Julie Hawkins

Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with Kd = 170 nM at the neutral pH of plasma, but with a Kd as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.

Pharmacologic Inhibition of Site 1 Protease Activity Inhibits Sterol Regulatory Element-Binding Protein Processing and Reduces Lipogenic Enzyme Gene Expression and Lipid Synthesis in Cultured Cells and Experimental Animals

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 μM PF-429242 (Bioorg Med Chem Lett 17:4411–4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC50 of 0.5 μM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.

Function and distribution of circulating human PCSK9 expressed extrahepatically in transgenic mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is predominantly expressed in liver and regulates cholesterol metabolism by down regulating liver LDL receptor (LDLR) proteins. Here we report transgenic overexpression of human PCSK9 in kidney increased plasma levels of PCSK9 and subsequently led to a dramatic reduction in liver LDLR proteins. The regulation of LDLR by PCSK9 displayed tissue specificity, with liver being the most responsive tissue. Even though the PCSK9 transgene was highly expressed in kidney, LDLR proteins were suppressed to a lower extent in this tissue than in liver. Adrenal LDLR proteins were not regulated by elevated plasma PCSK9. hPCSK9 transgene expression and subsequent reduction of liver LDLR led to increases in plasma total cholesterol, LDL cholesterol, and ApoB, which were further increased by a high-fat, high-cholesterol diet. We also observed that the size distribution of hPCSK9 in transgenic mouse plasma was heterogeneous. In chow-fed mice, the majority of PCSK9 proteins were in free forms; however, feeding a high-fat, high-cholesterol diet resulted in a shift of hPCSK9 distribution toward larger complexes. PCSK9 distribution in human plasma also exhibited heterogeneity and individual variability in the percentage of PCSK9 in free form and in large complexes. We provide strong evidence to support that human PCSK9 proteins secreted from extrahepatic tissue are able to promote LDLR degradation in liver and increase plasma LDL. Our data also suggest that LDLR protein regulation by PCSK9 has tissue specificity, with liver being the most responsive tissue.

Discovery of imidazo[1,5-a]pyridines and -pyrimidines as potent and selective RORc inverse agonists.

The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.

Discovery of 1-{4-[3-Fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): a Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C (RORc or RORγ) Inverse Agonist

Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the previously reported tertiary sulfonamide 1 as a starting point, we engineered structural modifications that significantly improved human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile. The most advanced δ-sultam compound, GNE-3500 (27, 1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone), possessed favorable RORc cellular potency with 75-fold selectivity for RORc over other ROR family members and >200-fold selectivity over 25 additional nuclear receptors in a cell assay panel. The favorable potency, selectivity, in vitro ADME properties, in vivo PK, and dose-dependent inhibition of IL-17 in a PK/PD model support the evaluation of 27 in preclinical studies.

Identification of tertiary sulfonamides as RORc inverse agonists.

Screening a nuclear receptor compound subset in a RORc biochemical binding assay revealed a benzylic tertiary sulfonamide hit. Herein, we describe the identification of compounds with improved RORc biochemical inverse agonist activity and cellular potencies. These improved compounds also possessed appreciable selectivity for RORc over other nuclear receptors.

Design and synthesis of novel muscarinic agonists containing the 1,2,4-triazine ring as an ester bioisostere

Abstract Replacement of the ester group in methyl quinuclidine-3-car☐ylate1 with a 1,2,4-triazine ring afforded the high affinity muscarinic partial agonist6a. Analogues7a,7b and7d which incorporate the 1-azabicyclo[2.2.1]heptane ring also display high affinity for muscarinic receptors.