Kui bioloog kohtab keemikut: HIV-1 inhibiitorite otsingul

Väitekirja elektrooniline versioon ei sisalda publikatsioone.HIV-1 on pandeemiline viirus ning nakatatud inimeste arv maailmas suureneb pidevalt. Vaktsiin selle vastu puudub, kuid nakatatud patsientide raviks on kliinilise kasutuse loa saanud ligi 30 erinevat ühendit. Erinevatesse klassidesse kuuluvate inhibiitorite kombineeritud kasutamine (ART) on kujunenud HIV-ga nakatunud patsientide ravimise aluseks. ART ravi kõige olulisemaks puuduseks on ravimite toksilised kõrvaltoimed ning uute resistentsete viiruse tüvede teke. Antud väitekirja põhieesmärgiks oli leida efektiivseid mittetoksilisi ühendeid, mis suruvad maha HIV-1 replikatsiooni (nii metsik-tüüpi viiruse kui ka resistentsete tüvede oma) keskendudes eelkõige pöördtranskriptaasi inhibiitoritele. Kolm gruppi keemilisi ühendeid testiti antiretroviirus-vastase toime suhtes: atsüklilised tümidiini nukleosiidi analoogid, bimorfoliinid ja nende derivaadid ning sahariidhüdrasoonid. Samuti tõestati eksperimentaalselt eelneva in silico skriiningu käigus saadud tulemusi. Käesoleva töö tulemusena töötati välja meetod HIV-1 inhibiitorite skriinimiseks ViraPower lentiviiruse ekspressioonisüsteemi (Invitrogen) põhjal. Meetod on kiire, lihtne, usaldusväärne ning on kõlblik kasutamiseks väljaspool kõrgenenud ohutustasemega laborit (BSL3). Kõige tugevam toime meie laboris uuritud ühenditest oli mitte-nukleosiidsel pöördtranskriptaasi inhibiitoril (NNRTI), mis omas tuntud ravimi Nevirapiiniga sarnast aktiivsust, kuid oli kahjuks mitteaktiivne viiruse resistentsete vormide vastu. Antud ühend leiti ratsionaalse ravimidisaini strateegiat kasutades. Antud töö tulemused võimaldasid täiustada olemasolevat in silico skriiningu meetodit, mis võimaldab tulevikus kavandada uusi ning efektiivsemaid HIV-1 inhibiitoreid.HIV-1 is a pandemic virus and the numbers of infected people are constantly increasing all over the world. There is no vaccine available, but about 30 compounds have been approved by FDA for the treatment of HIV-infected patients. ART (antiretroviral therapy) consists of a combination of at least 3 inhibitors with different mechanism of action. The main drawback of such treatment is side effects of the drugs and the appearance of new resistant forms of the virus. The aim of this work was to find non-toxic compounds which can effectively suppress HIV-1 replication (both wild-type and resistant forms of the virus). We focused our efforts mostly on the discovery of novel reverse transcriptase inhibitors. Three groups of compounds were tested for their antiretroviral activity (acyclic thymine nucleoside analogues, bimorpholines and their derivatives, and saccharide hydrazones); also we experimentally verified the results of the previous in silico screening. As a result of this work, we developed an assay for HIV-1 inhibitors′ screening. This assay is based on ViraPower Lentiviral Expression System (Invitrogen) and is simple, fast, reliable and can be used outside BSL3 facilities. The most potent compound, found by us so far, acts as a non-nucleoside reverse transcriptase inhibitor (NNRTI) and has activity comparable to the activity of a known NNRTI nevirapine, but is unfortunately inactive against the resistant forms of the virus. This compound was found using ration drug design strategy. Most importantly, the results of this work allowed us to improve the existing in silico screening method, which could result in more potent HIV-1 inhibitors in the future

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50) : 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50 : 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide: RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.Peer reviewe

Synthesis of Novel Saccharide Hydrazones

<div><p></p><p>Synthesis of important heterocyclic hydrazine derivatives N-aminopyrrolidine, N-aminopiperidine, and N-aminoazepane from hydrazine hydrate and dihalogenides were examined and optimized. These heterocyclic hydrazine derivatives were used in condensation reactions with six different monosaccharides to form corresponding hydrazones. Biological evaluations of these novel compounds, which are simple acyclic nucleoside analogs, were done. L-Arabinose N-aminoazepane hydrazone showed minor anti-HIV activity, giving a starting point for further structural modifications.</p></div

