Disha Patel, Janet Antwi, Pratibha C Koneru, Erik Serrao, Stefano Forli, Jacques J Kessl, Lei Feng, Nanjie Deng, Ronald M Levy, James R Fuchs, Arthur J Olson, Alan N Engelman, Joseph D Bauman, Mamuka Kvaratskhelia, Eddy Arnold

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal LEDGF/p75 binding pocket. The most active fragment 5 inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 μM and impaired HIV-1 infection of T cells at an EC50 of 36 μM. These findings are significant because the identified lead fragment with a relatively small molecular weight (221 Da) provides an optimal building block for developing a new class of inhibitors. Furthermore, while structurally distinct thiophenecarboxylic acid-derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead fragment 5 inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs.