Discovery of a Novel Series of Potent, Selective, Orally Available, and Brain-Penetrable C1s Inhibitors for Modulation of the Complement Pathway.

A novel series of non-amidine-based C1s inhibitors have been explored. Starting from high-throughput screening hit 3, isoquinoline was replaced with 1-aminophthalazine to enhance C1s inhibitory activity while exhibiting good selectivity against other serine proteases. We first disclose a crystal structure of a complex of C1s and a small-molecule inhibitor (4e), which guided structure-based optimization around the S2 and S3 sites to further enhance C1s inhibitory activity by over 300-fold. Improvement of membrane permeability by incorporation of fluorine at the 8-position of 1-aminophthalazine led to identification of (R)-8 as a potent, selective, orally available, and brain-penetrable C1s inhibitor. (R)-8 significantly inhibited membrane attack complex formation induced by human serum in a dose-dependent manner in an in vitro assay system, proving that selective C1s inhibition blocked the classical complement pathway effectively. As a result, (R)-8 emerged as a valuable tool compound for both in vitro and in vivo assessment.

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides.

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.

Structure-activity relationships and key structural feature of pyridyloxybenzene-acylsulfonamides as new, potent, and selective peroxisome proliferator-activated receptor (PPAR) γ Agonists.

In our search for a novel class of non-TZD, non-carboxylic acid peroxisome proliferator-activated receptor (PPAR) γ agonists, we explored alternative lipophilic templates to replace benzylpyrazole core of the previously reported agonist 1. Introduction of a pentylsulfonamide group into arylpropionic acids derived from previous in-house PPARγ ligands succeeded in the identification of 2-pyridyloxybenzene-acylsulfonamide 2 as a lead compound. Docking studies of compound 2 suggested that a substituent para to the central benzene ring should be incorporated to effectively fill the Y-shaped cavity of the PPARγ ligand-binding domain (LBD). This strategy led to significant improvement of PPARγ activity. Further optimization to balance in vitro activity and metabolic stability allowed the discovery of the potent, selective and orally efficacious PPARγ agonist 8f. Structure-activity relationship study as well as detailed analysis of the binding mode of 8f to the PPARγ-LBD revealed the essential structural features of this series of ligands.

Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl Moiety.

The therapeutic potential of monoacylglycerol lipase (MAGL) inhibitors in central nervous system-related diseases has attracted attention worldwide. However, the availability of reversible-type inhibitor is still limited to clarify the pharmacological effect. Herein, we report the discovery of novel spiro chemical series as potent and reversible MAGL inhibitors with a different binding mode to MAGL using Arg57 and His121. Starting from hit compound 1 and its co-crystal structure with MAGL, structure-based drug discovery (SBDD) approach enabled us to generate various spiro scaffolds like 2a (azetidine-lactam), 2b (cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl moiety in 1 with higher lipophilic ligand efficiency (LLE). Optimization of the left hand side afforded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL inhibitory activity (IC50 6.2 nM), good oral absorption, blood-brain barrier penetration, and significant pharmacodynamic changes (2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3-10 mg/kg, po. in mice.

Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.

Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation.

Progranulin (PGRN) haploinsufficiency associated with loss-of-function mutations in the granulin gene causes frontotemporal dementia (FTD). This suggests that increasing PGRN levels could have promising therapeutic implications for patients carrying GRN mutations. In this study, we explored the therapeutic potential of sortilin1 (SORT1), a clearance receptor of PGRN, by generating and characterizing monoclonal antibodies against SORT1. Anti-SORT1 monoclonal antibodies were generated by immunizing Sort1 knockout mice with SORT1 protein. The antibodies were classified into 7 epitope bins based on their competitive binding to the SORT1 protein and further defined by epitope bin-dependent characteristics, including SORT1-PGRN blocking, SORT1 down-regulation, and binding to human and mouse SORT1. We identified a positive correlation between PGRN up-regulation and SORT1 down-regulation. Furthermore, we also characterized K1-67 antibody via SORT1 down-regulation and binding to mouse SORT1 in vivo and confirmed that K1-67 significantly up-regulated PGRN levels in plasma and brain interstitial fluid of mice. These data indicate that SORT1 down-regulation is a key mechanism in increasing PGRN levels via anti-SORT1 antibodies and suggest that SORT1 is a potential target to correct PGRN reduction, such as that in patients with FTD caused by GRN mutation.

