Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.

Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3ß-hydroxy-5-cholenoic acid (3ß-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3ß-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3ß-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3ß-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.

Protein Modification at Tyrosine with Iminoxyl Radicals.

Post-translational modifications (PTMs) of proteins are a biological mechanism for reversibly controlling protein function. Synthetic protein modifications (SPMs) at specific canonical amino acids can mimic PTMs. However, reversible SPMs at hydrophobic amino acid residues in proteins are especially limited. Here, we report a tyrosine (Tyr)-selective SPM utilizing persistent iminoxyl radicals, which are readily generated from sterically hindered oximes via single-electron oxidation. The reactivity of iminoxyl radicals with Tyr was dependent on the steric and electronic demands of oximes; isopropyl methyl piperidinium oxime 1f formed stable adducts, whereas the reaction of tert-butyl methyl piperidinium oxime 1o was reversible. The difference in reversibility between 1f and 1o, differentiated only by one methyl group, is due to the stability of iminoxyl radicals, which is partly dictated by the bond dissociation energy of oxime O-H groups. The Tyr-selective modifications with 1f and 1o proceeded under physiologically relevant, mild conditions. Specifically, the stable Tyr-modification with 1f introduced functional small molecules, including an azobenzene photoswitch, to proteins. Moreover, masking critical Tyr residues by SPM with 1o, and subsequent deconjugation triggered by the treatment with a thiol, enabled on-demand control of protein functions. We applied this reversible Tyr modification with 1o to alter an enzymatic activity and the binding affinity of a monoclonal antibody with an antigen upon modification/deconjugation. The on-demand ON/OFF switch of protein functions through Tyr-selective and reversible covalent-bond formation will provide unique opportunities in biological research and therapeutics.

Palladium-Catalyzed C-H Heteroarylation of 2,5-Disubstituted Imidazoles.

We developed a palladium-catalyzed C-H N-heteroarylation of N-protected-2,5-disubstituted imidazoles at the C4-position using N-heteroaryl halides as a coupling partner. Intensive reaction condition screening led us to identify fluorinated bathophenanthroline 7 as the optimum ligand for the palladium catalyst. This reaction will enhance lead optimization of drug candidates by facilitating the synthesis of heterobiaryl compounds containing an imidazole ring.

Expanding Automated Multiconformer Ligand Modeling to Macrocycles and Fragments.

Small molecule ligands exhibit a diverse range of conformations in solution. Upon binding to a target protein, this conformational diversity is generally reduced. However, ligands can retain some degree of conformational flexibility even when bound to a receptor. In the Protein Data Bank (PDB), a small number of ligands have been modeled with distinct alternative conformations that are supported by X-ray crystallography density maps. However, the vast majority of structural models are fit to a single ligand conformation, potentially ignoring the underlying conformational heterogeneity present in the sample. We previously developed qFit-ligand to sample diverse ligand conformations and to select a parsimonious ensemble consistent with the density. While this approach indicated that many ligands populate alternative conformations, limitations in our sampling procedures often resulted in non-physical conformations and could not model complex ligands like macrocycles. Here, we introduce several improvements to qFit-ligand, including the use of routines within RDKit for stochastic conformational sampling. This new sampling method greatly enriches low energy conformations of small molecules and macrocycles. We further extended qFit-ligand to identify alternative conformations in PanDDA-modified density maps from high throughput X-ray fragment screening experiments. The new version of qFit-ligand improves fit to electron density and reduces torsional strain relative to deposited single conformer models and our previous version of qFit-ligand. These advances enhance the analysis of residual conformational heterogeneity present in ligand-bound structures, which can provide important insights for the rational design of therapeutic agents.

Extensive exploration of structure activity relationships for the SARS-CoV-2 macrodomain from shape-based fragment merging and active learning.

The macrodomain contained in the SARS-CoV-2 non-structural protein 3 (NSP3) is required for viral pathogenesis and lethality. Inhibitors that block the macrodomain could be a new therapeutic strategy for viral suppression. We previously performed a large-scale X-ray crystallography-based fragment screen and discovered a sub-micromolar inhibitor by fragment linking. However, this carboxylic acid-containing lead had poor membrane permeability and other liabilities that made optimization difficult. Here, we developed a shape-based virtual screening pipeline - FrankenROCS - to identify new macrodomain inhibitors using fragment X-ray crystal structures. We used FrankenROCS to exhaustively screen the Enamine high-throughput screening (HTS) collection of 2.1 million compounds and selected 39 compounds for testing, with the most potent compound having an IC50 value equal to 130 µM. We then paired FrankenROCS with an active learning algorithm (Thompson sampling) to efficiently search the Enamine REAL database of 22 billion molecules, testing 32 compounds with the most potent having an IC50 equal to 220 µM. Further optimization led to analogs with IC50 values better than 10 µM, with X-ray crystal structures revealing diverse binding modes despite conserved chemical features. These analogs represent a new lead series with improved membrane permeability that is poised for optimization. In addition, the collection of 137 X-ray crystal structures with associated binding data will serve as a resource for the development of structure-based drug discovery methods. FrankenROCS may be a scalable method for fragment linking to exploit ever-growing synthesis-on-demand libraries.

The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2.

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.

Systematic Study of Heteroarene Stacking Using a Congeneric Set of Molecular Glues for Procaspase-6.

Heteroaromatic stacking interactions are important in drug binding, supramolecular chemistry, and materials science, making protein-ligand model systems of these interactions of considerable interest. Here we studied 30 congeneric ligands that each present a distinct heteroarene for stacking between tyrosine residues at the dimer interface of procaspase-6. Complex X-ray crystal structures of 10 analogs showed that stacking geometries were well conserved, while high-accuracy computations showed that heteroarene stacking energy was well correlated with predicted overall ligand binding energies. Empirically determined KD values in this system thus provide a useful measure of heteroarene stacking with tyrosine. Stacking energies are discussed in the context of torsional strain, the number and positioning of heteroatoms, tautomeric state, and coaxial orientation of heteroarene in the stack. Overall, this study provides an extensive data set of empirical and high-level computed binding energies in a versatile new protein-ligand system amenable to studies of other intermolecular interactions.