Application des dérivés d'amino-benzosubérone (inhibition sélective des aminopeptidases mono ou bimétalliques)

Les aminopeptidases sont des cibles thérapeutiques importantes pour plusieurs maladies, car elles sont impliquées dans divers processus physiologiques et pathologiques comme la progression tumorale, l'angiogenèse, et certaines infections (virales, bactériennes, et parasitaires). Il en existe deux classes : les aminopeptidases avec un ion métallique (Aminopeptidase N [APN ou CD13] et leukotrien A4 hydrolase [LTA4H]) et les aminopeptidases avec deux ions métalliques (Aminopeptidase de l'Aeromonas proteolytica [APaero], Leucine Aminopeptidase cytosolique [LAPc] et Méthionine aminopeptidase 1 ou 2 [MetAP]). Deux types de composés dérivés des amino-benzosubérones ont été envisagés pour inhiber sélectivement chacune de ces classes d'aminopeptidases. L'étude des relations structures-activités (RSA) nous a permis de découvrir une molécule très puissante et sélective de l'APN (Ki 60 pM). L'APN est une enzyme monométallique considérée aujourd'hui comme une nouvelle cible pour la lutte contre le cancer car son inhibition bloque le processus de l'angiogenèse et donc la progression tumorale. L'étude d'une nouvelle classe de molécules trisubstituées dérivées des amino benzosubérones a abouti à la découverte d'une seconde molécule active et sélective des enzymes bimétalliques notamment l'APaero (Ki 10 nM).The aminopeptidases are important therapeutic targets for several diseases, because they are implied in various physiological and pathological processes like the tumoral progression, the angiogenesis, and certain infections (viral, bacterial, and parasitic). There are two classes: aminopeptidases with one metal ion (Aminopeptidase N [APN or CD13] and leukotriene A4 hydrolase [LTA4H]) and aminopeptidases with two metal ions (Aminopeptidase of Aeromonas proteolytica [APaero], cytosolic leucine aminopeptidase [LAPc] and Methionine aminopeptidase 1 or 2 [MetAP]). Two types of compounds of amino-benzosuberone derivatives were envisaged to inhibit selectively each one of these classes of aminopeptidases. The study of the structure-activity relationship (SAR) enabled us to discover a very powerful and selective molecule of the APN (Ki 60 pM). The APN is a monometallic enzyme considered today as a new target for the fight against cancer because its inhibition blocks the angiogenesis process and thus the tumoral progression. The study of a new class of trisubstituted molecules derived from the amino-benzosuberone led us to discover another molecule which is active and selective of the bimetallic enzymes in particular APaero (Ki 10 nM).MULHOUSE-SCD Sciences (682242102) / SudocSudocFranceF

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism

Conception et synthèse d'inhibiteurs de l'Aminopeptidase membranaire N ([EC. 3.4.11.2], APN ou CD13)

La lutte contre le cancer est l'un des défis majeurs du XXème siècle. Pour que les tumeurs puissent se développer dans l'organisme, elles ont besoin d'un apport en nutriment par le biais de vaisseaux sanguins pour se faire, elles vont avoir recours au processus angiogénique. Lors de ce processus, les cellules endothéliales qui tapissent la paroi des vaisseaux sanguins vont se multiplier et créer de nouveaux vaisseaux sanguins qui vont permettre la vascularisation des tumeurs. L'angiogenèse constitue donc aujourd'hui un axe de recherche pour la lutte contre la progression tumorale et donc contre le cancer. Lors de ce développement tumoral, une enzyme, l'aminopeptidase neutre APN est surexprimée sur les parois des cellules endothéliales. Différentes études ont été menées et montrent que l'inhibition de cette enzyme bloque la progression tumorale. Mon travail au sein de l'équipe du Pr Céline Tarnus consistait en la conception et la synthèse d'inhibiteurs de l'APN. Une relation structure activité de nos composés vis-à-vis de l'APN a tout d'abord été effectuée. Le développement de synthèse du composé le plus actif ont été faite, puis la synthèse d'inhibiteurs d'APN ayant pour objectif l'utilisation de la BNCT a été abordée.The fight against the cancer is one of the most important struggles of this century. For the development of the tumors inside the body, they need to receive nutriments by the blood vessels and they use the angiogenic process. During this process, the endothelial cells being shown on the wall of the blood vessel will multiply and design new blood vessel, which will allow the tumor's vascularisation. Today, the angiogenesis is an axis of research for the fight against the cancer. During the tumoral development, the aminopeptidase N (APN) is overexpressed on the wall of endothelial cells. Various studies have shown that the inhibition of this enzyme stops the tumoral progression. My work in the Pr. Céline Tarnus Team consists in the conception and the synthesis of APN's inhibitors. In a first time, a structure activity relationship has been realized. Syntheses of a subnamolar compound have been developed, and then the synthesis of APN's inhibitors with the use of BNCT has been got onto.MULHOUSE-SCD Sciences (682242102) / SudocSudocFranceF

Selective aminopeptidase-N (CD13) inhibitors with relevance to cancer chemotherapy

International audienc

Selective inhibition of PfA-M1, over PfA-M17, by an amino-benzosuberone derivative blocks malaria parasites development in vitro and in vivo

