Construction of Five Tryptophan Isomers and Application of the Isomers to Solid-Phase Total Syntheses of Lysocin E Derivatives.

Tryptophan (Trp) plays a unique role in peptides and proteins as its indole ring possesses an electron-rich character and an N1-H hydrogen-bond donor. Because of its non-rotationally symmetric structure, synthetic alterations of the orientation of the indole ring would modulate the intrinsic structures and functions of peptides and proteins. Here we developed synthetic routes to the five Trp isomers in which the C3-substitution of the indole ring was changed to the C2/4/5/6/7-substitutions, and applied the five monomers to Fmoc-based solid-phase peptide synthesis. Specifically, the five monomers were prepared via Negishi cross-coupling reactions of C2/4/5/6/7-iodoindoles. To demonstrate the applicability of the monomers to the solid-phase synthesis, the five Trp isomers of macrocyclic antibiotic lysocin E were selected as target molecules and synthesized through peptide elongation, on-resin macrocyclization, and global deprotection. The Trp isomers displayed markedly weaker antibacterial activity than the parent natural product, revealing the biological importance of the precise three-dimensional shape of the original Trp residue of lysocin E. The present methods for the preparation and application of these five Trp isomers provide a new strategy for analyzing and modifying the specific functions of numerous Trp-containing peptides and proteins beyond this study.

Lysocin E Targeting Menaquinone in the Membrane of Mycobacterium tuberculosis Is a Promising Lead Compound for Antituberculosis Drugs.

Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.

Burden of tuberculosis and hepatitis co-infection among people living with HIV in Nepal: a systematic review and meta-analysis.

People living with HIV (PLHIV) are prone to tuberculosis (TB) and hepatitis co-infections, which cause substantial burden on morbidity and mortality. However, data on the burden of HIV co-infection from a specific low- and middle-income country are limited. To address this gap in evidence, a meta-analysis of published literature and country surveillance report was conducted to estimate the burden of TB, hepatitis B (HBV) and hepatitis C (HCV) co-infection among PLHIV in Nepal. Twenty-three studies, including 5900 PLHIV, were included in the meta-analysis. The pooled prevalence of HIV-TB, HIV-HBV and HIV-HCV co-infection was 19% (95% CI, 10-28%), 3% (2-5%) and 19% (4-33%), respectively. Low CD4 cell count (pooled odds ratio [OR] 4.38, 95% CI 1.11-17.25), smoking (3.07, 1.48-6.37) and alcohol drinking (3.12, 1.52-6.43) were significantly correlated with HIV-TB co-infection. The odds of HCV co-infection was greater in PLHIV, who were male (5.39, 1.54-18.89) and drug users (166.26, 15.94-1734.44). PLHIV who were on antiretroviral therapy had a reduced risk of HCV co-infection (0.49, 0.36-0.66) than the general PLHIV population. The burden of TB and hepatitis co-infection among PLHIV in Nepal was high. Regular screening of PLHIV for co-infections and prompt initiation of treatment are essential to reduce the transmission of infection and improve quality of life.

YjbH regulates virulence genes expression and oxidative stress resistance in Staphylococcus aureus.

We previously reported that disruption of the yjbI gene reduced virulence of Staphylococcus aureus. In this study, we found virulence in both silkworms and mice was restored by introducing the yjbH gene but not the yjbI gene to both yjbI and yjbH genes-disrupted mutants, suggesting that yjbH, the gene downstream to the yjbI gene in a two-gene operon-yjbIH, is responsible for this phenomenon. We further observed a decrease in various surface-associated proteins and changes in cell envelope glycostructures in the mutants. RNA-seq analysis revealed that disruption of the yjbI and the yjbH genes resulted in differential expression of a broad range of genes, notably, significant downregulation of genes involved in virulence and oxidative stress. Administration of N-acetyl-L-cysteine, a free-radical scavenger, restored the virulence in both the mutants. Our findings suggested that YjbH plays a role in staphylococcal pathogenicity by regulating virulence gene expression, affecting the bacterial surface structure, and conferring resistance to oxidative stress in a host.

Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library.

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus Smith strain is a historical strain widely used for research purposes in animal infection models for testing the therapeutic activity of antimicrobial agents. We found that it displayed higher sensitivity toward lysocin E, a menaquinone (MK) targeting antibiotic, compared to other S. aureus strains. Therefore, we further explored the mechanism of this hypersensitivity. METHODS: MK production was analyzed by high-performance liquid chromatography and mass spectrometric analysis. S. aureus Smith genome sequence was completed using a hybrid assembly approach, and the MK biosynthetic genes were compared with other S. aureus strains. The hepT gene was cloned and introduced into S. aureus RN4220 strain using phage mediated recombination, and lysocin E sensitivity was analyzed by the measurement of colony-forming units. RESULTS: We found that Smith strain produced MKs with the length of the side chain ranging between 8 and 10, as opposed to other S. aureus strains that produce MKs 7-9. We revealed that Smith strain possessed the classical pathway for MK biosynthesis like the other S. aureus. HepT, a polyprenyl diphosphate synthase involved in chain elongation of isoprenoid, in Smith strain harbored a Q25P substitution. Introduction of hepT from Smith to RN4220 led to the production of MK-10 and an increased sensitivity toward lysocin E. CONCLUSION: We found that HepT was responsible for the definition of isoprenoid chain length of MKs and antibiotic sensitivity.

Prevalence and correlates of substance use among health care students in Nepal: a cross sectional study.

BACKGROUND: Substance use among health care students threatens professional standards and the delivery of quality services, potentially placing the public at risk. Therefore, our study aims to determine the prevalence and correlates associated with substance use among Nepalese health care students. METHOD: A cross-sectional survey using a self-administered health professional questionnaire was conducted among pharmacy, nursing, and public health students at three universities in Nepal in 2010. RESULTS: We analyzed data from 407 respondents (response rate, 82%) with a mean age of 22 years (standard deviation = 3.71). The overall lifetime prevalence of substance use (i. e., illegal use of prescription drugs and illegal drug use) was 42.8%. Marijuana was the most commonly used illegal drug (8.8%) and minor opiates (e.g., codeine cough syrups) were the most widely used illegal prescription drugs (32.4%). Substance use was directly associated with cigarette smoking, peer influence, and heavy drinking. In addition, respondents reported some major and minor dysfunctions because of their substance use. CONCLUSION: The prevalence of substance use among health care students at the three universities in Nepal was high. Peer influence, cigarette smoking, and heavy drinking were significant predictors of substance use.

Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation.

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.