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Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution, and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib, and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

Supplementary information

Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution, and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib, and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

A new class of alkynyl isoquinoline antibacterial compounds, synthesized via Sonogashira coupling, with strong bactericidal activity against a plethora of Gram-positive bacteria including methicillin- and vancomycin-resistant Staphylococcus aureus (S. aureus) strains is presented. HSN584 and HSN739, representative compounds in this class, reduce methicillin-resistant S. aureus (MRSA) load in macrophages, whilst vancomycin, a drug of choice for MRSA infections, was unable to clear intracellular MRSA. Additionally, both HSN584 and HSN739 exhibited a low propensity to develop resistance. We utilized comparative global proteomics and macromolecule biosynthesis assays to gain insight into the alkynyl isoquinoline mechanism of action. Our preliminary data show that HSN584 perturb S. aureus cell wall and nucleic acid biosynthesis. The alkynyl isoquinoline moiety is a new scaffold for the development of potent antibacterial agents against fatal multidrug-resistant Gram-positive bacteria. Published version

Selpercatinib (LOXO-292, LY3527723) and pralsetinib (BLU-667) are first-in-class RET-targeted cancer therapy drugs. However, secondary RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET Tyrosine kinase inhibitors (TKIs). While the G810C/R/S/V mutations located at the RET kinase solvent-front site were detected in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. We profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. We found that alkynyl nicotinamide compounds such as HSN608 have better drug-like properties than alkynyl benzamides. HSN608 inhibited RET and RET V804M gatekeeper mutant, and all six G810 mutants with low nanomolar IC50s in the BaF3/KIF5B-RET mutant cell model. In cell derived xenograft (CDX) tumors driven by KIF5B-RET(G810C), HSN608 caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib, and identifies an alkylnyl nicotinamide-based RET TKI for inhibiting selpercatinib/pralsetinib-resistant G810 mutants. Citation Format: Ujjwol Khatri, Neetu Dayal, Xueqing Hu, Elizabeth Larocque, Nimishetti Naganna, Tao Shen, Xuan Liu, Frederick W. Holtsberg, M. Javad Aman, Herman O. Sintim, Jie Wu. Targeting RET solvent-front mutants with an alkynyl nicotinamide-based inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3851.