Using Data Science for Mechanistic Insights and Selectivity Predictions in a Non-Natural Biocatalytic Reaction.

The study of non-natural biocatalytic transformations relies heavily on empirical methods, such as directed evolution, for identifying improved variants. Although exceptionally effective, this approach provides limited insight into the molecular mechanisms behind the transformations and necessitates multiple protein engineering campaigns for new reactants. To address this limitation, we disclose a strategy to explore the biocatalytic reaction space and garner insight into the molecular mechanisms driving enzymatic transformations. Specifically, we explored the selectivity of an "ene"-reductase, GluER-T36A, to create a data-driven toolset that explores reaction space and rationalizes the observed and predicted selectivities of substrate/mutant combinations. The resultant statistical models related structural features of the enzyme and substrate to selectivity and were used to effectively predict selectivity in reactions with out-of-sample substrates and mutants. Our approach provided a deeper understanding of enantioinduction by GluER-T36A and holds the potential to enhance the virtual screening of enzyme mutants.

Comparison of activity, structure, and dynamics of SF-1 and LRH-1 complexed with small molecule modulators.

Steroidogenic factor-1 (SF-1) is a phospholipid-sensing nuclear receptor expressed in the adrenal glands, gonads, and hypothalamus which controls steroidogenesis and metabolism. There is significant therapeutic interest in SF-1 because of its oncogenic properties in adrenocortical cancer. Synthetic modulators are attractive for targeting SF-1 for clinical and laboratory purposes due to the poor pharmaceutical properties of its native phospholipid ligands. While small molecule agonists targeting SF-1 have been synthesized, no crystal structures have been reported of SF-1 in complexes with synthetic compounds. This has prevented the establishment of structure-activity relationships that would enable better characterization of ligand-mediated activation and improvement in current chemical scaffolds. Here, we compare the effects of small molecules in SF-1 and its close homolog, liver receptor homolog-1 (LRH-1), and identify several molecules that specifically activate LRH-1. We also report the first crystal structure of SF-1 in complex with a synthetic agonist that displays low nanomolar affinity and potency for SF-1. We use this structure to explore the mechanistic basis for small molecule agonism of SF-1, especially compared to LRH-1, and uncover unique signaling pathways that drive LRH-1 specificity. Molecular dynamics simulations reveal differences in protein dynamics at the pocket mouth as well as ligand-mediated allosteric communication from this region to the coactivator binding interface. Our studies, therefore, shed important insight into the allostery driving SF-1 activity and show potential for modulation of LRH-1 over SF-1.

Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A.

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

Differential Modulation of Nuclear Receptor LRH-1 through Targeting Buried and Surface Regions of the Binding Pocket.

Liver receptor homologue-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviating intestinal inflammation and metabolic dysregulation in the liver. LRH-1 contains a large ligand-binding pocket, but generating synthetic modulators has been challenging. We have had recent success generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding and residues at the mouth of the pocket to mimic interactions made by phospholipids. Here, we unite these two designs into one molecule to synthesize the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and structural studies, we show that selective modulation can be driven through contacting deep versus surface polar regions in the pocket. While deep pocket contacts convey high affinity, contacts with the pocket mouth dominate allostery and provide a phospholipid-like transcriptional response in cultured cells.

A phospholipid mimetic targeting LRH-1 ameliorates colitis.

Phospholipids are ligands for nuclear hormone receptors (NRs) that regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions is a robust strategy to improve agonists of liver receptor homolog-1 (LRH-1), a therapeutic target for colitis. Conventional LRH-1 modulators only partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. We show that the phospholipid-mimicking groups interact with the targeted residues in crystal structures and improve binding affinity, LRH-1 transcriptional activity, and conformational changes at a key allosteric site. The best phospholipid mimetic markedly improves colonic histopathology and disease-related weight loss in a murine T cell transfer model of colitis. This evidence of in vivo efficacy for an LRH-1 modulator in colitis represents a leap forward in agonist development.

Hydroarylation of Arenes via Reductive Radical-Polar Crossover.

A photocatalytic system for the dearomative hydroarylation of benzene derivatives has been developed. Using a combination of an organic photoredox catalyst and an amine reductant, this process operates through a reductive radical-polar crossover mechanism where aryl halide reduction triggers a regioselective radical cyclization event, followed by anion formation and quenching to produce a range of complex spirocyclic cyclohexadienes. This light-driven protocol functions at room temperature in a green solvent system (aq. MeCN) without the need for precious metal-based catalysts or reagents or the generation of stoichiometric metal byproducts.

Development of a Versatile and Sensitive Direct Ligand Binding Assay for Human NR5A Nuclear Receptors.

As regulators of steroidogenesis, development, and metabolism, the nuclear receptor 5A (NR5A) subfamily members steroidogenic factor 1 (SF-1) and liver receptor homologue 1 (LRH-1) are important pharmacological targets for cancers and metabolic diseases. Evaluation of small molecule modulators and candidate endogenous ligands for these orphan receptors has been hindered by the lack of accessible, robust direct-binding assays. Here, we leverage the potency of our new NR5A agonist (6N) to create a high-affinity probe for fluorescence polarization competition assays by conjugating 6N to fluorescein (FAM). The 6N-FAM probe tightly binds the NR5A receptors and detects direct binding of synthetic and phospholipid ligands. For 25 LRH-1 agonists, affinity predicts potency in cellular activation assays, demonstrating the potential for this assay in drug discovery. Moreover, phospholipids dilauroylphosphatidylcholine and phosphatidylinositol(4,5)phosphate bind with high affinity, demonstrating this assay is robust for evaluation of candidate endogenous ligands for human NR5A receptors.

Development of the First Low Nanomolar Liver Receptor Homolog-1 Agonist through Structure-guided Design.

As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, liver receptor homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases (IBD). Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of IBD, the new agonist increases expression of LRH-1-controlled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility.

Development of Hybrid Phospholipid Mimics as Effective Agonists for Liver Receptor Homologue-1.

The orphan nuclear receptor Liver Receptor Homologue-1 (LRH-1) is an emerging drug target for metabolic disorders. The most effective known LRH-1 modulators are phospholipids or synthetic hexahydropentalene compounds. While both classes have micromolar efficacy, they target different portions of the ligand binding pocket and activate LRH-1 through different mechanisms. Guided by crystallographic data, we combined aspects of both ligand classes into a single scaffold, resulting in the most potent and efficacious LRH-1 agonists to date.

Crystal Structures of the Nuclear Receptor, Liver Receptor Homolog 1, Bound to Synthetic Agonists.

Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face π-π-stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.