BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.

Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.

Starting from our previous finding of 14 known drugs as inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC50 values in a kinetic assay. Free-energy perturbation (FEP) calculations for Mpro-ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to Mpro. Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment of COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization.

A 5-μM docking hit has been optimized to an extraordinarily potent (55 pM) non-nucleoside inhibitor of HIV reverse transcriptase. Use of free energy perturbation (FEP) calculations to predict relative free energies of binding aided the optimizations by identifying optimal substitution patterns for phenyl rings and a linker. The most potent resultant catechol diethers feature terminal uracil and cyanovinylphenyl groups. A halogen bond with Pro95 likely contributes to the extreme potency of compound 42. In addition, several examples are provided illustrating failures of attempted grafting of a substructure from a very active compound onto a seemingly related scaffold to improve its activity.

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC50 values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds like rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4-nM activity against the wild-type virus. The compounds also performed well with EC50 values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogs were also investigated; the most potent compounds in these cases have EC50 values towards wild-type HIV-1 near 10 nM and high-nM activities towards the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88-Å resolution for the complex of the parent 2-cyanoindolizine 10b and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogs were also measured to be ca. 40 µg/ml, which is similar to that for the approved drug efavirenz and ca. 1000-fold greater than for rilpivirine.