α‑Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD) Inhibitors as Novel Modulators of De Novo Nicotinamide Adenine Dinucleotide (NAD⁺) Biosynthesis

NAD⁺ has a central function in linking cellular metabolism to major cell-signaling and gene-regulation pathways. Defects in NAD⁺ homeostasis underpin a wide range of diseases, including cancer, metabolic disorders, and aging. Although the beneficial effects of boosting NAD⁺ on mitochondrial fitness, metabolism, and lifespan are well established, to date, no therapeutic enhancers of de novo NAD⁺ biosynthesis have been reported. Herein we report the discovery of 3-[[[5-cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]­thio]­methyl]­phenylacetic acid (TES-1025, <b>22</b>), the first potent and selective inhibitor of human ACMSD (IC₅₀ = 0.013 μM) that increases NAD⁺ levels in cellular systems. The results of physicochemical-property, ADME, and safety profiling, coupled with in vivo target-engagement studies, support the hypothesis that ACMSD inhibition increases de novo NAD⁺ biosynthesis and position <b>22</b> as a first-class molecule for the evaluation of the therapeutic potential of ACMSD inhibition in treating disorders with perturbed NAD⁺ supply or homeostasis

Hyper-acetylation by SIRT1 stabilizes SPDEF and triggers Paneth and goblet cells maturation.

<p><b>A,</b> SPDEF target genes (<i>Slug, uPA, Ccl6</i>) are induced in <i>Sirt1^int−/−</i> intestines. GIF1 (<i>Nrg3, Pax6, ChgA</i>) and SOX9 (<i>Igfbp4</i>) target genes, as well as <i>Spdef</i> are not changed. <b>B,</b> In vitro acetylation/deacetylation assays demonstrates that p300 acetylates SPDEF and SIRT1, but not SIRT6 and SIRT7, deacetylates SPDEF. <b>C</b>, Nano-LC-MS/MS shows SIRT1-dependent in vitro deacetylation of AcK294 (left panel). Sequence alignment showing the evolutionary conserved K294 residue (right panel). <b>D,</b> SIRT1, but not SIRT1G261A, deacetylates SPDEF in HEK293 immunoprecipitates. The SPDEFK294Q mutant is not acetylated. Tubulin was used as loading control. <b>E–F</b> SPDEF, SPDEFK294Q, and SIRT1 were transfected in the HEK293 cell line and visualized by immunoblotting before (0 min) and after cycloheximide (CHX) treatment. HSP90 is used as loading control (left panels in E and F). The relative stability of SPDEF or SPDEFK294Q was calculated by ImageJ (right panels in E and F). <b>G,</b> PC3 cells were co-transfected with an E-Cadherin promoter luciferase reporter construct, wild type or SPDEFK294Q in presence or absence of SIRT1. SIRT1 represses SPDEF-dependent reporter activation. The acetylated-mimic SPDEFK294Q mutant is constitutively activate and not affected by SIRT1. <b>H</b>, Immunoblotting of crypt enriched fractions from <i>Sirt1^int−/−</i> and <i>Sirt1^L2/L2</i> mice show increased SPDEF protein levels in <i>Sirt1^int−/−</i> intestines. β-Actin was used as loading control. Results are expressed as mean±SEM. *P<0.05; **P<0.01; ***P<0.001.</p

Intestinal <i>Sirt1</i> deletion impacts on the development of colorectal cancer.

<p><b>A–B,</b> Schematic representation of the AOM/DSS protocol (top left panel) and representative image of colons from <i>Sirt1^int−/−</i> and control mice after CAC induction (Bar = 200 µm) (bottom left panel). <i>Sirt1^int−/−</i> mice show significantly less tumors (right panel). <b>B</b>, Representative picture of a colon section from a <i>Sirt1^int−/−</i> and control mouse after CAC induction (left panels). Tumor size (right panel). <b>C,</b> Colon length at the time of sacrifice (AOM/DSS). <b>D,</b> Percentage of body weight change. For the AOM/DSS experiment 8 mice for each genotype were used. *P<0.05; **P<0.01; ***P<0.001. <b>E–F,</b> Principal Coordinate Analysis (PCA) of bacterial sequences from colon tissue performed using unweighted UniFrac distance matrix. <b>E</b>, <i>Sirt1^int−/−</i> colon tissue before and after AOM treatment (PERMANOVA p = 0.003, ANOSIM p = 0.016). <b>F</b>, <i>Sirt1^L2/L2</i> colon tissue with and without AOM treatment (PERMANOVA p = 0.067, ANOSIM p = 0.038). <b>G–H,</b> Most statistically significant OTUs before and after AOM in both <i>Sirt1^L2/L2</i> (<b>G</b>), and <i>Sirt1^int−/−</i> (<b>H</b>), colon tissues; *Indicates <i>Helicobacter</i> and <i>Desulfovibrio</i> (see also Table S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102495#pone.0102495.s005" target="_blank">File S1</a>). <b>I,</b> PCA of bacterial sequences from colon tissue after AOM (PERMANOVA p = 0.767, ANOSIM p = 0.167).</p