Preclinical characterization of TOS-358, a potent and selective covalent inhibitor of wild-type and mutant PI3Kα with superior anticancer activity

Phosphoinositide 3-kinase alpha (PI3Kα) is the most frequently mutated oncogene in cancer, inferring a critical role for this protein in neoplasia. The molecular biology of PI3Kα reveals it to be a protein that integrates a large and diverse set of cellular signals. In the development of small molecule inhibitors of this target protein it has been demonstrated that deep but also durable inhibition is critical to potent anti-cancer activity, an area traditionally challenging for reversible competitive and allosteric inhibitors. TOS-358 was developed to inhibit covalently both wild-type and mutant PI3Kα with observed IC50 s of 2.2 nM for WT PI3Kα and 4.1 nM for mutant PI3Ka (H1047R). TOS-358 is highly selective in a kinome-wide screen and selective for PI3Kα over other isoforms. We confirmed covalent binding to PI3Kα by NanoBRET in washout experiments, where it was observed that TOS-358 maintained 90% binding at 100 nM 6 hours after washout; in contrast, binding of Alpelisib at 100 nM was completely lost over the same time frame. Irreversible binding was further established using TR-FRET assay in which Kinact/KI was found to be 5.6 × 107 M−1s−1. Importantly, we also noted that TOS-358 produced sustained inhibition of phosphorylated AKT(S473) to 48 hours while allosteric inhibitors lose >60% inhibition. TOS-358 mediated cell growth inhibition has been evaluated in a panel of 120 cell lines and compared with Alpelisib in the same panel. This analysis revealed approximately 50% more cell lines to be responsive to TOS-358 compared with Alpelisib. There was no strong association of response and PI3Kα mutation status, and in fact for both TOS-358 and Alpelisib there was a higher frequency of WT PI3Kα cell lines responding to either treatment. TOS-358 activity has been tested in multiple different cell-derived and patient-derived xenograft cancer models and was found to produce reproducible and substantial tumor growth inhibition. Indeed, in several PDX models, TOS-358 induced tumor regressions while the clinical stage molecules Alpelisib and Inavolisib were unable to generate similar tumor regressions despite pharmacokinetic exposures comparable to, or in excess of, TOS-358. Finally, TOS-358 generated little or no glucose impact in mice and dogs at exposures that produced superior efficacy in these cancer models. Taken together, our in vitro and in vivo data reveal TOS-358 to be a potent, selective covalent inhibitor of PI3Kα with superior anticancer activity to comparator molecules. Conflict of interest: Ownership: The authors are employees of and may hold equity in Totus Medicines.