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Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 A X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound 13, replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -β by the inhibitors in HEK 293T cells.

[Image: see text] Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure–activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.

Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements focus on the distance between the G' region of EF-G and the N-terminal region of L11 (L11-NTD), located within the GTPase activation center of the ribosome. We demonstrate that single-turnover ribosome-dependent EF-G GTPase proceeds according to a kinetic scheme in which rapid G' to L11-NTD movement requires prior GTP hydrolysis and, via branching pathways, either precedes P(i) release (major pathway) or occurs simultaneously with it (minor pathway). Such movement retards P(i) release, with the result that P(i) release is essentially rate-determining in single-turnover GTPase. This is the most significant difference between the EF-G.GTPase activities of vacant and translocating ribosomes [Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) Mol. Cell 11, 1517-1523], which are otherwise quite similar. Both the G' to L11-NTD movement and P(i) release are strongly inhibited by thiostrepton but not by FA. Contrary to the standard view that FA permits only a single round of GTP hydrolysis [Bodley, J. W., Zieve, F. J., and Lin, L. (1970) J. Biol. Chem. 245, 5662-5667], we find that FA functions rather as a slow inhibitor of EF-G.GTPase, permitting a number of GTPase turnovers prior to complete inhibition while inducing a closer approach of EF-G to the GAC than is seen during normal turnover.

Ribosome recycling factor (RRF) and elongation factor-G (EF-G) are jointly essential for recycling bacterial ribosomes following termination of protein synthesis. Here we present equilibrium and rapid kinetic measurements permitting formulation of a minimal kinetic scheme that accounts quantitatively for RRF and EF-G interaction on the Escherichia coli ribosome. RRF and EF-G (a) each form a binary complex on binding to a bare ribosome which undergoes isomerization to a more stable complex, (b) form mixed ternary complexes on the ribosome in which the affinity for each factor is considerably lower than its affinity for binding to a bare ribosome, and (c) each bind to two sites per ribosome, with EF-G having considerably higher second-site affinity than RRF. Addition of EF-G to the ribosome-RRF complex induces rapid RRF dissociation, at a rate compatible with the rate of ribosome recycling in vivo, but added RRF does not increase the lability of ribosome-bound EF-G. Added thiostrepton slows the initial binding of EF-G, and prevents both formation of the more stable EF-G complex and EF-G-induced RRF dissociation. These findings are relevant for the mechanism of post-termination complex disassembly.