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The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl 1 ]RANTES(2-68), known as AOP–RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure–activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES { N -nonanoyl, des-Ser 1 [ l -thioproline 2 , l -cyclohexylglycine 3 ]-RANTES(2–68)}, which is 50 times more potent than AOP–RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.

The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized “buttons” containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.

Cell-free human immunodeficiency virus type 1 (HIV-1) can initiate infections, but contact between infected and uninfected T cells can enhance viral spread through intercellular structures called virological synapses (VS). The relative contribution of VS to cell-free viral transfer has not been carefully measured. Using an ultrasensitive, fluorescent virus transfer assay, we estimate that when VS between HIV-expressing Jurkat T cells and primary CD4 + T cells are formed, cell-associated transfer of virus is 18,000-fold more efficient than uptake of cell-free virus. Furthermore, in contrast to cell-free virus uptake, the VS deposits virus rapidly into focal, trypsin-resistant compartments in target T cells. This massive virus internalization requires Env-CD4 receptor interactions but is resistant to inhibition by patient-derived neutralizing antisera that inhibit homologous cell-free virus. Deleting the Env cytoplasmic tail does not abrogate VS-mediated transfer, but it renders the VS sensitive to neutralizing antibodies, suggesting that the tail limits exposure of VS-neutralizing epitopes on the surface of infected cells. Dynamic live imaging of the VS reveals that HIV-expressing cells are polarized and make sustained, Env-dependent contacts with target cells through uropod-like structures. The polarized T-cell morphology, Env-CD4 coordinated adhesion, and viral transfer from HIV-infected to uninfected cells suggest that VS allows HIV-1 to evade antibody neutralization and to disseminate efficiently. Future studies will discern to what extent this massive viral transfer contributes to productive infection or viral dissemination through the migration of virus-carrying T cells.

N-terminal proteolytic processing modulates the biological activity and receptor specificity of RANTES/CCL5. Previously, we showed that an unidentified protease associated with monocytes and neutrophils digests RANTES into a variant lacking three N-terminal residues (4-68 RANTES). This variant binds CCR5 but exhibits lower chemotactic and antiviral activities than unprocessed RANTES. In this study, we characterize cathepsin G as the enzyme responsible for this processing. Cell-mediated production of the 4-68 variant was abrogated by Eglin C, a leukocyte elastase and cathepsin G inhibitor, but not by the elastase inhibitor elastatinal. Further, anti-cathepsin G antibodies abrogated RANTES digestion in neutrophil cultures. In accordance, reagent cathepsin G specifically digested recombinant RANTES into the 4-68 variant. AOP-RANTES and Met-RANTES were also converted into the 4-68 variant upon exposure to cathepsin G or neutrophils, while PSC-RANTES was resistant to such cleavage. Similarly, macaque cervicovaginal lavage samples digested Met-RANTES and AOP-RANTES, but not PSC-RANTES, into the 4-68 variant and this processing was also inhibited by anti-cathepsin G antibodies. These findings suggest that cathepsin G mediates a novel pathway for regulating RANTES activity and may be relevant to the role of RANTES and its analogs in preventing HIV infection.

Prostratin is an unusual non-tumour promoting phorbol ester with potential as an inductive adjuvant therapy for highly active antiretroviral therapy (HAART) due to its ability to up-regulate viral expression from latent provirus. In addition, prostratin is also able to inhibit de novo HIV infection most probably because it induces down-regulation of HIV receptors from the surface of target cells. In this study, we investigate the mechanisms by which prostratin down-regulates HIV receptor and co-receptor surface expression in lymphocytic and monocytic cell lines. Our results indicate that prostratin induces down-regulation of surface expression of CD4 and CXCR4, but not CCR5, in various cell lines. Down-regulation of CD4 and CXCR4 by prostratin is achieved by internalization through receptor-mediated endocytosis and/or macropinocytosis, which is then followed by degradation of these molecules. Because prostratin is a protein kinase C (PKC) activator, we next examined the potential contribution of distinct PKC isoforms to down-regulate CD4 and CXCR4 in response to prostratin stimulation. Although exposure of cells to prostratin or phorbol-myristate-acetate (PMA) induces the translocation of several PKC isoforms to the plasma membrane, the use of specific PKC inhibitors revealed that novel PKCs are the main mediators of the prostratin-induced CD4 down-regulation, whereas both conventional and novel PKCs contribute to CXCR4 down-regulation. Altogether these results showed that prostratin, through the activation of conventional and/or novel PKC isoforms, rapidly reduces cell surface expression of CD4 and CXCR4, but not CCR5, by inducing their internalization and degradation.

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other β-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC 50 ) ranged from 0.04 nM with HIV-1 A-92RW009 to 1.3 nM with HIV-1 B-BaL . Experiments performed on the same isolate (HIV-1 B-BaL ) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC 50 values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC 50 values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.