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An Inhibitor’s-Eye View of the ATP-Binding Site of CDKs in Different Regulatory States
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† Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom
‡ Department of Chemical Sciences, University of Padua, via Marzolo 1, 35131 Padova, Italy
*E-mail: [email protected]
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ACS Chemical Biology

Cite this: ACS Chem. Biol. 2014, 9, 6, 1251–1256
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https://doi.org/10.1021/cb500135f
Published March 26, 2014

Copyright © 2014 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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We have used a chemically diverse panel of kinase inhibitors to assess the chemical similarity of the ATP-binding sites of cyclin-dependent kinase (CDK) subfamily members in a range of activation states. Using this approach, we find that different activation states of a particular CDK may differ from each other as much as different CDKs in the same activation state. We also find that inhibitors discriminate more effectively among CDK family members in their monomeric state than in their cyclin-bound state, providing direct evidence for the belief that selective binding to inactive kinase states might be more readily achieved than selective binding to active states.

Copyright © 2014 American Chemical Society

Subjects

what are subjects

Protein kinases represent attractive targets for therapeutic intervention against a wide variety of human diseases, most notably cancer (1, 2) and chronic inflammatory diseases. (3) A number of drugs that selectively target the protein kinase ATP binding site have been successfully introduced into the clinic, and many more are in clinical trials. (2, 4) However, a significant requirement for the further development of therapeutically useful compounds is a more complete understanding of the factors that dictate inhibitor selectivity across the protein kinase family. (5, 2, 6)
The eukaryotic serine/threonine and tyrosine protein kinase family is characterized by a conserved fold in which residues from both the N- and C-terminal lobes contribute to the active site. (7, 8) The identities of the residues that line the ATP binding pocket and the structural plasticity of the protein kinase fold constitute two key elements that together determine the inhibitor-binding profile of a protein kinase. Both of these elements have been successfully exploited to generate clinically useful drugs. (1, 2)
The cyclin-dependent kinases (CDKs) constitute a subfamily of ca. 13 members in humans (9) that play important roles in both the control of cell cycle progression (CDKs 1, 2, 3, 4, and 6) and in the regulation of transcription (CDKs 7, 8, 9, 12, and 13). (10-12) CDK2 has provided a structural paradigm for the CDK family and has been widely exploited for structure-aided CDK inhibitor design. (13, 14) The prevailing structural model for CDK regulation by cyclin and CDK inhibitor (CKI) binding and by phosphorylation has been elaborated through a series of structures of CDK2/cyclin A complexes. (15, 16) Monomeric CDK2 is inactive as a result of the disposition of active site residues, in turn dependent on the pose of the C-helix, and the conformation of the activation segment. (17) Cyclin A binding and Thr160 phosphorylation within the activation segment rearrange the CDK2 active site to orientate key ATP binding and catalytic residues and create the peptide substrate binding site. (15, 18) This model for the mechanism of regulation appears not to apply across the entire CDK subfamily. The determination of structures for CDK4/cyclin D3 (19) and CDK4/cyclin D1 phosphorylated on Thr172 (pCDK4/D) (20) revealed that CDK4 adopts an inactive C-helix out conformation despite being cyclin-bound. Two further examples are CDK5 (21) and CDK8 (22) that both adopt active conformations upon p25 and cyclin C binding, respectively, in the absence of activation loop phosphorylation.
Differential scanning fluorimetry (DSF) can be used to characterize inhibitor binding. (23) Here we define ΔTm as the difference between the apparent melting temperature (Tm) of the protein in the presence of the ligand and the Tm of the protein alone. The value is related to the concentration and binding affinity of the ligand for the protein and to thermodynamic properties of the ligand binding event. (23) The profile of ΔTm values measured for a protein in the presence of a sufficiently diverse range of inhibitors is characteristic of the inhibitor binding properties of that protein and therefore represents a fingerprint of the physicochemical properties of the active site. (24) We have used such fingerprints to assess the chemical similarity of a number of CDK subfamily members, namely, CDK2, CDK4, CDK7, and CDK9. Additionally, we have explored the inhibitor binding profiles of CDKs in different activation states to assess the extent to which CDK activation affects inhibitor binding.

Results and Discussion

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The discriminative power of this fingerprinting approach relies on including a sufficiently diverse panel of inhibitors to sense the chemical diversity apparent in the family of kinase active sites. To validate the use of our selected inhibitor set (Figure 1a and Supplementary Table 1) for this purpose, we confirmed that our set is able to target representative kinases from across the kinome. This analysis was achieved by mapping each inhibitor onto those kinases in the kinome phylogenetic tree that they are best documented to target (Supplementary Methods and Figure 1b). The approximately uniform distribution obtained confirms ours to be a relatively unbiased set of inhibitors.

Figure 1

Figure 1. Explored chemical space. (a) Representative inhibitor scaffolds. Examples of the inhibitor scaffolds included in the panel are shown. All inhibitor structures are provided in the Supporting Information (Supplementary Table 1). (b) Chemical space explored by the panel of inhibitors used in this study. The target(s) in the human kinome for each of the inhibitors of the panel are identified by a red rectangle on the kinome tree. This figure was adapted from the phylogenetic tree published in ref 9.

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We included four members of the CDK subfamily in the study. CDK2 was prepared in four different conformational states: monomeric unphosphorylated (CDK2), monomeric phosphorylated on Thr160 (pCDK2), unphosphorylated in complex with human cyclin A175–432 (CDK2/A), and Thr160-phosphorylated in complex with human cyclin A175–432 (pCDK2/A). CDK4 was characterized both as an inactive, nonphosphorylated monomer (CDK4) and as the fully activated binary complex (CDK4/cyclin D3 phosphorylated on Thr172, pCDK4/D). CDK7 and CDK9 were prepared in either a monomeric unphosphorylated (CDK7) or a cyclin-bound phosphorylated (CDK9/cyclin T1 phosphorylated on T186, pCDK9/T) state, respectively (Figure 2a). The identities of the CDKs together with their activation states define a search space within the functional kinome that has been explored by this study and is summarized in Figure 2b. CDK2, CDK7, CDK4, and CDK9 are highly homologous across their entire sequence (Supplementary Figure 1), and this similarity is particularly marked at the catalytic site (Figure 2c,d).

Figure 2

Figure 2. CDK space explored in this study. (a) Representative melting curves for cyclin-dependent kinase (CDK)/cyclin complexes used to derive ΔTm values in the presence of an ATP-competitive inhibitor. The shift in Tm upon staurosporine binding (red) is illustrated for each of the CDK/cyclin complexes in this study. Black curves, CDK/cyclin complexes in the absence of added inhibitor. Curves with closed triangle symbols correspond to pCDK9/T, closed diamonds to CDK7, closed circles to CDK4, closed squares to pCDK4/D, open squares to CDK2/A, open diamonds to CDK2, and open triangles to pCDK2. (b) The full set of different phosphorylated and/or cyclin-associated states of the four CDKs characterized in this study are shown, with those members for which measurements were made highlighted by shaded boxes. Color intensity for these protein species reflects activation state (more saturated, more active). (c) The ATP binding site of CDKs. The CDK2 ATP-binding site residues from a pCDK2/A structure (PDB code: 1QMZ) are colored according to the degree of sequence conservation between the 4 CDK sequences, calculated as described in the Supplementary Methods, from blue (highly conserved) to red (highly variable). The ATP molecule is represented in ball-and-stick mode for context. (d) The ATP-binding site pocket is highly conserved in CDK2, CDK7, CDK4, and CDK9. Residues that constitute the first shell of the CDK2 ATP-binding site (CDK2 numbering) were identified as defined in Supplementary Methods and used to construct a pseudosequence alignment. This alignment is displayed together with the associated consensus sequence, with X representing any residue, and ϕ a hydrophobic amino acid. Sequence motifs that are highly conserved across the protein kinase family are identified by numbered bars. Bar 1, the glycine-rich loop (residues 11–16 in CDK2) that defines the general consensus sequence GXGXXG that recognizes the phosphate moieties of ATP; bar 2, the “hinge” sequence composed of residues 80–84 that links the N- and C-terminal lobes and contributes to the ATP adenine binding site; bar 3, the “DFG motif” that defines the start of the activation segment. Asterisks below E51 and D127 highlight the conserved glutamate in the C-helix and the catalytic aspartate, respectively. A full sequence alignment is presented in Supplementary Figure 1.

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We have adopted the linear correlation coefficient of the inhibitory profiles of two kinases as a measure of the chemical similarity of those kinases (Tm values tabulated in Supplementary Table 1, Supplementary Figure 2). By this criterion, we find that the set of kinases studied in the monomeric, unphosphorylated state (CDK2, CDK4, and CDK7) demonstrate significantly different inhibitory profiles, with a lowest correlation coefficient of 0.39 (CDK4 vs CDK7) and a highest correlation coefficient of 0.74 (CDK2 vs CDK7) (Figure 3a). That the inhibitor binding fingerprint discriminates so effectively between highly related CDK subfamily members confirms that it provides a sensitive measure of active site similarity.

Figure 3

Figure 3. Comparison of the inhibitor-binding profiles of various cyclin-dependent kinase (CDK) subfamily members in different states of activation. Each block represents a CDK in a particular activation state: CDK (monomeric, unphosphorylated), pCDK (monomeric, phosphorylated), CDK/[A or D or T] (unphosphorylated binary complex between a CDK and the corresponding cyclin), and pCDK/[A or D or T] (phosphorylated binary complex between a CDK and the corresponding cyclin). The internal consistency of inhibitory fingerprints for each CDK/state pair, evaluated over two or three repeat measurements, is indicated by a value within the corresponding block. The correlation of fingerprints between different CDK/state pairs is indicated by a value on the line that connects them. (a) Comparison of the inhibitor fingerprints of CDK subfamily members in the monomeric form. (b) Comparison of the inhibitor fingerprints of CDK subfamily members in the phosphorylated binary forms. (c) Comparison of CDK2 inhibitor fingerprints in different activation states. Color intensity as in Figure 2b reflects activation state (more saturated, more active).

