Identification and characterization of pan-mutant selective PI3Kalpha inhibitors with the capability of PI3Kalpha H1047R protein degradation

Introduction: PIK3CA, a PI3K isoform, is one of the most frequently mutated genes in human cancers. PIK3CA mutations are highly enriched at “hotspot” sites in the helical (E542K, E545K) and kinase (H1047R/L) domains. Orthosteric inhibitors of PI3Kalpha, such as alpelisib and inavolisib, have been approved for the treatment of patients with advanced HR+/HER2− breast cancer. Selective targeting of mutant PI3Kalpha is anticipated to enhance antitumor efficacy and mitigate the toxicity associated with wild-type PI3Kalpha inhibition in normal tissue. Allosteric and mutant-selective PI3Kalpha inhibitors, including RLY-2608 (NCT06982521) and LY4064809 (STX-478, NCT07174336), have been successfully developed in the clinical phase III stage. Results: Several allosteric and potent pan-mutant PI3Kalpha inhibitors developed by TYK Medicines exhibited high selectivity over wild-type (WT) PI3Kalpha. Our observations revealed that the compounds inhibited AKT phosphorylation in the T47D (PI3Kalpha H1047R) cell line with an IC 50 in the single-digit nanomolar range, demonstrating nearly 200 times more selectivity compared to SK-BR-3 (PI3Kalpha WT) cells at same time. In in vitro antiproliferative assays, the compounds displayed superior antiproliferative activity against various PI3Kalpha mutant cells, including T47D, HCC1954 (PI3Kalpha H1047R), and MCF7 (PI3Kalpha E545K), compared to STX-478 and RLY-2608. For instance, TY-3659 was four and thirteen times more selective in T47D and MCF7 cells, respectively, than RLY-2608, aligning with the PI3Kalpha kinase inhibition assays. In addition, TY-3659 effectively degraded PI3Kalpha mutant protein in HCC1954 and MDA MB 453 (PI3Kalpha H1047R) cells. In the mouse HCC1954 and Cal33 (PI3Kalpha H1047R) CDX models, the compounds exhibited potent tumor-inhibitory activity, excellent tolerance, good DMPK properties, and desirable drug-like characteristics. No effects on fasting plasma glucose levels were observed after four days of treatment in the insulin tolerance test. In conclusion, we identified potent allosteric and pan-mutant selective PI3Kalpha inhibitors capable of degrading PI3Kalpha mutant protein. Our findings suggest that TYK compounds can significantly enhance the therapeutic index of PI3Kalpha-mutant cancers without causing PI3Kalpha WT-related toxicity. # Chengshan Niu and Shengli Dong contributed equally to this work. *Correspondence authors