In our search for novel compounds active against HIV-1 we revealed that certain antimicrobial peptides, the cationic theta-defensins from primates, could effectively inhibit HIV-1 infection in vitro. Human retrocyclins are unique theta-defensins [unreadable] due to a premature termination codon in their signal sequences, the mRNA transcripts but not the peptides are produced in vivo. We discovered retrocyclins, utilized solid-phase peptide synthesis to recreate these evolutionarily [unreadable]lost[unreadable] human theta-defensins based on their genetic code, and determined that they are active against a broad range of microbes including all strains of HIV-1, HIV-2 and SIV tested. Retrocyclins prevent HIV-1 entry by binding to the heptad repeat 2 (HR2) region of HIV-1 gp41, which precludes six-helix bundle (6HB) formation and subsequent viral fusion. Retrocyclins are macrocyclic, thus resistant to exoproteases, are small (18 residues) and are stabilized by three intramolecular disulfide bonds, which in part confer their resistance to boiling temperatures. We have also reported that retrocyclins retained complete activity in the presence of cervicovaginal fluids and tissues and were neither toxic nor proinflammatory to that mucosa. Moreover, in our initial studies, although HIV-1 could generate resistance to the retrocyclin RC-101, unlike other small molecule entry inhibitors, the level of resistance was modest and could be abrogated by either increasing retrocyclin concentration or by substituting amino acids in the retrocyclin primary structure. The current project is the natural evolution of our collective studies designed to characterize the anti-HIV mechanism of retrocyclins and develop next-generation retrocyclins. To continue these studies, we propose two specific aims. Aim 1 proposes to develop next generation retrocyclin analogs that prevent or significantly delay resistance in HIV. Herein, we will design and optimize next generation retrocyclin peptides, characterize retrocyclin peptides that prevent or significantly delay the emergence of resistant HIV-1, assess whether HIV-1 gains resistance to retrocyclins through a universal pattern of gp41 mutations, and determine if there are secondary consequences of gp41[unreadable]s evolved resistance to fusion inhibitors such as retrocyclins. Aim 2 will evaluate the effects of retrocyclins on bacterial and host determinants of HIV infection. Specifically, we will determine whether retrocyclins work in concert with cationic polypeptides of cervicovaginal fluid (CVF) to prevent HIV-1 infection, characterize the effects of retrocyclins on bacterial vaginosis (BV)-mediated suppression of innate immunity of the human cervicovaginal mucosa, and evaluate whether retrocyclins remain structurally intact upon interacting with cervicovaginal tissues and BV-related bacteria. Our proposed studies to examine mechanisms of viral resistance to retrocyclins as well as the effect of physiological factors of the cervicovaginal mucosa on the anti-HIV activity of retrocyclins will mimic the natural usage of retrocyclins in the prevention of HIV-1 infection in humans.