The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.

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.

We have studied the role of the N-terminal extracellular domain of the human immunodeficiency virus type 1 (HIV-1) coreceptor, CXCR-4, in the entry and fusion of syncytium-inducing strains of HIV-1. Progressive deletions were introduced in the N-terminal extracellular domain of CXCR-4 and the effect on infection by different isolates was tested. Infection of cells expressing the different CXCR-4 deletion mutants by HIV-1 LAI and 89.6 was reduced only about twofold. In contrast, the HIV-1 GUN-1 and RF isolates were substantially more impaired in their ability to mediate cell-free infection and cell-cell fusion. Since LAI and RF are T-cell line-tropic viruses while 89.6 and GUN-1 are dual tropic, no clear correlation between tropism and requirements for CXCR-4 N-terminal sequences emerged. We also introduced point mutations at the two N-linked glycosylation sites. The isolates tested (LAI, RF, GUN-1, and 89.6) were not affected by the removal of predicted N-linked glycosylation sites in CXCR-4. We conclude that distinct virus strains interact differently with the CXCR-4 coreceptor and that the N-terminal extracellular domain is not the sole functional domain important for HIV-1 entry.