WEE1 and PKMYT1 kinases play essential roles in cell cycle checkpoints and DNA damage response. Genetic interaction has been documented between WEE1 and PKMYT1, and clinical trials have reported clinical activity with WEE1 inhibitors adavosertib (adavo), azenosertib (azeno) and the PKMYT1 inhibitor lunresertib (lunres). Here, we report the discovery and characterization of ACR-2316, a dual inhibitor of WEE1 and PKMYT1 specifically designed for optimal selectivity through co-crystallography and superior single agent activity uniquely enabled by Acrivon Predictive Precision Proteomics (AP3).

Mass spectrometry-based AP3 profiling was conducted across several novel WEE1 and PKMYT1 inhibitor leads generated through co-crystallography-based rational drug design. Selective leads originating from one series were optimized using AP3 for biological structure-activity relationship analysis. AP3 profiling revealed WEE1 inhibitor-upregulated phosphorylation sites across a subset of phosphoproteins that were oppositely regulated by PKMYT1 inhibition, including CDK1-T14, a direct PKMYT1 phosphorylation site, as well as CHK1-S296. A particular lead compound, ACR-2316, demonstrated a desirable potent, balanced ratio of cellular WEE1 and PKMYT1 target engagement (95% inhibition of pCDK1/2-Y15 and 14% inhibition of pCDK1-T14 at 30 nM in ACHN cells), resulting in superior activation of the mitotic kinases CDK1, CDK2, and PLK1 compared to adavo and lunres, based on annotated kinase substrate relationships. ACR-2316 is more selective than adavo, azeno, and lunres based on >200 kinases profiled by AP3, and 468 kinases assessed by KINOMEscan. ACR-2316 induced a drastic S-G2/M accumulation distinct from adavo or lunres. In a panel of 20 human tumor cell lines, ACR-2316 demonstrated >5-fold greater potency in all cell lines tested (CellTiterGlo) compared to azeno and lunres (mean IC50 = 70, 414 and ≥496 nM, respectively). Superior anti-cancer activity of ACR-2316 was observed in 16 ovarian cancer patient-derived xenograft (PDX) models tested ex vivo (CellTiter-Glo 3D) compared to azeno and lunres (median IC50 = 11, 264 and 2260 nM, respectively). ACR-2316 demonstrated superior, durable, dose-dependent efficacy compared to azeno and lunres in human cell line derived xenograft and PDX models and was well tolerated. Tumor regression with ACR-2316 was associated with strong WEE1 (>85%) and intermediate PKMYT1 (30%) inhibition in tumors at 60 mg/kg. Additionally, ACR-2316 resulted in rapid and deep regression in xenograft tumors progressing on azeno.

In conclusion, ACR-2316 is a potentially first-in-class, selective dual WEE1/PKMYT1 inhibitor with superior preclinical single-agent activity compared to clinical benchmark WEE1 or PKMYT1 inhibitors. ACR-2316 is progressing through IND-enabling studies in preparation for clinical monotherapy development.