Discovery of a novel series of medium-sized cyclic enteropeptidase inhibitors.

Enteropeptidase is located in the duodenum that involved in intestinal protein digestion. We have reported enteropeptidase inhibitors with low systemic exposure. The aim of this study was to discover novel enteropeptidase inhibitors showing more potent in vivo efficacy while retaining low systemic exposure. Inhibitory mechanism-based drug design led us to cyclize ester 2 to medium-sized lactones, showing potent enteropeptidase inhibitory activity and improving the ester stability, thus increasing fecal protein output in vivo. Optimization on the linker between two benzene rings resulted in discovery of ether lactone 6b, exhibiting further enhanced enteropeptidase inhibitory activity and long duration of inhibitory state. Oral administration of 6b in mice significantly elevated fecal protein output compared with the lead 2. In addition, 6b showed low systemic exposure along with low intestinal absorption. Furthermore, we identified the 10-membered lactonization method for scale-up synthesis of 6b, which does not require high-dilution conditions.

Fully automated immunoassay for cholesterol uptake capacity to assess high-density lipoprotein function and cardiovascular disease risk.

High-density lipoprotein (HDL) cholesterol efflux capacity (CEC), which is a conventional metric of HDL function, has been associated with coronary heart disease risk. However, the CEC assay requires cultured cells and takes several days to perform. We previously established a cell-free assay to evaluate cholesterol uptake capacity (CUC) as a novel measure of HDL functionality and demonstrated its utility in coronary risk stratification. To apply this concept clinically, we developed a rapid and sensitive assay system based on a chemiluminescent magnetic particle immunoassay. The system is fully automated, providing high reproducibility. Measurement of CUC in serum is completed within 20 min per sample without HDL isolation, a notably higher throughput than that of the conventional CEC assay. CUC decreased with myeloperoxidase-mediated oxidation of HDL or in the presence of N-ethylmaleimide, an inhibitor of lecithin: cholesterol acyltransferase (LCAT), whereas CUC was enhanced by the addition of recombinant LCAT. Furthermore, CUC correlated with CEC even after being normalized by ApoA1 concentration and was significantly associated with the requirement for revascularization due to the recurrence of coronary lesions. Therefore, our new assay system shows potential for the accurate measurement of CUC in serum and permits assessing cardiovascular health.

Mirror-image streptavidin with specific binding to L-biotin, the unnatural enantiomer.

The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro folding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin-biotin system, even for in vivo use.

Silinanyl Rhodamines and Silinanyl Fluoresceins for Super-Resolution Microscopy.

Single-molecule localization microscopy (SMLM) enables the visualization of biomolecules at unprecedented resolution and requires control of the fluorescent blinking (ON/OFF) states of fluorophores to detect single-molecule fluorescence without overlapping of the signals. Although SMLM probes based on the intramolecular spirocyclization of Si-xanthene fluorophores have been developed, fluorophores with lower ON/OFF ratios are required for SMLM visualization of high-density structures. Here, we describe a silinane structure that lowers the ON/OFF ratio of Si-xanthene fluorophores. On the basis of Mulliken population analysis, we replaced the dimethylsilane moiety in Si-rhodamine with a silinane moiety to increase the partial charge at the 9-position of the carbon atom in the Si-xanthene ring and to promote the ring-closure reaction. Evaluation of fluorescence properties in a solution and in single-molecule imaging indicated that introducing the silinane sufficiently stabilized the nonfluorescent spirocyclic forms, thus decreasing the fluorescence ON/OFF ratio. This novel substitution was applied to Si-rhodamines with various amine structures and to an Si-fluorescein to expand the color palette. We demonstrated SMLM observation of microtubules in fixed HeLa cells using the developed fluorophores in two color channels. The results demonstrated the feasibility of extending the design strategies of SMLM probes based on Si-xanthenes through modification of the substituents on the Si atom.

Sodium butyrate, a histone deacetylase inhibitor, regulates Lymphangiogenic factors in oral cancer cell line HSC-3.

