Structure-based lead optimization to improve potency and selectivity of a novel tetrahydroimidazo[1,2-a]pyridine-5-carboxylic acid series of heparanase-1 inhibitor.

Heparanase-1 (HPSE1) is an endo-ß-d-glucuronidase that is the only mammalian enzyme known to cleave heparan sulfate (HS) of heparan sulfate proteoglycans (HSPG), a key component of the glycocalyx layer of the vascular endothelium matrix. Inhibition of HPSE1 has therapeutic potential for cancer and proteinuric kidney diseases. We previously reported that 2 showed a moderate potency as an HPSE1 inhibitor and an issue of selectivity against exo-ß-d-glucuronidase (GUSß) and glucocerebrosidase (GBA) remained. A structure-based lead optimization of 2 using X-ray co-crystal structure analysis and fragment molecular orbital calculation resulted in 4e, which showed a more than 7-fold increase in HPSE1 inhibitory activity. The subsequent introduction of a methyl group into the 6-hydroxy group of 4e resulted in 18 with reduced inhibitory activities against GUSß and GBA while maintaining the inhibitory activity against HPSE1. The inhibitory activities of 18 against serum HPSE1 in mice were significant and lasted for 4 h at doses of 3, 30, and 100 mg/kg. Compound 18 could be a novel lead compound for HPSE1 inhibitors with improved inhibitory activity against HPSE1 and increased HPSE1 selectivity over GUSß and GBA.

Fibroblast growth factor 19 treatment ameliorates disruption of hepatic lipid metabolism in farnesoid X receptor (Fxr)-null mice.

Human fibroblast growth factor 19 (FGF19) is an enterohepatic hormone that is involved in the regulation of hepatic metabolism of bile acids, lipids, and glucose. Farnesoid X receptor (Fxr)-null mice exhibit steatosis-like symptoms, showing higher hepatic lipid levels than with the wild-type mice. We investigated the influence of FGF19 treatment on hepatic lipogenesis in Fxr-null mice. Recombinant FGF19 treatment (400 µg/kg/d) for 3 d prevented the accumulation of lipid droplets and decreased serum alanine aminotransferase activity and hepatic lipid levels, including those of triglycerides and free fatty acids. The treatment significantly decreased the hepatic mRNA levels of acetyl-CoA carboxylase 1 (Acc1), Cd36, and sterol regulatory element-binding protein-1c (Srebp-1c) as well as those of acetyl-CoA carboxylase 2 (Acc2), stearoyl CoA desaturase 1 (Scd1), and Cyp7a1. FGF19 treatment (4 µg/kg/d) for 3 d also decreased the hepatic free fatty acid levels and mRNA levels of Acc1, Cd36, and Srebp-1c. These results indicate that FGF19-mediated signaling ameliorates disrupted hepatic lipogenesis in Fxr-null mice.

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE.

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.