Structure-guided design and synthesis of P1' position 1-phenylcycloalkylamine-derived pentapeptidic BACE1 inhibitors

Previously, we reported potent pentapeptidic BACE1 inhibitors with the hydroxymethylcarbonyl isostere as a substrate transition-state mimic. To improve the in vitro potency, we further reported pentapeptidic inhibitors with carboxylic acid bioisosteres at the P(4) and P1' positions. In the current study, we screened new P1' position 1-phenylcycloalkylamine analogs to find non-acidic inhibitors that possess double-digit nanomolar range IC(50) values. An extensive structure-activity relationship study was performed with various amine derivatives at the P1' position. The most potent inhibitor of this pentapeptide series, KMI-1830, possessing 1-phenylcyclopentylamine at the P1' position had an IC(50) value of 11.6 nM against BACE1 in vitro enzymatic assay.     

Investigation toward multi-epitope vaccine candidates using native chemical ligation

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.     

Synthesis and structure of poly(binaphthyl salen manganese complex) and its application to asymmetric epoxidation

Poly(binaphthyl salen manganese complex)es 3-Mn were synthesized from a 3,3'-diformylbinaphthol derivative, alpha,omega-diamines, and Mn(OAc)2. Their helical structures were well-supported by their IR, UV, and CD spectra. The catalysis of 3-Mn in an asymmetric epoxidation was investigated.     

Occurrence of both a-type and o-type cytochromes as the functional terminal oxidases in Rhodopseudomonas spheroides

1. 1. The functional terminal oxidase of the light-anaerobically grown Rhodopseudomonas spheroides cells was found to be the o-type cytochrome, whereas that of the dark-aerobically grown cells was the a-type cytochrome. When the dark-aerobically grown cells were further incubated under a semianaerobic condition in the dark, the content of the o-type cytochrome was increased in these cells, while the synthesis of the a-type cytochrome appeared to be repressed. In Rhodospirillum rubrum cells, grown either aerobically in the dark or anaerobically in the light, cytochrome o was the sole functional terminal oxidase.

2. 2. Reactions with the a-type and o-type cytochromes from Rhodopseudomonas spheroides and also with the o-type cytochrome from Rhodospirillum rubrum were compared using reduced yeast cytochrome c as substrate. The reaction with the a-type cytochrome was far less sensitive to NaN₃ and hydroxylamine than those with the o-type cytochromes, whereas all the reactions were inhibited by KCN in apparently the same manner.

Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.     

Electron microscopic study on the initiatory cell-activation of procambium at commencement of germination in radish cotyledons

Most fine structural studies of seed germination have been begun at 24 hr after imbibition of water and lasted for several days. However, studies on the initial period beginning with water uptake are few. In the present study, fine structural changes in radish seed cotyledonal tissues which occur within the first 24 hr of germination were investigated. Remarkable morphological and developmental changes of the cell organelles were profound in procambial cells in a matter of hours, while no cytological changes were noticed yet in mesophyll (storage tissue) cells. The endoplasmic reticulum and Golgi bodies were somewhat proliferated, a rapid development even, of proplastids occurred, and the increment of mitochondria in numbers and profiles were particularly noteworthy. From these observations, it was interpreted that, although the procambium accounts for only a small portion of the cotyledons, the initiatory activation of procambial cells, being sensitive to the starting of water uptake, might provide certain facility for leading the activation of the more lethargic storage tissue in the beginning of germination process.     

Synthesis and antiviral property of allophenylnorstatine-based HIV protease inhibitors incorporating D-cysteine derivatives as P2/P3 moieties

We designed several HIV protease inhibitors with various d-cysteine derivatives as P(2)/P(3) moieties based on the structure of clinical drug candidate, KNI-764. Herein, we report their synthesis, HIV protease inhibitory activity, HIV IIIB cell inhibitory activity, cellular toxicity, and inhibitory activity against drug-resistant HIV strains. KNI-1931 showed distinct selectivity against HIV proteases and high potency against drug-resistant strains, surpassing those of Ritonavir and Nelfinavir.