Biochemical and metabolic characterization of a G6PC2 inhibitor.

Three glucose-6-phosphatase catalytic subunits, that hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate, have been identified, designated G6PC1-3, but only G6PC1 and G6PC2 have been implicated in the regulation of fasting blood glucose (FBG). Elevated FBG has been associated with multiple adverse clinical outcomes, including increased risk for type 2 diabetes and various cancers. Therefore, G6PC1 and G6PC2 inhibitors that lower FBG may be of prophylactic value for the prevention of multiple conditions. The studies described here characterize a G6PC2 inhibitor, designated VU0945627, previously identified as Compound 3. We show that VU0945627 preferentially inhibits human G6PC2 versus human G6PC1 but activates human G6PC3. VU0945627 is a mixed G6PC2 inhibitor, increasing the Km but reducing the Vmax for G6P hydrolysis. PyRx virtual docking to an AlphaFold2-derived G6PC2 structural model suggests VU0945627 binds two sites in human G6PC2. Mutation of residues in these sites reduces the inhibitory effect of VU0945627. VU0945627 does not inhibit mouse G6PC2 despite its 84% sequence identity with human G6PC2. Mutagenesis studies suggest this lack of inhibition of mouse G6PC2 is due, in part, to a change in residue 318 from histidine in human G6PC2 to proline in mouse G6PC2. Surprisingly, VU0945627 still inhibited glucose cycling in the mouse islet-derived ßTC-3 cell line. Studies using intact mouse liver microsomes and PyRx docking suggest that this observation can be explained by an ability of VU0945627 to also inhibit the G6P transporter SLC37A4. These data will inform future computational modeling studies designed to identify G6PC isoform-specific inhibitors.

Steroidogenic Factor-1 form and function: From phospholipids to physiology.

Steroidogenic Factor-1 (SF-1, NR5A1) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, consisting of a DNA-binding domain (DBD) connected to a transcriptional regulatory ligand binding domain (LBD) via an unstructured hinge domain. SF-1 is a master regulator of development and adult function along the hypothalamic pituitary adrenal and gonadal axes, with strong pathophysiological association with endometriosis and adrenocortical carcinoma. SF-1 was shown to bind and be regulated by phospholipids, one of the most interesting aspects of SF-1 regulation is the manner in which SF-1 interacts with phospholipids: SF-1 buries the phospholipid acyl chains deep in the hydrophobic core of the SF-1 protein, while the lipid headgroups remain solvent-exposed on the exterior of the SF-1 protein surface. Here, we have reviewed several aspects of SF-1 structure, function and physiology, touching on other transcription factors that help regulate SF-1 target genes, non-canonical functions of SF-1, the DNA-binding properties of SF-1, the use of mass spectrometry to identify lipids that associate with SF-1, how protein phosphorylation regulates SF-1 and the structural biology of the phospholipid-ligand binding domain. Together this review summarizes the form and function of Steroidogenic Factor-1 in physiology and in human disease, with particular emphasis on adrenal cancer.

Modular Protein-DNA Cocrystals as Precise, Programmable Assembly Scaffolds.

High-precision nanomaterials to entrap DNA-binding molecules are sought after for applications such as controlled drug delivery and scaffold-assisted structural biology. Here, we engineered protein-DNA cocrystals to serve as scaffolds for DNA-binding molecules. The designed cocrystals, isoreticular cocrystals, contain DNA-binding protein and cognate DNA blocks where the DNA-DNA junctions stack end-to-end. Furthermore, the crystal symmetry allows topology preserving (isoreticular) expansion of the DNA stack without breaking protein-protein contacts, hence providing larger solvent channels for guest diffusion. Experimentally, the resulting designed isoreticular cocrystal adopted an interpenetrating I222 lattice, a phenomenon previously observed in metal-organic frameworks (MOFs). The interpenetrating lattice crystallized dependably in the same space group despite myriad modifications at the DNA-DNA junctions. Assembly was modular with respect to the DNA inserted for expansion, providing an interchangeable DNA sequence for guest-specified scaffolding. Also, the DNA-DNA junctions were tunable, accommodating varied sticky base overhang lengths and terminal phosphorylation. As a proof of concept, we used the interpenetrating scaffold crystals to separately entrap three distinct guest molecules during crystallization. Isoreticular cocrystal design offers a route to a programmable scaffold for DNA-binding molecules, and the design principles may be applied to existing cocrystals to develop scaffolding materials.

New High-Throughput Screen Discovers Novel Ligands of Full-Length Nuclear Receptor LRH-1.

Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is a lipid-regulated transcription factor and an important drug target for several liver diseases. Advances toward LRH-1 therapeutics have been driven recently by structural biology, with fewer contributions from compound screening. Standard LRH-1 screens detect compound-induced interaction between LRH-1 and a transcriptional coregulator peptide, an approach that excludes compounds that regulate LRH-1 through alternative mechanisms. Here, we developed a FRET-based LRH-1 screen that simply detects compound binding to LRH-1, applying it to discover 58 new compounds that bind the canonical ligand-binding site in LRH-1 (2.5% hit rate), also supported by computational docking. Four independent functional screens identified 15 of these 58 compounds to also regulate LRH-1 function in vitro or in living cells. Although one of these 15 compounds, abamectin, directly binds LRH-1 and regulates full-length LRH-1 in cells, abamectin failed to regulate the isolated ligand-binding domain in standard coregulator peptide recruitment assays using PGC1α, DAX-1, or SHP. Abamectin treatment of human liver HepG2 cells selectively regulated endogenous LRH-1 ChIP-seq target genes and pathways associated with known LRH-1 functions in bile acid and cholesterol metabolism. Thus, the screen reported here can discover compounds not likely to have been identified in standard LRH-1 compound screens but which bind and regulate full-length LRH-1 in cells.