Identification of RP-6685, an Orally Bioavailable Compound that Inhibits the DNA Polymerase Activity of Polθ

Abstract

DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2^-/- mouse tumor xenograft model.

Figure 1:

Compound 2 is the starting point for our hit-to lead efforts

Figure 2:

DSF of Compound 9 and Compound 14 with WT Polθ.

Figure 3:. Compound 14’s MOA towards human Polθ.

Kinetic parameters were determined using a real time kinetic assay. (A) shows the inhibition of Polθ by compound 14 for concentrations of the enzyme varying from 5 to 0.5 nM. (B) shows dNTP dependency for the enzyme at various concentrations of compound 14. It displays the dependency of the enzyme velocity on various inhibitor (0 to 40 nM) and dNTP concentrations.

Figure 4.. The crystal structure of compound 14 bound to an engineered construct of the polymerase domain of PolƟ.

A. Ribbon representation of the polymerase domain of Polθ bound to DNA, ddGTP, and compound 14 (PDB 8E23). B. Detailed crosseye stereo view of compound 14 bound to the fingers subdomain of PolƟ. PolƟ backbone is shown as ribbon with binding pocket side chains shown as sticks. Carbon, oxygen, nitrogen, and sulfur, atoms are coloured royal blue, red, blue, and yellow. Compound 14 is shown as sticks with carbon oxygen, and nitrogen atoms coloured cyan, red, blue.C. A LigPlot of the interaction of 14 with PolƟ. D. Surface view of compound 14 bound to the fingers subdomain PolƟ. Protein surface is coloured grey with protein and compound 14 atoms coloured as in B. E. Molecular model of the binding of compound 15 to Polθ. Flexible ligand alignment of 15 was performed on the 14 co-crystal structure, followed by energy minimization of 15 in MOE. Compound 15 is shown as sticks with orange carbon atoms and solvent-accessible surface shown as an orange mesh. Compound 14 is shown as sticks with cyan carbon atoms and solvent-accessible surface shown as a cyan mesh.

Figure 4.. The crystal structure of compound 14 bound to an engineered construct of the polymerase domain of PolƟ.

A. Ribbon representation of the polymerase domain of Polθ bound to DNA, ddGTP, and compound 14 (PDB 8E23). B. Detailed crosseye stereo view of compound 14 bound to the fingers subdomain of PolƟ. PolƟ backbone is shown as ribbon with binding pocket side chains shown as sticks. Carbon, oxygen, nitrogen, and sulfur, atoms are coloured royal blue, red, blue, and yellow. Compound 14 is shown as sticks with carbon oxygen, and nitrogen atoms coloured cyan, red, blue.C. A LigPlot of the interaction of 14 with PolƟ. D. Surface view of compound 14 bound to the fingers subdomain PolƟ. Protein surface is coloured grey with protein and compound 14 atoms coloured as in B. E. Molecular model of the binding of compound 15 to Polθ. Flexible ligand alignment of 15 was performed on the 14 co-crystal structure, followed by energy minimization of 15 in MOE. Compound 15 is shown as sticks with orange carbon atoms and solvent-accessible surface shown as an orange mesh. Compound 14 is shown as sticks with cyan carbon atoms and solvent-accessible surface shown as a cyan mesh.

Figure 5.. Compound 14 binds to a closed conformation of the fingers subdomain of PolƟ.

A. Detailed view of the fingers subdomain of Polθ bound to DNA, ddGTP, and compound 14 (PDB 8E23).. Atom colouring as in Figure 4. B. Superposition of the compound 14-Polθ co-crystal structure with the structure of Polθ in a closed fingers conformation (PDB =4X0Q) C. Superposition of the compound 14-Polθ co-crystal structure with the structure of Polθ in an open fingers conformation (PDB =6XBU)

Figure 5.. Compound 14 binds to a closed conformation of the fingers subdomain of PolƟ.

A. Detailed view of the fingers subdomain of Polθ bound to DNA, ddGTP, and compound 14 (PDB 8E23).. Atom colouring as in Figure 4. B. Superposition of the compound 14-Polθ co-crystal structure with the structure of Polθ in a closed fingers conformation (PDB =4X0Q) C. Superposition of the compound 14-Polθ co-crystal structure with the structure of Polθ in an open fingers conformation (PDB =6XBU)

Figure 5.. Compound 14 binds to a closed conformation of the fingers subdomain of PolƟ.

A. Detailed view of the fingers subdomain of Polθ bound to DNA, ddGTP, and compound 14 (PDB 8E23).. Atom colouring as in Figure 4. B. Superposition of the compound 14-Polθ co-crystal structure with the structure of Polθ in a closed fingers conformation (PDB =4X0Q) C. Superposition of the compound 14-Polθ co-crystal structure with the structure of Polθ in an open fingers conformation (PDB =6XBU)

Figure 6:. The Crystal structure of Compound 37 bound to PolƟ.

