Kinomics-structural biology and chemogenomics of kinase inhibitors and targets

Classifying kinases based entirely on small molecule selectivity data is a new approach to drug discovery that allows scientists to understand relationships between targets. This approach combines the understanding of small molecules and targets, and thereby assists the researcher in finding new targets for existing molecules or understanding selectivity and polypharmacology of molecules in related targets. Currently, structural information is available for relatively few of the protein kinases encoded in the human genome (7% of the estimated 518); however, even the current knowledge base, when paired with structure-based design techniques, can assist in the identification and optimization of novel kinase inhibitors across the entire protein class. Chemogenomics attempts to combine genomic data, structural biological data, classical dendrograms, and selectivity data to explore, define, and classify the medicinally relevant kinase space. Exploitation of this information in the discovery of kinase inhibitors defines practical kinase chemogenomics (kinomics). In this paper, we review the available information on kinase targets and their inhibitors, and present the relationships between the various classification schema for kinase space. In particular, we present the first dendrogram of kinases based entirely on small molecule selectivity data. We find that the selectivity dendrogram differs from sequence-based clustering mostly in the higher-level groupings of the smaller clusters, and remains very comparable for closely homologous targets. Highly homologous kinases are, on average, inhibited comparably by small molecules. This observation, although intuitive, is very important to the process of target selection, as one would expect difficulty in achieving inhibitor selectivity for kinases that share high sequence identity.     

Combinatorial Clustering of Residue Position Subsets Predicts Inhibitor Affinity across the Human Kinome

The protein kinases are a large family of enzymes that play fundamental roles in propagating signals within the cell. Because of the high degree of binding site similarity shared among protein kinases, designing drug compounds with high specificity among the kinases has proven difficult. However, computational approaches to comparing the 3-dimensional geometry and physicochemical properties of key binding site residue positions have been shown to be informative of inhibitor selectivity. The Combinatorial Clustering Of Residue Position Subsets (ccorps) method, introduced here, provides a semi-supervised learning approach for identifying structural features that are correlated with a given set of annotation labels. Here, ccorps is applied to the problem of identifying structural features of the kinase atp binding site that are informative of inhibitor binding. ccorps is demonstrated to make perfect or near-perfect predictions for the binding affinity profile of 8 of the 38 kinase inhibitors studied, while only having overall poor predictive ability for 1 of the 38 compounds. Additionally, ccorps is shown to identify shared structural features across phylogenetically diverse groups of kinases that are correlated with binding affinity for particular inhibitors; such instances of structural similarity among phylogenetically diverse kinases are also shown to not be rare among kinases. Finally, these function-specific structural features may serve as potential starting points for the development of highly specific kinase inhibitors.     

Kinase selectivity profiling by inhibitor affinity chromatography

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.     

Kinase selectivity profiling by inhibitor affinity chromatography

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.     

Proteomic analysis of kinase inhibitor selectivity and function

Small molecule inhibitors of protein kinases have become highly popular tools in signal transduction research, despite the fact that rather limited data about their respective selectivities have been available. We established an efficient chemical proteomics method to characterize the cellular targets of the widely used inhibitor SB 203580, which was deemed to be rather specific for p38 kinase. Our results revealed several protein kinases as high affinity targets of SB 203580 and therefore imply a far more complicated cellular mode of action of this inhibitor than previously assumed. This raises the important question whether a lack of selectivity is inherent to many other "specific" inhibitors of protein kinases and warrants their evaluation employing experimental approaches adapted from our described proteomic technique.     

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.     

Measuring and interpreting the selectivity of protein kinase inhibitors

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.     

Analysis of water patterns in protein kinase binding sites

Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors.     

Design, synthesis and characterization of "clickable" 4-anilinoquinazoline kinase inhibitors

Immobilized kinase inhibitors have emerged as powerful reagents for the determination of kinase inhibitor selectivity and for the enrichment of protein targets from cellular lysates. Here, we report the design and synthesis of a set of "clickable" 4-anilinoquinazoline kinase inhibitors. We demonstrate that the attachment of a flexible tether that contains a bio-orthogonal azide functionality does not adversely affect the potency or selectivity of these inhibitors. Furthermore, we demonstrate the utility of these inhibitors through the generation of an affinity matrix for the enrichment of interacting proteins from cellular lysates.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

The structure of CYP101D2 unveils a potential path for substrate entry into the active site

We describe in the present paper mutations of the catalytic subunit α of PKA (protein kinase A) that introduce amino acid side chains into the ATP-binding site and progressively transform the pocket to mimic that of Aurora protein kinases. The resultant PKA variants are enzymatically active and exhibit high affinity for ATP site inhibitors that are specific for Aurora kinases. These features make the Aurora-chimaeric PKA a valuable tool for structure-based drug discovery tasks. Analysis of crystal structures of the chimaera reveal the roles for individual amino acid residues in the binding of a variety of inhibitors, offering key insights into selectivity mechanisms. Furthermore, the high affinity for Aurora kinase-specific inhibitors, combined with the favourable crystallizability properties of PKA, allow rapid determination of inhibitor complex structures at an atomic resolution. We demonstrate the utility of the Aurora-chimaeric PKA by measuring binding kinetics for three Aurora kinase-specific inhibitors, and present the X-ray structures of the chimaeric enzyme in complex with VX-680 (MK-0457) and JNJ-7706621 [Aurora kinase/CDK (cyclin-dependent kinase) inhibitor].     

