Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors.

In recent years, dipeptidyl peptidase IV inhibitors have been noted as valuable agents for treatment of type 2 diabetes. Herein, we report the discovery of a novel potent DPP-4 inhibitor with 3H-imidazo[4,5-c]quinolin-4(5H)-one as skeleton. After efficient optimization of the lead compound 2a at the 7- and 8-positions using a docking study, we found 28 as a novel DPP-4 inhibitor with excellent selectivity against various DPP-4 homologues. Compound 28 showed strong DPP-4 inhibitory activity compared to marketed DPP-4 inhibitors. We also found that a carboxyl group at the 7-position could interact with the residue of Lys554 to form a salt bridge. Additionally, introduction of a carboxyl group to 7-position led to both activity enhancement and reduced risk for hERG channel inhibition and induced phospholipidosis. In our synthesis of compounds with 7-carboxyl group, we achieved efficient regioselective synthesis using bulky ester in the intramolecular palladium coupling reaction.

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): a potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A.

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC(50)=1.1 nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.

Discovery of new chemotype dipeptidyl peptidase IV inhibitors having (R)-3-amino-3-methyl piperidine as a pharmacophore.

Structures containing the (R)-3-amino-3-methyl piperidine unit as a new pharmacophore moiety have been shown to possess moderate inhibitory activity for DPP-4 with good pharmacokinetics profile. One of these compounds was found to have good oral bioavailability and PK/PD profile in ZF-rat.

PDX-1 protein is internalized by lipid raft-dependent macropinocytosis.

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell-surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4-64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4-64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.

Efficacy of ASP2151, a helicase-primase inhibitor, against thymidine kinase-deficient herpes simplex virus type 2 infection in vitro and in vivo.

ASP2151 was developed as a novel inhibitor of herpes simplex virus (HSV) and varicella-zoster virus helicase-primase. The anti-HSV activity of ASP2151 toward a clinical HSV isolate with acyclovir (ACV)-resistant/thymidine kinase (TK)-deficiency was characterized in vitro and in vivo using a plaque reduction assay and the ear pinna infection in mice. The IC(50) ranged from 0.018 to 0.024 µg/ml, indicating the susceptibility of TK-deficient HSV-2 was similar to that of wild-type HSV-2 strains. Anti-HSV activity of ASP2151 in vivo was evaluated in mice infected with wild-type HSV-2 and TK-deficient HSV-2. ASP2151 significantly reduced the copy numbers of wild-type HSV-2 and TK-deficient HSV-2 at the inoculation ear pinna, while valacyclovir significantly reduced the copy number of wild type HSV-2 but not that of TK-deficient HSV-2 in the inoculated ear pinna. Thus, ASP 2151 showed therapeutic efficacy in mice infected with both wild-type and TK-deficient HSV-2. In conclusion, ASP2151 is a promising novel herpes helicase-primase inhibitor that indicates the feasibility of ASP2151 for clinical application for the treatment of HSV infections, including ACV-resistant/TK-deficient HSV infection.

Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes.

BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified.

Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function.

Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH2-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.