Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase.

ABSTRACT: Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

SUBMITTER: Ditzler MA

PROVIDER: S-EPMC3561961 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Structure-based evaluation of non-nucleoside inhibitors with improved potency and solubility that target HIV reverse transcriptase variants.

Project description:The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ?10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 ?g/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series.

| S-EPMC4378236 | biostudies-literature

SAMHD1 specifically affects the antiviral potency of thymidine analog HIV reverse transcriptase inhibitors.

Project description:Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4(+) T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4(+) T cells infected with HIV-2 compared to infection with the HIV-2?Vpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes.

| S-EPMC4136047 | biostudies-literature

Murine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptase.

Project description:Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information.

| S-EPMC2443788 | biostudies-literature

Diaryl ethers with carboxymethoxyphenacyl motif as potent HIV-1 reverse transcriptase inhibitors with improved solubility.

Project description:In search of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved solubility, two series of novel diaryl ethers with phenacyl moiety were designed and evaluated for their HIV-1 reverse transcriptase inhibition potentials. All compounds exhibited good to excellent results with IC50 at low micromolar to submicromolar concentrations. Two most active compounds (7e and 7?g) exhibit inhibitory potency comparable or even better than that of nevirapine and rilpivirine. Furthermore, SupT1 and CD4+ cell infectivity assays for the most promising (7e) have confirmed its strong antiviral potential while docking studies indicate a novel binding interactions responsible for high activity.

| S-EPMC6009982 | biostudies-literature

Reverse transcriptase inhibitors in Aicardi-Goutières syndrome

Project description:Aicardi-Goutières syndrome (AGS) is a genetically heterogeneous encephalopathy whose pathology is linked to an abnormal type I interferon response induced by self-derived nucleic acids. Data indicate that endogenous retroelements represent one source of interferon-stimulatory self-nucleic acid. No effective therapies are available for this disorder. In this pilot study involving patients with AGS due to mutations in TREX1, RNASEH2A, RNASEH2B or SAMHD1 three nucleoside analogue reverse transcriptase inhibitors (RTIs) were administered over 12 months. Transcription profiling was done by RNA-seq.

2018-12-12 | E-MTAB-7087 | biostudies-arrayexpress

Emerging reverse transcriptase inhibitors for HIV-1 infection.

Project description:INTRODUCTION:There are 36.7 million people living with HIV with 20.9 million having access to antiretroviral therapy (ART). Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) remain the 'backbone' of ART. However, the currently available nine NRTIs and five non-nucleoside reverse transcriptase inhibitors (NNRTIs) have significant side effects and resistance profiles. Areas covered: We summarize the mechanisms of resistance and other limitations of the existing NRTIs/NNRTIs. GS-9131, MK-8591, Elsulfavirine and Doravirine are four new agents that are furthest along in development. Expert opinion: ART development has evolved with several new promising agents. Longer-acting agents, like MK-8591 are extremely attractive to enhance drug adherence and patient satisfaction. Doravirine offers an NNRTI effective against common mutations that has fewer side effects, limitations on dosing and drug interactions. GS-9131 is very potent and active against a variety of NRTI mutants but it is too early in its development to understand its full risks and benefits. Finally, Elsulfavirine has a long half-life and preliminary data suggests fewer side effects than the most commonly used NNRTI, efavirenz. Each of these new agents shows promise and potential to improve ART in the future. The newer generation of reverse transcriptase inhibitors have longer half-lives, more favorable adverse effect profiles, and fewer drug interactions.

| S-EPMC6158299 | biostudies-literature

Structural insights into the recognition of nucleoside reverse transcriptase inhibitors by HIV-1 reverse transcriptase: First crystal structures with reverse transcriptase and the active triphosphate forms of lamivudine and emtricitabine.

