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 (IC 50 = 22 nM, K D = 40 nM, K I = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC 50 ∼ 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.

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers

“…Our initial screens with WRN helicase assays (DNA unwinding, ATPase) identified only covalent hits, as reported for other WRN hit-finding campaigns 23 – 25 . We characterized them as allosteric, ATP-competitive inhibitors targeting Cys727.…”

Heteroaryl 2‐Sulfonamide Synthesis by SN2 Reaction–Oxidation Cascade

“…Inclusion of free alcohol in the substrate ( 6) was also tolerated in the reaction. Other 2-heteroaromatics, including pyrimidines with electron-donating functionalities (7 and 8), pyridines with various substituents (9)(10)(11), and benzothiazole (12) all served as effective substrates furnishing products in good yields. Next, the investigation extended to more challenging substrates such as secondary amines and anilines.…”

“…[8] In 2022, H3 Biomedicine discovered a series of pyrimidine sulfonamides as potential inhibitors for Werner syndrome helicase (WRN) by multiplexed high-throughput screening and finally identified H3B-968 as the most potent hit (Scheme 3). [9] However, rapid and large-scale synthesis of H3B-968 and other pyrimidine sulfonamide analogues are found to be challenging. Reported approaches including those for accessing general aryl sulfonamides [3,4] and heteroaryl 2-sulfonamides [7] are not productive in synthesis of H3B-968, resulting in generally less than 10 % yield.…”

James German and the Quest to Understand Human RECQ Helicase Deficiencies

“…Shortly after these Pan-Cancer Atlas results were published, several groups identified a synthetic lethal interaction between WRN loss and mismatch repair deficiency in human epithelial cancers [57][58][59] [reviewed in Chan 2022 monograph]. This observation led to a flurry of work to identify potent, specific WRN inhibitors in addition to the original small molecule inhibitors identified by Bob Brosh and colleagues [60,61]. Subsequent work has provided some mechanistic insight by identifying a role of WRN in resolving large palindromic AT-rich DNA sequences [62][63][64].…”