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One application of this measure is to assess the chemical similarity of the active sites of orthologous proteins from different species. For example, the correlation coefficients obtained for the comparison of human monomeric, unphosphorylated CDKs with the closest CDK orthologue from Plasmodium falciparum, protein kinase 5 (PfPK5), range from 0.42 (PfPK5 vs CDK4) to 0.69 (PfPK5 vs CDK2). This result demonstrates that the active sites of orthologous proteins from highly diverged species may be more similar to each other than are the active sites of close paralogues within a species.
For context, the equivalent comparison of each of the monomeric, unphosphorylated CDKs with an “unrelated” kinase, P. falciparum protein kinase 7 (PfPK7), (25) yields correlation coefficients in the range 0.33 (PfPK7 vs CDK2) to 0.31 (PfPK7 vs CDK2/A).
Next we investigated the apparent active-site similarity of a set of fully activated CDK subfamily members (pCDK2/A, pCDK4/D, and pCDK9/T). Although this comparison also yielded quantitatively different inhibitor fingerprints, our results suggest that the inhibitor-binding properties of the set of fully activated CDKs are more similar to each other than are those of the set of inactive monomeric forms (Figure 3b). The lowest correlation coefficient of 0.73 and the highest correlation coefficient of 0.78 were measured for comparisons of pCDK2/A vs pCDK9/T and pCDK2/A vs pCDK4/D, respectively. This result has two implications: first, it demonstrates that the inhibitor binding properties of CDK subfamily members depend not only on their respective sequence but also on the conformational state in which they are found. Second, it demonstrates that, in adopting an active conformation, two different CDKs assume more similar inhibitor binding properties. This latter point is most directly demonstrated by comparison of the correlations coefficients CDK2 vs CDK4 (CC = 0.69) and pCDK2/A vs pCDK4/D (CC = 0.78).
The inhibitor fingerprints of four different CDK2 activation states were recorded, and the resulting comparison is shown in Figure 3c. As expected from comparative structural studies, (26, 27) phosphorylation of the activation segment has little effect on the inhibitor-binding fingerprint (correlation coefficient for the comparison CDK2 vs pCDK2 is 0.94 and for the comparison of CDK2/A vs pCDK2/A is 0.96). Indeed, sequence and structural comparisons demonstrate that phosphorylation of CDK2 or CDK2/A on Thr160 introduces only minor changes to the identity and structure of the amino acids that line the extended inhibitor binding site (Supplementary Figure 3).
By contrast, when pCDK2 associates with cyclin A, the CDK2 inhibitor-binding fingerprint changes significantly (correlation coefficient for pCDK2 vs pCDK2/A is 0.79). Interestingly, the equivalent comparison for unphosphorylated CDK2 demonstrates somewhat less of a response: when cyclin A associates with the unphosphorylated form of CDK2, it perturbs its inhibitor binding profile to a lesser extent (correlation coefficient for CDK2 vs CDK2/A = 0.91). Comparison of the ATP binding site topology highlights a similar degree of conservation between the closest pairs considered, as summarized in Supplementary Figure 3.
For the CDKs analyzed in this study, the abundance of each conformational state within a cell will depend on a large number of factors that include CDK identity, cell type, cell environment, and cell cycle stage. Targeting a specific conformation of a protein kinase is a validated route to the design of selective and potent inhibitors and has been particularly successful in the design of tyrosine kinase inhibitors. (28) Cell cycle CDKs of differing phosphorylation status and in monomeric and cyclin-bound forms can be detected in cells, suggesting that such states are accessible for targeting. (29)
In summary, by using DSF we have demonstrated that it is possible to discriminate between closely related CDKs by comparing their inhibitor-binding profiles. We have shown that activation state can have a significant effect on inhibitor-binding profile, with cyclin binding having more profound consequences than phosphorylation at least in the case of CDK2. Our results further suggest that the inhibitor binding properties of a set of fully activated, cyclin-bound CDKs are more similar to each other than are those of a set of inactive, monomeric forms. These data provide direct evidence that, as has been proposed elsewhere, (13) protein kinases may be more amenable to selective inhibitor binding in their inactive states.

Methods

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Inhibitor Set

A full description of the inhibitor set is provided in the Supporting Information.

CDK Expression and Purification

Human CDK4 and cyclin D3, (19) CDK9 and cyclin T1 (30) were all expressed in insect cells and purified as described. CDK2 phosphorylated on Thr160 in association with cyclin A2 (pCDK2/A), (18) unphosphorylated CDK2 in association with cyclin A2 (CDK2/A), monomeric Thr160pCDK2 (pCDK2), (26) monomeric unphosphorylated CDK2 (CDK2), and CDK7 were prepared as described. (31) Monomeric CDK4 was expressed in Sf9 cells as a GST fusion and purified by affinity chromatography followed by 3C cleavage of the GST tag and subsequent size-exclusion chromatography. The proteins were concentrated to 2 mg mL–1 and flash frozen in liquid nitrogen prior to storage at −80 °C until further use.

Differential Scanning Fluorimetry

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene) essentially as described. (32, 33) A full description of the method is provided in the Supporting Information.

Kinase Targets of the Molecules Contained in the Inhibitor Set

The kinase targeted by each of the inhibitors was identified from either the supplier’s information or by literature analysis, mainly via the work reported in refs 34−36.

Computation

Residues corresponding to the first coordination shell of the ATP-binding sites were identified in ref 37. Sequence conservation scores were calculated using the ConSurf webserver (http://consurf.tau.ac.il).

Supporting Information

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This material is available free of charge via the Internet at http://pubs.acs.org.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
    • Martin E. M. Noble - Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom;  Email: [email protected]
  • Authors
    • Aude Echalier - Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom
    • Alison J. Hole - Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom
    • Graziano Lolli - Department of Chemical Sciences, University of Padua, via Marzolo 1, 35131 Padova, Italy
    • Jane A. Endicott - Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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We thank I. Taylor for technical assistance, E. Lowe and D. Staunton for crystallography and biophysics support, and TTP LabTech for help with inhibitor dispensing. We also thank R. Griffin and colleagues in the Department of Chemistry, Newcastle University and S. Knapp (SGC, Oxford) for providing reagents. We are grateful to S. Baumli (Merck Serono) for the gift of purified pCDK9/T and help with thermal denaturation data collection. This research was supported by Framework Program 6 (FP6) of the European Commission (PROKINASE Project, A.E.), and the MRC (J.A.E., A.H., and M.E.M.N.). A.J.H. was supported by a Wellcome Trust studentship (Grant 083113/Z/07/A).

Abbreviations

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CDK

cyclin-dependent kinase

DSF

differential scanning fluorimetry

pCDK4/D

Thr172-phosphorylated CDK4/cyclin D3

pCDK9/T

Thr186-phosphorylated CDK9/cyclin T1

pCDK2/A

Thr160-phosphorylated CDK2/cyclin A2

References

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This article references 37 other publications.

