction with erythrocytes of four [V^VO₂L₂]⁻ complexes, with L = picolinate (pic), 5-cyanopicolinate (picCN), 3-aminopyrazine-2-carboxylate (przNH₂), and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (dhp), was studied. The thermodynamic stability at physiological pH is: [V^VO₂(dhp)₂]⁻ > [V^VO₂(przNH₂)₂]⁻ > [V^VO₂(pic)₂]⁻ > [V^VO₂(picCN)₂]⁻. With picCN and pic, V exists at physiological pH as H₂V^VO₄^–, with przNH₂ as a mixture of H₂V^VO₄^– and [V^VO₂(przNH₂)₂]⁻ and with dhp as [V^VO₂(dhp)₂]⁻. In the systems with pic and picCN, H₂V^VO₄^– and the ligands cross the erythrocyte membrane independently, with dhp the uptake occurs by diffusion, whereas with przNH₂ both the mechanisms are active. Inside erythrocytes stable V^IVOL₂ complexes are formed, indicating that there is no relationship with the stability and redox state of the administered compounds and that, if the metal ion changes its oxidation state in the cytosol as V does, unstable complexes in the extracellular medium could become stable inside the cells and contribute to the pharmacological action.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC₅₀)) was established, as part of the lead selection process. Increasing the sp³ carbon content of 1 resulted in 10e (0.19 μM EC₅₀ against T. brucei and 990 μM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC₅₀: 0.013 μM; aqueous solubility: 880 μM; and CEC₅₀: 0.18 μM). Compound 22a reduced parasitemia 10⁹ fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Phosphodiesterase 10A (PDE10A) is a newly identified therapeutic target for central-nervous-system disorders. 2-(2-(3-(4-([¹⁸F]Fluoroethoxy)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([¹⁸F]MNI-659, [¹⁸F]5) is a useful positron-emission-tomography (PET) ligand for imaging of PDE10A in the human brain. However, the radiolabeled metabolite of [¹⁸F]5 can accumulate in the brain. In this study, using [¹⁸F]5 as a lead compound, we designed four new ¹⁸F-labeled ligands ([¹⁸F]6–9) to find one more suitable than [¹⁸F]5. Of these, 2-(2-(3-(4-([¹⁸F]fluoromethoxy-d₂)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([¹⁸F]9) exhibited high in vitro binding affinity (K_i = 2.9 nM) to PDE10A and suitable lipophilicity (log D = 2.2). In PET studies, the binding potential (BP_ND) of [¹⁸F]9 (5.8) to PDE10A in the striatum of rat brains was significantly higher than that of [¹⁸F]5 (4.6). Furthermore, metabolite analysis showed much lower levels of contamination with radiolabeled metabolites in the brains of rats given [¹⁸F]9 than in those given [¹⁸F]5. In conclusion, [¹⁸F]9 is a useful PET ligand for PDE10A imaging in brain.

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase–target pair deemed unsuitable: the von Hippel–Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure–activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target–ligase combinations.

Wnt/β-catenin signaling pathway is implicated in the etiology and progression of metabolic disorders. Although lines of genetic evidence suggest that blockage of this pathway yields favorable outcomes in treating such ailments, few inhibitors have been used to validate the promising genetic findings. Here, we synthesized and characterized a novel class of triazole-based Wnt/β-catenin signaling inhibitors and assessed their effects on energy metabolism. One of the top inhibitors, compound 3a, promoted Axin stabilization, which led to the proteasome degradation of β-catenin and subsequent inhibition of the Wnt/β-catenin signaling in cells. Treatment of hepatocytes and high fat diet-fed mice with compound 3a resulted in significantly decreased hepatic lipid accumulation. Moreover, compound 3a improved glucose tolerance of high fat diet-fed mice without noticeable toxicity, while downregulating the genes involved in the glucose and fatty acid anabolisms. The new inhibitors are expected to be further developed for the treatment of metabolic disorders.

Protein–protein interactions (PPIs) regulate all signaling pathways for cellular function. Developing molecules that modulate PPIs through the interface of their protein surfaces has been a significant challenge and there has been little success controlling PPIs through standard molecular library screening approaches. PPIs control the cell’s protein-folding machinery, and this machinery relies on a multiprotein complex formed with heat shock protein 70 (Hsp70). Described is the design, synthesis, and biological evaluation of molecules aimed to regulate the interaction between two proteins that are critical to the protein-folding machinery: heat shock protein 70 (Hsp70) and cochaperone heat shock organizing protein (HOP). We report the first class of compounds that directly regulate these two protein–protein interactions and inhibit protein folding events.

