HIV-1 RT Inhibitors with a Novel Mechanism of Action: NNRTIs that Compete with the Nucleotide Substrate

HIV-1 reverse transcriptase (RT) inhibitors currently used in antiretroviral therapy can be divided into two classes: (i) nucleoside analog RT inhibitors (NRTIs), which compete with natural nucleoside substrates and act as terminators of proviral DNA synthesis, and (ii) non-nucleoside RT inhibitors (NNRTIs), which bind to a hydrophobic pocket close to the RT active site. In spite of the efficiency of NRTIs and NNRTIs, the rapid emergence of multidrug-resistant mutations requires the development of new RT inhibitors with an alternative mechanism of action. Recently, several studies reported the discovery of novel non-nucleoside inhibitors with a distinct mechanism of action. Unlike classical NNRTIs, they compete with the nucleotide substrate, thus forming a new class of RT inhibitors: nucleotide-competing RT inhibitors (NcRTIs). In this review, we discuss current progress in the understanding of the peculiar behavior of these compounds

RNA Biol

The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba2+, Mn2+, Co2+ and Zn2+ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion

A proposal for a new HIV-1 DLS structural model

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play essential roles at various stages of the viral life cycle. Through a novel assay we had recently developed, we reported on the necessary and sufficient region for RNA dimerization in the HIV-1 virion. Using this system, we performed further detailed mapping of the functional base pairs necessary for HIV-1 DLS structure. Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions. Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation. Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies

HIV-1 Polymerase Inhibition by Nucleoside Analogs: Cellular- and Kinetic Parameters of Efficacy, Susceptibility and Resistance Selection

Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment

Etude structurale et biophysique du complexe forme entre la cytidine deaminase humaine apobec3g et le facteur d'infectivité virale du VIH-1

Le génome des lentivirus, dont celui du VIH-1, code pour des protéines auxiliaires dont la protéine Vif (Viral infectivity factor) qui interagit avec l ARN génomique lors de l assemblage du virus. Vif est nécessaire à la réplication du virus dans les cellules non permissives. Cette restriction est due à la présence dans ces cellules d un facteur contre carré par la présence de Vif. Ce facteur appartient à une famille d enzymes qui déaminent des cytosines en uraciles dans l ADN et/ou l ARN, APOBEC. Sans Vif, APOBEC-3G et APOBEC-3F sont encapsidés dans la particule virale et altèrent la séquence du génome du VIH-1. Vif induit la dégradation d APOBEC-3G/3F par le protéasome, empêchant son encapsidation dans les particules virales. Il n existe aucune information structurale sur la protéine Vif et seule la structure du site de déamination en C-terminal d APOBEC-3G est connue. Pour comprendre les interactions entre A3/Vif, nous avons tenté de déterminer la structure cristallographique de ce complexe. Nos tentatives de surproduction et de purification des protéines Vif et APOBEC seules sont restées vaines. Notre hypothèse de travail repose principalement sur la spécificité de reconnaissance entre Vif et A3G, nous avons choisi une stratégie de co-expression. La co-expression A3/Vif avec nos constructions n aillant pas été concluante, nous nous sommes tournés vers la stratégie co-ESPRIT qui permet de tester l expression et la solubilité de 30000 constructions et d identifier un domaine soluble de l une des protéines. Nos études montrent que Vif est capable d interagir avec le même domaine de TAR que la protéine Tat. Tat est la cible d inhibiteurs empêchant son interaction avec TAR, nous avons testé l inhibition de l interaction entre Vif et TAR par ces composés. Les informations apportées par une structure 3D du complexe serviraient de base pour l élaboration de molécules capables de contrer la dégradation d APOBEC.Unlike others retroviruses, the genome of lentiviruses encode several regulatory proteins. The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif functions to counteract anti-HIV-1 cellular factors in non permissive cells (fig. 1), APOBEC-3G and APOBEC-3F (APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide-like). APOBEC-3G/F are human antiviral cytidine deaminases and RNA editing enzymes that deaminate dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. This hypermutation results either into a degradation of U-rich DNA strands by uracyl-DNA glycosylase (thus inhibiting the viral genome reverse transcription) or into the production of aberrant viral proteins. The Vif protein interacts with APOBEC-3G/F and thereby serves as an adapter to recruit APOBEC3G/F to a cullin5 ECS E3 ubiquitin ligase. Formation of this complex result in the polyubiquitination and subsequent proteasome-mediated degradation of APOBEC-3G/F in virus producing cells. The mechanism of Vif/APOBEC-3 recognition is however still unknown. To date little structural data is available on HIV-1 Vif protein and APOBEC-3G/F proteins. This lack of information is due to the difficulty of expressing high levels of soluble protein using either prokaryotic or baculovirus expression systems. We used a co-expression strategy in order to solve this expression problem. Co-expression offers the possibility of co-folding protein partners that would otherwise be insoluble if expressed alone, just as APOBEC-3G/F and Vif, and can enable in vivo reconstitution with higher yields of the desired complex. The goal of our studies was the determination of the X-ray structure of the Vif-APOBEC-3G/F interaction. Understanding this molecular recognition might be useful to direct structure-based design of drugs that acts by inhibiting the action of Vif and lead to a new class of anti-HIV drugs.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

De l'hôpital à la ville (assurer la continuité des soins pour les patients vivant avec un cancer-Rôles du pharmacien et élaboration d'un carnet de coordination)

Le pharmacien, tant hospitalier qu officinal, a une place incontournable à chaque étape de la prise en charge du patient atteint de cancer : prévention, éducation thérapeutique et prise en charge des effets indésirables en ville, consultation pharmaceutique, contrôle des chimiothérapies et soins de support à l hôpital. De façon plus générale, chaque professionnel de santé (pharmacien, médecin, infirmier(ère), kinésithérapeute ) possède des compétences particulières dans son domaine, qu il est intéressant et essentiel de mutualiser afin d assurer une meilleure prise en charge du patient vivant avec un cancer. Afin d assurer la continuité des soins, et dans le cadre de consultations pharmaceutiques, initiées à l HIA percy pour la prise en charge des nausées et vomissements chimio-induits, nous avons élaboré et mis en place un carnet de coordination Ville-Hôpital. Ainsi, cette étude a également porté sur les différents rôlmes du pharmacien dans la prise en charge du patient vivant avec un cancer et sur la conception et la mise en place d un carnet de coordination Ville-Hôpital. Elle se bornera aux cancers broncho-pulmonaire aux cancers digestifs.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Les prescriptions médicamenteuses dans l hypertension artérielle essentielle (les médecins généralistes fondent-ils leur décision sur les données de la science ?)

PARIS7-Xavier Bichat (751182101) / SudocSudocFranceF