  1. 1
    Zhang, J., Yang, P. L., and Gray, N. S. (2009) Targeting cancer with small molecule kinase inhibitors Nat. Rev. Cancer 9, 28– 39
    Google Scholar
    1
    Targeting cancer with small molecule kinase inhibitors
    Zhang, Jianming; Yang, Priscilla L.; Gray, Nathanael S.
    Nature Reviews Cancer (2009), 9 (1), 28-39CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
    A review. Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiol. constraints on growth and survival. To date, 11 kinase inhibitors have received US Food and Drug Administration approval as cancer treatments, and there are considerable efforts to develop selective small mol. inhibitors for a host of other kinases that are implicated in cancer and other diseases. Herein we discuss the current challenges in the field, such as designing selective inhibitors and developing strategies to overcome resistance mutations. This Review provides a broad overview of some of the approaches currently used to discover and characterize new kinase inhibitors.
  2. 2
    Fabbro, D., Cowan-Jacob, S. W., Mobitz, H., and Martiny-Baron, G. (2012) Targeting cancer with small-molecular-weight kinase inhibitors Methods Mol. Biol. (N. Y.) 795, 1– 34
    Google Scholar
    2
    Targeting cancer with small-molecular-weight kinase inhibitors
    Fabbro Doriano; Cowan-Jacob Sandra W; Mobitz Henrik; Martiny-Baron Georg
    Methods in molecular biology (Clifton, N.J.) (2012), 795 (), 1-34 ISSN:.
    Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.
  3. 3
    Cohen, P. (2009) Targeting protein kinases for the development of anti-inflammatory drugs Curr. Opin. Cell Biol. 21, 317– 324
    Google Scholar
    3
    Targeting protein kinases for the development of anti-inflammatory drugs
    Cohen, Philip
    Current Opinion in Cell Biology (2009), 21 (2), 317-324CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)
    A review. In recent years, protein kinases have become the pharmaceutical industry's most studied class of drug target, and some 10 protein kinase inhibitors have so far been approved for the treatment of cancer. However, whether safe drugs that modulate protein kinase activities can also be developed for the treatment of chronic diseases, where they may need to be taken for decades, is an issue that is still unresolved. A no. of compds. that inhibit the p38α MAPK have entered clin. trials for the treatment of rheumatoid arthritis and psoriasis, but side effects have prevented their progression to Phase III clin. trials. Here the author briefly reviews the potential problems in targeting p38 MAPK and discusses other protein kinases that regulate the innate immune system, such as Tpl2, MAPKAP-K2/3, MSK1/2, and IRAK4, which may be better targets for the treatment of chronic inflammatory diseases, and NIK, which is an attractive target for the treatment of multiple myeloma, a late stage B-cell malignancy.
  4. 4
    Janne, P. A., Gray, N., and Settleman, J. (2009) Factors underlying sensitivity of cancers to small-molecule kinase inhibitors Nat. Rev. Drug Discovery 8, 709– 723
    Google Scholar
    There is no corresponding record for this reference.
  5. 5
    Karaman, M. W., Herrgard, S., Treiber, D. K., Gallant, P., Atteridge, C. E., Campbell, B. T., Chan, K. W., Ciceri, P., Davis, M. I., Edeen, P. T., Faraoni, R., Floyd, M., Hunt, J. P., Lockhart, D. J., Milanov, Z. V., Morrison, M. J., Pallares, G., Patel, H. K., Pritchard, S., Wodicka, L. M., and Zarrinkar, P. P. (2008) A quantitative analysis of kinase inhibitor selectivity Nat. Biotechnol. 26, 127– 132
    Google Scholar
    5
    A quantitative analysis of kinase inhibitor selectivity
    Karaman, Mazen W.; Herrgard, Sanna; Treiber, Daniel K.; Gallant, Paul; Atteridge, Corey E.; Campbell, Brian T.; Chan, Katrina W.; Ciceri, Pietro; Davis, Mindy I.; Edeen, Philip T.; Faraoni, Raffaella; Floyd, Mark; Hunt, Jeremy P.; Lockhart, Daniel J.; Milanov, Zdravko V.; Morrison, Michael J.; Pallares, Gabriel; Patel, Hitesh K.; Pritchard, Stephanie; Wodicka, Lisa M.; Zarrinkar, Patrick P.
    Nature Biotechnology (2008), 26 (1), 127-132CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biol. consequences of multikinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. The authors present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global anal. of the results, the authors introduce the concept of a selectivity score as a general tool to quantify and differentiate the obsd. interaction patterns. The authors further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.
  6. 6
    Smyth, L. A. and Collins, I. (2009) Measuring and interpreting the selectivity of protein kinase inhibitors J. Chem. Biol. 2, 131– 151
    Google Scholar
    6
    Measuring and interpreting the selectivity of protein kinase inhibitors
    Smyth Lynette A; Collins Ian
    Journal of chemical biology (2009), 2 (3), 131-51 ISSN:1864-6158.
    Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.
  7. 7
    Huse, M. and Kuriyan, J. (2002) The conformational plasticity of protein kinases Cell 109, 275– 282
    Google Scholar
    7
    The conformational plasticity of protein kinases
    Huse, Morgan; Kuriyan, John
    Cell (Cambridge, MA, United States) (2002), 109 (3), 275-282CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    A review with 48 refs. Protein kinases operate in a large no. of distinct signaling pathways, where the tight regulation of their catalytic activity is crucial to the development and maintenance of eukaryotic organisms. The catalytic domains of different kinases adopt strikingly similar structures when they are active. By contrast, crystal structures of inactive kinases have revealed a marked plasticity in the kinase domain that allows the adoption of distinct conformations in response to interactions with specific regulatory domains or proteins.
  8. 8
    Cowan-Jacob, S. W. (2006) Structural biology of protein tyrosine kinases Cell. Mol. Life Sci. 63, 2608– 2625
    Google Scholar
    8
    Structural biology of protein tyrosine kinases
    Cowan-Jacob, S. W.
    Cellular and Molecular Life Sciences (2006), 63 (22), 2608-2625CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
    A review. The current understanding of the structure, reaction mechanism, and modes of regulation of the protein tyrosine kinase family owes a great deal to structural biol. Structures are now available for >20 different tyrosine kinase domains, many of these in multiple conformational states. They form the basis for the design of expts. to further investigate the role of different structural elements in the normal function and regulation of the protein and in the pathogenesis of many human diseases. Once thought to be too similar to be specifically inhibited by a small mol., structural differences between kinases allow the design of compds. which inhibit only an acceptable few. Here, the author provides a general overview of protein tyrosine kinase structural biol., including a discussion of the strengths and limitations of the investigative methods involved.
  9. 9
    Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome Science 298, 1912– 1934
    Google Scholar
    9
    The Protein Kinase Complement of the Human Genome
    Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S.
    Science (Washington, DC, United States) (2002), 298 (5600), 1912-1916, 1933-1934CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive anal. of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome anal. through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.
  10. 10
    Morgan, D. O. (2007) The Cell Cycle, Principles of Control (Primers in Biology), New Science Press Ltd., London.
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Bartkowiak, B., Liu, P., Phatnani, H. P., Fuda, N. J., Cooper, J. J., Price, D. H., Adelman, K., Lis, J. T., and Greenleaf, A. L. (2010) CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1 Genes Dev. 24, 2303– 2316
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Chen, H. H., Wong, Y. H., Geneviere, A. M., and Fann, M. J. (2007) CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing Biochem. Biophys. Res. Commun. 354, 735– 740
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Noble, M. E., Endicott, J. A., and Johnson, L. N. (2004) Protein kinase inhibitors: insights into drug design from structure Science 303, 1800– 1805
    Google Scholar
    13
    Protein Kinase Inhibitors: Insights into Drug Design from Structure
    Noble, Martin E. M.; Endicott, Jane A.; Johnson, Louise N.
    Science (Washington, DC, United States) (2004), 303 (5665), 1800-1805CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A review. Protein kinases are targets for treatment of a no. of diseases. This review focuses on kinase inhibitors that are in the clinic or in clin. trials and for which structural information is available. Structures have informed drug design and have illuminated the mechanism of inhibition. We review progress with the receptor tyrosine kinases (growth factor receptors EGFR, VEGFR, and FGFR) and nonreceptor tyrosine kinases (Bcr-Abl), where advances have been made with cancer therapeutic agents such as Herceptin and Gleevec. Among the serine-threonine kinases, p38, Rho-kinase, cyclin-dependent kinases, and Chk1 have been targeted with productive results for inflammation and cancer. Structures have provided insights into targeting the inactive or active form of the kinase, for targeting the global constellation of residues at the ATP site or less conserved addnl. pockets or single residues, and into targeting noncatalytic domains.
  14. 14
    Ali, S., Heathcote, D. A., Kroll, S. H., Jogalekar, A. S., Scheiper, B., Patel, H., Brackow, J., Siwicka, A., Fuchter, M. J., Periyasamy, M., Tolhurst, R. S., Kanneganti, S. K., Snyder, J. P., Liotta, D. C., Aboagye, E. O., Barrett, A. G., and Coombes, R. C. (2009) The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity Cancer Res. 69, 6208– 6215
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Pavletich, N. P. (1999) Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors J. Mol. Biol. 287, 821– 828
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Echalier, A., Endicott, J. A., and Noble, M. E. (2010) Recent developments in cyclin-dependent kinase biochemical and structural studies Biochim. Biophys. Acta 1804, 511– 519
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    De Bondt, H. L., Rosenblatt, J., Jancarik, J., Jones, H. D., Morgan, D. O., and Kim, S. H. (1993) Crystal structure of cyclin-dependent kinase 2 Nature 363, 595– 602
    Google Scholar
    17
    Crystal structure of cyclin-dependent kinase 2
    De Bondt, Hendrik L.; Rosenblatt, Jody; Jancarik, Jarmila; Jones, Heather D.; Morgan, David O.; Kim, Sung Hou
    Nature (London, United Kingdom) (1993), 363 (6430), 595-602CODEN: NATUAS; ISSN:0028-0836.
    Cyclin-dependent kinase 2 (CDK2) is a member of a highly conserved family of protein kinases that regulate the eukaryotic cell cycle. The crystal structures of the human CDK2 apoenzyme and its Mg2+ ATP complex have been detd. to 2.4-Å resoln. The structure is bi-lobate, like that of the cAMP-dependent protein kinase, but contains a unique helix-loop segment that interferes with ATP and protein substrate binding and probably plays a key part in the regulation of all cyclin-dependent kinases.
  18. 18
    Brown, N. R., Noble, M. E., Endicott, J. A., and Johnson, L. N. (1999) The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases Nat. Cell Biol. 1, 438– 443
    Google Scholar
    18
    The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases
    Brown, Nick R.; Noble, Martin E. M.; Endicott, Jane A.; Johnson, Louise N.
    Nature Cell Biology (1999), 1 (7), 438-443CODEN: NCBIFN; ISSN:1465-7392. (Macmillan Magazines Ltd)
    Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require assocn. with a cyclin and phosphorylation by a sep. protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.
  19. 19
    Takaki, T., Echalier, A., Brown, N. R., Hunt, T., Endicott, J. A., and Noble, M. E. (2009) The structure of CDK4/cyclin D3 has implications for models of CDK activation Proc. Natl. Acad. Sci. U.S.A. 106, 4171– 4176
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Day, P. J., Cleasby, A., Tickle, I. J., O’Reilly, M., Coyle, J. E., Holding, F. P., McMenamin, R. L., Yon, J., Chopra, R., Lengauer, C., and Jhoti, H. (2009) Crystal structure of human CDK4 in complex with a D-type cyclin Proc. Natl. Acad. Sci. U.S.A. 106, 4166– 4170
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Tarricone, C., Dhavan, R., Peng, J., Areces, L. B., Tsai, L. H., and Musacchio, A. (2001) Structure and regulation of the CDK5-p25(nck5a) complex Mol. Cell 8, 657– 669
    Google Scholar
    21
    Structure and regulation of the CDK5-p25nck5a complex
    Tarricone, Cataldo; Dhavan, Rani; Peng, Junmin; Areces, Liliana B.; Tsai, Li-Huei; Musacchio, Andrea
    Molecular Cell (2001), 8 (3), 657-669CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
    CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase, p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equiv. to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.
  22. 22
    Schneider, E. V., Bottcher, J., Blaesse, M., Neumann, L., Huber, R., and Maskos, K. (2011) The structure of CDK8/CycC implicates specificity in the CDK/cyclin family and reveals interaction with a deep pocket binder J. Mol. Biol. 412, 251– 266
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Lo, M. C., Aulabaugh, A., Jin, G., Cowling, R., Bard, J., Malamas, M., and Ellestad, G. (2004) Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery Anal. Biochem. 332, 153– 159
    Google Scholar
    23
    Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery
    Lo, Mei-Chu; Aulabaugh, Ann; Jin, Guixian; Cowling, Rebecca; Bard, Jonathan; Malamas, Michael; Ellestad, George
    Analytical Biochemistry (2004), 332 (1), 153-159CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compd. libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temp. (ΔTm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, com. available device for temp. control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titrn. calorimetry and with the IC50 values from enzymic assays. The potential pitfalls in the data anal. of thermal shift assays are also discussed.
