Kinase hinge binding scaffolds and their hydrogen bond patterns.

Protein kinases constitute a major class of intracellular signaling molecules, and describe some of the most prominent drug targets. Kinase inhibitors commonly employ small chemical scaffolds that form hydrogen bonds with the kinase hinge residues connecting the N- and C-terminal lobes of the catalytic domain. In general the satisfied hydrogen bonds are required for potent inhibition, therefore constituting a conserved feature in the majority of inhibitor-kinase interactions. From systematically analyzing the kinase scaffolds extracted from Pfizer crystal structure database (CSDb) we recognize that large number of kinase inhibitors of diverse chemical structures are derived from a relatively small number of common scaffolds. Depending on specific substitution patterns, scaffolds may demonstrate versatile binding capacities to interact with kinase hinge. Afforded by thousands of ligand-protein binary complexes, the hinge hydrogen bond patterns were analyzed with a focus on their three-dimensional configurations. Most of the compounds engage H6 NH for hinge recognition. Dual hydrogen bonds are commonly observed with additional recruitment of H4 CO upstream and/or H6 CO downstream. Triple hydrogen bonds accounts for small number of binary complexes. An unusual hydrogen bond with a non-canonical H5 conformation is observed, requiring a peptide bond flip by a glycine residue at the H6 position. Additional hydrogen bonds to kinase hinge do not necessarily correlate with an increase in potency; conversely they appear to compromise kinase selectivity. Such learnings could enhance the prospect of successful therapy design.

Scaffold mining of kinase hinge binders in crystal structure database.

Protein kinases are the second most prominent group of drug targets, after G-protein-coupled receptors. Despite their distinct inhibition mechanisms, the majority of kinase inhibitors engage the conserved hydrogen bond interactions with the backbone of hinge residues. We mined Pfizer internal crystal structure database (CSDb) comprising of several thousand of public as well as internal X-ray binary complexes to compile an inclusive list of hinge binding scaffolds. The minimum ring scaffolds with directly attached hetero-atoms and functional groups were extracted from the full compounds by applying a rule-based filtering procedure employing a comprehensive annotation of ATP-binding site of the human kinase complements. The results indicated large number of kinase inhibitors of diverse chemical structures are derived from a relatively small number of common scaffolds, which serve as the critical recognition elements for protein kinase interaction. Out of the nearly 4,000 kinase-inhibitor complexes in the CSDb we identified approximately 600 unique scaffolds. Hinge scaffolds are overwhelmingly flat with very little sp3 characteristics, and are less lipophilic than their corresponding parent compounds. Examples of the most common as well as the uncommon hinge scaffolds are presented. Although the most common scaffolds are found in complex with multiple kinase targets, a large number of them are uniquely bound to a specific kinase, suggesting certain scaffolds could be more promiscuous than the others. The compiled collection of hinge scaffolds along with their three-dimensional binding coordinates could serve as basis set for hinge hopping, a practice frequently employed to generate novel invention as well as to optimize existing leads in medicinal chemistry.

Kinase inhibition that hinges on halogen bonds.

A major challenge for the discovery of protein kinase inhibitors is to identify potent, selective, and novel pharmacophores. In this issue, Fedorov et al. (2011) describes KH-CB19, an ATP-competitive inhibitor of cdc2-like kinase that interacts with the ATP hinge region through a halogen-bonding motif.

The design, annotation, and application of a kinase-targeted library.

We present here a workflow for designing a kinase-targeted library (KTL) with the goal of capturing known kinase inhibitor chemical space. We validated our design retrospectively using recent, high-throughput screening data and found significant enrichment of kinase inhibitor hits while retaining majority of the active kinase inhibitor series. To further assist kinase projects in triaging KTL screen hits, we also developed a methodology to systematically annotate known kinase inhibitors in the KTL with regard to their binding modes.

Inhibition of interleukin-1beta converting enzyme (ICE or caspase 1) by aspartyl acyloxyalkyl ketones and aspartyl amidooxyalkyl ketones.

