Structural basis of the TPX-0022/c-Met interaction. (A) Chemical structure of TPX-0022. (B) The potency of TPX-0022 against c-Met was analysed using an in vitro kinase assay kit. (C) Cellular activity determination of TPX-0022 using NCI-H1993 cells. Data in (B/C) are represented as the mean ± SD of n = 3 independent experiments. (D) The overall structure of the c-Met/TPX-0022 complex. c-Met kinase is colored gray. TPX-0022 is colored yellow with the electron density map (2mFo-DFc) at 1σ showing the density. The hinge loop (red) and A-loop (green) are highlighted. (E) Surface presentation of c-Met shows the binding pocket of TPX-0022. (F) Hydrophobic interaction between c-Met and TPX-0022. (G) Role of residues Y1230, K1110 and D1228 in the interaction with TPX-0022. (H) Hydrogen bond interactions between c-Met and TPX-0022. Salt bridges and hydrogen bonds are indicated as black dotted lines.

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... targeting the semaphorin domain and kinase inhibitors targeting the kinase domain are effective strategies for the development of c-Met-targeted drugs. Small molecule inhibitors targeting the c-Met kinase domain have made inspiring progress in recent years. Six inhibitors have been approved by the FDA or NMPA for clinical treatment of cancer ( Fig. S1) [15][16][17][18][19]. The indications for these approved drugs are mainly for NSCLC harboring MET gene alterations [11]. Meanwhile, some c-Met inhibitors are under clinical trials for gastric cancer and hepatocellular carcinoma [10]. TPX-0022 (also known as elzovantinib), developed by Turning Point Therapeutics, is an orally ...

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... an orally bioavailable c-Met inhibitor that also targets CSF1R and c-Src kinases [20,21]. Unlike most linear c-Met inhibitors, TPX-0022 possesses a compact multicyclic scaffold, which could offer unique drug-like profiles such as desirable conformational rigidity, improved oral bioavailability, enhanced metabolic stability and cell permeability (Fig. 1A) [22]. TPX-0022 was granted fast track approval and orphan drug designation by the FDA for the treatment of gastric cancer in 2021. Preclinical data indicated that TPX-0022 blocked both HGF-and CSF1-mediated signaling and showed activity in multiple MET-and CSF1R-dependent cell lines and xenograft models [21]. An ongoing phase I/II ...

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... first performed a kinase assay to verify the binding activity of TPX-0022 to c-Met. The results indicated that TPX-0022 potently inhibited c-Met with an IC 50 of 2.7 nM (Fig. 1B). In the NSCLC NCI-H1993 cell line harboring c-Met alteration, TPX-0022 effectively inhibited cell proliferation with an IC 50 of 0.92 nM (Fig. 1C). The intracellular inhibitory activity of TPX-0022 was stronger than that of the kinase assay, possibly due to differences in experimental methods or the inhibition of multiple kinases by ...

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... first performed a kinase assay to verify the binding activity of TPX-0022 to c-Met. The results indicated that TPX-0022 potently inhibited c-Met with an IC 50 of 2.7 nM (Fig. 1B). In the NSCLC NCI-H1993 cell line harboring c-Met alteration, TPX-0022 effectively inhibited cell proliferation with an IC 50 of 0.92 nM (Fig. 1C). The intracellular inhibitory activity of TPX-0022 was stronger than that of the kinase assay, possibly due to differences in experimental methods or the inhibition of multiple kinases by the ...

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... further analyse the molecular interactions between TPX-0022 and c-Met, we determined the co-crystal structure of TPX-0022 with c-Met at 2.7 Å. The statistics of data collection and model refinement are listed in Table S1. As shown in Fig. 1D, c-Met exhibits a typical protein tyrosine kinase structure, consisting of a bilobed architecture (N-lobe and Clobe). TPX-0022 binds to the ATP-binding cavity of c-Met in a type-I inhibitor binding mode (Fig. 1D, Fig. S2A). TPX-0022 bent into a "Ushaped" binding conformation and wrapped around Met1211 (Fig. 1E). It establishes ...