  24. 24
    Najmanovich, R. J., Allali-Hassani, A., Morris, R. J., Dombrovsky, L., Pan, P. W., Vedadi, M., Plotnikov, A. N., Edwards, A., Arrowsmith, C., and Thornton, J. M. (2007) Analysis of binding site similarity, small-molecule similarity and experimental binding profiles in the human cytosolic sulfotransferase family Bioinformatics 23, e104– 109
    Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Merckx, A., Echalier, A., Langford, K., Sicard, A., Langsley, G., Joore, J., Doerig, C., Noble, M., and Endicott, J. (2008) Structures of P. falciparum protein kinase 7 identify an activation motif and leads for inhibitor design Structure 16, 228– 238
    Google Scholar
    25
    Structures of P. falciparum Protein Kinase 7 Identify an Activation Motif and Leads for Inhibitor Design
    Merckx, Anais; Echalier, Aude; Langford, Kia; Sicard, Audrey; Langsley, Gordon; Joore, Jos; Doerig, Christian; Noble, Martin; Endicott, Jane
    Structure (Cambridge, MA, United States) (2008), 16 (2), 228-238CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    Malaria is a major threat to world health. The identification of parasite targets for drug development is a priority, and parasitic protein kinases suggest themselves as suitable targets as many display profound structural and functional divergences from their host counterparts. In this paper, we describe the structure of the orphan protein kinase, Plasmodium falciparum protein kinase 7 (PFPK7). Several Plasmodium protein kinases contain extensive insertions, and the structure of PFPK7 reveals how these may be accommodated as excursions from the canonical eukaryotic protein kinase fold. The constitutively active conformation of PFPK7 is stabilized by a structural motif in which the role of the conserved phosphorylated residue that assists in structuring the activation loop of many protein kinases is played by an arginine residue. We identify two series of PFPK7 ATP-competitive inhibitors and suggest further developments for the design of selective and potent PFPK7 lead compds. as potential antimalarials.
  26. 26
    Brown, N. R., Noble, M. E., Lawrie, A. M., Morris, M. C., Tunnah, P., Divita, G., Johnson, L. N., and Endicott, J. A. (1999) Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity J. Biol. Chem. 274, 8746– 8756
    Google Scholar
    26
    Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity
    Brown, Nicholas R.; Noble, Martin E. M.; Lawrie, Alison M.; Morris, May C.; Tunnah, Paul; Divita, Gilles; Johnson, Louise N.; Endicott, Jane A.
    Journal of Biological Chemistry (1999), 274 (13), 8746-8756CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The authors prepd. phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystn. using CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 was more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibited histone H1 kinase activity corresponding to ∼0.3% of that obsd. with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements showed that Thr-160 phosphorylation increased the affinity of CDK2 for both the histone substrate and ATP and decreased its affinity for ADP. By contrast, phosphorylation of CDK2 had a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 were solved at 2.1-Å resoln. The structures were similar, with the major difference occurring in the activation segment, which was disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystn. suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity obsd.
  27. 27
    Russo, A. A., Jeffrey, P. D., and Pavletich, N. P. (1996) Structural basis of cyclin-dependent kinase activation by phosphorylation Nat. Struct. Biol. 3, 696– 700
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Fang, Z., Grutter, C., and Rauh, D. (2013) Strategies for the selective regulation of kinases with allosteric modulators: exploiting exclusive structural features ACS Chem. Biol. 8, 58– 70
    Google Scholar
    28
    Strategies for the Selective Regulation of Kinases with Allosteric Modulators: Exploiting Exclusive Structural Features
    Fang, Zhizhou; Gruetter, Christian; Rauh, Daniel
    ACS Chemical Biology (2013), 8 (1), 58-70CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    A review. The modulation of kinase function has become an important goal in modern drug discovery and chem. biol. research. In cancer-targeted therapies, kinase inhibitors have been experiencing an upsurge, which can be measured by the increasing no. of kinase inhibitors approved by the FDA in recent years. However, lack of efficacy, limited selectivity, and the emergence of acquired drug resistance still represent major bottlenecks in the clinic and challenge inhibitor development. Most known kinase inhibitors target the active kinase and are ATP competitive. A second class of small org. mols., which address remote sites of the kinase and stabilize enzymically inactive conformations, is rapidly moving to the forefront of kinase inhibitor research. Such allosteric modulators bind to sites that are less conserved across the kinome and only accessible upon conformational changes. These mols. are therefore thought to provide various advantages such as higher selectivity and extended drug target residence times. This review highlights various strategies that have been developed to utilizing exclusive structural features of kinases and thereby modulating their activity allosterically.
  29. 29
    Schachter, M. M., Merrick, K. A., Larochelle, S., Hirschi, A., Zhang, C., Shokat, K. M., Rubin, S. M., and Fisher, R. P. (2013) A Cdk7-Cdk4 T-loop phosphorylation cascade promotes G1 progression Mol. Cell 50, 250– 260
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Baumli, S., Lolli, G., Lowe, E. D., Troiani, S., Rusconi, L., Bullock, A. N., Debreczeni, J. E., Knapp, S., and Johnson, L. N. (2008) The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation EMBO J. 27, 1907– 1918
    Google Scholar
    30
    The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation
    Baumli, Sonja; Lolli, Graziano; Lowe, Edward D.; Troiani, Sonia; Rusconi, Luisa; Bullock, Alex N.; Debreczeni, Judit E.; Knapp, Stefan; Johnson, Louise N.
    EMBO Journal (2008), 27 (13), 1907-1918CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
    The pos. transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have detd. the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26° of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clin. trials, binds to the ATP site of CDK9, inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.
  31. 31
    Lolli, G., Lowe, E. D., Brown, N. R., and Johnson, L. N. (2004) The crystal structure of human CDK7 and its protein recognition properties Structure 12, 2067– 2079
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor Biochemistry 44, 5258– 5266
    Google Scholar
    32
    Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor
    Matulis, Daumantas; Kranz, James K.; Salemme, F. Raymond; Todd, Matthew J.
    Biochemistry (2005), 44 (13), 5258-5266CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equil. binding ligands increase protein thermal stability by an amt. proportional to the concn. and affinity of the ligand. Binding consts. (Kb) were measured by examg. the systematic effect of ligand concn. on protein stability. The precise ligand effects depend on the thermodn. of protein stability: in particular, the unfolding enthalpy. An extension of current theor. treatments was developed for tight binding inhibitors, where ligand effect on Tm can also reveal binding stoichiometry. A thermodn. anal. of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30°; the heat capacity of protein unfolding was estd. from the dependence of calorimetric enthalpy on Tm. The binding affinity of six sulfonamide inhibitors to two isoenzymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titrn. calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.
  33. 33
    Bullock, A. N., Debreczeni, J. E., Fedorov, O. Y., Nelson, A., Marsden, B. D., and Knapp, S. (2005) Structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM-1) kinase J. Med. Chem. 48, 7604– 7614
    Google Scholar
    33
    Structural Basis of Inhibitor Specificity of the Human Protooncogene Proviral Insertion Site in Moloney Murine Leukemia Virus (PIM-1) Kinase
    Bullock, Alex N.; Debreczeni, Judit E.; Fedorov, Oleg Y.; Nelson, Adam; Marsden, Brian D.; Knapp, Stefan
    Journal of Medicinal Chemistry (2005), 48 (24), 7604-7614CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The kinase PIM-1 plays a pivotal role in cytokine signaling and is implicated in the development of a no. of tumors. The three-dimensional structure of PIM-1 is characterized by an unique hinge region which lacks a second hydrogen bond donor and makes it particularly important to det. how inhibitors bind to this kinase. We detd. the structures of PIM-1 in complex with bisindolylmaleimide (BIM-1) and established the structure-activity relationship (SAR) for this inhibitor class. In addn., we screened a kinase targeted library and identified a no. of high affinity inhibitors of PIM-1 such as imidazo[1,2-b]pyridazines, pyrazolo[1,5-a]pyrimidines, and members of the flavonoid family. In this paper we present an initial SAR of the identified scaffolds detd. on the basis of a thermostability shift assay, calorimetric binding data, and biochem. assays which may find applications for the treatment of PIM-1 dependent cancer types.
  34. 34
    Bain, J., McLauchlan, H., Elliott, M., and Cohen, P. (2003) The specificities of protein kinase inhibitors: an update Biochem. J. 371, 199– 204
    Google Scholar
    34
    The specificities of protein kinase inhibitors: an update
    Bain, Jenny; McLauchlan, Hilary; Elliott, Matthew; Cohen, Philip
    Biochemical Journal (2003), 371 (1), 199-204CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    We have previously examd. the specificities of 28 com. available compds., reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases. In the present study, we have extended this anal. to a further 14 compds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a no. of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiol. roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiol. roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC50 = 0.33 μM) and p38-regulated/activated kinase (PRAK; IC50 = 1.0 μM).
  35. 35
    Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C. J., McLauchlan, H., Klevernic, I., Arthur, J. S., Alessi, D. R., and Cohen, P. (2007) The selectivity of protein kinase inhibitors: a further update Biochem. J. 408, 297– 315
    Google Scholar
    35
    The selectivity of protein kinase inhibitors: a further update
    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; McLauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip
    Biochemical Journal (2007), 408 (3), 297-315CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    The specificities of 65 compds. reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compds. that we have studied in cells and other data in the literature, we recommend the use of the following small-mol. inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiol. roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory assocd. protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-mol. inhibitors of protein kinases to assess the physiol. roles of these enzymes. Despite being used widely, many of the compds. that we analyzed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.
  36. 36
    Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors Biochem. J. 351, 95– 105
    Google Scholar
    36
    Specificity and mechanism of action of some commonly used protein kinase inhibitors
    Davies, Stephen P.; Reddy, Helen; Caivano, Matilde; Cohen, Philip
    Biochemical Journal (2000), 351 (1), 95-105CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    The specificities of 28 com. available compds. reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examd. against a large panel of protein kinases. The compds. KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based expts. are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compds. with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one addnl. protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. The authors propose guidelines for the use of protein kinase inhibitors in cell-based assays.
  37. 37
    Pratt, D. J., Bentley, J., Jewsbury, P., Boyle, F. T., Endicott, J. A., and Noble, M. E. (2006) Dissecting the determinants of cyclin-dependent kinase 2 and cyclin-dependent kinase 4 inhibitor selectivity J. Med. Chem. 49, 5470– 5477
    Google Scholar
    37
    Dissecting the Determinants of Cyclin-Dependent Kinase 2 and Cyclin-Dependent Kinase 4 Inhibitor Selectivity
    Pratt, David J.; Bentley, Jo; Jewsbury, Philip; Boyle, F. Tom; Endicott, Jane A.; Noble, Martin E. M.
    Journal of Medicinal Chemistry (2006), 49 (18), 5470-5477CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Cyclin dependent kinases are a key family of kinases involved in cell cycle regulation and are an attractive target for cancer chemotherapy. The roles of four residues of the cyclin-dependent kinase active site in inhibitor selectivity were investigated by producing cyclin-dependent kinase 2 mutants bearing equiv. cyclin-dependent kinase 4 residues, namely F82H, L83V, H84D, and K89T. Assay of the mutants with a cyclin-dependent kinase 4-selective bisanilinopyrimidine shows that the K89T mutation is primarily responsible for the selectivity of this compd. Use of the cyclin-dependent kinase 2-selective 6-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine (NU6102) shows that K89T has no role in the selectivity, while the remaining three mutations have a cumulative influence. The results indicate that certain residues that are not frequently considered in structure-aided kinase inhibitor design have an important role to play.

