Discovery and Structure-Activity Relationship Study of (Z)-5-Methylenethiazolidin-4-one Derivatives as Potent and Selective Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Abstract

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 Å 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.

Figure 1.

(A) Scatter plot of high-throughput screen for the PI5P4Kα activity of in-house small molecule library with ~6000 compounds (shown are hits at <120% DMSO control). The results of two replicates are plotted against each other (normalized to DMSO). Negative controls (DMSO) are shown in dark gray, CVM-05–002 is depicted in green, and the 10% DSMO control cutoff (90% inhibition) is labeled. (B) Concentration– response curves of CVM-05–002, including counterscreen results. (C) Structure of identified lead compound, CVM-05–002.

Figure 2.

(A) Binding site of cocrystal structure of PI5P4Kα with bound inhibitor CVM-05–002 (dark gray). Key hydrogen bond interactions are depicted by dashed lines (numbers indicate distances in Å). (B) Key hydrogen bond interactions of the hinge-binding site are shown as dashed lines (numbers indicate distances in Å). (C) Proposed hydrogen bond network of the thiazolidinone moiety of CVM-05–002 with Lys209 and Thr232 (interactions are shown as dashed lines; numbers indicate distances in Å). PDB-ID: 6UX9.

Figure 3.

Kinome-wide selectivity profile for compounds CVM-05–002, 3, 13, and 31 (excluding mutant, atypical, and pathogen panels). The inhibitors were tested at 1 μM on a panel of 468 kinases. The results are displayed as red circles with their sizes correlating with the inhibitor’s binding affinity (percent DMSO control).

Figure 4.

(A) Western blot of the melting curve determination of PI5P4Kα and -β. HEK 293T cells were either treated for 1 h with DMSO or 10 μM CVM-05–002. Representative Western blot of two independent repeats. (B) Structures of selected compounds of the presented SAR study and their biochemical activities. (C) Quantified relative band intensity (%) of the isothermal CETSA experiment (Supporting Figure 10). The mean of four independent experiments is shown. ** p < 0.01, *** p < 0.001 by t-test.

Scheme 1.. Synthesis of Compound CVM-05–002 and Analogues^a

^aReagents and conditions: (a) phenylboronic acids/respective pinacol boronic acid esters, Pd(PPh₃)₂Cl₂, Cs₂CO₃, 1,4-dioxane/H₂O, 100 °C, 1.5 h, 9–93% yield; (b) (Z)-methylenethiazolidinone derivative, β-alanine, AcOH, 100 °C, 0.5 h, 12–33% yield, or maleimide, PPh₃, dichloromethane (DCM), rt, overnight, 21% yield, or oxazolidine-2,4-dione, piperidine, EtOH, 100 °C, overnight, 4% yield, or phenylboronic acids/respective pinacol boronic acid ester, Pd(PPh₃)₂Cl₂, Cs₂CO₃, 1,4-dioxane/H₂O, 100 °C, 1.5 h, 1–37% yield, or N-(3-hydroxyphenyl)acetamide, CuI, K₂CO₃, N,N-dimethylformamide (DMF), 120 °C, 2 d, 3% yield, or N-(3-bromophenyl)acetamide, Pd₂(dba)₃, X-Phos, K₂CO₃, 1,4-dioxane/H₂O, 80 °C, overnight, 7% yield; (c) 4-(methanesulfonylamino)phenylboronic acid pinacol ester, Pd(PPh₃)₂Cl₂, Cs₂CO₃, 1,4-dioxane/H₂O, 100 °C, 1.5 h, 19– 27% yield; (d) 2-iminothiazolidin-4-one, β-alanine, AcOH, 100 °C, 0.5 h, 4–22% yield.