SUMMARY: This research project builds on an already-initiated joint effort by laboratories at Drexel University in Philadelphia and the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, to pursue the development of new classes of HIV-1 therapeutic inhibitors targeting entry and to investigate the underlying hypothesis that antagonists of HIV-1 Env and host cell co-receptors can be covalently joined into bifunctional synergistic combinations that improve antiviral activity and decrease the susceptibility to function- compromising viral resistance. New agents for HIV-1 intervention remain urgently needed to reduce the global occurrence and spread of AIDS. HIV-1 infection of host cells is initiated by the interaction of the Env protein complex on the exposed surface of the virus with two cell receptors, CD4 and a co-receptor that is most commonly either CCR5 or CXCR4. We believe that combined inhibition of Env and co-receptors would be an effective means to suppress the progression of infection, including at the earliest stages of exposure to the virus, and in addition have the potential to inactivate infected cells expressing both Env and co-receptor proteins on their surface during virus proliferation. In our collaborating laboratories, we have been investigating novel peptide triazole (PT) gp120 antagonists and both CCR5 and CXCR4 co-receptor antagonists. Results obtained in a US-China project, initiated in 2013 funded by R01 AI106633-01, established the synthetic protocol to covalently combine CCR5 and gp120 antagonists to form the bifunctional chimera LJC240-linker4-UM15. Importantly, the chimera has a sub-nanomolar potency in inhibiting virus cell infection and combines both the antiEnv and antiCoR properties of the two component inhibitors. The chimera is specific for CCR5-expressing cells, has minimal if any general cellular toxicity and exhibits no detectable unwanted infection enhancement of CD4-negative cells. The results we have obtained in this first R01 period argue that coordinately acting inhibitors targeting Env and co-receptor can improve the potential use of these types of inhibitors for therapeutic intervention. In the work we propose here, we will expand the bifunctional chimera effort to improve antiviral potency and to include newly discovered and proteolytically-resistant macrocyclic PT's (cPT's) as well as different types of co-receptor inhibitors (CoRI's), in particular for both CXCR4 and CCR5. In addition, the mechanisms by which these chimeras can suppress cell infection as well as already-infected cells need to be better understood. In the Specific Aims of this project, we will [1] expand the synthetic effort to obtain diverse and potency-optimized PT-CoRI chimeras, [2] learn how they act mechanistically at the cell-virus interface and with infected cells, and [3] evaluate the ability of the bifunctional chimeras to better avoid virus mutagenic escape than individual inhibitor components. Overall, this project will lead to potent HIV-1 cell entry inhibitors and inactivators of HIV-1 virions and infected cells, and will expand our knowledge of Env-co-receptor engagement mechanisms.