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  6. Tejaswi Somarowthu, Rohan R. Patekar, Sandip B. Bharate. Identification of mitoxantrone as a potent inhibitor of CDK7/Cyclin H via structure-based virtual screening and In-Vitro validation by ADP-Glo kinase assay. Bioorganic Chemistry 2025, 155 , 108111. https://doi.org/10.1016/j.bioorg.2024.108111
  7. Navid Jamali, Roohollah Mohseni, Mansoor Zareei, Javad Saffari-Chaleshtori. The Molecular Docking and Molecular Dynamic Effects of Omeprazole on CDKs 2, 4, and 6 as a Potential CDK Inhibitor in Cancer Treatment. Current Cancer Therapy Reviews 2025, 21 (1) , 110-117. https://doi.org/10.2174/0115733947274344231116113325
  8. Elloise H. Stephenson, Jonathan M. G. Higgins. Pharmacological approaches to understanding protein kinase signaling networks. Frontiers in Pharmacology 2023, 14 https://doi.org/10.3389/fphar.2023.1310135
  9. Polina Rusina, Erik Gandalipov, Yana Abdusheva, Maria Panova, Alexandra Burdenkova, Vasiliy Chaliy, Maria Brachs, Oleg Stroganov, Ksenia Guzeeva, Igor Svitanko, Alexander Shtil, Fedor Novikov. Imidazole-4-N-acetamide Derivatives as a Novel Scaffold for Selective Targeting of Cyclin Dependent Kinases. Cancers 2023, 15 (15) , 3766. https://doi.org/10.3390/cancers15153766
  10. Polina V. Rusina, Alexey A. Lisov, Alexandra A. Denisova, Erik R. Gandalipov, Fedor N. Novikov, Alexander A. Shtil. Clinical CDK2 Inhibitors: Trends to Selectivity and Efficacy. Recent Patents on Anti-Cancer Drug Discovery 2023, 18 (2) , 102-107. https://doi.org/10.2174/1574892817666220617091700
  11. Gina Bouchard, Fernando Jose Garcia-Marques, Loukia Georgiou Karacosta, Weiruo Zhang, Abel Bermudez, Nicholas McIlvain Riley, Sushama Varma, Lindsey Catherine Mehl, Jalen Anthony Benson, Joseph B. Shrager, Carolyn Ruth Bertozzi, Sharon J. Pitteri, Amato J. Giaccia, Sylvia Katina Plevritis. Multiomics Analysis of Spatially Distinct Stromal Cells Reveals Tumor-Induced O-Glycosylation of the CDK4–pRB Axis in Fibroblasts at the Invasive Tumor Edge. Cancer Research 2022, 82 (4) , 648-664. https://doi.org/10.1158/0008-5472.CAN-21-1705
  12. Yi Liu, Leilei Fu, Junhao Wu, Ming Liu, Guan Wang, Bo Liu, Lan Zhang. Transcriptional cyclin-dependent kinases: Potential drug targets in cancer therapy. European Journal of Medicinal Chemistry 2022, 229 , 114056. https://doi.org/10.1016/j.ejmech.2021.114056
  13. Mingfei Wu, Jianfei Han, Zhicheng Liu, Yilong Zhang, Cheng Huang, Jun Li, Zeng Li. Identification of novel CDK 9 inhibitors based on virtual screening, molecular dynamics simulation, and biological evaluation. Life Sciences 2020, 258 , 118228. https://doi.org/10.1016/j.lfs.2020.118228
  14. Shovonlal Bhowmick, Nora Abdullah AlFaris, Jozaa Zaidan ALTamimi, Zeid A. ALOthman, Tahany Saleh Aldayel, Saikh Mohammad Wabaidur, Md Ataul Islam. Screening and analysis of bioactive food compounds for modulating the CDK2 protein for cell cycle arrest: Multi-cheminformatics approaches for anticancer therapeutics. Journal of Molecular Structure 2020, 1216 , 128316. https://doi.org/10.1016/j.molstruc.2020.128316
  15. Marc Hafner, Caitlin E. Mills, Kartik Subramanian, Chen Chen, Mirra Chung, Sarah A. Boswell, Robert A. Everley, Changchang Liu, Charlotte S. Walmsley, Dejan Juric, Peter K. Sorger. Multiomics Profiling Establishes the Polypharmacology of FDA-Approved CDK4/6 Inhibitors and the Potential for Differential Clinical Activity. Cell Chemical Biology 2019, 26 (8) , 1067-1080.e8. https://doi.org/10.1016/j.chembiol.2019.05.005
  16. Przemysław Czeleń. Investigation of the Inhibition Potential of New Oxindole Derivatives and Assessment of Their Usefulness for Targeted Therapy. Symmetry 2019, 11 (8) , 974. https://doi.org/10.3390/sym11080974
  17. Przemysław Czeleń, Beata Szefler. The Immobilization of ChEMBL474807 Molecules Using Different Classes of Nanostructures. Symmetry 2019, 11 (8) , 980. https://doi.org/10.3390/sym11080980
  18. Amy B Heptinstall, IWS Adiyasa, Céline Cano, Ian R Hardcastle. Recent Advances in CDK Inhibitors for Cancer Therapy. Future Medicinal Chemistry 2018, 10 (11) , 1369-1388. https://doi.org/10.4155/fmc-2017-0246
  19. , , Mathew P. Martin, Jane A. Endicott, Martin E.M. Noble. Structure-based discovery of cyclin-dependent protein kinase inhibitors. Essays in Biochemistry 2017, 61 (5) , 439-452. https://doi.org/10.1042/EBC20170040
  20. Gabrielle Stetz, Amanda Tse, Gennady M. Verkhivker, . Ensemble-based modeling and rigidity decomposition of allosteric interaction networks and communication pathways in cyclin-dependent kinases: Differentiating kinase clients of the Hsp90-Cdc37 chaperone. PLOS ONE 2017, 12 (11) , e0186089. https://doi.org/10.1371/journal.pone.0186089
  21. Jimma Likisa Lenjisa, Solomon Tadesse, Nishat Zareen Khair, Malika Kumarasiri, Mingfeng Yu, Hugo Albrecht, Robert Milne, Shudong Wang. Cdk5 in Oncology: Recent Advances and Future Prospects. Future Medicinal Chemistry 2017, 9 (16) , 1939-1962. https://doi.org/10.4155/fmc-2017-0097
  22. G. M. Verkhivker. Network-based modelling and percolation analysis of conformational dynamics and activation in the CDK2 and CDK4 proteins: dynamic and energetic polarization of the kinase lobes may determine divergence of the regulatory mechanisms. Molecular BioSystems 2017, 13 (11) , 2235-2253. https://doi.org/10.1039/C7MB00355B
  23. Tinghan Li, Tianwei Weng, Minzan Zuo, Zhihui Wei, Ming Chen, Zhiyu Li. Recent Progress of Cyclin-Dependent Kinase Inhibitors as Potential Anticancer Agents. Future Medicinal Chemistry 2016, 8 (17) , 2047-2076. https://doi.org/10.4155/fmc-2016-0129
  24. Carl S. Rye, Nicola E. A. Chessum, Scott Lamont, Kurt G. Pike, Paul Faulder, Julie Demeritt, Paul Kemmitt, Julie Tucker, Lorenzo Zani, Matthew D. Cheeseman, Rosie Isaac, Louise Goodwin, Joanna Boros, Florence Raynaud, Angela Hayes, Alan T. Henley, Emmanuel de Billy, Christopher J. Lynch, Swee Y. Sharp, Robert te Poele, Lisa O’ Fee, Kevin M. Foote, Stephen Green, Paul Workman, Keith Jones. Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9. MedChemComm 2016, 7 (8) , 1580-1586. https://doi.org/10.1039/C6MD00159A
  25. Marion Peyressatre, Camille Prével, Morgan Pellerano, May Morris. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors. Cancers 2015, 7 (1) , 179-237. https://doi.org/10.3390/cancers7010179
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Cite this: ACS Chem. Biol. 2014, 9, 6, 1251–1256
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https://doi.org/10.1021/cb500135f
Published March 26, 2014