A Crosseye stereo view of compound 37 bound to PolQ (PDB 8E24). PolƟ backbone is shown as ribbon with side chains shown as sticks. Carbon, oxygen, nitrogen, and sulfur, atoms are coloured royal blue, red, blue, and yellow, respectively. Compound 37 is shown as sticks with carbon oxygen, nitrogen, and florine atoms coloured cyan, red, blue and grey, respectively. Dashed lines highlight a hydrogen bond interaction.B. A LigPlot of the interaction of 37 with PolƟ.

Figure 7:

DSB assay. A) Type of DNA repair observed at AAVS1 cut site. Non-homologous end-joining (open bars) and microhomology-mediated end-joining (filled bars). B) Inhibition of MMEJ-mediated DNA repair by RP-6685.

Figure 8:

A) TLR schematic. B) TLR assay with Ctrl siRNA and PolQ siRNA. C) Inhibition of Alt-EJ-mediated DNA repair by RP-6685.

Figure 9:. The Efficacy and Tolerability of RP-6885 in an Isogenic HCT116wt and BRCA2−/− Xenograft Model.

Mice implanted with either HCT116 or HCT116 (BRCA2 ^−/−) tumor cells were treated orally BID with Vehicle or RP-6685 at 80 mg/kg for 21 days. A, B Mean tumor volume; C, D body weight change ± SEM ( n = 8 mice per group). * Statistical significance relative to vehicle control at days 8 and 21 was established by unpaired t-test with Welch’s correction (GraphPad Prism v9). *P < 0.05

Figure 10.. The pharmacokinetics of RP-6685 in CD-1 nude mice on days 1 and 7 of oral administration in CD-1 nude mice.

RP-6685 was administered BID (at time 0 and 8h) to n=3 CD-1 nude mice at the indicated doses and whole blood sampled at various time points on Day 1 and Day 7 after time 0. RP-6685 free plasma concentrations were determined taking into consideration plasma protein binding and the blood/plasma ratio and are indicated as mean ± SEM at each time point. The AUC was calculated using WinNonLin. The dashed line indicates the IC₅₀ of RP-6685 in an Incucyte viability assay with HCT-116 (BRCA2 ^−/−) tumor cells.

Scheme 1.

(a) LDA, THF or ether, −78 °C, 30 min then ethyl trifluoroacetate 2h at rt; (b) ethyl 2-hydrazinoacetate, isopropanol or toluene, 75 °C, 2h;(c) LiOH, H₂O, 70 °C, 1h (d) N-Me aniline, DIPEA, HATU or T3P, DMF or THF rt.

Scheme 2.

(a) Hydrazine, Ethanol, 120 °C microwave, 120 °C (b) NaH (60%), Ethyl bromoacetate, DMF, 0 °C to rt, 1h; (c) LiOH, MeOH, THF, H₂O, rt 3h (d) N-Me aniline, T3P, DIPEA, THF, 70 °C, 1h.

Scheme 3.

(a) methane sulfonic acid, NaN₃, rt; (b) LiOH, MeOH, THF, H₂O, rt 3h; (c) N-Me aniline, HATU, DIPEA, DMF, rt.

Scheme 4.

(a) CrO₃, AcOH, o/w; (b) N-Me aniline, HATU, DIPEA, DMF, rt; (c) LiOH, MeOH, THF, H₂O, rt 3h; (d) NaBH₄, DCM, isopropanol, rt.

Scheme 5.

(a) ethyl 2-hydrazinoacetate, ethanol, 75 °C, 12h; (b) LiOH, MeOH, THF, H₂O, rt 3h; (c) N-Me aniline, T3P, DIPEA, THF, 80 °C, 6h.

Scheme 6.

(a) NaH, 30 min rt, DMF; (b) propargyl bromide, 2h rt; (c) 2-[2,4-bis(trifluoromethyl)phenyl]acetic acid, T3P, DIPEA, THF rt, 1h; (d) Aryl bromide, Pd(PPh₃)₄, CuI, TEA, DMF, 70 °C, 4h.

Scheme 7.

(a) 2-[2,4-bis(trifluoromethyl)phenyl]acetic acid, 4-anilinobut-2-yn-1-ol, T3P, DIPEA, THF rt, 1h; (b) 4-methylbenzenesulfonyl chloride, N,N-diethylethanamine N,N-dimethylpyridin-4-amine, DCM, 0 °C; (c) sodium azide, DMF, rt; (d) Ph₃P, THF, rt (e) carboxylic acid, DIPEA, HATU or T3P, DMF or THF rt.

Scheme 8.

(a) tert-Butyl methyl malonate, NaH, 1h, rt, THF; (b) 2-chloro-3,5-bis(trifluoromethyl)pyridine, 60 °C, o/n (c) TFA / DCM, 2h rt; (d) LiOH, Dioxane/MeOH/H₂O 80 °C, 20min; (e) Int-56, T3P, DIPEA, THF rt, 1h; (f) Aryl bromide, Pd(PPh₃)₄, CuI, TEA, DMF, 70 °C, 4h

Scheme 9:

(a) Aryl bromide, Pd(PPh₃)₄, CuI, TEA, DMF, 70 °C, 4h; (b) 62, T3P, DIPEA, THF rt, 1h; (c) DCM/TFA, rt.