Design, Synthesis, and Evaluation of Polyamine Deacetylase Inhibitors, and High-Resolution Crystal Structures of Their Complexes with Acetylpolyamine Amidohydrolase

Polyamines are essential aliphatic polycations that bind to nucleic acids and accordingly are involved in a variety of cellular processes. Polyamine function can be regulated by acetylation and deacetylation, just as histone function can be regulated by lysine acetylation and deacetylation. Acetylpolyamine amidohydrolase (APAH) from <i>Mycoplana ramosa</i> is a zinc-dependent polyamine deacetylase that shares approximately 20% amino acid sequence identity with human histone deacetylases. We now report the X-ray crystal structures of APAH–inhibitor complexes in a new and superior crystal form that diffracts to very high resolution (1.1–1.4 Å). Inhibitors include previously synthesized analogues of <i>N</i>⁸-acetylspermidine bearing trifluoromethylketone, thiol, and hydroxamate zinc-binding groups [Decroos, C., Bowman, C. M., and Christianson, D. W. (2013) <i>Bioorg. Med. Chem. 21</i>, 4530], and newly synthesized hydroxamate analogues of shorter, monoacetylated diamines, the most potent of which is the hydroxamate analogue of <i>N</i>-acetylcadaverine (IC₅₀ = 68 nM). The high-resolution crystal structures of APAH–inhibitor complexes provide key inferences about the inhibition and catalytic mechanism of zinc-dependent deacetylases. For example, the trifluoromethylketone analogue of <i>N</i>⁸-acetylspermidine binds as a tetrahedral gem-diol that mimics the tetrahedral intermediate and its flanking transition states in catalysis. Surprisingly, this compound is also a potent inhibitor of human histone deacetylase 8 with an IC₅₀ of 260 nM. Crystal structures of APAH–inhibitor complexes are determined at the highest resolution of any currently existing zinc deacetylase structure and thus represent the most accurate reference points for understanding structure–mechanism and structure–inhibition relationships in this critically important enzyme family

Synthèse et évaluation métabolique de sondes moléculaires destinées à l'Oxymétrie et l'imagerie in vivo par résonance paramagnétique électronique

La concentration en O2 est un paramètre physiologique primordial. L'oxymétrie in vivo est importante pour l'aide au diagnostic, au traitement et au pronostic dans le cas du cancer. L'oxymétrie par RPE repose sur l'utilisation de sondes radicalaires dont la largeur du signal est directement proportionnelle à la concentration en O2. Les radicaux persistants tris-(p-carboxyltétrathiaaryl)méthyle (TAM) possèdent les propriétés requises pour être utilisés comme sondes oxymétriques. Cependant, aucune donnée sur le métabolisme de ces composés utilisés in vivo n'était disponible au début de notre travail. Nous avons étudié leur réactivité et leur métabolisme in vitro. Nous avons montré que les radicaux superoxyde et alkylperoxyle oxydent ces radicaux en quinone-méthides par un mécanisme de décarboxylation oxydative spécifique d'oxydants à trois électrons. En présence de microsomes hépatiques, il se forme deux métabolites: le triarylméthane par réduction et la quinone-méthide par oxydation impliquant les cytochromes P45O et leur réductase. Les peroxydases en présence d'hydroperoxyde transforment les TAM en quinone-méthide avec la formation intermédiaire du cation triarylméthyle TAM+. Ce cation TAM+ est réactif vis-a-vis de divers nucléophiles d'importance biologique en formant des adduits covalents. Enfin, nous avons developpé une nouvelle méthode générale d'accès à de nouveaux derivés TAM non symétriques par réaction entre le cation TAM+ et différents nucléophiles (ipso-substitution nucléophile aromatique dépendant d'une décarboxylation oxydative). Nos résultats ouvrent de nouvelles perspectives pour le développement de sondes plus stables utilisables in vivo.O2 concentration is an essential physiological parameter. In vivo oximetry might be helpful for the diagnosis, the prognosis, as well as for the treatment in the case of cancer. EPR oximetry is based on the use of paramagnetic probes the linewidth of which is directly proportional to O2 concentration. Persistent tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals have required properties to be used as EPR oximetry probes. However, at the beginning of our work, no data was available about the metabolism of these probes used in vivo. We studied their reactivity and their metabolism in vitro. We showed that superoxide and alkylperoxyle radicals efficiently oxidize these radicals to the corresponding quinone-methide by an oxidative decarboxylation mechanism, specific of three electron oxidants. In the presence of hepatic microsomes, two major metabolites are formed: the triarylmethane by reduction and the quinone-methide by oxidation, involving cytochromes P45O and their reductase. Peroxydases in the presence of hydroperoxide catalyze the oxidation of TAM into the quinone-methide with the intermediate formation of the trityl cation TAM+. This cation TAM+ appeared to be reactive toward biological nucleophiles by forming covalent adducts. Finally, we also developed a new general method to access to new dissymetric TAM radicals through a reaction between trityl cation TAM+ and various nucleophiles (ipso-nucleophilic aromatic substitution coupled to an oxidative decarboxylation). Our results offer new perspectives for the development of stable probes for in vivo use.PARIS5-BU Saints-Pères (751062109) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Oxidation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes: evidence for their oxidative decarboxylation and molecular origin of their specific ability to react with O2˙−

International audienc

Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant

International audienc

Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens

International audienc