Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell

The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility

Understanding multivalent effects in glycosidase inhibition using C-glycoside click clusters as molecular probes

The synthesis of the first examples of multivalent C-glycosides based on C60-fullerene or β-cyclodextrin cores by way of Cu(I)-catalyzed azide–alkyne cycloadditions is reported. These compounds were designed as molecular probes to understand the mechanisms underlying the outstanding multivalent effects observed in glycosidase inhibition. The inhibition results obtained support a multivalent-binding model based on two scenarios both involving nonspecific interactions and varying by the presence or the absence of active site specific interactions. The magnitude of the multivalent effect obtained depends on the identity of the glycosidase involved and more specifically on the accessibility of its catalytic active site. Large inhibitory multivalent effects can be obtained when both glycosidase active sites and non-catalytic sites at the protein surface are involved in binding events. On the other hand, nonspecific interactions alone are not sufficient to achieve relative affinity enhancements exceeding a simple statistical effect (i.e., a relative inhibition potency not better than one on a valence-corrected basis).Ministerio de Economía y Competitividad de España (MINECO) y Fondos Europeos de Desarrollo Regional (FEDER y FSE). SAF2013-44021-

Structural Basis of Outstanding Multivalent Effects in Jack Bean α-Mannosidase Inhibition

Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic‐level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high‐resolution crystal structures of the Jack bean α‐mannosidase (JBα‐man) in apo and inhibited states. The three‐dimensional structure of JBα‐man in complex with the multimeric cyclopeptoid‐based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.Instituto de Física de Líquidos y Sistemas Biológico

Synthesis and study of molecules targeting the HIV-1 RNA dimerization site

Comme pour tous les rétrovirus, le génome du VIH-1 se compose d'un dimère d'ARN simples brins homologues. La dimérisation de l'ARN est une étape clé pour l'infectivité virale. Elle est initiée par une tige-boucle auto complémentaire, nommée site d'initiation à la dimérisation (DIS). Des modélisations ont conduit à la conception de molécules pouvant se fixer sur cette nouvelle cible thérapeutique. Ainsi, des dimères constitués de deux molécules de néamine reliées par un bras espaceur fixé sur l'amine en position 1 de la néamine ont été synthétisés. Pour cela, il a été nécessaire de protéger sélectivement les fonctions de la néamine à l'exception de l'amine en position 1, puis de faire réagir cette amine sur différents bras espaceurs afin de former des dimères. Plusieurs dimères ayant des bras espaceurs de différentes longueurs ont été synthétisés, puis déprotégés afin d'évaluer leur activité sur le DIS. Les dimères se fixent effectivement sur le DIS in vitro et ils possèdent également une activité ex vivo.As any retrovirus, HIV-1 has his genome organized as a dimer of homologous single stranded RNA. Dimerization is a crucial step for virion infectivity, which is initiated by a self-complementary hairpin, called dimerization initiation site (DIS). Modelling studies led to the design of molecules that could bind this new therapeutical target. Dimers formed by two molecules of neamine linked by a spacer bound on amine in position 1 were synthesized. In this goal, it was necessary to protect selectively the different functions of neamine, except amine in position 1, in order to make it react on different spacers to build up dimers. Several dimers having spacers of different lengths have been synthesized, deprotected and their activity has been evaluated on the DIS. They indeed bind the DIS in vitro and they also exhibit an activity ex vivo

Synthèse et étude de molécules ciblant le site de dimérisation de l ARN du VIH-1

Comme pour tous les rétrovirus, le génome du VIH-1 se compose d'un dimère d ARN simples brins homologues. La dimérisation de l'ARN est une étape clé pour l'infectivité virale. Elle est initiée par une tige-boucle auto complémentaire, nommée site d initiation à la dimérisation (DIS). Des modélisations ont conduit à la conception de molécules pouvant se fixer sur cette nouvelle cible thérapeutique. Ainsi, des dimères constitués de deux molécules de néamine reliées par un bras espaceur fixé sur l'amine en position 1 de la néamine ont été synthétisés. Pour cela, il a été nécessaire de protéger sélectivement les fonctions de la néamine à l'exception de l'amine en position 1, puis de faire réagir cette amine sur différents bras espaceurs afin de former des dimères. Plusieurs dimères ayant des bras espaceurs de différentes longueurs ont été synthétisés, puis déprotégés afin d'évaluer leur activité sur le DIS. Les dimères se fixent effectivement sur le DIS in vitro et ils possèdent également une activité ex vivo.As any retrovirus, HIV-1 has his genome organized as a dimer of homologous single stranded RNA. Dimerization is a crucial step for virion infectivity, which is initiated by a self-complementary hairpin, called dimerization initiation site (DIS). Modelling studies led to the design of molecules that could bind this new therapeutical target. Dimers formed by two molecules of neamine linked by a spacer bound on amine in position 1 were synthesized. In this goal, it was necessary to protect selectively the different functions of neamine, except amine in position 1, in order to make it react on different spacers to build up dimers. Several dimers having spacers of different lengths have been synthesized, deprotected and their activity has been evaluated on the DIS. They indeed bind the DIS in vitro and they also exhibit an activity ex vivo.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Tight-binding inhibition of jack bean α-mannosidase by glycoimidazole clusters

The best multivalent effects observed in glycosidase inhibition have been achieved so far with jack bean α-mannosidase (JBα-man) using iminosugar clusters based on weakly binding mismatching active-site-directed inhibiting epitopes (inhitopes) in the d-gluco series. Here, we synthesize and evaluate as JBα-man inhibitors a series of mono- to 14-valent glycoimidazoles with inhitopes displaying inhibition values up to the range of hundreds of nMs to study the impact of inhitope affinity on the multivalent effect. The most potent inhibitor of the series, a 14-valent mannoimidazole derivative, inhibits JBα-man with a nanomolar Ki value (2 ± 0.5 nM) and binding enhancements observed are, at best, relatively small (up to 25-fold on a valency-corrected basis). The results of this study support the fact that JBα-man-inhitope affinity and the strength of the inhibitory multivalent effect evolve in the opposite direction. The major impact of the glycoimidazole-based inhitope is found on the binding scenario; most of the synthesized mannoimidazole clusters as well as a 14-valent glucoimidazole derivative prove to be tight binding inhibitors of JBα-man.</p

A Convergent Strategy for the Synthesis of Second-Generation Iminosugar Clusters Using “Clickable” Trivalent Dendrons

International audienceA convergent strategy to access high‐valency iminosugar clusters based on two successive CuI‐catalysed azide–alkyne cycloadditions is reported. The key step of this approach relies on the grafting of azide‐armed trivalent iminosugar dendrons onto polyalkyne “clickable” scaffolds, thus tripling their initial valency. As a proof of concept, the synthesis of cyclodextrin‐based clusters containing the highest number of peripheral iminosugar ligands reported to date has been achieved. Evaluation of these compounds as mannosidase inhibitors revealed one of the strongest multivalent effects observed so far in glycosidase inhibition