Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay

Abstract

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC₅₀ = 22 nM, K_D = 40 nM, K_I = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC₅₀ ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

Figure 1

Summary of full-length WRN high-throughput screening campaign using a catch-all multiplexed assay to measure ATPase, helicase, and exonuclease activities from the same well. (A) Helicase (top) and exonuclease (bottom) readout Z′-factors across all plates from the primary screen. (B) Screening funnel utilized to filter primary hits and determine hit selectivity for FL-WRN versus the individual helicase and exonuclease domains.

Figure 2

Evidence for covalent modification of WRN by H3B-859. (A)  Chemical structure for starting lead H3B-859. The pyrimidine ring is numbered according to convention. (B) Real-time DNA unwinding progress curves with 0.6 nM WRN helicase domain, 0.5 μM Hel-10bp, 5 μM Trap-10bp, 120 μM ATP, and the indicated concentrations of H3B-859. (C) Intact mass analysis of WRN helicase domain (expected MW = 51026.6 Da) with DMSO. (D) Intact mass analysis after overnight treatment with a 10-fold excess of H3B-859. Alkylation by H3B-859 is expected to shift the mass by 253 Da. Masses corresponding to unmodified, monoalkylated, and dialkylated proteins are indicated in the spectrum as 0×, 1×, and 2×, respectively.  (E) Proposed mechanism of inhibition by 2-sulfonylpyrimidine. A cysteine from WRN undergoes nucleophilic aromatic substitution to form the indicated adduct with departure of the sulfinic acid leaving group.

Figure 3

Evidence for covalent modification of WRN by 2-sulfonamide pyrimidines. The top panels show real-time DNA unwinding progress curves with varying concentrations of H3B-219 (A) and H3B-960 (B). Assays contained 0.6 nM WRN helicase domain, 0.5 μM Hel-10bp, 5 μM Trap-10bp, 120 μM ATP, and the indicated compound concentrations. The bottom panels show the intact mass spectra of WRN helicase domain (expected MW = 50939.5 Da) treated overnight with 10-fold excess of H3B-219 (C) or H3B-960 (D). Alkylation by H3B-219 and H3B-960 is expected to shift the mass by 253 or 307 Da adduct, respectively. For H3B-960, the indicated masses at 1× and 2× correspond to the mono- and dialkylated protein.

Figure 4

Binding and selectivity data for 2-sulfonyl/sulfonamide pyrimidines. (A) Evidence for binding of H3B-960 to WRN helicase domain by isothermal titration calorimetry. Selectivity of H3B-859 (B) and H3B-219 (C) for WRN (black) versus BLM (red) and RecQL1 helicase domain (blue). Eleven-point dose responses were evaluated with the ADP-Glo assay as described in the Experimental Section. WRN IC₅₀ values were 865 and 171 nM for H3B-859 and H3B-219, respectively, whereas IC₅₀ values for BLM and RecQL1 helicase domain were greater than 50 μM.

Figure 5

Covalent inactivation of WRN helicase domain. (A) Proposed two-step mechanism where the initial E–I* complex is a reversible binding event (K_I) prior to the inactivation step that forms the covalent E–I complex (k_inact). The compounds H3B-859 (B), H3B-219 (C), and H3B-960 (D) follow this two-step mechanism. Real-time DNA unwinding assays were run with 0.5 nM WRN helicase domain, 120 μM ATP, 500 nM Hel-10bp, and 5 μM Trap-10bp.

Figure 6

Quantification of the abundance of WRN peptides containing C727 in DMSO controls versus samples treated with a 10-fold excess of H3B-219 (left) or H3B-960 (right) reveals significant depletion of the peptides treated with compound under multiple proteolytic conditions. Total protein quantification (representative example for samples treated with chymotrypsin shown) indicated that the depletion was not due to differences in sample concentration, suggesting that peptides containing C727 become alkylated upon compound treatment.

Figure 7

Location of proposed site of modification, C727, relative to ADP and the Walker motif K577 in the WRN crystal structure (PDB 6YHR). Covalent modification of the C727 allosteric site could impact the conformation of key residues involved in ATP binding and hydrolysis.