Transition State Analogs of Human DNPH1 Reveal Two Electrophile Migration Mechanisms

DNPH1 is responsible for eliminating the epigenetically
modified
nucleotide, 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate
(hmdUMP), preventing formation of hmdUTP, a mutation-inducing nucleotide.
Loss of DNPH1 activity sensitizes PARP inhibition-resistant BRCA-deficient
cancers by causing incorporation of hmdUTP into DNA. Hydrolysis of
hmdUMP by DNPH1 proceeds through a covalent intermediate between Glu104
and 2-deoxyribose 5-phosphate, followed by hydrolysis, a reaction
cycle with two transition states. We describe synthesis and characterization
of transition state mimics for both transition states of DNPH1. Both
transition states prefer inhibitors with cationic charge at the anomeric
center and provide a foundation for inhibitor design. Ground-state
complexes show reaction coordinate nucleophiles poised 3.3–3.7
Å from the anomeric carbon while transition state analogs tighten
the reaction coordinate to place the nucleophiles 2.7–2.8 Å
from the anomeric carbon. Crystal structures of DNPH1 with transition
state analogs reveal transition states where the electrophilic ribocation
migrates between the leaving groups and attacking nucleophiles.