PROTAC D16-M1P2 is an orally active CRBN -mediated selective PKMYT1 PROTAC degrader, with a DC50 of 0.7 nM in CCNE1-amplified HCC1569 breast cancer cells. PROTAC D16-M1P2 induces PKMYT1 degradation via the ubiquitin-proteasome system, directly inhibits PKMYT1 kinase activity, suppresses Thr14 phosphorylation of CDK1, and depends on functional CRBN and ubiquitination-like modification processes. PROTAC D16-M1P2 can be used for the research of CCNE1 -amplified breast cancer, FBXW7 -mutated cholangiocarcinoma, and PPP2R1A -deficient cancers [1] [2] [3]. (Pink: PKMYT1 ligand; Blue: Cereblon E3 ligase ligand; Black: linker).
In Vivo, PROTAC D16-M1P2 (40-120 mg/kg; p.o.; twice daily; 21 days) exhibits dose-dependent antitumor efficacy in CCNE1-amplified HCC1569 xenografts, achieving 35.1% TGI at 40 mg/kg and 66.4% TGI at 120 mg/kg, with corresponding dose-dependent PKMYT1 degradation and pCDK1T14 inhibition[1].
In Vitro, PROTAC D16-M1P2 (100 μM (3-fold serial dilutions, 10 gradients); 15 min (compound + PKMYT1); 60 min (tracer addition); 60 min (GST-Tb addition)) potently binds purified recombinant human PKMYT1 with an IC50 range of 4.1-5.0 nM in a competitive HTRF binding assay[1]. PROTAC D16-M1P2 inhibits purified recombinant human PKMYT1 enzymatic activity with an IC50 of 7.6 nM in an ADP-Glo kinase assay[1]. PROTAC D16-M1P2 (10 min (compound + kinase); 60 min (kinase reaction with substrate and ATP)) shows over 50-fold selectivity for purified recombinant human PKMYT1 over BRAF, RAF1, and SRC, with IC50 values of 485.3 nM, 373.1 nM, and 398.5 nM respectively[1]. PROTAC D16-M1P2 (30 min (compound + cells); 2 h (tracer addition)) engages intracellular PKMYT1 in transiently transfected HEK293 cells with an EC50 of 12.0 nM in a NanoBRET assay[1]. PROTAC D16-M1P2 (1 µs) (in both R- and S-oxa-azaspirodecane linker configurations) selectively accommodates the PKMYT1-CRBN-M4 conformation, forming moderately stable ternary complexes with docking scores of -10.85 kcal/mol (R) and -10.70 kcal/mol (S) that maintain sustained ubiquitination accessibility over 1 µs MD simulations[3]. PROTAC D16-M1P2 (100 ns) has linker length distributions compatible with the productive PKMYT1-CRBN-M4 conformation, consistent with its potent PKMYT1 degradation activity (DC50 = 0.70 nM)[3]. PROTAC D16-M1P2 (0.01-1000 nM; 24 h) induces potent, dose-dependent PKMYT1 degradation (DC50 0.7 nM, Dmax 90%) and pCDK1 (Thr14) inhibition (IC50 9.0 nM) in CCNE1-amplified HCC1569 breast cancer cells after 24 h treatment[1]. PROTAC D16-M1P2 (1 nM; 100 nM; 2, 4, 8, 24 h) induces rapid, time-dependent PKMYT1 degradation and pCDK1(Thr14) inhibition in CCNE1-amplified HCC1569 breast cancer cells, with 100 nM treatment producing robust pathway suppression while 1 nM treatment only induces partial degradation without significant signaling inhibition[1]. PROTAC D16-M1P2 (100 nM; 24 h pretreatment; 0, 2, 4, 16, 24 h post-washout incubation) induces durable PKMYT1 degradation and pCDK1Thr14 inhibition in CCNE1-amplified HCC1569 breast cancer cells, with effects sustained for at least 24 h after compound washout[1]. PROTAC D16-M1P2 (0.1 μM; 24 h) mediates PKMYT1 degradation in CCNE1-amplified HCC1569 breast cancer cells via a ubiquitin-proteasome system-dependent mechanism requiring both PKMYT1 and CRBN binding[1]. PROTAC D16-M1P2 (10 nM; 100 nM; 8 h) exhibits high proteome-wide selectivity for PKMYT1 in CCNE1-amplified HCC1569 breast cancer cells, with PKMYT1 being the only significantly degraded protein after 8 h treatment at 10 nM or 100 nM[1]. PROTAC D16-M1P2 (7 days) inhibits proliferation of human tumor cell lines with CCNE1 amplification, FBXW7 mutation, or PPP2R1A loss with 12-fold selectivity over wild-type cell lines, with an average IC50 of 177 nM in biomarker-positive cells after 7 days of treatment[1].
References:
[1]. Wang Y, et al. Discovery of a bifunctional PKMYT1-targeting PROTAC empowered by AI-generation. Nature communications. 2025 Nov 28;16(1):10759.
