Search

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug-target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

Over 20 years after the approval of the first-in-class protein kinase inhibitor imatinib, the biological function of a significant fraction of the human kinome remains poorly understood while most research continues to be focused on few well-validated targets. Given the strong genetic evidence for involvement of many kinases in health and disease, the understudied fraction of the kinome holds a large and unexplored potential for future therapies. Specific chemical probes are indispensable tools to interrogate biology enabling proper preclinical validation of novel kinase targets. In this Perspective, we highlight recent case studies illustrating the development of high-quality chemical probes for less-studied kinases and their application in target validation. We spotlight emerging techniques and approaches employed in the generation of chemical probes for protein kinases and beyond and discuss the associated challenges and opportunities.

Simple Summary The two human p70 ribosomal S6 kinases, S6K1 and S6K2, have been associated with a variety of cellular processes and human pathologies, especially cancer. Thus far, only S6K1 was thoroughly studied and selectively addressed by small molecule inhibitors. Despite growing evidence suggesting S6K2 as a promising anticancer target, this isoform has been severely neglected, which can partly be attributed to the lack of isoform-selective inhibitors to study its function. By exploiting a cysteine residue exclusive to S6K2, we were able to generate the first known isoform-selective S6K2 inhibitor. Besides its excellent selectivity against S6K1 and other human kinases, the compound showed weak intrinsic reactivity and promising in vitro metabolic stability. Our proof-of-concept study provides a basis for the development of high quality S6K2 chemical probes to validate this kinase as a target for therapeutic interventions. Abstract The ribosomal protein S6 kinase beta 2 (S6K2) is thought to play an important role in malignant cell proliferation, but is understudied compared to its closely related homolog S6 kinase beta 1 (S6K1). To better understand the biological function of S6K2, chemical probes are needed, but the high similarity between S6K2 and S6K1 makes it challenging to selectively address S6K2 with small molecules. We were able to design the first potent and highly isoform-specific S6K2 inhibitor from a known S6K1-selective inhibitor, which was merged with a covalent inhibitor engaging a cysteine located in the hinge region in the fibroblast growth factor receptor kinase (FGFR) 4 via a nucleophilic aromatic substitution (SNAr) reaction. The title compound shows a high selectivity over kinases with an equivalently positioned cysteine, as well as in a larger kinase panel. A good stability towards glutathione and Nα-acetyl lysine indicates a non-promiscuous reactivity pattern. Thus, the title compound represents an important step towards a high-quality chemical probe to study S6K2-specific signaling.

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint securing proper chromosome segregation. It is being evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer with several reversible inhibitors currently undergoing clinical trials. While long drug–target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors are known. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor RMS-07 targeting a cysteine (Cys604) in the kinase's hinge region present only in few other protein kinases. The compound showed excellent MPS1 inhibitory potency and high selectivity against all protein kinases harboring an equivalent cysteine as well as in a larger differential scanning fluorimetry-based screening panel. Covalent binding was confirmed by mass spectrometry and X-ray crystal structure. We expect this tool compound to open new avenues for the design of MPS1-specific covalent chemical probes or drugs.

Here we describe the synthesis of N-(6-chloro-3-nitropyridin-2-yl)5-(1-methyl-1H-pyrazol-4-yl)isoquinolin-3-amine via a three-step procedure including a Buchwald–Hartwig arylamination with benzophenone imine and a highly regioselective nucleophilic aromatic substitution. The title compound was analyzed by nuclear magnetic resonance spectroscopy (1H, 13C, HSQC, HMBC, COSY, DEPT90 and NOESY), high resolution mass spectrometry (ESI-TOF-HRMS) and infrared spectroscopy (ATR-IR) and its structure was confirmed by single crystal X-ray diffraction. The inhibitory potency of the title compound was evaluated for selected kinases harboring a rare cysteine in the hinge region (MPS1, MAPKAPK2 and p70S6Kβ/S6K2).

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compounds displayed KD values of 190 nM and 401 nM for VRK1 and VRK2, respectively. Differences in compound binding mode and substituent preferences between the two VRKs were identified by the series structure-activity relationship combined with the crystallographic analysis of key compounds. We expect that our results will serve as a starting point for the design of specific and potent inhibitors against each of the two VRKs based on a pyridine scaffold.