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 used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open reading frames or y-genes) and conserved in the compact genome of Mycoplasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to screen for essential E. coli genes. To verify this phenotype, we constructed conditional mutants in genes for which no null mutants could be obtained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ). The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Bacillus subtilis. This low number of essential genes was unexpected and might be due to a characteristic of the versatile genomes of E. coli and B. subtilis that is comparable to the phenomenon of nonorthologous gene displacement. The gene ygjD, encoding a sialoglycoprotease, was eliminated from a minimal genome computationally derived from a comparison of the Haemophilus influenzae and M. genitalium genomes. We show that ygjD and its ortholog ydiE are essential in E. coli and B. subtilis, respectively. Thus, we include this gene in a minimal genome. This study systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown function required for survival on complex media.

C-C chemokine receptor 5 (CCR5) is the primary coreceptor for human immunodeficiency virus type 1 (HIV-1) infection. Native chemokines that bind to CCR5 inhibit HIV-1 infection, albeit weakly, but chemically modified chemokines inhibit infection more efficiently. We have investigated the inhibitory mechanism of three N-terminally modified RANTES variants (AOP-, NNY-, and PSC-RANTES) with the MT-2 human T-cell line stably expressing either native or mutated CCR5. The RANTES analogues showed the same rank order (PSC > NNY > AOP) in their capacity to induce prolonged CCR5 internalization, inhibit surface reexpression, and prevent HIV-1 infection on MT-2 cells expressing wild-type CCR5 or CCR5 with four C-terminal serine phosphorylation sites mutated to alanine. None of the RANTES analogues caused internalization of a Cterminal cytoplasmic domain deletion mutant of CCR5, and each derivative had equal potency in inhibiting HIV-1 infection of MT-2 cells expressing this mutant. We conclude that the C-terminal cytoplasmic residues of CCR5 are necessary for receptor sequestration by RANTES analogues but that the process and the relative activity of each derivative are not dependent upon phosphorylation of the C-terminal serine residues. Two mechanisms of antiviral activity are demonstrated: receptor blockade and receptor sequestration. Potency correlates with the ability to induce CCR5 sequestration but not with receptor binding, suggesting that sequestration may make the greater contribution to antiviral activity.

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