Design, Synthesis, and Evaluation of (4 R)-1-{3-[2-(18F)Fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one ([18F]T-401) as a Novel Positron-Emission Tomography Imaging Agent for Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase involved in endocannabinoid and inflammatory signaling. Positron-emission tomography (PET) imaging of MAGL serves to validate target engagement of therapeutic MAGL inhibitors as well as to investigate MAGL levels under normal and disease conditions. However, PET radioligands with reversible binding kinetics for MAGL, which allow quantitative assessment of MAGL, are hitherto unavailable. In this study, we designed and synthesized fluoro-containing PET probes starting from a recently identified piperazinyl pyrrolidine-2-one derivative with reversible binding to MAGL. By tailoring the lipophilicity of the molecule to optimize nonspecific binding and blood-brain barrier permeability, we successfully identified two compounds that show high uptake to regions enriched with MAGL. PET imaging of wild-type and MAGL-deficient mice as well as a macaque monkey indicated that [18F]5 ((4 R)-1-{3-[2-(18F)fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one, [18F]T-401) specifically binds to MAGL with adequate reversibility, yielding a high contrast for MAGL within an appropriate imaging time.

Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound ( R)-3t. Oral administration of compound ( R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.

Perilipin-mediated lipid droplet formation in adipocytes promotes sterol regulatory element-binding protein-1 processing and triacylglyceride accumulation.

Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

The peroxisome proliferator-activated receptor gamma regulates expression of the perilipin gene in adipocytes.

Recent studies have shown that lipid droplets are covered with a proteinaceous coat, although the functions and identities of the component proteins have not yet been well elucidated. The first identified lipid droplet-specific proteins are the perilipins, a family of proteins coating the surfaces of lipid droplets of adipocytes. The generation of perilipin-null mice has revealed that although they consume more food than control mice, they have normal body weight and are resistant to diet-induced obesity. In one study (Martinez-Botas, J., Anderson, J. B., Tessier, D., Lapillonne, A., Chang, B. H. J., Quast, M. J., Gorenstein, D., Chen, K. H., and Chan, L. (2000) Nat. Genet. 26, 474-479) it was reported that in an animal model obesity was reversible by breeding perilipin -/- alleles into Lepr db/db obese mice, ostensibly by increasing the metabolic rate of the mice. To understand the exact mechanisms that drive the exclusive expression of the perilipin gene in adipocytes, we analyzed the 5'-flanking region of the mouse gene. Treatment of differentiating 3T3-L1 adipocytes with an agonist of proliferator-activated receptor (PPAR) gamma, the putative "master regulator" of adipocyte differentiation, significantly augmented perilipin gene expression. Reporter assays using the -2.0-kb promoter revealed that this region contains a functional PPARgamma-responsive element. Gel mobility shift and chromatin immunoprecipitation assays showed that endogenous PPARgamma protein binds to the perilipin promoter. PPARgamma2, an isoform exclusively expressed in adipocytes, was found to be the most potent regulator from among the PPAR family members including PPARalpha and PPARgamma1. These results make evident the fact that perilipin gene expression in differentiating adipocytes is crucially regulated by PPARgamma2, providing new insights into the adipogenic action of PPARgamma2 and adipose-specific gene expression, as well as potential anti-obesity pharmaceutical agents targeted to a reduction of the perilipin gene product.

Sterol regulatory element-binding protein-2 interacts with hepatocyte nuclear factor-4 to enhance sterol isomerase gene expression in hepatocytes.

In the course of an effort to identify unknown targets genes for sterol regulatory element-binding proteins (SREBPs) by PCR, the gene for ATP citrate-lyase was determined to be one such gene. (Sato, R., Okamoto, A., Inoue, J., Miyamoto, W., Sakai, Y., Emoto, N., Shimano, H., and Maeda, M. (2000) J. Biol. Chem. 275, 12497-12502). We here report that gene expression of sterol Delta8-isomerase (SI), which catalyzes the conversion of the 8-ene isomer into the 7-ene isomer in the last steps of the cholesterol biosynthetic pathway, is regulated by SREBPs, mainly by SREBP-2. Luciferase assays using the promoter of the human SI gene revealed that a 200-base pair segment upstream region from the transcription start site contains functional elements required for the activity of the SREBPs, Sp1 and NF-Y. Interestingly, SI gene expression was well regulated by sterols in Caco-2 and HepG2 cells, in contrast with HEK293 and HeLa cells. Overexpression of hepatocyte nuclear factor (HNF)-4 in HEK293 cells augmented expression of SREBP-responsive genes including the SI gene, whereas inactivation of HNF-4 by small interfering RNAs in HepG2 cells reduced the SI gene promoter activity. The in vitro pull-down and in vivo co-immunoprecipitation experiments showed the direct interaction between SREBP-2 and HNF-4. These data provide a novel pathway by which HNF-4 potentiates the SREBP functions and stimulates expression of SREBP-responsive genes in enterohepatic cells.