Dynamic combinatorial chemistry (DCC) has emerged as a promising strategy for template-driven selection of high-affinity ligands for biological targets from equilibrating combinatorial libraries. However, only a few examples using disulfide-exchange-based DCC are reported for nucleic acid targets. Herein, we have demonstrated that gold-coated magnetic nanoparticle-conjugated DNA targets can be used as templates for dynamic selection of ligands from an imine-based combinatorial library. The implementation of DCC using DNA nanotemplates enables efficient identification of the lead compounds, from the dynamic combinatorial library via magnetic decantation. It further allows quick separation of DNA nanotemplates for reuse in DCC reactions. The identified lead compound exhibits significant quadruplex versus duplex DNA selectivity and suppresses promoter activity of c-MYC gene that contains G-quadruplex DNA forming sequence in the upstream promoter region. Further cellular experiments indicated that the lead compound is able to permeate into cell nuclei and trigger a DNA damage response in cancer cells.

Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.

The final stages of polio eradication are proving more difficult than the early phases, and the development of effective drugs and treatments is considered a priority; thus, the research is ongoing. A screening of our in-house chemical library against poliovirus Sabin strains led to the identification of compounds 5 and 6 as hits active at submicromolar concentrations. Derivatives of these compounds were synthesized as a preliminary structure–activity-relationship study. Among them, 7 and 11 were highly active against poliovirus Sabin 1–3. Compound 11 was also very potent against a large panel of wild and vaccine-derived polioviruses. Time-of-addition experiments suggest that 5 and 7 could be active at an early stage of viral replication, whereas 11 was active at same concentration at all stages of viral replication. A ligand-based approach was applied to find the common structural features shared by the new compounds and already-known poliovirus inhibitors.

We previously developed a novel series of vinyl sulfones as nuclear factor erythroid 2-related factor 2 (Nrf2) activators with therapeutic potential for Parkinson’s disease (PD). However, the previously developed lead compound (1) exhibited undesirable druglike properties. Here, we optimized vinyl sulfones by introducing nitrogen heterocycles to improve druglike properties. Among the synthesized compounds, 17e was the most promising drug candidate with good druglike properties. Compound 17e showed superior effects on Nrf2 activation in cell-based assays compared to compound 1 (17e: half-maximal effective concentration (EC₅₀) = 346 nM; 1: EC₅₀ = 530 nM). Compound 17e was further confirmed to induce expression of Nrf2-dependent antioxidant enzymes at both mRNA and protein levels. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of PD, 17e significantly attenuated loss of tyrosine hydroxylase-immunopositive dopaminergic neurons, suppressed microglial activation, and alleviated PD-associated motor dysfunction. Thus, 17e is a novel Nrf2 activator with excellent druglike properties and represents a potential therapeutic candidate for PD.

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Na_v1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund’s adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia and models of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The observed efficacy could be correlated with the mouse dorsal root ganglion exposure and Na_V1.7 potency associated with 29.

We designed and synthesized quinazolin-2,4-dione-based hydroxamic acids to serve as selective competitive inhibitors of histone deacetylase-6 (HDAC6). The most potent and selective compound, 3d (IC₅₀, 4 nM, HDAC6; IC₅₀ > 10 μM, HDAC1), substantially increased acetylation of α-tubulin instead of histones in the lung cancer cell line, LL2. Paclitaxel in combination with 3d had a synergistic anticancer effect on reduction of programmed death-ligand 1 expression in LL/2 cells. When given orally, 3d was mainly found to locate in the liver and lungs, at a concentration 18- to 70-fold greater, respectively, than in plasma. As an orally active HDAC6 inhibitor, 3d (20 mg/kg) potentiated paclitaxel antitumor activity (percentage tumor growth inhibition, 67.5%) in a xenograft syngeneic non-small cell lung cancer mouse model.

