Potent Nonnucleoside Reverse Transcriptase Inhibitors Target HIV-1 Gag-Pol

Figueiredo, Anna; Moore, Katie L.; Mak, Johnson; Sluis‐Cremer, Nicolas; Béthune, Marie‐Pierre de; Tachedjian, Gilda

doi:10.1371/journal.ppat.0020119

Cited by 97 publications

(126 citation statements)

References 55 publications

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“…Some antiretroviral drugs, like potent NNRTIs, display weak activities (<1% of full potency) against secondary steps in the viral lifecycle (36). Low micromolar styrylquinoline-based IN inhibitors can inhibit recombinant IN nuclear transport in vitro (37) and reverse transcription and integration during HIV-1 infection (38), although the contributions of these different activities to compound potency have remained unclear (39).…”

Section: Discussionmentioning

confidence: 99%

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

Jurado

Wang

Slaughter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

184

413

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Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/ transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development. AIDS | antiretroviral therapy

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Section: Discussionmentioning

confidence: 99%

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

Jurado

Wang

Slaughter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

184

413

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“…Samples were centrifuged through a 20% w/v sucrose cushion (100,000 ϫ g, 1 h 4°C) to pellet viral particles. The pellet was lysed, and virion proteins were separated by SDS-PAGE and detected by Western blot analysis using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) and HIV human immune serum as described (33).…”

Section: Methodsmentioning

confidence: 99%

Decoding the Membrane Activity of the Cyclotide Kalata B1

Henriques

Huang

Rosengren

et al. 2011

Journal of Biological Chemistry

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158

116

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Cyclotides, a large family of cyclic peptides from plants, have a broad range of biological activities, including insecticidal, cytotoxic, and anti-HIV activities. In all of these activities, cell membranes seem likely to be the primary target for cyclotides. However, the mechanistic role of lipid membranes in the activity of cyclotides remains unclear. To determine the role of lipid organization in the activity of the prototypic cyclotide, kalata B1 (kB1), and synthetic analogs, their bioactivities and affinities for model membranes were evaluated. We found that the bioactivity of kB1 is dependent on the lipid composition of target cell membranes. In particular, the activity of kB1 requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups but is further modulated by nonspecific peptide-lipid hydrophobic interactions, which are favored in raft-like membranes. Negatively charged phospholipids do not favor high kB1 affinity. This lipid selectivity explains trends in antimicrobial and hemolytic activities of kB1; it does not target bacterial cell walls, which are negatively charged and lacking PE-phospholipids but can insert in the membranes of red blood cells, which have a low PE content and raft domains in their outer layer. We further show that the anti-HIV activity of kB1 is the result of its ability to target and disrupt the membranes of HIV particles, which are raft-like membranes very rich in PE-phospholipids.Cyclotides are plant-derived peptides characterized by a cyclic backbone and three disulfide bonds forming a cystine knot motif (1) (Fig. 1) that confers them with exceptional stability (2). Their natural function appears to be as host defense insecticidal agents (3), but many other activities have also been reported, including uterotonic (4), anti-HIV (5), hemolytic (6), antibacterial (7), anti-cancer (8), and pesticidal action (9). These activities reflect the ability of cyclotides to target cells of different compositions. More than 150 cyclotides have been characterized (10), and their diverse sequences, remarkable stability, and various bioactivities suggest that they have exciting potential as molecular frameworks in drug design (11).It is believed that the activity of cyclotides is mediated by their ability to target and disrupt cell membranes. Such a mechanism of action is consistent with their hemolytic properties (6) and ability to disrupt gut epithelial cells in lepidopteron larvae (3) and is further supported by a range of biophysical studies (12-15). The prototypic cyclotide kalata B1 (kB1) 7 is the most well studied cyclotide, and it has been shown to bind to (12) and disrupt phospholipid bilayers by a pore-forming mechanism (15). Furthermore, in a previous study we showed that the all-D enantiomer (D-kB1) is bioactive, suggesting that activity does not require recognition by a chiral protein receptor (9). In combination, these reports suggest that cyclotides target cell membranes by interacting directly with lipids.Differences in bioactivity are...

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“…Previous studies have shown that EFV acts as a chemical enhancer of RT dimerization (27)(28)(29). Recent studies have indicated that EFV also inhibits the late stage of the HIV-1 life cycle, i.e., virus particle release, through the enhancement of Gag/Gag-Pol processing (30). They have also shown enhanced dimerization of the Pol fragments containing PR-RT in the presence of EFV in a yeast two-hybrid assay (30).…”

mentioning

confidence: 99%

“…Recent studies have indicated that EFV also inhibits the late stage of the HIV-1 life cycle, i.e., virus particle release, through the enhancement of Gag/Gag-Pol processing (30). They have also shown enhanced dimerization of the Pol fragments containing PR-RT in the presence of EFV in a yeast two-hybrid assay (30). Mutations of the tryptophan repeat motif in RT that impaired RT dimerization and polymerase activity (31) rescued the defect of HIV-1 particle release imposed by EFV (32).…”

mentioning

confidence: 99%

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

Sudo

Haraguchi

Hirai

et al. 2013

J Virol

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d Efavirenz (EFV), a nonnucleoside reverse transcriptase (RT) inhibitor, also inhibits HIV-1 particle release through enhanced Gag/Gag-Pol processing by protease (PR). To better understand the mechanisms of the EFV-mediated enhancement of Gag processing, we examined the intracellular localization of Gag/Gag-Pol processing products and their precursors. Confocal microscopy revealed that in the presence of EFV, the N-terminal p17 matrix (p17MA) fragment was uniformly distributed at the plasma membrane (PM) but the central p24 capsid (p24CA) and the Pol-encoded RT antigens were diffusely distributed in the cytoplasm, and all of the above were observed in puncta at the PM in the absence of EFV. EFV did not impair PM targeting of Gag/ Gag-Pol precursors. Membrane flotation analysis confirmed these findings. Such uniform distribution of p17MA at the PM was not seen by overexpression of Gag-Pol and was suppressed when EFV-resistant HIV-1 was used. Forster's fluorescence resonance energy transfer assay revealed that Gag-Pol precursor dimerization occurred mainly at the PM and that EFV induced a significant increase of the Gag-Pol dimerization at the PM. Gag-Pol dimerization was not enhanced when HIV-1 contained the EFV resistance mutation in RT. Bacterial two-hybrid assay showed that EFV enhanced the dimerization of PR-RT fragments and restored the dimerization impaired by the dimerization-defective mutation in the RT tryptophan repeat motif but not that impaired by the mutation at the PR dimer interface. Collectively, our data indicate that EFV enhances Gag-Pol precursor dimerization, likely after PM targeting but before complete particle assembly, resulting in uniform distribution of p17MA to and dissociation of p24CA and RT from the PM.

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Potent Nonnucleoside Reverse Transcriptase Inhibitors Target HIV-1 Gag-Pol

Cited by 97 publications

References 55 publications

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

Decoding the Membrane Activity of the Cyclotide Kalata B1

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

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