Antibacterial activity of the nitrovinylfuran G1 (Furvina) and its conversion products

2-Bromo-5-(2-bromo-2-nitrovinyl) furan (G1 or Furvina) is an antimicrobial with a direct reactivity against thiol groups. It is active against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. By reacting with thiol groups it causes direct damage to proteins but, as a result, is very short-living and interconverts into an array of reaction products. Our aim was to characterize thiol reactivity of G1 and its conversion products and establish how much of antimicrobial and cytotoxic effects are due to the primary activity of G1 and how much can be attributed to its reaction products. Stability of G1 in growth media as well as its conversion in the presence of thiols was characterized. The structures of G1 decomposition products were determined using NMR and mass-spectroscopy. Concentration-and time-dependent killing curves showed that G1 is bacteriostatic for Escherichia coli at the concentration of 16 mu g/ml and bactericidal at 32 mu g/ml. However, G1 is inefficient against non-growing E. coli. Addition of cysteine to medium reduces the antimicrobial potency of G1. Nevertheless, the reaction products of G1 and cysteine enabled prolonged antimicrobial action of the drug. Therefore, the activity of 2-bromo-5-(2-bromo-2-nitrovinyl) furan is a sum of its immediate reactivity and the antibacterial effects of the conversion products

TRIM5&alpha; Restriction of HIV-1-N74D Viruses in Lymphocytes Is Caused by a Loss of Cyclophilin A Protection

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein CPSF6. In primary human CD4+ T cells, HIV-1-N74D viruses exhibit an infectivity defect when compared to wild-type. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4+ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D viruses is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5&alpha; (TRIM5&alpha;hu) restriction in CD4+ T cells, we found that depletion of TRIM5&alpha;hu in CD4+ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5&alpha;hu. Accordingly, TRIM5&alpha;hu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In agreement with the notion that N74D capsids are defective in their ability to bind Cyp A, we found that HIV-1-N74D viruses were 20-fold less sensitive to TRIMCyp restriction when compared to wild-type viruses in OMK cells. Structural analysis revealed that N74D hexameric capsid protein in complex with PF74 is different from wild-type hexameric capsid protein in complex with PF74, which explains the defect of N74D capsids to interact with Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4+ T cells is due to a loss of Cyp A protection from TRIM5&alpha;hu restriction activity

Cyclophilin A Prevents HIV-1 Restriction in Lymphocytes by Blocking Human TRIM5α Binding to the Viral Core

International audienceDisruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand this mechanism, we utilize human Jurkat cells, peripheral blood mononuclear cells (PBMCs), and CD4+ T cells. Our results show that inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human tripartite motif 5α (TRIM5αhu), showing that TRIM5αhu restricts HIV-1 in CD4+ T cells. Accordingly, depletion of TRIM5αhu in CD4+ T cells rescues HIV-1 that fail to interact with CypA, such as HIV-1-P90A. We found that TRIM5αhu binds to the HIV-1 core. Disruption of CypA-capsid interactions fail to affect HIV-1-A92E/G94D infection, correlating with the loss of TRIM5αhu binding to HIV-1-A92E/G94D cores. Disruption of CypA-capsid interactions in primary cells has a greater inhibitory effect on HIV-1 when compared to Jurkat cells. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4+ T cells inhibits reverse transcription. Overall, our results reveal that CypA binding to the core protects HIV-1 from TRIM5αhu restriction

Sequences of DNA and LNA/DNA gapmer oligonucleotides.

<p>NH2 = 5’ amino modifier C6. This group was present only in the oligonucleotides used for melting curve determination and for the analysis of delivery into cells. + = prefix for LNA; X = 5-OH-dC; Y = 8-oxo-dG.</p><p>Sequences of DNA and LNA/DNA gapmer oligonucleotides.</p

Sequences of oligonucleotides used to determine the melting temperatures by FRET.

<p>NH2 = 5’ amino modifier C6; + = prefix for LNA; X = 5-OH-dC; Y = 8-oxo-dG.</p><p>Sequences of oligonucleotides used to determine the melting temperatures by FRET.</p