Most of the current FMS-like tyrosine kinase 3 (FLT3) inhibitors lack selectivity between FLT3 kinase and cKIT kinase as well as the FLT3 wt and internal tandem duplication (ITD) mutants. We report a new compound 27, which displays GI₅₀ values of 30–80 nM against different ITD mutants and achieves selectivity over both FLT3 wt (8-fold) and cKIT kinase in the transformed BaF3 cells (>300-fold). 27 potently inhibits the proliferation of the FLT3-ITD-positive acute myeloid leukemia cancer lines through suppression of the phosphorylation of FLT3 kinase and downstream signaling pathways, induction of apoptosis, and arresting the cell cycle into the G0/G1 phase. 27 also displays potent antiproliferative effect against FLT3-ITD-positive patient primary cells, whereas it does not apparently affect FLT3 wt primary cells. In addition, it also exhibits a good therapeutic window to PBMC compared to PKC412. In the in vivo studies, 27 demonstrates favorable PK profiles and suppresses the tumor growth in the MV4-11 cell inoculated mouse xenograft model.

Neuroinflammatory disorders, such as multiple sclerosis or experimental autoimmune encephalomyelitis (EAE), an established mouse model mimicking part of the human pathology, are characterized by inflammatory infiltrates containing T helper 1 (T_H1) and T_H17 cells, which cause demyelination and neurodegeneration. Disease onset and perpetuation are mediated by peripherally generated autoreactive T cells infiltrating into the central nervous system, where they are restimulated by antigen-presenting cells. Here, we show that newly designed peripherally active, potent, and selective κ-opioid receptor (KOR) agonists comprising the ethylenediamine KOR pharmacophore in a perhydroquinoxaline scaffold exhibit potent anti-inflammatory capacities in primary antigen presenting cells as well as T cells. In the EAE model, the secondary amine 12 and the triazole 14 were able to ameliorate disease severity and to delay disease onset by blocking effector T cell activation. Importantly, the beneficial effects were mediated via signaling through KOR because off-target effects were excluded by using KOR-deficient mouse mutants.

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide Na_V1.7 inhibitors that are selective for Na_V1.7 over Na_V1.5 and highly efficacious in in vivo models of pain and hNa_V1.7 target engagement. An analysis of the physicochemical properties of literature Na_V1.7 inhibitors suggested that acyl sulfonamides with high f_sp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against Na_V1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNa_V1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the Na_V1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious Na_V1.7 inhibitors reported to date.

The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein, we report the identification of a highly potent and selective ERK5 inhibitor BAY-885 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction, and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis, and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology.

We report herein the discovery of highly potent PROTAC degraders of androgen receptor (AR), as exemplified by compound 34 (ARD-69). ARD-69 induces degradation of AR protein in AR-positive prostate cancer cell lines in a dose- and time-dependent manner. ARD-69 achieves DC₅₀ values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1 AR+ prostate cancer cell lines, respectively. ARD-69 is capable of reducing the AR protein level by >95% in these prostate cancer cell lines and effectively suppressing AR-regulated gene expression. ARD-69 potently inhibits cell growth in these AR-positive prostate cancer cell lines and is >100 times more potent than AR antagonists. A single dose of ARD-69 effectively reduces the level of AR protein in xenograft tumor tissue in mice. Further optimization of ARD-69 may ultimately lead to a new therapy for AR+, castration-resistant prostate cancer.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

P-Glycoprotein is a well-known membrane transporter responsible for the efflux of an ample spectrum of anticancer drugs. Its relevance in the management of cancer chemotherapy is increased in view of its high expression in cancer stem cells, a population of cancer cells with strong tumor-promoting ability. In the present study, a series of compounds were synthesized through structure modulation of [4′-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-ylmethyl)biphenyl-4-ol] (MC70), modifying the phenolic group of the lead compound. Among them, compound 5b emerged for its activity against the transporter (EC₅₀ = 15 nM) and was capable of restoring doxorubicin antiproliferative activity at nontoxic concentration. Its behavior was rationalized through a molecular modeling study consisting of a well-tempered metadynamics simulation, which allowed one to identify the most favorable binding pose, and of a subsequent molecular dynamics run, which indicated a peculiar effect of the compound on the motion pattern of the transporter.

Human acid ceramidase (AC) is a lysosomal cysteine amidase, which has received a great deal of interest in recent years as a potential target for the development of new therapeutics against melanoma and glioblastoma tumors. Despite the strong interest in obtaining structural information, only the structures of the apo-AC enzyme in its zymogen and activated conformations are available. In this work, the crystal structure of AC in complex with the covalent carmofur inhibitor is presented. Carmofur is an antineoplastic drug containing an electrophilic carbonyl reactive group that targets the catalytic cysteine. This novel structural data explains the basis of the AC inhibition, provides insights into the enzymatic properties of the protein, and is a great aid toward the structure-based drug design of potent inhibitors for AC, providing the detailed mechanism, which has eluded the scientific community for more than 30 years, of carmofur’s mysterious 5-fluorouracil-independent antitumor activity.

