Computational Discovery of an Allosteric Pocket on APOBEC3B and the Impact of Metal Contamination on Inhibitor Validation

The APOBEC3 (A3) family of enzymes are zinc metalloenzymes that catalyze the conversion of 2'- deoxycytidine to 2'-deoxyuridine in single stranded DNA. APOBEC3B (A3B), a member of the A3 family, has emerged as a key driver of genomic instability in many cancer types by mutating host DNA to drive tumorigenesis and therapy resistance. Small molecule inhibitors of A3B would extend the durability of current therapies by limiting mutations that promote tumor escape and therapy resistance. Consequently, a computer aided drug discovery (CADD) campaign was employed to identify inhibitors of A3B. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the c-terminal domain of A3B and virtually screened the ChemBridge Diversity Set ([~]110,000 small molecules) against both the active and predicted allosteric sites. High scoring compounds were selected for in vitro testing and triage, resulting in 13 candidate inhibitors. Using cysteine reactive probes, one of the original hit compounds was mapped to the computationally identified allosteric pocket. However, after resynthesis of representative chemotypes and further investigation using in vitro assays, none of the chemotypes retained inhibitory activity. Further analysis revealed that most of the inhibition observed in the primary assay was due to metal contamination in the screening sample, which resulted in the removal of the catalytic zinc from the enzyme active site. Although validated A3B inhibitors were not discovered in this study, we report a ligandable allosteric site on A3B and several cautionary insights for researchers developing small molecule inhibitors of zinc metalloenzymes. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/591187v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b7e577org.highwire.dtl.DTLVardef@13a1a43org.highwire.dtl.DTLVardef@192309eorg.highwire.dtl.DTLVardef@b60a31_HPS_FORMAT_FIGEXP M_FIG C_FIG