Proteomic Analysis of Kinase Inhibitor Selectivity and Function

Small molecule inhibitors of protein kinases have become highly popular tools in signal transduction research, despite the fact that rather limited data about their respective selectivities have been available. We established an efficient chemical proteomics method to characterize the cellular targets of the widely used inhibitor SB203580, which was deemed to be rather specific for p38 kinase. Our results revealed several protein kinases as high affinity targets of SB 203580 and therefore imply a far more complicated cellular mode of action of this inhibitor than previously assumed. This raises the important question whether a lack of selectivity is inherent to many other “specific” inhibitors of protein kinases and warrants their evaluation employing experimental approaches adapted from our described proteomic technique.     

Tricyclic azepine derivatives: pyrimido[4,5-b]-1,4-benzoxazepines as a novel class of epidermal growth factor receptor kinase inhibitors

A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 microM in cellular phosphorylation assays (IC(50) 0.47-0.69 microM) and are highly selective against a small kinase panel. Such compounds demonstrate anti-EGFR activity within a class that is different from any known EGFR inhibitor scaffolds. They also provide a basis for the design of kinase inhibitors with the desired selectivity profile.     

Protein structure: discovering selective protein kinase inhibitors

A plethora of important targets for therapeutic intervention occurs in the protein kinase superfamily, one of the most thoroughly investigated groups of drug targets. Kinases have a deep hydrophobic ATP binding site that has been successfully exploited with the discovery of potent ATP-competitive drugs. However, most features of this pocket are well conserved in all protein kinases, which explains why kinase inhibitors typically exhibit a fairly indiscriminate spectrum of activity. Crystal structures of various protein kinases bound to their ligands are described, which begin to explain the observed selectivity profiles of kinase inhibitors. The insights gained from these structures suggest several approaches to improve inhibitor specificity and these approaches are summarized. The exciting potential of new high-throughput methods in structure determination that enable the systematic atomic-resolution investigation of large numbers of inhibitors bound to their various kinase targets will be discussed.     

Steered Molecular Dynamics Simulations Reveal the Likelier Dissociation Pathway of Imatinib from Its Targeting Kinases c-Kit and Abl

Development of small molecular kinase inhibitors has recently been the central focus in drug discovery. And type II kinase inhibitors that target inactive conformation of kinases have attracted particular attention since their potency and selectivity are thought to be easier to achieve compared with their counterpart type I inhibitors that target active conformation of kinases. Although mechanisms underlying the interactions between type II inhibitors and their targeting kinases have been widely studied, there are still some challenging problems, for example, how type II inhibitors associate with or dissociate from their targeting kinases. In this investigation, steered molecular dynamics simulations have been carried out to explore the possible dissociation pathways of typical type II inhibitor imatinib from its targeting protein kinases c-Kit and Abl. The simulation results indicate that the most favorable pathway for imatinib dissociation corresponds to the ATP-channel rather than the relatively wider allosteric-pocket-channel, which is mainly due to the different van der Waals interaction that the ligand suffers during dissociation. Nevertheless, the direct reason comes from the fact that the residues composing the ATP-channel are more flexible than that forming the allosteric-pocket-channel. The present investigation suggests that a bulky hydrophobic head is unfavorable, but a large polar tail is allowed for a potent type II inhibitor. The information obtained here can be used to direct the discovery of type II kinase inhibitors.     

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC₅₀ of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.     

Functional interrogation of the kinome using nucleotide acyl phosphates

The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.     

Developing kinase inhibitors for malaria: an opportunity or liability?

Highly druggable and essential to almost all aspects of cellular life, the protein and phosphoinositide kinase gene families offer a wealth of potential targets for pharmacological modulation for both noncommunicable and infectious diseases. Despite the success of kinase inhibitors in oncology and other disease indications, targeting kinases comes with significant challenges. Key hurdles for kinase drug discovery include selectivity and acquired resistance. The phosphatidylinositol 4-kinase beta inhibitor MMV390048 showed good efficacy in Phase 2a clinical trials, demonstrating the potential of kinase inhibitors for malaria treatment. Here we argue that the potential benefits of Plasmodium kinase inhibitors outweigh the risks, and we highlight the opportunity for designed polypharmacology to reduce the risk of resistance.     

A Targeted Library Screen Reveals a New Inhibitor Scaffold for Protein Kinase D

Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.     

Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): Optimization and selectivity profiling

We have recently developed a fragment based selection strategy for targeting kinases, where a small molecule warhead can be non-covalently tethered to a phage-displayed library of peptides. This approach was applied to the conversion of the promiscuous kinase inhibitor, staurosporine, into a potent bivalent ligand for cAMP-dependent protein kinase (PKA). Herein we report a systematic evaluation of this new bivalent ligand (BL); (a) Lineweaver–Burke analysis revealed that the BL, unlike substrate-based bivalent kinase inhibitors, displayed non-competitive inhibition with respect to the peptide substrate, suggesting an allosteric mechanism of action; (b) linker optimization of the BL, afforded one of the most potent, sub-nanomolar, inhibitors of PKA reported to date; (c) the BL was found to be modular, where attachment of active site targeted small molecule warheads in lieu of staurosporine could achieve similar gains in affinity; and (d) profiling studies of both the staurosporine derivative and the BL (amide isostere) against a panel of 90 kinases revealed almost unique enhancement in selectivity against PKA (>5-fold) compared to the starting staurosporine derivative. These combined results provide new insights for BL discovery, which has the potential to provide guidance toward the development of kinase selective reagents while uncovering new allosteric sites on kinases for therapeutic targeting.