Comparative Structure-Based Analysis of Predicted BZD9L1 Binding Modes Across Human Sirtuins.

Sirtuins (SIRTs) are NAD+-dependent enzymes implicated in cancer and other diseases, but the high conservation of their catalytic sites complicates the development of isoform-selective inhibitors. BZD9L1 is a benzimidazole-based sirtuin inhibitor with previously reported activity against SIRT1 and SIRT2. However, its potential interactions with other human sirtuin isoforms remain incompletely characterized. Here, we applied a comparative structure-based modelling framework integrating homology modelling, molecular docking, and targeted experimental assessment to investigate plausible binding modes of BZD9L1 across human SIRT1-7. Docking predicted that BZD9L1 could occupy the ADP-ribose cofactor-binding region of all seven isoforms, with broadly conserved orientations but differences in the predicted interaction networks. Hydrogen-bonding and π-mediated contacts predominated in the selected SIRT1-3 poses, whereas hydrophobic contacts were more apparent in several selected SIRT4-7 poses. The modest differences in docking scores were interpreted qualitatively and do not establish differential binding affinities or isoform selectivity. In colorectal cancer cells, BZD9L1 treatment altered acetyl-SOD2 levels, consistent with altered SIRT3-associated deacetylation in a cellular context. In a separate cell-free enzymatic assay, no measurable SIRT5 inhibition was detected under the conditions tested. These complementary assays provided distinct, independently interpreted readouts of SIRT3-associated cellular activity and SIRT5 enzymatic activity. Collectively, this study provides testable structural hypotheses regarding BZD9L1 recognition by human sirtuins and identifies interaction features that may guide subsequent biochemical and structure-based investigations.