Project 1 - The goal of Project 1 is to define the mechanism of action of three new chemokine analogueswith potent activity in blocking HIV-1 entry via CCR5. These fully recombinant molecules display threedistinct activity profiles: (group I) CCR5 blockade without signaling activity or receptor internalization; (groupII), CCR5 rapid internalization with signaling activity; and (group III), moderate CCR5 internalization andblockade without signaling activity. These new molecules have significant potential advantages in terms ofsafety and cost of production ..over currenynh[b^ fwither, irrtpr.pvejTients.,p,nthese bahdidate'microbicides requires more detailed knowledge of their mechanisms of action. We willdefine the route to intracellular sequestration for group II molecules, and compare with that of the group IIImolecules. We will examine a number of hypotheses to explain prolonged antiviral activity in the absence ofintracellular receptor sequestration (group I) or with moderate internalization (group III), including changes inCCR5 dimer formation, altered receptor localization in the membrane, allosteric effects, and receptorinternalization independent of G-protein-linked signaling. We will also examine the impact of CCR5, CCL5,and CCL3L1 genetic polymorphisms on CCR5 protein synthesis and turnover rate. These studies aredesigned to investigate variability in the susceptibility of primary target cells from normal human donors toeach of the new inhibitors. We will also use a panel of mutant CCR5 molecules to examine structuralcorrelates of activity. We will use this information on mechanism and target cell variability to develop newmolecules with even better activity profiles, and we will perform coordinated experiments with other projectsin this Program to relate our findings in cell-based models to the effects of current and new CCR5 inhibitorsin tissue explant and whole animal models. This approach will generate better and safer CCR5 inhibitors thatcan be produced on a scale suitable for stopping the spread of HIV/AIDS in the most-impacted areas.
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