Recombination between variants from genital tract and plasma: evolution of multidrug-resistant HIV type 1.

Multidrug-resistant (MDR) HIV-1 presents a challenge to the efficacy of antiretroviral therapy (ART). To examine mechanisms leading to MDR variants in infected individuals, we studied recombination between single viral genomes from the genital tract and plasma of a woman initiating ART. We determined HIV-1 RNA sequences and drug resistance profiles of 159 unique viral variants obtained before ART and semiannually for 4 years thereafter. Soon after initiating zidovudine, lamivudine, and nevirapine, resistant variants and intrapatient HIV-1 recombinants were detected in both compartments; the recombinants had inherited genetic material from both genital and plasma-derived viruses. Twenty-three unique recombinants were documented during 4 years of therapy, comprising ~22% of variants. Most recombinant genomes displayed similar breakpoints and clustered phylogenetically, suggesting evolution from common ancestors. Longitudinal analysis demonstrated that MDR recombinants were common and persistent, demonstrating that recombination, in addition to point mutation, can contribute to the evolution of MDR HIV-1 in viremic individuals.

Library-based discovery and characterization of daphnane diterpenes as potent and selective HIV inhibitors in Daphne gnidium.

Despite the existence of an extended armamentarium of effective synthetic drugs to treat HIV, there is a continuing need for new potent and affordable drugs. Given the successful history of natural product based drug discovery, a library of close to one thousand plant and fungal extracts was screened for antiretroviral activity. A dichloromethane extract of the aerial parts of Daphne gnidium exhibited strong antiretroviral activity and absence of cytotoxicity. With the aid of HPLC-based activity profiling, the antiviral activity could be tracked to four daphnane derivatives, namely, daphnetoxin (1), gnidicin (2), gniditrin (3), and excoecariatoxin (4). Detailed anti-HIV profiling revealed that the pure compounds were active against multidrug-resistant viruses irrespective of their cellular tropism. Mode of action studies that narrowed the site of activity to viral entry events suggested a direct interference with the expression of the two main HIV co-receptors, CCR5 and CXCR4, at the cell surface by daphnetoxin (1).

Thiated derivatives of 2',3'-dideoxy-3'-fluorothymidine: synthesis, in vitro anti-HIV-1 activity and interaction with recombinant drug resistant HIV-1 reverse transcriptase forms.

Various thiated analogues of thymine 2',3'-dideoxy-3'-fluoronucleoside (FLT) and their 5'-monophosphates and 5'-triphosphates were prepared with the use of modified multistep procedures. The thiated analogues of FLT and FLTMP were evaluated against the wild type and drug- and multidrug-resistant strains of HIV-1, using the replicative phenotyping format of the deCIPhR assay, and showed potent inhibition of drug-resistant HIV-1 strains at low cytotoxicity. Additionally, inhibition of recombinant drug resistant forms of reverse transcriptase from single and multiple HIV-1 mutants by the synthesized 5'-triphosphates was investigated. The strongest inhibition was observed for K103N and Δ67 mutants and the most potent anti-HIV-1 activity against drug resistant strains and the lowest cytotoxicity was exerted by S4FLTMP and FLTMP which may be regarded as potential anti-HIV/AIDS agents.

Replicative phenotyping adds value to genotypic resistance testing in heavily pre-treated HIV-infected individuals--the Swiss HIV Cohort Study.

BACKGROUND: Replicative phenotypic HIV resistance testing (rPRT) uses recombinant infectious virus to measure viral replication in the presence of antiretroviral drugs. Due to its high sensitivity of detection of viral minorities and its dissecting power for complex viral resistance patterns and mixed virus populations rPRT might help to improve HIV resistance diagnostics, particularly for patients with multiple drug failures. The aim was to investigate whether the addition of rPRT to genotypic resistance testing (GRT) compared to GRT alone is beneficial for obtaining a virological response in heavily pre-treated HIV-infected patients. METHODS: Patients with resistance tests between 2002 and 2006 were followed within the Swiss HIV Cohort Study (SHCS). We assessed patients' virological success after their antiretroviral therapy was switched following resistance testing. Multilevel logistic regression models with SHCS centre as a random effect were used to investigate the association between the type of resistance test and virological response (HIV-1 RNA <50 copies/mL or ≥1.5 log reduction). RESULTS: Of 1158 individuals with resistance tests 221 with GRT+rPRT and 937 with GRT were eligible for analysis. Overall virological response rates were 85.1% for GRT+rPRT and 81.4% for GRT. In the subgroup of patients with >2 previous failures, the odds ratio (OR) for virological response of GRT+rPRT compared to GRT was 1.45 (95% CI 1.00-2.09). Multivariate analyses indicate a significant improvement with GRT+rPRT compared to GRT alone (OR 1.68, 95% CI 1.31-2.15). CONCLUSIONS: In heavily pre-treated patients rPRT-based resistance information adds benefit, contributing to a higher rate of treatment success.

