In drug discovery, small molecules must often discriminate between healthy and diseased cells. This feat is usually accomplished by binding to a protein that is preferentially expressed in the target cell or on its surface. However, in many cases, the expression of an individual protein may not generate sufficient cyto-selectivity. Here, we demonstrate that bispecific molecules can better discriminate between similar cell types by exploiting their simultaneous affinity for two proteins. Inspired by the natural product FK506, we designed molecules that have affinity for both FKBP12 and HIV protease. Using cell-based reporters and live virus assays, we observed that these compounds preferentially accumulated in cells that express both targets, mimicking an infected lymphocyte. Treatment with FKBP12 inhibitors reversed this partitioning, while overexpression of FKBP12 protein further promoted it. The partitioning into the target cell type could be tuned by controlling the properties of the linker and the affinities for the two proteins. These results show that bispecific molecules create significantly better potential for cyto-selectivity, which might be especially important in the development of safe and effective antivirals and anticancer compounds.

Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.

Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and export of the viral RNA. These complexes are composed of many proteins and are dynamic, making them difficult to fully recapitulate in vitro Therefore, we developed a cell-based reporter assay to monitor the assembly of viral complexes for inhibitor screening. We screened a small-molecule library and identified multiple hits that inhibit the activity of the viral complexes. A subsequent chemistry effort was focused on a thieno[2,3-b]pyridine scaffold, examples of which inhibited HIV replication and the emergence from viral latency. Notable aspects of the effort to determine the structure-activity relationship (SAR) include migration to the regioisomeric thieno[2,3-c]pyridine ring system and the identification of analogs with single-digit nanomolar activity in both reporter and HIV infectivity assays, an improvement of >100-fold in potency over the original hits. These results validate the screening strategy employed and reveal a promising lead series for the development of a new class of HIV therapeutics.