POWER-QC – assessment of covalent enantiomer libraries for fragment-based drug design by harnessing the power of proteome-wide, site-specific chemoproteomics

Frontier"s drug discovery engine combines chemoproteomics, covalent fragment-based approaches, and AI and allows us to target >90% of the proteome. Screening fragments has emerged as a powerful approach in drug discovery, especially when utilizing a covalent strategy, in which a covalent bond can irreversibly capture an otherwise weak ligand. Frontier"s covalent fragment library is custom-built and leverages the concept of enantiomers or "enantiopairs", molecules with identical atomic composition but spatially arranged as non-superimposable mirror images of each other. Because the chemical reactivity of enantiomers is identical, discovery of enantiomer-specific (ES) hits provides immediate structure-activity relationships (SAR). Careful quality control (QC) of enantiomer libraries is crucial to minimize identification of false-positive ES hits, e.g. if one fragment has lost activity due to synthetic impurities or degradation. However, compound QC would not capture downstream problems such as sample preparation or enantio-selective interactions with efflux transporters. Here we introduce ProteOme-Wide Enantio-Ratio QC (POWER-QC) to complement standard compound QC by leveraging high-throughput mass spectrometry-based chemoproteomics. We apply POWER-QC to a subset of our covalent enantiomer library, assess its ability to flag compounds with potential QC problems, and demonstrate its utility to differentiate between true and false positive ES hits.

Proteome-wide mapping of site-level protein-compound engagements was accomplished by an optimized streamlined cysteine activity-based protein profiling (SLC-ABPP) protocol utilizing TMT-based quantification of DMSO/compound competition ratios (CR). Selectivity and reactivity scores were calculated from proteome-wide chemoproteomics data as hit ratios and as 95th percentiles of the CR distribution. Derived scores should be similar for the two members of an enantiopair, and metrics to quantify deviations were assessed. The significance of the extent of deviations between enantiopair members was determined empirically using known QC-failed examples.

We calculated proteome-wide scores for each compound which exhibited the expected distributions across different warhead classes, and which correlated well with estimated promiscuity scores derived from molecular physical properties of the compounds. 95% of a subset of our enantiomer fragment library passed POWER-QC, suggesting most enantiopairs showed concordant selectivity and reactivity. Comparison to compound purity assessments revealed only partial agreement, suggesting POWER-QC as a complementary QC method. We discovered several examples of ES hits to active sites of enzymes passing POWER-QC, providing promising starting points to modulate the catalytic activity of these enzymes.

POWER-QC is a valuable addition to Frontier"s toolbox and is routinely used in our hit triage process for cellular screening campaigns and orthogonal biochemical assays.