This project builds on our successful development of a series of molecules that prevent HIV from using the CCR5 coreceptor to enter its target cells. The new molecules are even more powerful than our original lead of this type, AOP-RANTES (IC50 values in vitro are now in the low picomolar range). The whole series appears to function by a novel and promising mechanism, that of receptor sequestration, rather than by receptor competition. Our new proposals, which aim at improving other aspects of the performance of these molecules than just potency and to service other investigators' projects in the Program, fall into five categories: (1) Our molecules have agonist activity on CCR5. Even though this requires higher concentrations than those necessary for antiviral activity, we wish to engineer a reduction of this activity. (2) Our molecules, as is typical of RANTES-derived chemokines, bind to CCR1 and CCR3 as well as to CCR5. We wish to narrow the specificity to CCR5 as far as possible. (3) In order to feed data into the design cycles for the above, we wish to establish more comprehensive pharmacological activity profiles of our lead compounds. We already have about 100 site-specific modifications of the lead and will be adding to that number. Potencies vary over several orders of magnitude. We will use the breadth of this resource to study stimulation of Ca2++ flux, inhibition of cAMP synthesis, activation of the MAP kinase pathway, and chemotaxis, in various leukocyte populations and cell lines. Data from the study of this panel will feed directly into our design cycles for engineering the properties for our next compounds. (4) We will use stable-isotope dilution techniques, developed here for another purpose, to carry out analyses for ourselves and other Program members of the pharmacokinetics of the current lead, PSC- RANTES. This will be in a variety of biological situations relevant to the preclinical and clinical projects of our partners. (5) We have already learned that it is best to target more than one element in the HIV cell entry process. Since we have the technical capacity to do so, we will synthesize and supply to other Program members existing molecules shown by other workers to inhibit through gp41.
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