Mécanismes de suppression de la résistance de la transcriptase inverse du VIH-1 aux analogues de nucléotides

Le virus d'immunodéficience humaine (VIH), isolé en 1983, est l'agent pathogène responsable de la maladie du SIDA. Dès 1987, l'arrivée de l'AZT sur le marché des antiviraux a permis de conforter l'idée que bloquer l'activité des enzymes du VIH pouvait servir à empêcher la multiplication virale. En effet, l'AZT est un analogue de nucléoside qui, une fois phosphorylé dans la cellule infectée, inhibe la polymérase virale : la transcriptase inverse. La découverte de l'activité antivirale de cette molécule a ouvert le champ des monothérapies anti-SIDA, basées au départ sur l'utilisation de nucléosides, puis élargies à d'autres molécules et à d'autres cibles enzymatiques telles que la protéase. A partir de 1996, la combinaison des traitements en tri-thérapies permet de ralentir de manière spectaculaire la progression de l'infection par le VIH. Toutefois, la faible efficacité des antirétroviraux facilite la sélection de mutations dans le génome du VIH entraînant une perte progressive de sensibilité aux traitements, en donnant aux enzymes cibles la capacité de contourner l'inhibition par les antiviraux. En particulier, les mutations dans le gène "pol" de la transcriptase inverse s'accumulent progressivement à travers les cycles de réplication, et conduisent irrémédiablement à l'échec thérapeutique. Dans quelle mesure la connaissance au niveau moléculaire des mécanismes de résistance de la transcriptase inverse peut être exploitée de façon à concevoir de nouvelles stratégies thérapeutiques ? Nous avons évalué le potentiel inhibiteur des boranophosphates, une nouvelle génération de nucléotides capables d'inhiber "in vitro" la transciptase inverse mutée résistante aux autres molécules existantes. Nous avons aussi évalué les bases rationnelles de la suppression de résistance par accumulation de mutations antagonistes. Ce phénomène complexe permettrait peut-être de choisir des combinaisons antivirales obligeant le virus à évoluer vers une "impasse génétique". La sélection de mutations de résistance se fait dans de nombreux cas au détriment de l'activité enzymatique de la transcriptase inverse. Nous avons donc déterminé comment certaines de ces mutations, une fois combinées, peuvent amplifier cette baisse d'activité, qui se répercute alors sur la capacité du virus à se répliquer.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Gln151 of HIV-1 reverse transcriptase acts as a steric gate towards clinically relevant acyclic phosphonate nucleotide analogues.

International audienceIn the treatment of HIV, the loose active site of the HIV-1 reverse transcriptase (RT) allows numerous nucleotide analogues to act as proviral DNA 'chain-terminators'. Acyclic nucleotide phosphonate analogues (ANPs) represent a particular class of nucleotide analogue that does not possess a ribose moiety. The structural basis for their substrate efficiency regarding viral DNA polymerases is poorly understood

Gln151 of HIV-1 reverse transcriptase acts as a steric gate towards clinically relevant acyclic phosphonate nucleotide analogues.

International audienceIn the treatment of HIV, the loose active site of the HIV-1 reverse transcriptase (RT) allows numerous nucleotide analogues to act as proviral DNA 'chain-terminators'. Acyclic nucleotide phosphonate analogues (ANPs) represent a particular class of nucleotide analogue that does not possess a ribose moiety. The structural basis for their substrate efficiency regarding viral DNA polymerases is poorly understood

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase▿

Nonobligate chain terminators, such as 2′-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3′-hydroxyl group can be excised in the presence of pyrophosphate (PPi), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2′-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2′-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2′-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2′-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation

The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception

International audienc

Borano-nucleotides: new analogues to circumvent HIV-1 RT-mediated nucleoside drug-resistance

International audienceAlpha-boranophosphates suppress RT-mediated resistance when the catalytic rate of incorporation (kpol) of the analogue 5'-triphosphate is responsable for drug resistance, such as in the case of K65R mutant and ddNTPs, and Q151M toward AZTTP and ddNTPs. This suppression is also observed with BH3-d4T and BH3-3TC toward their clinically relevant mutants Q151M and M184V. Moreover, the presence of the borano (BH3-) group renders the incorporation of the analogue independent from amino-acid substitutions in RT. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analogu

Acetaminophen recruits spinal p42/p44 mAPKs and GH/IGF-1 receptors to produce analgesia via the serotonergic system

