From PARP1 to TNKS2 Inhibition: A Structure-Based Approach

Abstract

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/β-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone 5 as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.

Figure 1

Analogues of MC2050 investigated in the study.

Figure 2

(A) Octahedral binding of Ni²⁺ (green sphere) in PARP1, with 2.2 ± 0.1 Å coordination distances. Red spheres represent water molecules. The anomalous density is contoured at 5σ. (B) Active site of the hPARP1c/MC2050 complex. The inhibitor (magenta) is overlaid with F_o – F_c omit density contoured at 3σ. (C and D) Binding mode of MC2050 in the active site of hPARP1c. (E) Structural overlay of PARP complexes with the inhibitors olaparib (green, 5DS3), rucaparib (orange, 4RV6), niraparib (yellow, 4R6E), and MC2050 (magenta). The NAM-binding site is indicated by the red dashed circle.

Figure 3

Structural comparison between the crystal structures of the human TNKS2 ARTD catalytic domain (light gray) and MC2050 bound to the active site of PARP1 (cyan). The structural superposition underscores differences in key residues in the AD-binding site, in particular Asp770 and Arg878 in PARP1 (cyan) are replaced by Phe1188/Phe1035 and His1201/His1048 in TNKS1/2 enzymes (residue numbering refers to PDB codes 4MSG(20) and 3U9Y, respectively).

Scheme 1. Synthetic Routes to Compounds 1–6

Reagents and conditions: (a) 1,3-dibromopropane, K₂CO₃, dry CH₃CN, reflux; (b) 2-(piperazin-1-yl)ethan-1-ol, K₂CO₃, NaI, dry DMF, 60 °C; (c) 2-mercapto-4(3H)-quinazolinone, DIAD, 1 M trimethylphosphine in toluene, dry DMF, microwave, 40 °C; (d) (i) 4-bromobutanoyl chloride, TEA, dry DCM, rt, (ii) toluene, reflux; (e) tert-butyl 4-oxopiperidine-1-carboxylate, pyrrolidine, dry MeOH, reflux; (f) 4 M HCl in dioxane, dry THF; (g) bromoacetyl chloride, TEA, dry DCM, rt; (h) benzyloxycarbonyl chloride, TEA, dry THF, rt; (i) CBr₄, triphenylphosphine, dry THF, rt; (j) 2-mercapto-4(3H)-quinazolinone, K₂CO₃, NaI, dry DMF, rt; (k) 2-bromoethan-1-ol, K₂CO₃, dry CH₃CN, reflux; (l) NH₃ aq., EDCI, HOBt, 4-methylmorpholine, dry THF, rt; (m) piperidin-4-one hydrochloride, concentrated H₂SO₄, CH₃COOH, rt; (n) EDCI, HOBt, TEA, dry DMF, rt; (o) 2 M potassium hydroxide, EtOH, rt.

Figure 4

(A) Bar diagram of antiproliferative effects on DLD-1 CRC cells of 2 and 5. (B) Colony forming assay on DLD-1 cancer cells. (A) untreated cells; (B) compound 2 at 1 μM; (C) compound 2 at 10 μM; (D) compound 5 at 0.1 μM; (E) compound 5 at 1 μM; (F) IWR-1 at 1 μM.