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  • Abstract

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    Figure 1

    Figure 1. Explored chemical space. (a) Representative inhibitor scaffolds. Examples of the inhibitor scaffolds included in the panel are shown. All inhibitor structures are provided in the Supporting Information (Supplementary Table 1). (b) Chemical space explored by the panel of inhibitors used in this study. The target(s) in the human kinome for each of the inhibitors of the panel are identified by a red rectangle on the kinome tree. This figure was adapted from the phylogenetic tree published in ref 9.

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    Figure 2

    Figure 2. CDK space explored in this study. (a) Representative melting curves for cyclin-dependent kinase (CDK)/cyclin complexes used to derive ΔTm values in the presence of an ATP-competitive inhibitor. The shift in Tm upon staurosporine binding (red) is illustrated for each of the CDK/cyclin complexes in this study. Black curves, CDK/cyclin complexes in the absence of added inhibitor. Curves with closed triangle symbols correspond to pCDK9/T, closed diamonds to CDK7, closed circles to CDK4, closed squares to pCDK4/D, open squares to CDK2/A, open diamonds to CDK2, and open triangles to pCDK2. (b) The full set of different phosphorylated and/or cyclin-associated states of the four CDKs characterized in this study are shown, with those members for which measurements were made highlighted by shaded boxes. Color intensity for these protein species reflects activation state (more saturated, more active). (c) The ATP binding site of CDKs. The CDK2 ATP-binding site residues from a pCDK2/A structure (PDB code: 1QMZ) are colored according to the degree of sequence conservation between the 4 CDK sequences, calculated as described in the Supplementary Methods, from blue (highly conserved) to red (highly variable). The ATP molecule is represented in ball-and-stick mode for context. (d) The ATP-binding site pocket is highly conserved in CDK2, CDK7, CDK4, and CDK9. Residues that constitute the first shell of the CDK2 ATP-binding site (CDK2 numbering) were identified as defined in Supplementary Methods and used to construct a pseudosequence alignment. This alignment is displayed together with the associated consensus sequence, with X representing any residue, and ϕ a hydrophobic amino acid. Sequence motifs that are highly conserved across the protein kinase family are identified by numbered bars. Bar 1, the glycine-rich loop (residues 11–16 in CDK2) that defines the general consensus sequence GXGXXG that recognizes the phosphate moieties of ATP; bar 2, the “hinge” sequence composed of residues 80–84 that links the N- and C-terminal lobes and contributes to the ATP adenine binding site; bar 3, the “DFG motif” that defines the start of the activation segment. Asterisks below E51 and D127 highlight the conserved glutamate in the C-helix and the catalytic aspartate, respectively. A full sequence alignment is presented in Supplementary Figure 1.

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    Figure 3

    Figure 3. Comparison of the inhibitor-binding profiles of various cyclin-dependent kinase (CDK) subfamily members in different states of activation. Each block represents a CDK in a particular activation state: CDK (monomeric, unphosphorylated), pCDK (monomeric, phosphorylated), CDK/[A or D or T] (unphosphorylated binary complex between a CDK and the corresponding cyclin), and pCDK/[A or D or T] (phosphorylated binary complex between a CDK and the corresponding cyclin). The internal consistency of inhibitory fingerprints for each CDK/state pair, evaluated over two or three repeat measurements, is indicated by a value within the corresponding block. The correlation of fingerprints between different CDK/state pairs is indicated by a value on the line that connects them. (a) Comparison of the inhibitor fingerprints of CDK subfamily members in the monomeric form. (b) Comparison of the inhibitor fingerprints of CDK subfamily members in the phosphorylated binary forms. (c) Comparison of CDK2 inhibitor fingerprints in different activation states. Color intensity as in Figure 2b reflects activation state (more saturated, more active).

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  • References

    This article references 37 other publications.

