Novel 1,4-Dihydropyridines as Specific Binders and Activators of SIRT3 Impair Cell Viability and Clonogenicity and Downregulate Hypoxia-Induced Targets in Cancer Cells

Abstract

The mitochondrial SIRT3 modulates several biological pathways such as cancer, metabolism, and hypoxia-related diseases. Recently, we discovered new 1,4-dihydropyridines, compounds 2 and 3, the latter being a SIRT3-specific activator. In the present work, a novel 2- and 3-related small series of compounds have been developed, with 3c displaying the strongest SIRT3 binding and activation, with a K D of 29 μM and 387% of enzyme activation. Differently, 3d was the best in enhancing glutamate dehydrogenase activity and deacetylating K68- and K122-acMnSOD in triple-negative MDA-MB-231 breast cancer cells. Tested in CAL-62 thyroid cancer and MDA-MB-231 cells, 3d displayed the strongest time- and dose-dependent reduction of cell viability and clonogenicity at a single-digit micromolar level, along with cell death, in both normoxia and hypoxia conditions. Moreover, 3d downregulated not only hypoxia-induced factors, such as HIF-1α, EPAS-1, and CA-IX, but also epithelial–mesenchymal transition master regulators and extracellular matrix components such as SNAIL1, ZEB1, SLUG, COL1A2, MMP2, and MMP9, markedly hampering MDA-MB-231 cell migration.

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“…Generally, the effect of the two compounds was clearly stronger in hypoxic conditions, highlighting the function of SIRT3 in the modulation of hypoxia-dependent gene expression. Of note, 22c and, to a better extent, 22d reduced the MDA-MB-231 cell migration after 48h of treatment at 50 μM …”

“…Notably, 22d shows reduced cell viability and colony formation in normoxia and hypoxia environments when tested on breast MDA-MB-231 and thyroid CAL-62 cancer cells with single-digit micromolar IC 50 values. 22d is also able to downregulate hypoxia-induced factors (HIF-1α, EPAS-1, and CA-IX) and epithelial-mesenchymal transition master regulators and extracellular matrix components (SNAIL1, ZEB1, SLUG, COL1A2, MMP2, and MMP9), other than impairing MDA-MB-231 cell migration …”

“…SIRT3 can also deacetylase the antioxidant enzyme SOD2 in the mitochondrial matrix. Studies demonstrate that SIRT3 deacetylases K53, K68, and K122 of SOD2. − Moreover, calorie restriction (CR) induces deacetylation and activation of SOD2, very likely via the upregulation of SIRT3. In particular, Qiu et al observed that SIRT3 KO mice fed with a caloric restriction diet for 6 months showed higher oxidative stress and damage than WT mice fed with the same diet.…”

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“…Compound 3D enhances the activity of glutamate dehydrogenase and reduces the deacetylation level of MnSOD by activating SIRT3 in thyroid cancer CAL-62 and triple-negative breast cancer MDA-MB-231 cells. Furthermore, compound 3D demonstrates significant anticancer effects through suppressing the proliferation and migration of cancer cells by reducing the expression of HIF-1α, endothelial PAS domain protein 1 (EPAS-1) and carbonic anhydrase IX (CA-IX), and the related proteins involved in epithelial mesenchymal transition 102 . Another SIRT3 activators developed by using a structure-based approach to small molecule drug design is compound 33c, which was derived through high-throughput screening and structural optimization of the initial compound amiodarone.…”

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“…DHP-8 was identified to be an allosteric enhancer of SIRT1 leading to the increased binding of SIRT1 to its targets and its cofactor NAD + . More recently, another 1,4-DHP, MC2789, was found to activate SIRT3 in cancer cell lines, resulting in elevated activation of manganese-dependent superoxide dismutase (MnSOD) [150]. This is especially advantageous, as MnSOD protects the mitochondria from oxidative stress and subsequent dysfunction [151].…”