Andrea Mancini

PC21 – Andrea Mancini

Department of Drug Chemistry and Technologies, Sapienza University of Rome, Italy

ORCID

an.mancini@uniroma1.it

From Fragments to Leads: Design and Synthesis of High-Affinity Sirtuin 6 Inhibitors via Structure-Guided Optimization
Andrea Mancini¹, Emanuele Fabbrizi,¹Weijie You, ² Wei Zheng, ^3,4 Francesco Fiorentino, ¹Daniela Trisciuoglio, ⁵ Francesco Spallotta, ⁶ Clemens Steegborn, ²Dante Rotili, ⁷Antonello Mai.¹ ¹ Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy ² Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany ³ Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA ⁴ Geriatric Research, Education, and Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, USA; ⁵ Institute of Molecular Biology and Pathology (IBPM-CNR), Rome, Italy ⁶ Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy; ⁷ Department of Sciences, Roma Tre University, Rome, Italy
Abstract As an important NAD⁺-dependent enzyme, SIRT6 has received significant attention since its discovery. Recent evidence has demonstrated that SIRT6 functions as a Lys deacetylase, deacylase, and mono-ADP-ribosyltransferase, participating in variety of cellular signaling pathways and regulating a wide range of physiological and pathological processes. Specifically, SIRT6 regulates the expression and activity of both pro-apoptotic (e.g., Bax) and anti-apoptotic factors (e.g., Bcl-2, survivin) in a context-dependent manner, with mounting evidence regarding its role in tumor initiation and progression. Given these key roles in various cancer types, we set out to develop novel SIRT6 inhibitors that may lay the ground for new anticancer drugs. After screening a fragment library using protein crystallography, we designed several putative hit compounds by merging the best fragments with different linkers. This allowed us to identify novel scaffolds for targeting the SIRT6 pocket; the resulting lead, compound S6023, showed an IC₅₀ = 5 µM. This compound selectively inhibits SIRT6 and is active in W138 cells, where it increases levels of the senescence markers p21 and γH2AX. To improve the potency of S6023, we developed over thirty analogues. Among these, we identified MC4637, which possesses a similar binding mode to S6023 according to its co-crystal structure with SIRT6. Notably, in the fluorogenic enzymatic FdL assay, MC4637 exhibited an IC₅₀ = 600 nM and did not significantly affect the activity of other SIRT isoforms (Sirt1-3 and Sirt5) even at the highest concentration tested (25 µM). The anticancer activity of these SIRT6 inhibitors was evaluated across a representative panel of human cancer cell lines, such as H1299, A549, and HT29. MC4637 emerged as the most potent inhibitor, displaying single digit micromolar IC₅₀ values after 48 h of treatment, consistent with its superior enzymatic potency. In pancreatic ductal adenocarcinoma (PDAC) models (BxPC-3 and Mia-PaCa-2), a dose-dependent reduction in proliferation was observed. BxPC-3 cells were particularly sensitive (1 µM), whereas Mia-PaCa-2 cells required higher concentrations (5 µM), reflecting genetic differences such as KRAS mutational status. The inactive analogue MC4692 showed no effect, confirming the specificity of MC4637.