[2]. Li L, et al. PKMYT1 in Cancer: Beyond Cell Cycle Checkpoints to Context-Dependent Therapeutic Vulnerability. Genes, chromosomes & cancer. 2026 Jul;65(7):e70151.
[3]. Nassar H, et al. Computational mapping of productive POI-E3 ligase conformations to guide de novo degrader design: application to WEE1 and PKMYT1 PROTACs. Journal of cheminformatics. 2026 Jul 24;18(1):103.
In Vivo, PROTAC D16-M1P2 (40-120 mg/kg; p.o.; twice daily; 21 days) exhibits dose-dependent antitumor efficacy in CCNE1-amplified HCC1569 xenografts, achieving 35.1% TGI at 40 mg/kg and 66.4% TGI at 120 mg/kg, with corresponding dose-dependent PKMYT1 degradation and pCDK1T14 inhibition[1].
In Vitro, PROTAC D16-M1P2 (100 μM (3-fold serial dilutions, 10 gradients); 15 min (compound + PKMYT1); 60 min (tracer addition); 60 min (GST-Tb addition)) potently binds purified recombinant human PKMYT1 with an IC50 range of 4.1-5.0 nM in a competitive HTRF binding assay[1]. PROTAC D16-M1P2 inhibits purified recombinant human PKMYT1 enzymatic activity with an IC50 of 7.6 nM in an ADP-Glo kinase assay[1]. PROTAC D16-M1P2 (10 min (compound + kinase); 60 min (kinase reaction with substrate and ATP)) shows over 50-fold selectivity for purified recombinant human PKMYT1 over BRAF, RAF1, and SRC, with IC50 values of 485.3 nM, 373.1 nM, and 398.5 nM respectively[1]. PROTAC D16-M1P2 (30 min (compound + cells); 2 h (tracer addition)) engages intracellular PKMYT1 in transiently transfected HEK293 cells with an EC50 of 12.0 nM in a NanoBRET assay[1]. PROTAC D16-M1P2 (1 µs) (in both R- and S-oxa-azaspirodecane linker configurations) selectively accommodates the PKMYT1-CRBN-M4 conformation, forming moderately stable ternary complexes with docking scores of -10.85 kcal/mol (R) and -10.70 kcal/mol (S) that maintain sustained ubiquitination accessibility over 1 µs MD simulations[3]. PROTAC D16-M1P2 (100 ns) has linker length distributions compatible with the productive PKMYT1-CRBN-M4 conformation, consistent with its potent PKMYT1 degradation activity (DC50 = 0.70 nM)[3]. PROTAC D16-M1P2 (0.01-1000 nM; 24 h) induces potent, dose-dependent PKMYT1 degradation (DC50 0.7 nM, Dmax 90%) and pCDK1 (Thr14) inhibition (IC50 9.0 nM) in CCNE1-amplified HCC1569 breast cancer cells after 24 h treatment[1]. PROTAC D16-M1P2 (1 nM; 100 nM; 2, 4, 8, 24 h) induces rapid, time-dependent PKMYT1 degradation and pCDK1(Thr14) inhibition in CCNE1-amplified HCC1569 breast cancer cells, with 100 nM treatment producing robust pathway suppression while 1 nM treatment only induces partial degradation without significant signaling inhibition[1]. PROTAC D16-M1P2 (100 nM; 24 h pretreatment; 0, 2, 4, 16, 24 h post-washout incubation) induces durable PKMYT1 degradation and pCDK1Thr14 inhibition in CCNE1-amplified HCC1569 breast cancer cells, with effects sustained for at least 24 h after compound washout[1]. PROTAC D16-M1P2 (0.1 μM; 24 h) mediates PKMYT1 degradation in CCNE1-amplified HCC1569 breast cancer cells via a ubiquitin-proteasome system-dependent mechanism requiring both PKMYT1 and CRBN binding[1]. PROTAC D16-M1P2 (10 nM; 100 nM; 8 h) exhibits high proteome-wide selectivity for PKMYT1 in CCNE1-amplified HCC1569 breast cancer cells, with PKMYT1 being the only significantly degraded protein after 8 h treatment at 10 nM or 100 nM[1]. PROTAC D16-M1P2 (7 days) inhibits proliferation of human tumor cell lines with CCNE1 amplification, FBXW7 mutation, or PPP2R1A loss with 12-fold selectivity over wild-type cell lines, with an average IC50 of 177 nM in biomarker-positive cells after 7 days of treatment[1].
References:
[1]. Wang Y, et al. Discovery of a bifunctional PKMYT1-targeting PROTAC empowered by AI-generation. Nature communications. 2025 Nov 28;16(1):10759.
[2]. Li L, et al. PKMYT1 in Cancer: Beyond Cell Cycle Checkpoints to Context-Dependent Therapeutic Vulnerability. Genes, chromosomes & cancer. 2026 Jul;65(7):e70151.
[3]. Nassar H, et al. Computational mapping of productive POI-E3 ligase conformations to guide de novo degrader design: application to WEE1 and PKMYT1 PROTACs. Journal of cheminformatics. 2026 Jul 24;18(1):103.