We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5 human immunodeficiency virus type 1 (HIV-1) including the potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, although mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue by residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1.Highly active antiretroviral therapy has brought about a major impact on the acquired immunodeficiency syndrome (AIDS) epidemics in industrially advanced nations (1, 2), however, eradication of HIV-1 2 appears to be currently impossible mainly because of the viral reservoirs remaining in blood and infected tissues (3). Successful antiviral drugs, in theory, exert their virus-specific effects by interacting with viral components such as viral genes or their transcripts without disturbing cellular metabolisms or functions (2). However, at present, no antiretroviral drugs or agents have been demonstrated to be completely specific for HIV-1 and devoid of toxicity or side effects in the therapy of AIDS (4). Limitations of antiviral therapy of AIDS are exacerbated by complicated regimens, emergence of drug-resistant HIV-1 variants (1), and a number of inherent adverse effects (5).Thus, identification of new antiretroviral drugs that have unique mechanisms of action and produce no or least minimal side effects remains an important therapeutic objective (2, 4). CCR5 is a member of the G protein-coupled, seven-transmembrane segment receptors, which comprise the largest superfamily of proteins in the body (6). In 1996, it was revealed that CCR5 serves as one of the two essential coreceptors for HIV-1 entry to human CD4 ϩ cells, thereby serving as an attractive target for possible intervention of HIV-1 infection (7-10). Aplaviroc (AVC; AK602/ONO4128/873140; Fig. 1), a novel spirodiketopiperazine derivative, represents a CCR5 inhibitor that specifically binds to human CCR5 with a high affinity, greatly blocks HIV-1-gp120/ CCR5 binding, and exerts potent activity against a wide spectrum of ...

In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.

The human monoclonal antibody, mAb 2F5, has broad HIV-1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high-affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme-linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end-capped, linear nonapeptide, Ac-LELDKWASL-amide, was sufficient to attain maximal affinity within the set of native gp41-sequence peptides assayed. Scanning single-residue alanine and d-residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high-affinity binding of the linear nonapeptide. Further studies of side-chain and backbone-modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu(3) and Trp(6) residues. The nine-residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15-residue, disulfide-bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C-peptide helix immediately adjacent to the N-terminal end of the ELDKWA epitope were examined through the synthesis of helix-initiated analogs. Circular dichroism (CD) studies indicated that the alpha-helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV-neutralizing actions of 2F5.

SummaryA cluster of promising epitopes for the development of HIV vaccines is located in the membraneproximal external region (MPER) of the gp41 subunit of the HIV envelope spike structure. The crystal structure of the peptide corresponding to the so-called ELDKWA epitope (HIV-1 HxB2 gp41 residues 662-668) in complex with the corresponding broadly neutralizing human monoclonal antibody 2F5 provides a target for structure-based vaccine design strategies aimed at finding macromolecular carriers able to present this MPER-derived epitope with optimal antigenic activity. To this end, a series of replica exchange molecular dynamics computer simulations was conducted to characterize the distributions of conformations of ELDKWA-based epitopes inserted on a rhinovirus carrier and to identify those with the highest fraction of conformations able to bind 2F5. The length, the hydrophobic character, and the precise site of insertion were found to be critical for achieving structural similarity to the target crystal structure. A construct was obtained with a high degree of complementarity to the corresponding determinant region of 2F5. This construct was employed to build a high-resolution structural model of the complex between the 2F5 antibody and the chimeric HRV14:HIV-1 ELDKWA virus particle. Additional simulations, conducted to study the conformational propensities of the ELDKWA region in solution, confirm the hypothesis that the ELDKWA region of gp41 is highly flexible and capable of assuming helical conformations, as in the post-fusion helical bundle structure, as well as β-turn conformations, as in the complex with the 2F5 antibody. These results also suggest that the ELDKWA epitope can be involved in intramolecular and likely intermolecular hydrophobic interactions. This tendency offers an explanation for the observation that mutations decreasing the hydrophobic character of the MPER in many cases result in conformational changes that increase the affinity of this region for the 2F5 antibody.