Identification of inhibitors of Tartrate-resistant acid phosphatase (TRAP/ACP5) activity by small molecule screening

Abstract

Tartrate-resistant acid phosphatase (TRAP/ACP5) occurs as two isoforms- TRAP 5a with low enzymatic activity due to a loop interacting with the active site and the more active TRAP isoform 5b generated upon proteolytic cleavage of this loop. TRAP has been implicated in several diseases, including cancer. Thus this study set out identifying small molecule inhibitors of TRAP activity. A microplate-based enzymatic assay for TRAP 5b was applied in a screen of 30,315 compounds, resulting in the identification of 90 primary hits. After removal of promiscuous compounds, unwanted groups and false-positives by orthogonal assays and three-concentration validation, the properties of 52 compounds were further investigated to better understand their mechanism of action. Full concentration-response curves for these compounds were established under different enzyme concentrations and (pre-) incubation times to remove compounds with inconsistent results and low potencies. Full concentration-response curves were also performed for both isoforms, to examine isoform prevalence. Filtering led to six prioritized compounds, representing different clusters. One of these, CBK289001 or (6S)-6-[3-(2H-1,3-benzodioxol-5-yl)-1,2,4-oxadiazol-5-yl]-N-(propan-2-yl)-1H,4H,5H,6H,7H-imidazo[4,5-c]pyridine-5-carboxamide demonstrated efficacy in a migration assay and IC₅₀ values from 4-125 μM. Molecular docking studies and analog testing were performed around CBK289001 to provide openings for further improvement towards more potent blockers of TRAP activity.

This article is protected by copyright. All rights reserved.

TRAP has been implicated in several diseases, including cancer and well-characterized inhibitors are majorly missing. In this study isoform-specific inhibitors for TRAP enzyme activity were identified by small molecule screening of a library containing drug-like compounds. Filtered by orthogonal validation and liability assessments, full-concentration responses and isoform selectivity were assessed for a selection of hit compounds. Six potential lead structures were characterized for molecular docking modes. The compound CBK289001 rendered valid as inhibiting TRAP-dependent migration in a cell system.

http://ift.tt/2FS1yH7