    1. 1
      Zhang, J., Yang, P. L., and Gray, N. S. (2009) Targeting cancer with small molecule kinase inhibitors Nat. Rev. Cancer 9, 28– 39
      1
      Targeting cancer with small molecule kinase inhibitors
      Zhang, Jianming; Yang, Priscilla L.; Gray, Nathanael S.
      Nature Reviews Cancer (2009), 9 (1), 28-39CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiol. constraints on growth and survival. To date, 11 kinase inhibitors have received US Food and Drug Administration approval as cancer treatments, and there are considerable efforts to develop selective small mol. inhibitors for a host of other kinases that are implicated in cancer and other diseases. Herein we discuss the current challenges in the field, such as designing selective inhibitors and developing strategies to overcome resistance mutations. This Review provides a broad overview of some of the approaches currently used to discover and characterize new kinase inhibitors.
    2. 2
      Fabbro, D., Cowan-Jacob, S. W., Mobitz, H., and Martiny-Baron, G. (2012) Targeting cancer with small-molecular-weight kinase inhibitors Methods Mol. Biol. (N. Y.) 795, 1– 34
      2
      Targeting cancer with small-molecular-weight kinase inhibitors
      Fabbro Doriano; Cowan-Jacob Sandra W; Mobitz Henrik; Martiny-Baron Georg
      Methods in molecular biology (Clifton, N.J.) (2012), 795 (), 1-34 ISSN:.
      Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.
    3. 3
      Cohen, P. (2009) Targeting protein kinases for the development of anti-inflammatory drugs Curr. Opin. Cell Biol. 21, 317– 324
      3
      Targeting protein kinases for the development of anti-inflammatory drugs
      Cohen, Philip
      Current Opinion in Cell Biology (2009), 21 (2), 317-324CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)
      A review. In recent years, protein kinases have become the pharmaceutical industry's most studied class of drug target, and some 10 protein kinase inhibitors have so far been approved for the treatment of cancer. However, whether safe drugs that modulate protein kinase activities can also be developed for the treatment of chronic diseases, where they may need to be taken for decades, is an issue that is still unresolved. A no. of compds. that inhibit the p38α MAPK have entered clin. trials for the treatment of rheumatoid arthritis and psoriasis, but side effects have prevented their progression to Phase III clin. trials. Here the author briefly reviews the potential problems in targeting p38 MAPK and discusses other protein kinases that regulate the innate immune system, such as Tpl2, MAPKAP-K2/3, MSK1/2, and IRAK4, which may be better targets for the treatment of chronic inflammatory diseases, and NIK, which is an attractive target for the treatment of multiple myeloma, a late stage B-cell malignancy.
    4. 4
      Janne, P. A., Gray, N., and Settleman, J. (2009) Factors underlying sensitivity of cancers to small-molecule kinase inhibitors Nat. Rev. Drug Discovery 8, 709– 723
      There is no corresponding record for this reference.
    5. 5
      Karaman, M. W., Herrgard, S., Treiber, D. K., Gallant, P., Atteridge, C. E., Campbell, B. T., Chan, K. W., Ciceri, P., Davis, M. I., Edeen, P. T., Faraoni, R., Floyd, M., Hunt, J. P., Lockhart, D. J., Milanov, Z. V., Morrison, M. J., Pallares, G., Patel, H. K., Pritchard, S., Wodicka, L. M., and Zarrinkar, P. P. (2008) A quantitative analysis of kinase inhibitor selectivity Nat. Biotechnol. 26, 127– 132
      5
      A quantitative analysis of kinase inhibitor selectivity
      Karaman, Mazen W.; Herrgard, Sanna; Treiber, Daniel K.; Gallant, Paul; Atteridge, Corey E.; Campbell, Brian T.; Chan, Katrina W.; Ciceri, Pietro; Davis, Mindy I.; Edeen, Philip T.; Faraoni, Raffaella; Floyd, Mark; Hunt, Jeremy P.; Lockhart, Daniel J.; Milanov, Zdravko V.; Morrison, Michael J.; Pallares, Gabriel; Patel, Hitesh K.; Pritchard, Stephanie; Wodicka, Lisa M.; Zarrinkar, Patrick P.
      Nature Biotechnology (2008), 26 (1), 127-132CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biol. consequences of multikinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. The authors present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global anal. of the results, the authors introduce the concept of a selectivity score as a general tool to quantify and differentiate the obsd. interaction patterns. The authors further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.
    6. 6
      Smyth, L. A. and Collins, I. (2009) Measuring and interpreting the selectivity of protein kinase inhibitors J. Chem. Biol. 2, 131– 151
      6
      Measuring and interpreting the selectivity of protein kinase inhibitors
      Smyth Lynette A; Collins Ian
      Journal of chemical biology (2009), 2 (3), 131-51 ISSN:1864-6158.
      Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.
    7. 7
      Huse, M. and Kuriyan, J. (2002) The conformational plasticity of protein kinases Cell 109, 275– 282
      7
      The conformational plasticity of protein kinases
      Huse, Morgan; Kuriyan, John
      Cell (Cambridge, MA, United States) (2002), 109 (3), 275-282CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      A review with 48 refs. Protein kinases operate in a large no. of distinct signaling pathways, where the tight regulation of their catalytic activity is crucial to the development and maintenance of eukaryotic organisms. The catalytic domains of different kinases adopt strikingly similar structures when they are active. By contrast, crystal structures of inactive kinases have revealed a marked plasticity in the kinase domain that allows the adoption of distinct conformations in response to interactions with specific regulatory domains or proteins.
    8. 8
      Cowan-Jacob, S. W. (2006) Structural biology of protein tyrosine kinases Cell. Mol. Life Sci. 63, 2608– 2625
      8
      Structural biology of protein tyrosine kinases
      Cowan-Jacob, S. W.
      Cellular and Molecular Life Sciences (2006), 63 (22), 2608-2625CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
      A review. The current understanding of the structure, reaction mechanism, and modes of regulation of the protein tyrosine kinase family owes a great deal to structural biol. Structures are now available for >20 different tyrosine kinase domains, many of these in multiple conformational states. They form the basis for the design of expts. to further investigate the role of different structural elements in the normal function and regulation of the protein and in the pathogenesis of many human diseases. Once thought to be too similar to be specifically inhibited by a small mol., structural differences between kinases allow the design of compds. which inhibit only an acceptable few. Here, the author provides a general overview of protein tyrosine kinase structural biol., including a discussion of the strengths and limitations of the investigative methods involved.
    9. 9
      Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome Science 298, 1912– 1934
      9
      The Protein Kinase Complement of the Human Genome
      Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S.
      Science (Washington, DC, United States) (2002), 298 (5600), 1912-1916, 1933-1934CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive anal. of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome anal. through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.
    10. 10
      Morgan, D. O. (2007) The Cell Cycle, Principles of Control (Primers in Biology), New Science Press Ltd., London.
      There is no corresponding record for this reference.
    11. 11
      Bartkowiak, B., Liu, P., Phatnani, H. P., Fuda, N. J., Cooper, J. J., Price, D. H., Adelman, K., Lis, J. T., and Greenleaf, A. L. (2010) CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1 Genes Dev. 24, 2303– 2316
      There is no corresponding record for this reference.
    12. 12
      Chen, H. H., Wong, Y. H., Geneviere, A. M., and Fann, M. J. (2007) CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing Biochem. Biophys. Res. Commun. 354, 735– 740
      There is no corresponding record for this reference.
    13. 13
      Noble, M. E., Endicott, J. A., and Johnson, L. N. (2004) Protein kinase inhibitors: insights into drug design from structure Science 303, 1800– 1805
      13
      Protein Kinase Inhibitors: Insights into Drug Design from Structure
      Noble, Martin E. M.; Endicott, Jane A.; Johnson, Louise N.
      Science (Washington, DC, United States) (2004), 303 (5665), 1800-1805CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review. Protein kinases are targets for treatment of a no. of diseases. This review focuses on kinase inhibitors that are in the clinic or in clin. trials and for which structural information is available. Structures have informed drug design and have illuminated the mechanism of inhibition. We review progress with the receptor tyrosine kinases (growth factor receptors EGFR, VEGFR, and FGFR) and nonreceptor tyrosine kinases (Bcr-Abl), where advances have been made with cancer therapeutic agents such as Herceptin and Gleevec. Among the serine-threonine kinases, p38, Rho-kinase, cyclin-dependent kinases, and Chk1 have been targeted with productive results for inflammation and cancer. Structures have provided insights into targeting the inactive or active form of the kinase, for targeting the global constellation of residues at the ATP site or less conserved addnl. pockets or single residues, and into targeting noncatalytic domains.
    14. 14
      Ali, S., Heathcote, D. A., Kroll, S. H., Jogalekar, A. S., Scheiper, B., Patel, H., Brackow, J., Siwicka, A., Fuchter, M. J., Periyasamy, M., Tolhurst, R. S., Kanneganti, S. K., Snyder, J. P., Liotta, D. C., Aboagye, E. O., Barrett, A. G., and Coombes, R. C. (2009) The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity Cancer Res. 69, 6208– 6215
      There is no corresponding record for this reference.
    15. 15
      Pavletich, N. P. (1999) Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors J. Mol. Biol. 287, 821– 828
      There is no corresponding record for this reference.
    16. 16
      Echalier, A., Endicott, J. A., and Noble, M. E. (2010) Recent developments in cyclin-dependent kinase biochemical and structural studies Biochim. Biophys. Acta 1804, 511– 519
      There is no corresponding record for this reference.
    17. 17
      De Bondt, H. L., Rosenblatt, J., Jancarik, J., Jones, H. D., Morgan, D. O., and Kim, S. H. (1993) Crystal structure of cyclin-dependent kinase 2 Nature 363, 595– 602
      17
      Crystal structure of cyclin-dependent kinase 2
      De Bondt, Hendrik L.; Rosenblatt, Jody; Jancarik, Jarmila; Jones, Heather D.; Morgan, David O.; Kim, Sung Hou
      Nature (London, United Kingdom) (1993), 363 (6430), 595-602CODEN: NATUAS; ISSN:0028-0836.
      Cyclin-dependent kinase 2 (CDK2) is a member of a highly conserved family of protein kinases that regulate the eukaryotic cell cycle. The crystal structures of the human CDK2 apoenzyme and its Mg2+ ATP complex have been detd. to 2.4-Å resoln. The structure is bi-lobate, like that of the cAMP-dependent protein kinase, but contains a unique helix-loop segment that interferes with ATP and protein substrate binding and probably plays a key part in the regulation of all cyclin-dependent kinases.
    18. 18
      Brown, N. R., Noble, M. E., Endicott, J. A., and Johnson, L. N. (1999) The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases Nat. Cell Biol. 1, 438– 443
      18
      The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases
      Brown, Nick R.; Noble, Martin E. M.; Endicott, Jane A.; Johnson, Louise N.
      Nature Cell Biology (1999), 1 (7), 438-443CODEN: NCBIFN; ISSN:1465-7392. (Macmillan Magazines Ltd)
      Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require assocn. with a cyclin and phosphorylation by a sep. protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.
    19. 19
      Takaki, T., Echalier, A., Brown, N. R., Hunt, T., Endicott, J. A., and Noble, M. E. (2009) The structure of CDK4/cyclin D3 has implications for models of CDK activation Proc. Natl. Acad. Sci. U.S.A. 106, 4171– 4176
      There is no corresponding record for this reference.
    20. 20
      Day, P. J., Cleasby, A., Tickle, I. J., O’Reilly, M., Coyle, J. E., Holding, F. P., McMenamin, R. L., Yon, J., Chopra, R., Lengauer, C., and Jhoti, H. (2009) Crystal structure of human CDK4 in complex with a D-type cyclin Proc. Natl. Acad. Sci. U.S.A. 106, 4166– 4170
      There is no corresponding record for this reference.
    21. 21
      Tarricone, C., Dhavan, R., Peng, J., Areces, L. B., Tsai, L. H., and Musacchio, A. (2001) Structure and regulation of the CDK5-p25(nck5a) complex Mol. Cell 8, 657– 669
      21
      Structure and regulation of the CDK5-p25nck5a complex
      Tarricone, Cataldo; Dhavan, Rani; Peng, Junmin; Areces, Liliana B.; Tsai, Li-Huei; Musacchio, Andrea
      Molecular Cell (2001), 8 (3), 657-669CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
      CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase, p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equiv. to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.
    22. 22
      Schneider, E. V., Bottcher, J., Blaesse, M., Neumann, L., Huber, R., and Maskos, K. (2011) The structure of CDK8/CycC implicates specificity in the CDK/cyclin family and reveals interaction with a deep pocket binder J. Mol. Biol. 412, 251– 266
      There is no corresponding record for this reference.
    23. 23
      Lo, M. C., Aulabaugh, A., Jin, G., Cowling, R., Bard, J., Malamas, M., and Ellestad, G. (2004) Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery Anal. Biochem. 332, 153– 159
      23
      Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery
      Lo, Mei-Chu; Aulabaugh, Ann; Jin, Guixian; Cowling, Rebecca; Bard, Jonathan; Malamas, Michael; Ellestad, George
      Analytical Biochemistry (2004), 332 (1), 153-159CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compd. libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temp. (ΔTm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, com. available device for temp. control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titrn. calorimetry and with the IC50 values from enzymic assays. The potential pitfalls in the data anal. of thermal shift assays are also discussed.