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... structure of TPX-0022 with c-Met at 2.7 Å. The statistics of data collection and model refinement are listed in Table S1. As shown in Fig. 1D, c-Met exhibits a typical protein tyrosine kinase structure, consisting of a bilobed architecture (N-lobe and Clobe). TPX-0022 binds to the ATP-binding cavity of c-Met in a type-I inhibitor binding mode (Fig. 1D, Fig. S2A). TPX-0022 bent into a "Ushaped" binding conformation and wrapped around Met1211 (Fig. 1E). It establishes hydrophobic contacts with residues I1084, V1092, A1108, L1157, M1211 and A1221 in the ATP pocket (Fig. 1F). In addition, the benzonitrile group of TPX-0022 forms a π-stacking interaction with the aromatic side chain of Y1230 of ...

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... are listed in Table S1. As shown in Fig. 1D, c-Met exhibits a typical protein tyrosine kinase structure, consisting of a bilobed architecture (N-lobe and Clobe). TPX-0022 binds to the ATP-binding cavity of c-Met in a type-I inhibitor binding mode (Fig. 1D, Fig. S2A). TPX-0022 bent into a "Ushaped" binding conformation and wrapped around Met1211 (Fig. 1E). It establishes hydrophobic contacts with residues I1084, V1092, A1108, L1157, M1211 and A1221 in the ATP pocket (Fig. 1F). In addition, the benzonitrile group of TPX-0022 forms a π-stacking interaction with the aromatic side chain of Y1230 of the A-loop, and the orientation of the Aloop is stabilized by a salt bridge between D1228 and ...

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... bilobed architecture (N-lobe and Clobe). TPX-0022 binds to the ATP-binding cavity of c-Met in a type-I inhibitor binding mode (Fig. 1D, Fig. S2A). TPX-0022 bent into a "Ushaped" binding conformation and wrapped around Met1211 (Fig. 1E). It establishes hydrophobic contacts with residues I1084, V1092, A1108, L1157, M1211 and A1221 in the ATP pocket (Fig. 1F). In addition, the benzonitrile group of TPX-0022 forms a π-stacking interaction with the aromatic side chain of Y1230 of the A-loop, and the orientation of the Aloop is stabilized by a salt bridge between D1228 and K1110 (Fig. 1G). (Fig. 1H). These van der Waals interactions help stabilize the c-Met/ TPX-0022 ...

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... 1E). It establishes hydrophobic contacts with residues I1084, V1092, A1108, L1157, M1211 and A1221 in the ATP pocket (Fig. 1F). In addition, the benzonitrile group of TPX-0022 forms a π-stacking interaction with the aromatic side chain of Y1230 of the A-loop, and the orientation of the Aloop is stabilized by a salt bridge between D1228 and K1110 (Fig. 1G). (Fig. 1H). These van der Waals interactions help stabilize the c-Met/ TPX-0022 ...

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... hydrophobic contacts with residues I1084, V1092, A1108, L1157, M1211 and A1221 in the ATP pocket (Fig. 1F). In addition, the benzonitrile group of TPX-0022 forms a π-stacking interaction with the aromatic side chain of Y1230 of the A-loop, and the orientation of the Aloop is stabilized by a salt bridge between D1228 and K1110 (Fig. 1G). (Fig. 1H). These van der Waals interactions help stabilize the c-Met/ TPX-0022 ...

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... addition to c-Met, TPX-0022 also shows potent inhibitory ability to c-Src and CSF1R, which is thought to have the potential to improve clinical efficacy [21]. Kinase assays indicated that TPX-0022 had similar inhibitory potency against c-Met and c-Src, with IC 50 values of 2.7 nM and 3.7 nM, respectively (Figs. 1B, 3A). To help elucidate the binding mechanism, we determined the structure of TPX-0022 in complex with c-Src at a resolution of 2.8 Å (Table ...

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... MET alterations. The most frequent on-target mutation sites include G1163, D1228 and Y1230 for type I inhibitors and L1195 and F1200 for type II inhibitors. In general, type I inhibitors had potent activity against mutations of L1195 and F1200 but low affinity against mutations of D1228 and Y1230, such as capmatinib, tepotinib and savolitinib (Fig. S10) [45]. TPX-0022 could also not overcome D1228N and Y1230C but still had moderate inhibition activity against Y1230H. The rigid structure of TPX-0022 may play a role in maintaining close proximity to Y1230H to form π-stacking interactions. The location of G1163 at the edge of the pocket and the compact multicyclic scaffold of TPX-0022 ...