Discovery of novel, highly potent and selective beta-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles.

Novel highly potent CXCR4 inhibitors with good pharmacokinetic properties were designed and optimized starting from the naturally occurring beta-hairpin peptide polyphemusin II. The design involved incorporating important residues from polyphemusin II into a macrocyclic template-bound beta-hairpin mimetic. Using a parallel synthesis approach, the potency and ADME properties of the mimetics were optimized in iterative cycles, resulting in the CXCR4 inhibitors POL2438 and POL3026. The inhibitory potencies of these compounds were confirmed in a series of HIV-1 invasion assays in vitro. POL3026 showed excellent plasma stability, high selectivity for CXCR4, favorable pharmacokinetic properties in the dog, and thus has the potential to become a therapeutic compound for application in the treatment of HIV infections (as an entry inhibitor), cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and in stem cell transplant therapy.

Natural polymorphisms in the protease gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure.

BACKGROUND: Genetic variation in the HIV-1 pol gene, which encodes the main targets for anti-HIV drugs, may favors different susceptibility and resistance pathways to antiretroviral agents. Several amino acid substitutions occur frequently in some non-B viruses at positions associated with drug resistance in clade B viruses. The clinical relevance of those polymorphisms is unclear. OBJECTIVE: To evaluate the effect of two natural protease (PR) polymorphisms, K20I and M36I, which are frequently found in non-B subtypes, on the virus replicative capacity in the presence and absence of protease inhibitors (PI). STUDY DESIGN: Infectious HIV-1 clones carrying K20I, M36I or K20I/M36I were designed. Their replication kinetics were analyzed by viral competition in the absence of PI. Susceptibility to six different PI was phenotypically assessed in clones and in recombinant viruses carrying non-B proteases from 16 drug-naive individuals. RESULTS: In the absence of drug, the M36I clone replicated more rapidly than wt (wild type) or the double mutant K20I/M36I. Natural polymorphisms 20I and/or 36I improved the virus replicative capacity under drug pressure, reducing the susceptibility to saquinavir and indinavir, with IC(50) values 2-3.5-fold higher than wt. All but one drug-naive individual carrying non-B viruses were fully susceptibility to all tested PI, suggesting that additional substitutions within the PR might compensate the reduced PI susceptibility caused by K20I and/or M36I. CONCLUSION: Natural PR polymorphisms in non-B HIV-1 variants can influence in vitro the virus replication capacity in the presence and/or absence or certain PI. Hypothetically, the improved viral replication of mutant 36I might favor a more rapid spreading of non-B subtypes of HIV-1.

The peptidyl-prolyl isomerase Pin1 regulates phospho-Ser77 retinoic acid receptor alpha stability.

Peptidyl-prolyl isomerases (PPIase) facilitate the cis-trans interconversion of the peptidyl-prolyl bond and in such way affect protein folding. Pin1 is a PPIase, which specifically recognizes phosphorylated S/T-P bonds. The transcription factor TFIIH mediates phosphorylation of the retinoic acid receptor alpha (RARalpha) at position Ser77. In the presence of retinoic acid ligand (RA), the Ser77 non-phosphorylated receptor is suggested to undergo degradation through the proteasome pathway. Here we provide evidence that Pin1 is able to selectively destabilize RARalpha in a ligand independent-manner. We show that this is caused by RARalpha ubiquitination, which in turn is phosphorylation dependent. The single mutation Ser77>A completely abolishes RARalpha degradation whereas the mutation Ser77>E rescues this effect. In addition, we correlate RARalpha stability to Ser77 phosphorylation required for the ligand independent transcriptional activity on fgf8 promoter. Finally, we show that the ligand-independent Ser77 phosphorylation requires the genuine ligand-binding domain.

Promoter of FGF8 reveals a unique regulation by unliganded RARalpha.

We previously reported that retinoids were inducing a complete switch in the expression of two isoforms from the fgf8 gene. In order to gain insight into the transcriptional mechanisms possibly involved in this regulation, we cloned and sequenced a fragment of genomic DNA encompassing 6 kb of the region 5' upstream of the fgf8 coding sequence and investigated its promoter elements. A comprehensive series of biochemical and cellular experiments determined two distinct functional regions cis-responsive to retinoids and/or their receptors: (i) a canonical RARE (type DR2) which is the cis target of a RARalpha-RXRalpha liganded heterodimer; and (ii) a completely novel type of response element composed of two half-binding sites separated by 87 nucleotides, which we demonstrate to be the target of an unliganded RARalpha homodimer phosphorylated on the Ser77 residue. Combined activities of these cis and trans-acting factors support a model of a complex regulation of fgf8 expression: by alternative binding to two distinct promoter elements, phosphorylated-unliganded-RARalpha homodimer or its liganded form have two distinct and mutually exclusive trans-activating activities, explaining the expression of two different isoforms of fgf8.