A series of acyloxyalkyl and amidooxyalkyl ketones appended to a carbobenzyloxy aspartic acid core have been prepared. The most potent of these new inhibitors was 4i with a K(i) of 0.5 microM. These two series provide an improved understanding of the binding requirements for the hydrophobic prime side of ICE.

Succinic acid amides as P2-P3 replacements for inhibitors of interleukin-1beta converting enzyme (ICE or caspase 1).

Succinic acid amides have been found to be effective P2-P3 scaffold replacements for peptidic ICE inhibitors. Heteroarylalkyl fragments occupying the P4 position provided access to compounds with nM affinities. Utilization of an acylal prodrug moiety was required to overcome biopharmaceutical issues which led to the identification of 17f, a potential clinical candidate.

Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry.

Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.

Statistical method on nonrandom clustering with application to somatic mutations in cancer.

BACKGROUND: Human cancer is caused by the accumulation of tumor-specific mutations in oncogenes and tumor suppressors that confer a selective growth advantage to cells. As a consequence of genomic instability and high levels of proliferation, many passenger mutations that do not contribute to the cancer phenotype arise alongside mutations that drive oncogenesis. While several approaches have been developed to separate driver mutations from passengers, few approaches can specifically identify activating driver mutations in oncogenes, which are more amenable for pharmacological intervention. RESULTS: We propose a new statistical method for detecting activating mutations in cancer by identifying nonrandom clusters of amino acid mutations in protein sequences. A probability model is derived using order statistics assuming that the location of amino acid mutations on a protein follows a uniform distribution. Our statistical measure is the differences between pair-wise order statistics, which is equivalent to the size of an amino acid mutation cluster, and the probabilities are derived from exact and approximate distributions of the statistical measure. Using data in the Catalog of Somatic Mutations in Cancer (COSMIC) database, we have demonstrated that our method detects well-known clusters of activating mutations in KRAS, BRAF, PI3K, and beta-catenin. The method can also identify new cancer targets as well as gain-of-function mutations in tumor suppressors. CONCLUSIONS: Our proposed method is useful to discover activating driver mutations in cancer by identifying nonrandom clusters of somatic amino acid mutations in protein sequences.

Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors.

The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported.

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.

Prediction of specificity-determining residues for small-molecule kinase inhibitors.

BACKGROUND: Designing small-molecule kinase inhibitors with desirable selectivity profiles is a major challenge in drug discovery. A high-throughput screen for inhibitors of a given kinase will typically yield many compounds that inhibit more than one kinase. A series of chemical modifications are usually required before a compound exhibits an acceptable selectivity profile. Rationalizing the selectivity profile for a small-molecule inhibitor in terms of the specificity-determining kinase residues for that molecule can be an important step toward the goal of developing selective kinase inhibitors. RESULTS: Here we describe S-Filter, a method that combines sequence and structural information to predict specificity-determining residues for a small molecule and its kinase selectivity profile. Analysis was performed on seven selective kinase inhibitors where a structural basis for selectivity is known. S-Filter correctly predicts specificity determinants that were described by independent groups. S-Filter also predicts a number of novel specificity determinants that can often be justified by further structural comparison. CONCLUSION: S-Filter is a valuable tool for analyzing kinase selectivity profiles. The method identifies potential specificity determinants that are not readily apparent, and provokes further investigation at the structural level.

Targeting the unactivated conformations of protein kinases for small molecule drug discovery.

BACKGROUND: The number of drugs in active clinical development or on the market that target the unactivated conformational states of protein kinases is growing and represents a significant portion of kinase research at biopharmaceutical companies. These non-classical kinase inhibitors have a mode of action which may overcome some of the liabilities of classical ATP-site inhibitors that substantially overlap the space that ATP occupies in the activated kinase. OBJECTIVE: This review will discuss state-of-the-art methods of inhibiting protein kinases by targeting the unactivated conformations of the enzyme with small molecules directed to the ATP binding region. METHODS: Biochemical and structural biology publications and public domain crystal structures were evaluated to identify key concepts in drug discovery for unactivated protein kinase inhibitors that target the ATP binding region. CONCLUSION: The potential for enhanced selectivity, potency and duration of pharmacological action may allow non-classical kinase therapeutics to be used for chronic dosing in non-life-threatening indications. Moreover, by targeting additional conformational space on the kinase protein it is possible that new chemical matter will be discovered such that current intellectual property limitations on traditional ATP-site chemical scaffolds may be circumvented.