Abstract Background Plasmodium falciparum M1 family aminopeptidase is currently considered as a promising target for anti-malarial chemotherapy. Several series of inhibitors developed by various research groups display IC50/Ki values down to nM range on native PfA-M1 or recombinant forms and block the parasite development in culture at µM to sub-µM concentrations. A handful of these inhibitors has been tested on murine models of malaria and has shown anti plasmodial in vivo activity. However, most of these inhibitors do also target the other neutral malarial aminopeptidase, PfA-M17, often with lower Ki values, which questions the relative involvement and importance of each enzyme in the parasite biology. Results An amino-benzosuberone derivative from a previously published collection of chemicals targeting specifically the M1-aminopeptidases has been identified; it is highly potent on PfA-M1 (Ki = 50 nM) and devoid of inhibitory activity on PfA-M17 (no inhibition up to 100 µM). This amino-benzosuberone derivative (T5) inhibits, in the µM range, the in vitro growth of two P. falciparum strains, 3D7 and FcB1, respectively chloroquino-sensitive and resistant. Evaluated in vivo, on the murine non-lethal model of malaria Plasmodium chabaudi chabaudi, this amino-benzosuberone derivative was able to reduce the parasite burden by 44 and 40% in a typical 4-day Peters assay at a daily dose of 12 and 24 mg/kg by intraperitoneal route of administration. Conclusions The evaluation of a highly selective inhibitor of PfA-M1, over PfA-M17, active on Plasmodium parasites in vitro and in vivo, highlights the relevance of PfA-M1 in the biological development of the parasite as well as in the list of promising anti-malarial targets to be considered in combination with current or future anti-malarial drugs

New fluoroketones as human renin inhibitors

AbstractRenin inhibition has been evaluated for a new class of fluorinated ketones, true analogues of peptides that have been retroinverted at the C-terminal position. The readily formed hydrate of the ketone is proposed to mimic the tetrahedral intermediate that occurs during the enzyme-catalyzed hydrolysis of amide linkage. From this series of compounds it appears that the number of reverted amide bonds is crucial in terms of activity. Furthermore, a shortening of the C-terminal part of our peptide analogues and the replacement of the leucine residue in P1 by a cyclohexylalanine leads to the tripeptide analogue 12 a potent renin inhibitor (IC50 = 3.5 × 10−9 M)

Wood Metabolomic Responses of Wild and Cultivated Grapevine to Infection with Neofusicoccum parvum, a Trunk Disease Pathogen

Grapevine trunk diseases (GTDs), which are associated with complex of xylem-inhabiting fungi, represent one of the major threats to vineyard sustainability currently. Botryosphaeria dieback, one of the major GTDs, is associated with wood colonization by Botryosphaeriaceae fungi, especially Neofusicoccum parvum. We used GC-MS and HPLC-MS to compare the wood metabolomic responses of the susceptible Vitis vinifera subsp. vinifera (V. v. subsp. vinifera) and the tolerant Vitis vinifera subsp. sylvestris (V. v. subsp. sylvestris) after artificial inoculation with Neofusicoccum parvum (N. parvum). N. parvum inoculation triggered major changes in both primary and specialized metabolites in the wood. In both subspecies, infection resulted in a strong decrease in sugars (fructose, glucose, sucrose), whereas sugar alcohol content (mannitol and arabitol) was enhanced. Concerning amino acids, N. parvum early infection triggered a decrease in aspartic acid, serine, and asparagine, and a strong increase in alanine and β-alanine. A trend for more intense primary metabolism alteration was observed in V. v. subsp. sylvestris compared to V. v. subsp. vinifera. N. parvum infection also triggered major changes in stilbene and flavonoid compounds. The content in resveratrol and several resveratrol oligomers increased in the wood of both subspecies after infection. Interestingly, we found a higher induction of resveratrol oligomer (putative E-miyabenol C, vitisin C, hopeaphenol, ampelopsin C) contents after wood inoculation in V. v. subsp. sylvestris

Synthesis of the first examples of iminosugar clusters based on cyclopeptoid cores

Cyclic N-propargyl α-peptoids of various sizes were prepared by way of macrocyclizations of linear N-substituted oligoglycines. These compounds were used as molecular platforms to synthesize a series of iminosugar clusters with different valency and alkyl spacer lengths by means of Cu(I)-catalysed azide–alkyne cycloadditions. Evaluation of these compounds as α-mannosidase inhibitors led to significant multivalent effects and further demonstrated the decisive influence of scaffold rigidity on binding affinity enhancements

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization

<div><p>Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in <i>Botryosphaeriaceae</i> fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of <i>Neofusicoccum parvum</i> and <i>Diplodia seriata</i>, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that <i>Botryosphaeriaceae</i> were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. <i>N</i>. <i>parvum</i> is able to degrade parietal polysaccharides, whereas <i>D</i>. <i>seriata</i> has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on <i>N</i>. <i>parvum</i> Bourgogne S-116. We further show that <i>Botryosphaeriaceae</i> are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for <i>D</i>. <i>seriata</i>. In conclusion, the different <i>Botryosphaeriaceae</i> isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable <i>Botryosphaeriaceae</i> to bypass chemical and physical barriers of the grapevine plant. The specific signature of <i>Botryosphaeriaceae</i> aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host.</p></div