    24. 24
      Najmanovich, R. J., Allali-Hassani, A., Morris, R. J., Dombrovsky, L., Pan, P. W., Vedadi, M., Plotnikov, A. N., Edwards, A., Arrowsmith, C., and Thornton, J. M. (2007) Analysis of binding site similarity, small-molecule similarity and experimental binding profiles in the human cytosolic sulfotransferase family Bioinformatics 23, e104– 109
      There is no corresponding record for this reference.
    25. 25
      Merckx, A., Echalier, A., Langford, K., Sicard, A., Langsley, G., Joore, J., Doerig, C., Noble, M., and Endicott, J. (2008) Structures of P. falciparum protein kinase 7 identify an activation motif and leads for inhibitor design Structure 16, 228– 238
      25
      Structures of P. falciparum Protein Kinase 7 Identify an Activation Motif and Leads for Inhibitor Design
      Merckx, Anais; Echalier, Aude; Langford, Kia; Sicard, Audrey; Langsley, Gordon; Joore, Jos; Doerig, Christian; Noble, Martin; Endicott, Jane
      Structure (Cambridge, MA, United States) (2008), 16 (2), 228-238CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Malaria is a major threat to world health. The identification of parasite targets for drug development is a priority, and parasitic protein kinases suggest themselves as suitable targets as many display profound structural and functional divergences from their host counterparts. In this paper, we describe the structure of the orphan protein kinase, Plasmodium falciparum protein kinase 7 (PFPK7). Several Plasmodium protein kinases contain extensive insertions, and the structure of PFPK7 reveals how these may be accommodated as excursions from the canonical eukaryotic protein kinase fold. The constitutively active conformation of PFPK7 is stabilized by a structural motif in which the role of the conserved phosphorylated residue that assists in structuring the activation loop of many protein kinases is played by an arginine residue. We identify two series of PFPK7 ATP-competitive inhibitors and suggest further developments for the design of selective and potent PFPK7 lead compds. as potential antimalarials.
    26. 26
      Brown, N. R., Noble, M. E., Lawrie, A. M., Morris, M. C., Tunnah, P., Divita, G., Johnson, L. N., and Endicott, J. A. (1999) Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity J. Biol. Chem. 274, 8746– 8756
      26
      Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity
      Brown, Nicholas R.; Noble, Martin E. M.; Lawrie, Alison M.; Morris, May C.; Tunnah, Paul; Divita, Gilles; Johnson, Louise N.; Endicott, Jane A.
      Journal of Biological Chemistry (1999), 274 (13), 8746-8756CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The authors prepd. phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystn. using CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 was more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibited histone H1 kinase activity corresponding to ∼0.3% of that obsd. with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements showed that Thr-160 phosphorylation increased the affinity of CDK2 for both the histone substrate and ATP and decreased its affinity for ADP. By contrast, phosphorylation of CDK2 had a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 were solved at 2.1-Å resoln. The structures were similar, with the major difference occurring in the activation segment, which was disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystn. suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity obsd.
    27. 27
      Russo, A. A., Jeffrey, P. D., and Pavletich, N. P. (1996) Structural basis of cyclin-dependent kinase activation by phosphorylation Nat. Struct. Biol. 3, 696– 700
      There is no corresponding record for this reference.
    28. 28
      Fang, Z., Grutter, C., and Rauh, D. (2013) Strategies for the selective regulation of kinases with allosteric modulators: exploiting exclusive structural features ACS Chem. Biol. 8, 58– 70
      28
      Strategies for the Selective Regulation of Kinases with Allosteric Modulators: Exploiting Exclusive Structural Features
      Fang, Zhizhou; Gruetter, Christian; Rauh, Daniel
      ACS Chemical Biology (2013), 8 (1), 58-70CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A review. The modulation of kinase function has become an important goal in modern drug discovery and chem. biol. research. In cancer-targeted therapies, kinase inhibitors have been experiencing an upsurge, which can be measured by the increasing no. of kinase inhibitors approved by the FDA in recent years. However, lack of efficacy, limited selectivity, and the emergence of acquired drug resistance still represent major bottlenecks in the clinic and challenge inhibitor development. Most known kinase inhibitors target the active kinase and are ATP competitive. A second class of small org. mols., which address remote sites of the kinase and stabilize enzymically inactive conformations, is rapidly moving to the forefront of kinase inhibitor research. Such allosteric modulators bind to sites that are less conserved across the kinome and only accessible upon conformational changes. These mols. are therefore thought to provide various advantages such as higher selectivity and extended drug target residence times. This review highlights various strategies that have been developed to utilizing exclusive structural features of kinases and thereby modulating their activity allosterically.
    29. 29
      Schachter, M. M., Merrick, K. A., Larochelle, S., Hirschi, A., Zhang, C., Shokat, K. M., Rubin, S. M., and Fisher, R. P. (2013) A Cdk7-Cdk4 T-loop phosphorylation cascade promotes G1 progression Mol. Cell 50, 250– 260
      There is no corresponding record for this reference.
    30. 30
      Baumli, S., Lolli, G., Lowe, E. D., Troiani, S., Rusconi, L., Bullock, A. N., Debreczeni, J. E., Knapp, S., and Johnson, L. N. (2008) The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation EMBO J. 27, 1907– 1918
      30
      The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation
      Baumli, Sonja; Lolli, Graziano; Lowe, Edward D.; Troiani, Sonia; Rusconi, Luisa; Bullock, Alex N.; Debreczeni, Judit E.; Knapp, Stefan; Johnson, Louise N.
      EMBO Journal (2008), 27 (13), 1907-1918CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
      The pos. transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have detd. the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26° of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clin. trials, binds to the ATP site of CDK9, inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.
    31. 31
      Lolli, G., Lowe, E. D., Brown, N. R., and Johnson, L. N. (2004) The crystal structure of human CDK7 and its protein recognition properties Structure 12, 2067– 2079
      There is no corresponding record for this reference.
    32. 32
      Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor Biochemistry 44, 5258– 5266
      32
      Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor
      Matulis, Daumantas; Kranz, James K.; Salemme, F. Raymond; Todd, Matthew J.
      Biochemistry (2005), 44 (13), 5258-5266CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equil. binding ligands increase protein thermal stability by an amt. proportional to the concn. and affinity of the ligand. Binding consts. (Kb) were measured by examg. the systematic effect of ligand concn. on protein stability. The precise ligand effects depend on the thermodn. of protein stability: in particular, the unfolding enthalpy. An extension of current theor. treatments was developed for tight binding inhibitors, where ligand effect on Tm can also reveal binding stoichiometry. A thermodn. anal. of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30°; the heat capacity of protein unfolding was estd. from the dependence of calorimetric enthalpy on Tm. The binding affinity of six sulfonamide inhibitors to two isoenzymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titrn. calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.
    33. 33
      Bullock, A. N., Debreczeni, J. E., Fedorov, O. Y., Nelson, A., Marsden, B. D., and Knapp, S. (2005) Structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM-1) kinase J. Med. Chem. 48, 7604– 7614
      33
      Structural Basis of Inhibitor Specificity of the Human Protooncogene Proviral Insertion Site in Moloney Murine Leukemia Virus (PIM-1) Kinase
      Bullock, Alex N.; Debreczeni, Judit E.; Fedorov, Oleg Y.; Nelson, Adam; Marsden, Brian D.; Knapp, Stefan
      Journal of Medicinal Chemistry (2005), 48 (24), 7604-7614CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The kinase PIM-1 plays a pivotal role in cytokine signaling and is implicated in the development of a no. of tumors. The three-dimensional structure of PIM-1 is characterized by an unique hinge region which lacks a second hydrogen bond donor and makes it particularly important to det. how inhibitors bind to this kinase. We detd. the structures of PIM-1 in complex with bisindolylmaleimide (BIM-1) and established the structure-activity relationship (SAR) for this inhibitor class. In addn., we screened a kinase targeted library and identified a no. of high affinity inhibitors of PIM-1 such as imidazo[1,2-b]pyridazines, pyrazolo[1,5-a]pyrimidines, and members of the flavonoid family. In this paper we present an initial SAR of the identified scaffolds detd. on the basis of a thermostability shift assay, calorimetric binding data, and biochem. assays which may find applications for the treatment of PIM-1 dependent cancer types.
    34. 34
      Bain, J., McLauchlan, H., Elliott, M., and Cohen, P. (2003) The specificities of protein kinase inhibitors: an update Biochem. J. 371, 199– 204
      34
      The specificities of protein kinase inhibitors: an update
      Bain, Jenny; McLauchlan, Hilary; Elliott, Matthew; Cohen, Philip
      Biochemical Journal (2003), 371 (1), 199-204CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      We have previously examd. the specificities of 28 com. available compds., reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases. In the present study, we have extended this anal. to a further 14 compds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a no. of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiol. roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiol. roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC50 = 0.33 μM) and p38-regulated/activated kinase (PRAK; IC50 = 1.0 μM).
    35. 35
      Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C. J., McLauchlan, H., Klevernic, I., Arthur, J. S., Alessi, D. R., and Cohen, P. (2007) The selectivity of protein kinase inhibitors: a further update Biochem. J. 408, 297– 315
      35
      The selectivity of protein kinase inhibitors: a further update
      Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; McLauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip
      Biochemical Journal (2007), 408 (3), 297-315CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      The specificities of 65 compds. reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compds. that we have studied in cells and other data in the literature, we recommend the use of the following small-mol. inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiol. roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory assocd. protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-mol. inhibitors of protein kinases to assess the physiol. roles of these enzymes. Despite being used widely, many of the compds. that we analyzed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.
    36. 36
      Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors Biochem. J. 351, 95– 105
      36
      Specificity and mechanism of action of some commonly used protein kinase inhibitors
      Davies, Stephen P.; Reddy, Helen; Caivano, Matilde; Cohen, Philip
      Biochemical Journal (2000), 351 (1), 95-105CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      The specificities of 28 com. available compds. reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examd. against a large panel of protein kinases. The compds. KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based expts. are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compds. with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one addnl. protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. The authors propose guidelines for the use of protein kinase inhibitors in cell-based assays.
    37. 37
      Pratt, D. J., Bentley, J., Jewsbury, P., Boyle, F. T., Endicott, J. A., and Noble, M. E. (2006) Dissecting the determinants of cyclin-dependent kinase 2 and cyclin-dependent kinase 4 inhibitor selectivity J. Med. Chem. 49, 5470– 5477
      37
      Dissecting the Determinants of Cyclin-Dependent Kinase 2 and Cyclin-Dependent Kinase 4 Inhibitor Selectivity
      Pratt, David J.; Bentley, Jo; Jewsbury, Philip; Boyle, F. Tom; Endicott, Jane A.; Noble, Martin E. M.
      Journal of Medicinal Chemistry (2006), 49 (18), 5470-5477CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Cyclin dependent kinases are a key family of kinases involved in cell cycle regulation and are an attractive target for cancer chemotherapy. The roles of four residues of the cyclin-dependent kinase active site in inhibitor selectivity were investigated by producing cyclin-dependent kinase 2 mutants bearing equiv. cyclin-dependent kinase 4 residues, namely F82H, L83V, H84D, and K89T. Assay of the mutants with a cyclin-dependent kinase 4-selective bisanilinopyrimidine shows that the K89T mutation is primarily responsible for the selectivity of this compd. Use of the cyclin-dependent kinase 2-selective 6-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine (NU6102) shows that K89T has no role in the selectivity, while the remaining three mutations have a cumulative influence. The results indicate that certain residues that are not frequently considered in structure-aided kinase inhibitor design have an important role to play.

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