To catalyze research on historically untargeted protein kinases, we created the PKIS, an annotated set of 367 small molecule kinase inhibitors. The set has been widely distributed to academic ...collaborators as an open access tool. It has been used to identify chemical starting points for development of chemical probes for orphan kinases and to investigate kinase signaling in high content phenotypic assays. Access to the set comes with few restrictions other than the requirement that assay results be released into the public domain for the benefit of the entire research community. Examples from the efforts of several collaborators are summarized.
A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, ...was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.
Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase ...(CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8
T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2
mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2
macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8
T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.
New strategies are urgently needed to counter the threat to human health posed by drug-resistant fungi. To explore an as-yet unexploited target space for antifungals, we screened a library of protein ...kinase inhibitors for the ability to reverse resistance of the most common human fungal pathogen, Candida albicans, to caspofungin, a widely used antifungal. This screen identified multiple 2,3-aryl-pyrazolopyridine scaffold compounds capable of restoring caspofungin sensitivity. Using chemical genomic, biochemical, and structural approaches, we established the target for our most potent compound as Yck2, a casein kinase 1 family member. Combination of this compound with caspofungin eradicated drug-resistant C. albicans infection while sparing co-cultured human cells. In mice, genetic depletion of YCK2 caused an ∼3-log10 decline in fungal burden in a model of systemic caspofungin-resistant C. albicans infection. Structural insights and our tool compound's profile in culture support targeting the Yck2 kinase function as a broadly active antifungal strategy.
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•Screen of kinase inhibitors identifies Yck2 kinase as a promising antifungal target•Clinical echinocandin resistance is reversed by pyrazolopyridine Yck2 inhibitors•Combination treatment eradicates fungus while sparing co-cultured human cells•Genetic depletion of YCK2 markedly impairs Candida albicans virulence in mice
New agents are needed to counter infection by drug-resistant fungi. We screened protein kinase inhibitors for the ability to reverse antifungal resistance. Chemogenomic, biochemical, and structural data established Yck2 as the target of our most potent hit. Findings support inhibiting this kinase as a promising therapeutic strategy.
A genomic signature designed to assess the activity of the estrogen-related receptor alpha (ERRα) was used to profile more than 800 breast tumors, revealing a shorter disease-free survival in ...patients with tumors exhibiting elevated receptor activity. Importantly, this signature also predicted the ability of an ERRα antagonist, XCT790, to inhibit proliferation in cellular models of breast cancer. Using a chemical genomic approach, it was determined that activation of the Her2/IGF-1R signaling pathways and subsequent C-MYC stabilization upregulate the expression of peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1β), an obligate cofactor for ERRα activity. PGC-1β knockdown in breast cancer cells impaired ERRα signaling and reduced cell proliferation, implicating a functional role for PGC-1β/ERRα in the pathogenesis of breast cancers.
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► ERRα signature predicts relapse-free survival in multiple breast cancer cohorts ► PGC-1β/ERRα signaling affects breast cancer cell proliferation ► Her2 and IGF-1 signaling activate the PGC1β/ERRα axis
Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have ...developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.
A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a ...cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein down-regulation through drug "off-targets" might be relevant for other inhibitors that serendipitously target pseudokinases.
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We demonstrate an innovative approach for optimization of kinase inhibitor potency and selectivity utilising kinase mini-panels and kinome-wide panels. We present a focused case study ...on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs.
We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G-associated kinase (GAK), together with a structurally related negative control SGC-GAK-1N (14). 11 was highly ...selective in an in vitro kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent RIPK2 inhibitor lacking GAK activity. Together, this chemical probe set can be used to interrogate GAK cellular biology.
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