Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41

Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.This work has been supported by the Spanish MINECO (project SAF2012-39760-C02, co-financed by the FEDER programme); Plan Nacional de Cooperacion Público-Privada, subprograma INNPACTO (project IPT-2012-0213-060000, co-financed by the FEDER programme), the Comunidad de Madrid (BIPEDD2-CM-S2010/BMDE2457) and by “The Centers of Excellence” of the K.U.Leuven (EF-05/ 15 and PF-10/18). The Spanish MICINN/MINECO are also acknowledged for a grant to E. Rivero-BucetaPeer Reviewe

Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly ¿-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes ¿-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.The Spanish MICINN/MINECO (Project: SAF 201239760-C02-01, co-financed by the FEDER programme); Plan Nacional de Cooperación Público-Privada. Subprograma INNPACTO (IPT-2012-0213-060000, co-financed by the FEDER programme) and the Comunidad de Madrid (BIPEDD2-CM-S2010/BMD-2457) are acknowledged for fi nancial support. The Spanish ICINN/MINECCO is also acknowledged for a grant to E. Rivero-Buceta. We thank Leentje Persoons, Frieda De Meyer, Leen Ingels, Stijn Delmotte, Katrien Geerts, and Inge Vliegen for excellent technical assistance. Financial support of KU Leuven (GOA 10/14; PF 10/18) and the FWO (G-0528.12N) was provided for the antiviral experiments. The integrase studies were supported by the Center for Cancer Research, the Intramural Program of the National Cancer Institute,NIH (Z01-BC 007333).Peer Reviewe

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

The role of N -glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents

BackgroundCarbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development.ResultsWe generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN+ cells and transmission of DC-SIGN-captured virions to CD4+ T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic IIIB and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, IIIB and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1ADA contained gp120 levels similar to the gp120 levels in WT HIV-1ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity.ConclusionsWe revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.status: publishe

Exposure of HIV-1 to a combination of two carbohydrate-binding agents markedly delays drug resistance development and selects for virus strains with compromised fitness

We investigated the effect of combining two potent carbohydrate-binding agents (CBAs) with a complementary resistance profile (based on different N-glycan deletion selections in the HIV envelope glycoprotein gp120) on drug resistance development and viral fitness.status: publishe

Several <i>N</i>-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

<div><p>The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 <i>N</i>-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and <i>N</i>-linked glycans in HIV-1 gp120, with a predominance of <i>N</i>-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, <i>N</i>-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, <i>N</i>-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and <i>N</i>-glycans was also found for the <i>N</i>-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of <i>N</i>-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved <i>N</i>-glycans or by inserting new <i>N</i>-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three <i>N</i>-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new <i>N</i>-glycans upstream of several disulphide bridges, at locations where there was a significant absence of <i>N</i>-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the <i>N</i>-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.</p></div

Infectivity of WT and mutant gp120 HIV-1 strains.

<p>At day 0, CD4⁺ T lymphocyte C8166 cells were infected with similar viral loads of WT or mutant virus strains, based on equal amounts of the p24 capsid protein. For 7 consecutive days, samples were harvested, fixed and subjected to flow cytometry to analyse eGFP expression. (A) Infectivity of mutant viruses lacking disulphide bridges in gp120, due to the mutation of one of the involved cysteines into an alanine. (B) Infectivity of mutant viruses lacking one <i>N</i>-glycosylation site in gp120 due to the mutation of the asparagine of the glycosylation motif into a glutamine. (C) The infectivity as shown in panel B was quantified using a linear regression to part of the curves, from day 1 post infection to the peak of the infectivity curve. Data are the means ± SEM of at least 2 independent experiments. The difference between WT and mutant virus was considered to be significant when the <i>p</i> value calculated using the student’s t-test was <0.05 (* = p<0.05).</p