Discovery of YJZ5118: A Potent and Highly Selective Irreversible CDK12/13 Inhibitor with Synergistic Effects in Combination with Akt Inhibition

Abstract

Cyclin-dependent kinases 12 and 13 (CDK12/13) have emerged as promising therapeutic targets for castration-resistant prostate cancer (CRPC) and other human cancers. Despite the development of several CDK12/13 inhibitors, challenges remain in achieving an optimal balance of potency, selectivity and pharmacokinetic properties. Here, we report the discovery of YJZ5118, a novel, potent and highly selective covalent inhibitor of CDK12/13 with reasonable pharmacokinetic profiles. YJZ5118 effectively inhibited CDK12 and CDK13 with IC₅₀ values of 39.5 and 26.4 nM, respectively, while demonstrating high selectivity over other CDKs. Mass spectrometry analysis, cocrystal structure determination, and pulldown-proteomic experiments confirmed the compound's covalent binding mode with CDK12/13. Functionally, YJZ5118 efficiently suppressed the transcription of DNA damage response genes, induced DNA damage, and triggered apoptosis. Moreover, the compound significantly inhibited the proliferation of multiple tumor cell lines, particularly prostate cancer cells. Notably, YJZ5118 exhibited synergistic effects with Akt inhibitors both in vitro and in vivo.

Figure 1.

Representative compounds suppressing/degrading CDK12/13 kinases.

Figure 2.

Structure-based design of new irreversible CDK12/13 inhibitors 11. Chemical structures and predicted binding modes of compounds 2 and 14a with CDK12 (PDB: 5ACB) were shown. Hydrogen bonds were indicated by yellow dashed lines.

Figure 3.

(A) PK profiles of compound YJZ5118 in mice; (B) Kinase inhibitory IC₅₀ values of compounds 2 and YJZ5118 against CDK family members.

Figure 4.

(A) Mass spectral peaks corresponding to CDK12 and CDK12 covalently labeled with compound YJZ5118; (B) Co-crystal structure of CDK12/CCNK and compound YJZ5118 (PDB: 9JK1). Compound YJZ5118 is shown in cyan stick structure. The key residues of CDK12 kinase are shown in gray sticks. Hydrogen bonds to key amino acids are indicated by yellow dashed lines; (C) Peptide to spectrum matches (PSM) proteomic analysis of pull-down experiments in whole cell lysate, and the structure of compound YJZ9149 (biotinylated compound of YJZ5118); (D, E) Washout experiments using compound YJZ5118 and reversible inhibitors 10 and 2 in VCaP cells.

Figure 5.

(A) Immunoblots of pSer2 of RNA polymerase II CTD and cleaved PARP in VCaP cells treated with compound YJZ5118 or 3 at selected concentrations and time points. α-Tubulin is used as a loading control; (B) Scatter plot showing Log2 fold changes in gene expression vs. gene length in Log2 scale for each protein-coding gene in VCaP cells (p < 2.2e−16, F-test). Differentially expressed genes are indicated (FDR < 0.1 and Log2 FC > 1); (C) Analysis of indicated gene expression by qPCR at selected time points with 100 nM of compound YJZ5118 in VCaP cells. Data are presented as mean values ± SD of triplicate points. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by t test; (D) Left: Representative images from comet assay of VCaP cells after 12 h of treatment with vehicle or compound YJZ5118 (100 nM) stained with propidium iodide. Scale bar represents 50 μm; Right: Tail moments obtained from comet assay of VCaP cells after treatment with vehicle or compound YJZ5118. Horizontal bars denote the median. For each condition, 50 cells were analyzed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by t test; (E) The apoptosis rate of VCaP cells was analyzed by flow cytometry using Annexin V/PI staining; (F) IC₅₀ values of compound YJZ5118 in a panel of human-derived cancer or normal cell lines after 5 days of treatment.

Figure 6.

(A) Immunoblots of the indicated proteins at selected concentrations in VCaP cells after treated with compound YJZ5118 for 24 h. α-Tubulin is used as a loading control; (B) 22RV1 cells were treated with compounds YJZ5118 and/or MK2206 at varied concentrations to determine the effect on cell growth and drug synergism, with assessments using the Loewe method. Red peaks in the 3D plots denote synergy with the average synergy scores noted above; (C) Real-time growth curves of 22RV1 cells upon treatment with compound YJZ5118 and/or Akt inhibitors. Data are presented as mean +/− SD (n = 3) from one of three independent experiments.

Figure 7.

(A) Tumor volume (measured twice weekly using calipers) measurements of treated with compound YJZ5118 (i.p., q.d.), uprosertib (p.o., 5x/week) or combination; (B) Waterfall plot depicting the change in tumor volume; (C) Percentage of mouse body weight throughout the treatment period. Data are presented as mean ± SEM; (D) Expression of indicated genes (Real-Time quantitative PCR assay) in tumors after the treatment of compound YJZ5118 for 27 days in a VCaP CRPC model. Data are presented as mean values ± SD of triplicate points. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, by t test; (E) Immunoblots of the noted proteins from tumors after 27 days of treatment with compound YJZ5118. α-Tubulin is the loading control; (F) Representative H&E and pAKT staining for tumors after 27 days of treatment.

Scheme 1.

Synthesis of Compounds 14a-m and YJZ9149^{a a}Reagents and conditions: (a) 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos), tris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃), tert-BuONa, toluene, 100 °C, 12 h, 82%; (b) N,N-diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), 95 °C, 4 h, 79%; (c) trifluoroacetic acid (TFA), dichloromethane (DCM), 50 °C, reflux, 3 h, 60–77%; (d) Cs₂CO₃, DMF, 60 °C, 1 h, 80%; (e) 10% Pd-C/H₂, MeOH, room temperature (rt), 2 h; (f) DCM, DIPEA, acryloyl chloride, 0 °C, 35–54% (two steps); (g) Xantphos, Pd₂(dba)₃, tert-BuONa, toluene, DMF, 100 °C, 40–64%; (h) 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one, K₂CO₃, tetrakis(triphenylphosphine)palladium (Pd(PPh₃)₄), 1,4-dioxane/H₂O (10:1), 100 °C, 10 h, 52%; (i) Fe, conc. HCl (aq), 70 °C, 2 h; (j) 2-(7-Azabenzotriazol-1-yl)-N,’,N’,N’-tetramethyluronium hexafluorophosphate (HATU), 17-oxo-21-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azahenicosanoic acid, DCM, triethylamine (Et₃N), rt, 20 min, 67%.