Preclinical Lead Optimization of a 1,2,4-Triazole Based Tankyrase Inhibitor

Abstract

Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans-cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC₅₀ inhibition in WNT/β-catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.

Figure 1

Lead compound 1 and the building blocks defined as West (red), South (blue), East (green), and linker (pink).

Scheme 1. (a, Left) Synthesis of Targets with South and West Variations and (b, Right) Synthesis of Targets with East Variations

Scheme 2. Synthesis of 18 and 51

Figure 2

(a) Selected key compounds in the optimization campaign and respective TNKS2 and HEK293 IC₅₀ values. Mean ± SD values for three independent experiments are shown. Moieties are in color when differing from 1. (b) ADME data of key compounds. (c) Cellular efficacy and mouse po PK 5 mg/kg of the short-listed compounds.

Figure 3

Cocrystal structures of TNKS2 with inhibitors. (a) Binding mode of 105 with TNKS2 catalytic domain (PDB code 6TKN). (b) Binding mode of 13 with TNKS2 catalytic domain (PDB code 6TG4). (c) Binding mode of 10 with TNKS2 catalytic domain (PDB code 6TKM). (d) Binding mode of 88 with TNKS2 catalytic domain (PDB code 6TKS). The dashed lines in black represent hydrogen bonds, and the red spheres represent water molecules. The σ_A weighted 2F_o – F_c electron density maps around the ligands are contoured at (1.4–1.7)σ. Structures were solved with molecular replacement using the structure of TNKS2 (PDB code 5NOB) as a starting model.

Figure 4

Atropisomerism of 1 (stereochemistry arbitrarily assigned) on chiral SFC and TNKS2 and HEK293 IC₅₀ values.

Figure 5

13 can inhibit WNT/β-catenin signaling activity and act as an antiproliferative agent in COLO 320DM cells. (a) Representative immunoblots of cytoplasmic TNKS1/2, AXIN1, AXIN2 and cytoplasmic and nuclear transcriptionally active β-catenin (non-phospho) and β-catenin. Actin or lamin B1 document equals protein loading, and ∗ indicates that the same actin immunoblot is used as loading control for both AXIN2 and β-catenin. For (a) and (b), control = 0.01% DMSO. (b) Real-time RT-qPCR analyses of WNT/β-catenin signaling target genes (AXIN2, DKK1, NKD1, and APCDD1). For (b) and (c), ANOVA tests (Holm–Sidak method, versus control) are indicated by ∗∗∗ (P < 0.001), and ANOVA on ranks tests (Tukeys test versus control) are indicated by † (P < 0.05). Mean values ± standard deviations for combined data from a minimum of three independent experiments with three replicates each are shown. (c) MTS colorimetric cell growth assay for various doses of 13 in APC^mutated COLO 320DM (black) and APC^wild-type RKO (gray) cells. Control = 0. 1% DMSO. Dotted lines depict 50% (GI₅₀) and 25% (GI₂₅) growth inhibition levels. Mean values ± standard deviations for one representative experiment out of three independent experiments are shown.