Project description:The retrovirus HIV-1 has been a major health issue since its discovery in the early 80s. In 2017, over 37 million people were infected with HIV-1, of which 1.8 million were new infections that year. Currently, the most successful treatment regimen is the highly active antiretroviral therapy (HAART), which consists of a combination of three to four of the current 26 FDA-approved HIV-1 drugs. Half of these drugs target the reverse transcriptase (RT) enzyme that is essential for viral replication. One class of RT inhibitors is nucleoside reverse transcriptase inhibitors (NRTIs), a crucial component of the HAART. Once incorporated into DNA, NRTIs function as a chain terminator to stop viral DNA replication. Unfortunately, treatment with NRTIs is sometimes linked to toxicity caused by off-target side effects. NRTIs may also target the replicative human mitochondrial DNA polymerase (Pol γ), causing long-term severe drug toxicity. The goal of this work is to understand the discrimination mechanism of different NRTI analogues by RT. Crystal structures and kinetic experiments are essential for the rational design of new molecules that are able to bind selectively to RT and not Pol γ. Structural comparison of NRTI-binding modes with both RT and Pol γ enzymes highlights key amino acids that are responsible for the difference in affinity of these drugs to their targets. Therefore, the long-term goal of this research is to develop safer, next generation therapeutics that can overcome off-target toxicity.

| S-EPMC6699100 | biostudies-literature

Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against "nucleotide-competing reverse transcriptase inhibitors".

Project description:Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). Although the structure of these compounds differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the polymerase active site. NRTI-associated mutations that are clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1. Here we have studied the underlying biochemical mechanism. RT enzymes containing the isolated mutations M184V and Y115F cause 2-3-fold increases in IC(50) values, while the combination of the two mutations causes a >15-fold increase. K65R can partially counteract these effects. Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of the enzyme to discriminate against the inhibitor. Studies with other strategic mutations at residues Phe-61 and Ala-62, as well as the use of chemically modified templates shed further light on the putative binding site of the inhibitor and ternary complex formation. An abasic site residue at position n, i.e. opposite the 3'-end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect. Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleoside triphosphates (dNTPs). We therefore propose to refer to members of this class of compounds as "nucleotide-competing RT inhibitors" (NcRTIs).

| S-EPMC2573056 | biostudies-literature

Approved HIV reverse transcriptase inhibitors in the past decade

Project description:HIV reverse transcriptase (RT) inhibitors are the important components of highly active antiretroviral therapies (HAARTs) for anti-HIV treatment and pre-exposure prophylaxis in clinical practice. Many RT inhibitors and their combination regimens have been approved in the past ten years, but a review on their drug discovery, pharmacology, and clinical efficacy is lacking. Here, we provide a comprehensive review of RT inhibitors (tenofovir alafenamide, rilpivirine, doravirine, dapivirine, azvudine and elsulfavirine) approved in the past decade, regarding their drug discovery, pharmacology, and clinical efficacy in randomized controlled trials. Novel RT inhibitors such as islatravir, MK-8504, MK-8507, MK8583, IQP-0528, and MIV-150 will be also highlighted. Future development may focus on the new generation of novel antiretroviral inhibitors with higher bioavailability, longer elimination half-life, more favorable side-effect profiles, fewer drug–drug interactions, and higher activities against circulating drug-resistant strains. Graphical abstract Approved anti-HIV regimens in the past decade. Most regimens are composed of two nucleoside/nucleotide reverse transcriptase inhibitors plus one integrase inhibitor, one non-nucleoside reverse transcriptase inhibitor, or one protease inhibitor.Image 1

| S-EPMC9279714 | biostudies-literature

Solution structural dynamics of HIV-1 reverse transcriptase heterodimer.

Project description:Crystal structures and simulations suggest that conformational changes are critical for the function of HIV-1 reverse transcriptase. The enzyme is an asymmetric heterodimer of two subunits, p66 and p51. The two subunits have the same N-terminal sequence, with the p51 subunit lacking the C-terminal RNase H domain. We used hydrogen exchange mass spectrometry to probe the structural dynamics of RT. H/D exchange revealed that the fingers and palm subdomains of both subunits form the stable core of the heterodimer. In the crystal structure, the tertiary fold of the p51 subunit is more compact than that of the polymerase domain of the p66 subunit, yet both subunits show similar flexibility. The p66 subunit contains the polymerase and RNase H catalytic sites. H/D exchange indicated that the RNase H domain of p66 is very flexible. The beta-sheet beta12-beta13-beta14 lies at the base of the thumb subdomain of p66 and contains highly conserved residues involved in template/primer binding and NNRTI binding. Using the unique ability of hydrogen exchange mass spectrometry to resolve slowly interconverting species, we found that beta-sheet beta12-beta13-beta14 undergoes slow cooperative unfolding with a t(1/2) of <20 s. The H/D exchange results are discussed in relation to existing structural, simulation, and sequence information.

| S-EPMC2814253 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data