AIM: Tumor angiogenesis is a focus of molecularly-targeted therapies. This study investigated the effect of sodium butyrate (SB), a histone deacetylase inhibitor, on the synthesis of antiangiogenic and lymphangiogenic factors in oral squamous cell carcinoma. DESIGN: Gene alterations in HSC-3 cells were assessed using cDNA microarrays before and after treatment with SB. The mRNA and protein expression of lymphangiogenic factors were also assessed by quantitative PCR, western blotting and immunocytochemistry. RESULTS: Microarray analysis revealed that treatment with SB led to altered expression of angiogenesis-related gene expression. The quantitative polymerase chain reaction showed that platelet-derived growth factor-B, angiopoietin-2, vascular endothelial growth factor (VEGF)-C, and VEGFD were down-regulated. Western blotting and immunocytochemistry confirmed reduced protein synthesis of VEGFC. CONCLUSION: SB inhibits expression of lymphangiogenic factors in HSC-3 cells. Within the limitations of the present study, SB may have potential as an anti-metastatic pro-drug for oral cancer.

Discovery of a piperidine-4-carboxamide CCR5 antagonist (TAK-220) with highly potent Anti-HIV-1 activity.

We incorporated various polar groups into previously described piperidine-4-carboxamide CCR5 antagonists to improve their metabolic stability in human hepatic microsomes. Introducing a carbamoyl group into the phenyl ring of the 4-benzylpiperidine moiety afforded the less lipophilic compound 5f, which possessed both high metabolic stability and good inhibitory activity of HIV-1 envelope-mediated membrane fusion (IC(50) = 5.8 nM). Further optimization to increase potency led to the discovery of 1-acetyl-N-{3-[4-(4-carbamoylbenzyl)piperidin-1-yl]propyl}-N-(3-chloro-4-methylphenyl)piperidine-4-carboxamide (5m, TAK-220), which showed high CCR5 binding affinity (IC(50) = 3.5 nM) and potent inhibition of membrane fusion (IC(50) = 0.42 nM), as well as good metabolic stability. Compound 5m strongly inhibited the replication of CCR5-using HIV-1 clinical isolates in human peripheral blood mononuclear cells (mean EC(50) = 1.1 nM, EC(90) = 13 nM) and exhibited a good pharmacokinetic profile in monkeys (BA = 29%). This compound has been chosen as a clinical candidate for further development.

CCR5 antagonists as anti-HIV-1 agents. Part 3: Synthesis and biological evaluation of piperidine-4-carboxamide derivatives.

Replacement of the 5-oxopyrrolidin-3-yl fragment in the previously reported lead structure with a 1-acetylpiperidin-4-yl group led to the discovery of a novel series of potent CCR5 antagonists. Introduction of small hydrophobic substituents on the central phenyl ring increased the binding affinity, providing low to sub-nanomolar CCR5 antagonists. The selected compound 11f showed excellent antiviral activity against CCR5-using HIV-1 replication in human peripheral blood mononuclear cells (EC50=0.59 nM) and an acceptable pharmacokinetic profile in dogs.

CCR5 antagonists as anti-HIV-1 agents. Part 2: Synthesis and biological evaluation of N-[3-(4-benzylpiperidin-1-yl)propyl]-N,N'-diphenylureas.

We have previously reported the novel lead compound 1a as a CCR5 antagonist for treatment of HIV-1 infection. SAR studies on incorporating various acyl groups as a replacement for the 5-oxopyrrolidine-3-carbonyl group of the lead structure resulted in the discovery of N-[3-(4-benzylpiperidin-1-yl)propyl]-N,N'-diphenylurea (4a) with significantly improved CCR5 binding affinity. Substitutions (4-Cl, 4e,f; 4-Me, 4i) on the N'-phenyl ring further increased the binding affinity. Introduction of polar substituents on the phenyl ring of the 4-benzylpiperidine moiety enhanced the inhibitory activity of the HIV-1 envelope-mediated membrane fusion (4v,w), suggesting that polar substituents at this position can interfere effectively with HIV-1 cell entry.

Palladium-Catalyzed Intramolecular Allylic Alkylation Reaction in Marine Natural Product Synthesis: Enantioselective Synthesis of (+)-Methyl Pederate, a Key Intermediate in Syntheses of Mycalamides.

A novel preparation of (+)-methyl pederate (4), a key intermediate in syntheses of mycalamides (1), marine natural products from a New Zealand sponge of the genus Mycale, is described. The key step involves palladium-catalyzed intramolecular allylic alkylation of the carbonate 21, derived from (+)-(4R,5R,E)-5-(tert-butyldimethylsiloxy)-4-methyl-2-hexenol (13), yielding lactones 5 in 87% yield. Demethoxycarbonylation of the cyclization products 5 and further functional group transformations led to (+)-methyl pederate (4).