Structure-Based Identification and Neutralization Mechanism of Tyrosine Sulfate Mimetics That Inhibit HIV-1 Entry

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.
ACS Chemical Biology (Impact Factor: 5.33). 08/2011; 6(10):1069-77. DOI: 10.1021/cb200068b
Source: PubMed


Tyrosine sulfate-mediated interactions play an important role in HIV-1 entry. After engaging the CD4 receptor at the cell surface, the HIV-1 gp120 glycoprotein binds to the CCR5 co-receptor via an interaction that requires two tyrosine sulfates, at positions 10 and 14 in the CCR5-N terminus. Building on previous structure determinations of this interaction, here we report the targeting of these tyrosine sulfate binding sites for drug design through in silico screening of small molecule libraries, identification of lead compounds, and characterization of biological activity. A class of tyrosine sulfate-mimicking small molecules containing a "phenyl sulfonate-linker-aromatic" motif was identified that specifically inhibited binding of gp120 to the CCR5-N terminus as well as to sulfated antibodies that recognize the co-receptor binding region on gp120. The most potent of these compounds bound gp120 with low micromolar affinity and its CD4-induced conformation with K(D)'s as tight as ∼50 nM. Neutralization experiments suggested the targeted site to be conformationally inaccessible prior to CD4 engagement. Primary HIV-1 isolates were weakly neutralized, preincubation with soluble CD4 enhanced neutralization, and engineered isolates with increased dependence on the N terminus of CCR5 or with reduced conformational barriers were neutralized with IC(50) values as low as ∼1 μM. These results reveal the potential of targeting the tyrosine sulfate interactions of HIV-1 and provide insight into how mechanistic barriers, evolved by HIV-1 to evade antibody recognition, also restrict small-molecule-mediated neutralization.

Download full-text


Available from: Mark K Louder
  • Source
    • "Although protein-protein interfaces are often relatively featureless and devoid of traditional cavities into which a small molecule can dock, the realization that the gp120 coreceptor binding site displays a restricted number of functionally important basic residues has very recently attracted the attention of many studies. Many of them reported that anionic molecules target the CD4i epitope, as shown by their ability to competitively inhibit mAb 17b binding with IC 50 in the 1–100 mM range (Acharya et al., 2011; Brower et al., 2009; Cohen et al., 2008; Cormier et al., 2000; Crublet et al., 2008; Dervillez et al., 2010; Farzan et al., 2000; Kwong et al., 2011; Seitz et al., 2010). HS belongs to this class of CD4i domain targeting molecules (Crublet et al., 2008), and a highly sulfated and regular sequence comprising 12 monosaccharide units has been recently prepared. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The HIV-1 envelope gp120, which features both the virus receptor (CD4) and coreceptor (CCR5/CXCR4) binding sites, offers multiple sites for therapeutic intervention. However, the latter becomes exposed, thus vulnerable to inhibition, only transiently when the virus has already bound cellular CD4. To pierce this defense mechanism, we engineered a series of heparan sulfate mimicking tridecapeptides and showed that one of them target the gp120 coreceptor binding site with μM affinity. Covalently linked to a CD4-mimetic that binds to gp120 and renders the coreceptor binding domain available to be targeted, the conjugated tridecapeptide now displays nanomolar affinity for its target. Using solubilized coreceptors captured on top of sensorchip we show that it inhibits gp120 binding to both CCR5 and CXCR4 and in peripheral blood mononuclear cells broadly inhibits HIV-1 replication with an IC(50) of 1 nM.
    Full-text · Article · Jan 2012 · Chemistry & biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.
    No preview · Article · Mar 2012 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Since the discovery of human immunodeficiency virus (HIV) as a causative agent of acquired immune deficiency syndrome (AIDS) various strategies were employed to counter its devastating actions. One such concept relies on the prevention of HIV entry into host's "competent" cells by means of compounds known as entry inhibitors. HIV entry inhibitors comprise a group of immensely diverse compounds ranging from proteins/antibodies to small organic molecules and capable of targeting various stages of viral entry. Although already in clinical use, this approach to HIV therapy is still being investigated to produce new promising antiviral compounds. Here, we review the latest advances in this area.
    Full-text · Article · Oct 2012 · Current pharmaceutical design
Show more