Synthesis and structure-activity relationships of N-6 substituted analogues of 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of Wee1 and Chk1 checkpoint kinases.

A series of N-6 substituted 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones were prepared from N-substituted (5-methoxyphenyl)ethenylindoles. The target compounds were tested for their ability to inhibit the G2/M cell cycle checkpoint kinases, Wee1 and Chk1. Analogues with neutral or cationic N-6 side chains were potent dual inhibitors. Acidic side chains provided potent (average IC(50) 0.057 microM) and selective (average ratio 223-fold) Wee1 inhibition. Co-crystal structures of inhibitors bound to Wee1 show that the pyrrolo[3,4-c]carbazole scaffold binds in the ATP-binding site, with N-6 substituents involved in H-bonding to conserved water molecules. HT-29 cells treated with doxorubicin and then target compounds demonstrate an active Cdc2/cyclin B complex, inhibition of the doxorubicin-induced phosphorylation of tyrosine 15 of Cdc2 and abrogation of the G2 checkpoint.

4-Phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione inhibitors of the checkpoint kinase Wee1. Structure-activity relationships for chromophore modification and phenyl ring substitution.

High-throughput screening has identified a novel class of inhibitors of the checkpoint kinase Wee1, which have potential for use in cancer chemotherapy. These inhibitors are based on a 4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione template and have been shown by X-ray crystallography to bind at the ATP site of the enzyme. An extensive study of the effects of substitution around this template has been carried out, which has identified substituents which lead to improvements in potency and selectivity for Wee1. While retention of the maleimide ring and pendant 4-phenyl group is necessary for potency, replacement of the carbazole nitrogen by oxygen is well tolerated and results in improved Wee1 selectivity against the related checkpoint kinase Chk1. Wee1 potency and selectivity are also enhanced by the incorporation of lipophilic functionality at the 2'-position of the 4-phenyl ring, and Wee1 selectivity against Chk1 is favored by C3-C5 alkyl substitution of the carbazole nitrogen. These studies provide a basis for the design of active analogues of the pyrrolocarbazole lead with improved physical properties.

Design, synthesis, and biological activity of novel polycyclic aza-amide FKBP12 ligands.

Since the discovery that FK-506 promotes neurite outgrowth, considerable attention has been focused on the development of potent nonimmunosuppressive ligands for FK-506 binding proteins (FKBPs). Such neuroimmunophilin agents have been reported to show neuroregenerative activity in a variety of cell and animal models including neurite outgrowth, age-related cognitive decline, Parkinson's disease, peripheral nerve injury, optic nerve degeneration, and diabetic neuropathy. We have designed and synthesized a unique series of tetracyclic aza-amides that have been shown to be potent FKBP12 rotamase inhibitors. The structure-activity relationships established in this study have demonstrated diverse structural modifications that result in potent rotamase inhibitory activity.

Structure-based design of nonpeptide inhibitors of interleukin-1beta converting enzyme (ICE, caspase-1).

A novel class of reversible inhibitors of Interleukin-1beta-converting enzyme (ICE, caspase-1) were discovered by iterative structure-based design. Guided by the X-ray crystal structure of analogues 1, 7 and 10 bound to ICE, we have designed a nonpeptide series of small molecule inhibitors. These compounds incorporate an arylsulfonamide moiety which replaces Val-His unit (P3-P2 residues) amino acids of the native substrate. The synthesis of the core structure, structure-activity relationships (SARs), and proposed binding orientation based on molecular modeling studies for this series of ICE inhibitors are described.