Class I histone deacetylase (HDAC) enzymes 1, 2, and 3 organize chromatin as the catalytic subunits within seven distinct multiprotein corepressor complexes and are established drug targets. We report optimization studies of benzamide-based Von Hippel–Lindau (VHL) E3-ligase proteolysis targeting chimeras (PROTACs) and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand, we identified PROTACs 7 , 9 , and 22 with submicromolar DC 50 values for HDAC1 and/or HDAC3 in HCT116 cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2 degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate that HDAC1/2 degradation by PROTACs correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects.

Ultraspiracle (USP) is the invertebrate homologue of the mammalian retinoid X receptor (RXR). RXR plays a uniquely important role in differentiation, development, and homeostasis through its ability to serve as a heterodimeric partner to many other nuclear receptors. RXR is able to influence the activity of its partner receptors through the action of the ligand 9-cis retinoic acid. In contrast to RXR, USP has no known high-affinity ligand and is thought to be a silent component in the heterodimeric complex with partner receptors such as the ecdysone receptor. Here we report the 2.4-A crystal structure of the USP ligand-binding domain. The structure shows that a conserved sequence motif found in dipteran and lepidopteran USPs, but not in mammalian RXRs, serves to lock USP in an inactive conformation. It also shows that USP has a large hydrophobic cavity, implying that there is almost certainly a natural ligand for USP. This cavity is larger than that seen previously for most other nuclear receptors. Intriguingly, this cavity has partial occupancy by a bound lipid, which is likely to resemble the natural ligand for USP.

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

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Infections of vascular prostheses are still a major risk in surgery. The current work presents an in vitro evaluation of novel slow release antibiotic coatings based on new gentamicin fatty acid salts for polytetrafluoroethylene grafts. These grafts were coated with gentamicin sodium dodecyl sulfate, gentamicin laurate and gentamicin palmitate. Drug release kinetics, anti-infective characteristics, biocompatibility and haemocompatibility of developed coatings were compared to commercially available gelatin sealed PTFE grafts (SEALPTFE™) and knitted silver coated Dacron(®) grafts (InterGard(®)). Each gentamicin fatty acid coating showed a continuous drug release in the first eight hours followed by a low continuous release. Grafts coated with gentamicin fatty acids reduced bacterial growth even beyond pathologically relevant high concentrations. Cytotoxicity levels depending on drug formulation bringing up gentamicin palmitate as the most promising biocompatible coating. Thrombelastography studies, ELISA assays and an amidolytic substrate assay confirmed haemocompatibility of developed gentamicin fatty acid coatings comparable to commercially available grafts.

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Co-repressor proteins mediate transcriptional repression by nuclear receptors in the absence of ligand. The identification of a co-repressor-receptor interaction motif, and the finding that co-repressors and co-activators compete for the same site on the receptor, suggests a simple mechanism for the switch from repression to activation upon ligand binding. Defects in this mechanism result in dominant-negative receptors that repress transcription. Such receptors have been implicated in several clinically important diseases, including thyroid hormone resistance and diabetes mellitus.