A series of novel quinoline–chalcone derivatives were designed, synthesized, and evaluated for their antiproliferative activity. Among them, compound 24d exhibited the most potent activity with IC₅₀ values ranging from 0.009 to 0.016 μM in a panel of cancer cell lines. Compound 24d also displayed a good safety profile with an LD₅₀ value of 665.62 mg/kg by intravenous injection, and its hydrochloride salt 24d-HCl significantly inhibited tumor growth in H22 xenograft models without observable toxic effects, which was more potent than that of CA-4. Mechanism studies demonstrated that 24d bound to the colchicine site of tubulin, arrested the cell cycle at the G2/M phase, induced apoptosis, depolarized mitochondria, and induced reactive oxidative stress generation in K562 cells. Moreover, 24d has potent in vitro antimetastasis and in vitro and in vivo antivascular activities. Collectively, our findings suggest that 24d deserves to be further investigated as a potent and safe antitumor agent for cancer therapy.

Sialic acid sugars on mammalian cells regulate numerous biological processes, while aberrant expression of sialic acid is associated with diseases such as cancer and pathogenic infection. Inhibition of the sialic acid biosynthesis may therefore hold considerable therapeutic potential. To effectively decrease the sialic acid expression, we synthesized C-5-modified 3-fluoro sialic acid sialyltransferase inhibitors. We found that C-5 carbamates significantly enhanced and prolonged the inhibitory activity in multiple mouse and human cell lines. As an underlying mechanism, we have identified that carbamate-modified 3-fluoro sialic acid inhibitors are more efficiently metabolized to their active cytidine monophosphate analogues, reaching higher effective inhibitor concentrations inside cells.

Structure–activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2-a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Recently, we have discovered that the registered pesticide, tolfenpyrad, unexpectedly and potently inhibits the development of the L4 larval stage of the parasitic nematode Haemonchus contortus with an IC₅₀ value of 0.03 μM while displaying good selectivity, with an IC₅₀ of 37.9 μM for cytotoxicity. As a promising molecular template for medicinal chemistry optimization, we undertook anthelmintic structure–activity relationships for this chemical. Modifications of the left-hand side (LHS), right-hand side (RHS), and middle section of the scaffold were explored to produce a set of 57 analogues. Analogues 25, 29, and 33 were shown to be the most potent compounds of the series, with IC₅₀ values at a subnanomolar level of potency against the chemotherapeutically relevant fourth larval (L4) stage of H. contortus. Selected compounds from the series also showed promising activity against a panel of other different parasitic nematodes, such as hookworms and whipworms.

JAK family kinases are important mediators of immune cell signaling and Janus Kinase 3 (JAK3) has long been indicated as a potential target for autoimmune disorders. Intensive efforts to develop highly selective JAK3 inhibitors have been underway for many years. However, because of JAK3’s strong binding preference to adenosine 5′-triphosphate (ATP), a number of inhibitors exhibit large gaps between enzymatic and cellular potency, which hampers efforts to dissect the roles of JAK3 in cellular settings. Using a targeted covalent inhibitor approach, we discovered compound 32, which overcame ATP competition (1 mM) in the enzymatic assay, and demonstrated significantly improved inhibitory activity for JAK3-dependent signaling in mouse CTLL-2 and human peripheral blood mononuclear cells. Compound 32 also exhibited high selectivity within the JAK family and good pharmacokinetic properties. Thus, it may serve as a highly valuable tool molecule to study the overlapping roles of JAK family kinases in complex biological settings. Our study also suggested that for covalent kinase inhibitors, especially those targeting kinases with low K_m ATP values, the reversible interactions between molecules and proteins should be carefully optimized to improve the overall potency.

Heparin is a polypharmacological agent with anticoagulant activity. Periodate oxidation of the nonsulfated glucuronic acid residue results in non-anticoagulant heparin derivative (NACH) of reduced molecular weight. Similar treatment of a low molecular weight heparin, dalteparin, also removes its anticoagulant activity, affording a second heparin derivative (D-NACH). A full structural characterization of these two derivatives reveals their structural differences. SPR studies display their ability to bind to several important heparin-binding proteins, suggesting potential new therapeutic applications.