International audienceThe mechanism of action of acetaminophen is currently widely discussed. Direct inhibition of cyclooxygenase isoforms remains the commonly advanced hypothesis. We combined behavioral studies with molecular techniques to investigate the mechanism of action of acetaminophen in a model of tonic pain in rats. We show that acetaminophen indirectly stimulates spinal 5-hydroxytryptamine (5-HT)(1A) receptors in the formalin test, thereby increasing transcript and protein levels of low-affinity neurotrophin receptor, insulin-like growth factor-1 (IGF-1) receptor alpha subunit, and growth hormone receptor and reducing the amount of somatostatin 3 receptor (sst3R) mRNA. Those cellular events seem to be important for the antinociceptive activity of acetaminophen. Indeed, down-regulation of sst3R mRNA depends on acetaminophen-elicited, 5-HT1A receptor-dependent increase in neuronal extracellular signal-regulated kinase 1/2 (ERK1/2) activities that mediate antinociception. In addition, spinal growth hormone (GH) and IGF-1 receptors would also be involved in the antinociceptive activity of the analgesic at different degrees. Our results show the involvement of specific 5-HT1A receptor-dependent cellular events in acetaminophen-produced antinociception and consequently indicate that inhibition of cyclooxygenase activities is not the exclusive mechanism involved. Furthermore, we propose that the mechanisms of 5-HT1A receptor-elicited antinociception and the role of the spinal ERK1/2 pathway in nociception are more intricate than suspected so far and that the GH/IGF-1 axis is an interesting new player in the regulation of spinal nociception

Activation of TREK-1 by morphine results in analgesia without adverse side effects

International audienceMorphine is the gold-standard pain reliever for severe acute or chronic pain but it also produces adverse side effects that can alter the quality of life of patients and, in some rare cases, jeopardize the vital prognosis. Morphine elicits both therapeutic and adverse effects primarily through the same m opioid receptor subtype, which makes it difficult to separate the two types of effects. Here we show that beneficial and deleterious effects of morphine are mediated through different signalling pathways downstream from m opioid receptor. We demonstrate that the TREK-1 K þ channel is a crucial contributor of morphine-induced analgesia in mice, while it is not involved in morphine-induced constipation, respiratory depression and dependence-three main adverse effects of opioid analgesic therapy. These observations suggest that direct activation of the TREK-1 K þ channel, acting downstream from the m opioid receptor, might have strong analgesic effects without opioid-like adverse effects

Properties of subependymal cerebrospinal fluid contacting neurones in the dorsal vagal complex of the mouse brainstem

Cerebrospinal fluid (CSF) contacting neurones have been observed in various brain regions such as the hypothalamus, the dorsal nucleus of the raphe and around the central canal (cc) of the spinal cord but their functional role remains unclear. At the level of the spinal cord, subependymal cerebrospinal fluid contacting neurones (S-CSF-cNs) present a peculiar morphology with a soma close to the ependymal layer, a process projecting towards the cc and ending with a bud and a cilium. These neurones were recently shown to express polycystin kidney disease 2-like 1 (PKD2L1 or TRPP3) channels that are members of the polycystin subtype of the transient receptor potential (TRP) channel superfamily and that have been proposed as either chemo- or mechanoreceptors in several tissues. Using immunohistological techniques and whole-cell electrophysiological recordings in brain slices obtained from PKD2L1:EGFP transgenic adult mice, we looked for and determined the functional properties of S-CSF-cNs in the dorsal vagal complex (DVC), a hindbrain structure controlling autonomic functions such as blood pressure, energy balance and food intake. Here, we demonstrate that S-CSF-cNs received GABAergic and/or glycinergic synaptic entries and were also characterised by the presence of non-selective cationic channels of large conductance that could be detected even under whole-cell configuration. The channel activity was not affected by Psalmopoeus cambridgei toxin 1, a blocker of acid sensing ion channels (ASICs), but was blocked by amiloride and by a strong extracellular acidification. In contrast, extracellular alkalinisation and hypo-osmotic shocks increased channel activity. Based on these properties, we suggest that the single-channel activity recorded in medullar S-CSF-cNs is carried by PKD2L1 channels. Our study therefore reinforces the idea that PKD2L1 is a marker of S-CSF-cNs and points toward a role for S-CSF-cNs in the detection of circulating signals and of modifications in the extracellular environment