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Abstract: Small molecule drugs targeting kinases have revolutionized treatment options for millions of patients worldwide, especially in oncology. These targeted treatments have less side effects because they inhibit a specific dysfunctional kinase usually with relatively narrow selectivity. However, kinase inhibitors do have well‐established liabilities, most prominently the emergence of drug resistance. Moreover, the majority of kinases are multidomain and multifunctional proteins that in addition to their enzymatic activity have scaffolding and other roles, and inhibitors seldom address these alternative functions. Recently, small molecule mediated targeted protein degradation emerged as a new pharmacological strategy. The majority of small molecule degraders are bispecific molecules called proteolysis targeting chimeras (PROTACs), and their mechanism of action is based on simultaneous recruitment of the target of interest and an E3 ligase, resulting in target polyubiquitination and eventual destruction by the proteasome. Over the last couple of years, PROTAC strategy has been developed and validated for a range of targets, including kinases. Here, we introduce the targeted protein degradation strategy, provide an overview of representative kinase PROTACs, and describe design rationales, efficacy and specificity. We also discuss their potential advantages, as well as comment on some of the limitations of this emerging pharmacological modality. [ABSTRACT FROM AUTHOR]

Selective small-molecule inhibitors of protein kinases can serve as powerful tools to elucidate biological function. Efforts to develop potential drug candidates have yielded a wealth of kinase inhibitors. However, selecting the optimal kinase inhibitor for a particular application can be challenging. While the optimal inhibitor will be application specific, we have attempted to summarize some of the best reported inhibitors for various kinases. [ABSTRACT FROM AUTHOR]

Selective small-molecule inhibitors of protein kinases can serve as powerful tools to elucidate biological function. Efforts to develop potential drug candidates have yielded a wealth of kinase inhibitors. However, selecting the optimal kinase inhibitor for a particular application can be challenging. While the optimal inhibitor will be application specific, we have attempted to summarize some of the best reported inhibitors for various kinases. [ABSTRACT FROM AUTHOR]

The author discusses the design strategies to improve the coverage of kinome and development of its selective inhibitors. The author investigated the crystal structure of the protein kinase domain (PKA), the location of ATP-binding site on protein kinases, and the selectivity of type III inhibitors.

The TAIRE family of kinases are an understudied branch of the CDK kinase family, that have been implicated in a number of cancers. This manuscript describes the design, synthesis and SAR of covalent CDK14 inhibitors, culminating in identification of FMF-04-159-2, a potent, covalent CDK14 inhibitor with a TAIRE kinase biased selectivity profile. [ABSTRACT FROM AUTHOR]

Acute myelogenous leukemia (AML) is often associated with activating mutations in the receptor tyrosine kinase, Flt3, including internal tandem duplications (ITDs) within the regulatory juxtamembrane region. Previous studies have linked Flt3-ITD to the activation of the Fes protein tyrosine kinase in AML, and RNAi-knockdown studies suggest that Fes may be required for Flt3 function. In this study, we tested Fes inhibitors from three different chemical classes for their growth-suppressive activity against Flt3-ITD+ myeloid leukemia cell lines (MV4-11, MOLM-13 and MOLM-14) vs. myeloid cells with wild-type Flt3 (THP-1). All Fes inhibitors selectively inhibited the growth of Flt3-ITD+ AML cells, with IC50 values for diaminopyrimidine and pyrrolopyridine inhibitors ranging from 19 to 166 nM. In contrast, a pyrazolopyrimidine inhibitor was less potent in Flt3-ITD+ AML cells, with IC50 values in the 1.0 μM range. In vitro kinase assays showed that the most potent inhibitors of Flt3-ITD+ AML cell proliferation blocked both Fes and Flt3-ITD kinase activity, while the pyrazolopyrimidine was more selective for Fes vs. Flt3-ITD. All three inhibitors induced significant apoptosis in Flt3-ITD+ AML cells, with potency equivalent to or greater than the established Flt3-ITD inhibitor, tandutinib. Transformation of TF-1 cells with Flt3-ITD resulted in constitutive activation of endogenous Fes, and rendered the cells highly sensitive to all three Fes inhibitors with IC50 values in the 30–500 nM range. The pyrrolopyridine compound also induced apoptotic responses in patient-derived Flt3-ITD+ AML bone marrow cells but not in normal bone marrow mononuclear cells. These results demonstrate that Fes kinase activity contributes to Flt3-ITD signaling in AML, and suggests that dual inhibition of both Flt3 and Fes may provide a therapeutic advantage for the treatment of Flt3-ITD+ AML. [ABSTRACT FROM AUTHOR]

Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases. Western blotting and RNA sequencing demonstrated that treatment of infected cells with XMD7 compounds resulted in a defect in the production of the major HCMV transcriptional transactivator IE2 proteins (IE2-86, IE2-60 and IE2-40) and an overall reduction in transcription from the viral genome. However, production of certain viral proteins was not compromised by treatment with XMD7 compounds. Thus, these novel anti-HCMV compounds likely inhibited transcription from the viral genome and suppressed production of a subset of viral proteins by inhibiting IE2 protein production. [ABSTRACT FROM AUTHOR]

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. [ABSTRACT FROM AUTHOR]

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. [ABSTRACT FROM AUTHOR]

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes. [ABSTRACT FROM AUTHOR]

Activation of the ERK pathway is a hallmark of cancer, and targeting of upstream signaling partners led to the development of approved drugs. Recently, SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induces a so-far-unknown binding pocket that accommodates the piperazine-phenyl-pyrimidine decoration. This new binding pocket was created by an inactive conformation of the phosphate-binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed two canonical but distinct type I binding modes. Notably, the new binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell-based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity. [ABSTRACT FROM AUTHOR]

Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To date, the majority of clinical and preclinical kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential “kinase cysteinome” and discuss strategies for the efficient development of new covalent inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson’s disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC50s of around 10nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3–1.0μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg. [Copyright &y& Elsevier]

Abstract: Here we describe the synthesis and characterization of a number of 3-amino-1H-indazol-6-yl-benzamides that were designed to target the ‘DFG-out’ conformation of the kinase activation loop. Several compounds such as 4 and 11 exhibit single-digit nanomolar EC50s against FLT3, c-Kit and the gatekeeper T674M mutant of PDGFRα. [Copyright &y& Elsevier]

Abstract: A series of alkyne-containing type II inhibitors with potent inhibitory activity of T315I Bcr-Abl has been identified. The most active compound 4 exhibits an EC50 of less than 1nM against wild-type Bcr-Abl and an EC50 of 10nM against T315I mutant but is broadly active against a number of other kinases. [Copyright &y& Elsevier]

Abstract: The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed. [Copyright &y& Elsevier]

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens. [ABSTRACT FROM AUTHOR]

The SAR of a series of benzopyrimidodiazepinone inhibitors of TNK2 was developed, starting from the potent and selective compound XMD8-87. A diverse set of anilines was introduced in an effort to improve the in vivo PK profile and minimize the risk of quinone diimine formation. [ABSTRACT FROM AUTHOR]

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development. • Our study highlights a multi-targeted strategy to identify druggable cysteines • We identified 23 covalent kinase targets across multiple spatial locations • Nine of these have previously not been covalently targeted by an inhibitor • Our findings highlight potential means to advance covalent drug discovery efforts The current work by Rao et al. describes using a promiscuous ligand as a tool to identify new targets for drug discovery. The findings from this study highlight previously unknown targets against which irreversible inhibitors can be developed. These targets are typically deregulated in diseases including cancer. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 14 (CDK14) and other TAIRE family kinases (CDKs 15–18) are proteins that lack functional annotation but are frequent off-targets of clinical kinase inhibitors. In this study we develop and characterize FMF-04-159-2, a tool compound that specifically targets CDK14 covalently and possesses a TAIRE kinase-biased selectivity profile. This tool compound and its reversible analog were used to characterize the cellular consequences of covalent CDK14 inhibition, including an unbiased investigation using phospho-proteomics. To reduce confounding off-target activity, washout conditions were used to deconvolute CDK14-specific effects. This investigation suggested that CDK14 plays a supporting role in cell-cycle regulation, particularly mitotic progression, and identified putative CDK14 substrates. Together, these results represent an important step forward in understanding the cellular consequences of inhibiting CDK14 kinase activity. • A covalent, cell-permeable inhibitor of CDK14 was developed • Methods for assessing cellular engagement of TAIRE family kinases are described • Kinase motif analysis and phospho-proteomics identified putative CDK14 substrates Cyclin-dependent kinase 14 (CDK14) is an understudied kinase that lacks functional annotation. The authors develop and characterize a covalent CDK14 inhibitor, FMF-04-159-2, and use it to interrogate the role of CDK14 in the cell cycle and phospho-signaling. [ABSTRACT FROM AUTHOR]