Benjamin C. Whitehurst , Niall A. Anderson , Argyrides Argyrou , Peter Astles , Bernard Barlaam , Elaine B. Cadogan , Luca Carlino , Gavin W. Collie , Alex Edwards , Linda Kitching , Yaqin Li , Alexander G. Milbradt , Jenni Nikkilä , Sarah Northall , Sara Pahlén , Saleha Patel , Wendy Savory , Markus Schade , Jonathan A. Spencer , Darren Stead , Christopher J. Stubbs , Aquan Wang , Wenxin Wang

ACS Medicinal Chemistry Letters

DOI: 10.1021/acsmedchemlett.5c00651

Abstract

DNPH1 is a hydrolase enzyme that degrades the noncanonical nucleotide 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate (hmdUMP), thus acting as a nucleotide pool sanitizer by preventing its aberrant incorporation into DNA. Recent studies have shown that loss of DNPH1 enhances the sensitivity of homologous recombination repair-deficient cancer cells to PARP inhibitors, highlighting its potential as an attractive therapeutic target. Herein we report the design and prosecution of an integrated hit finding strategy combining high-throughput screening, DNA-encoded library screening, and fragment-based lead generation which enabled the discovery of the first non-nucleotide ligands for DNPH1. We compare four hit compounds which differ markedly in their chemical structures, physicochemical properties, and binding modes and summarize parallel hit-to-lead workup efforts. We also provide discussion of the merits of an integrated approach for hit discovery when applied to challenging novel targets such as DNPH1.

Summary

DNPH1 is a nucleotide-pool sanitizing hydrolase whose deletion selectively sensitizes homologous-recombination-deficient tumors to PARP inhibitors. To enable small-molecule validation of this synthetic-lethal target, AstraZeneca executed a fully integrated hit-finding program that combined high-throughput screening (HTS, 1.8 M compounds), DNA-encoded library (DEL, 7.1 billion compounds) affinity selection and fragment-based lead generation (FBLG). The campaign delivered four structurally distinct, non-nucleotide chemotypes—thiadiazine, imidazole, triazole and tetrahydro-isoquinoline (THIQ)—that were biophysically validated (IC₅₀ 2–24 µM; SPR Kd 2–9 µM) and structurally characterized by X-ray crystallography. Subsequent parallel optimization showed that only the thiadiazine series could be advanced to low-nM, cell-permeable inhibitors (e.g. compound 10: IC₅₀ 0.5 nM, cellular TE IC₅₀ 61 nM) and to potent PROTAC degraders (e.g. 11: DC₅₀ 28 nM). DEL-derived triazole ligands also furnished early PROTACs (e.g. 13) that achieved >90 % DNPH1 degradation before the more drug-like quinazoline/quinoline series became available. Imidazole and THIQ cores could not be driven below ~1 µM potency, illustrating the necessity of an acidic anchor for high-affinity binding and the penalty of stabilizing a folded bioactive conformation.

Highlights

• First non-nucleotide ligands for DNPH1 discovered through a tri-platform approach (HTS + DEL + FBLG).
• Four validated chemotypes reveal divergent binding modes within a flexible, dimeric catalytic site.
• Thiadiazine → quinazoline core hop overcame permeability hurdles, yielding nM cell-active inhibitors and efficient PROTACs.
• DEL screen accelerated biology by enabling direct-to-biology PROTAC synthesis before lead-optimization completion.
• Structural and SAR data demonstrate that strong engagement of the phosphate-binding pocket (charged H-bond) is critical for sub-µM potency.
• Integrated screening maximized chemical coverage and mitigated single-technology failure (FBLG produced no confirmed hits).

Conclusion

By concurrently deploying HTS, DEL and FBLG, the team rapidly generated a diversified hit collection against the previously ligand-naïve target DNPH1. Crystal structures illuminated both opportunities and limitations: loop plasticity and the requirement for polar anchoring complicated optimization of neutral scaffolds, whereas acid-bearing thiadiazines were successfully morphed into quinazoline/quinoline analogues with single-digit nM enzymatic potency, robust cellular activity and efficient target degradation. A DEL-derived triazole further enabled early PROTAC proof-of-concept, underscoring the strategic value of exploiting the DNA-conjugation vector. Overall, the work delivers chemical tools that confirm DNPH1 as a druggable node in DNA-damage response pathways and exemplifies how an integrated discovery engine can de-risk and accelerate prosecution of challenging, novel targets within industrial timelines.