Synthesis and mechanistic study of ultrashort peptides that inhibits Alzheimer's Aß-aggregation-induced neurotoxicity.

Misfolding/aggregation of ß-amyloid peptide lead to the formation of toxic oligomers or accumulation of amyloid plaques, which is a seminal step in the progression of Alzheimer's disease (AD). Despite continuous efforts in the development of therapeutic agents, the cure for AD remains a major challenge. Owing to specific binding affinity of structure-based peptides, we report the synthesis of new peptide-based inhibitors derived from the C-terminal sequences, Aß38-40 and Aß40-42. Preliminary screening using MTT cell viability assay and corroborative results from ThT fluorescence assay revealed a tripeptide showing significantly effective inhibition towards Aß1-42 aggregation and induced toxicity. Peptide 3 exhibited excellent cell viability of 94.3 % at 2 µM and of 100 % at 4 µM and 10 µM. CD study showed that peptide 3 restrict the conformation transition of Aß1-42 peptide towards cross-ß-sheet structure and electron microscopy validated the absence of Aß aggregates as indicated by the altered morphology of Aß1-42 in the presence of peptide 3. The HRMS-ESI, DLS and ANS studies were performed to gain mechanistic insights into the effect of inhibitor against Aß aggregation. This Aß-derived ultrashort motif provides impetus for the development of peptide-based anti-AD agents.

Synthesis, biological evaluation and mechanistic studies of 4-(1,3-thiazol-2-yl)morpholine-benzimidazole hybrids as a new structural class of antimicrobials.

In spite of several attempts to develop newer pharmacophores as potential antimicrobial agents, the benzimidazole scaffold is still considered as one of the most sought after structural component towards the design of compounds that act against a wide spectrum of microbes. Herein, we report the design and synthesis of a new structural class of 4-(1,3-thiazol-2-yl)morpholine-benzimidazole hybrids as antimicrobial agents. The most potent analog, 6g shows IC50 of 1.3 µM, 2.7 µM, 10.8 µM, 5.4 µM and 10.8 µM against Cryptococcus neoformans, Candida albicans, Candida parapsilosis, Escherichia coli and Staphylococcus aureus, respectively. Interestingly 6g exhibits selectivity towards the cryptococcal cells with fungicidal behavior. Propidium iodide uptake study shows permeabilization of pathogenic cells in the presence of 6g. Flow cytometric analysis confirms that cell death is predominantly due to apoptosis. Moreover, electron microscopic analysis specifies that it shrinks, disrupts and initiate pore(s) formation in the cell membrane leading to cell lysis.

Synthetic amino acids-based short amphipathic peptides exhibit antifungal activity by targeting cell membrane disruption.

Availability of a limited number of antifungal drugs created a necessity to develop new antifungals with distinct mode of action. Investigation on a new series of peptides led us to identify Boc-His-Trp-His[1-(4-tert-butylphenyl)] (10g) as the most promising inhibitor exhibiting IC50 value of 4.4 µg/mL against Cryptococcus neoformans. Analog 10g exhibit high selectivity to fungal cells and was nonhemolytic and noncytotoxic at its minimum inhibitory concentration. 10g produced fungicidal effect on growing cryptococcal cells and displayed synergistic effect with amphotericin B. Overall cationic character of 10g resulted in interaction with negatively charged fungal membrane while hydrophobicity enhanced penetration inside the cryptococcal cells causing hole(s) formation and disruption to the membrane as evident by the scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy analyses. Flow cytometric investigation revealed rapid death of fungal cells by apopotic pathway.

Anticryptococcal activity and mechanistic studies of short amphipathic peptides.

Cryptococcus neoformans, an opportunistic fungal pathogen, causes cryptococcosis in immunocompromised persons. A series of modified L-histidines-containing peptides are synthesized that exhibit promising activity against C. neoformans. Analog 11d [L-His(2-adamantyl)-L-Trp-L-His(2-phenyl)-OMe] produced potency with an IC50 of 3.02 µg/ml (MIC = 5.49 µg/ml). This peptide is noncytotoxic and nonhaemolytic at the MIC and displays synergistic effects with amphotericin B at subinhibitory concentration. Mechanistic investigation of 11d using microscopic tools indicates cell wall and membrane disruption of C. neoformans, while flow cytometric analysis confirms cell death by apoptosis. This study indicates that 11d exhibits antifungal potential and acts via the rapid onset of action.

Antifungal evaluation and mechanistic investigations of membrane active short synthetic peptides-based amphiphiles.

The quest for new class of peptide-based antibiotics has steered this research towards the design and synthesis of short sequences possessing modified amphiphilic histidine along with hydrophobic tryptophan residues. The new structural class of dipeptides Trp-His(1-Bn)-OMe/NHBn and tripeptides His(1-Bn)-Trp-His(1-Bn)-OMe/NHBn demonstrated promising antifungal and antibacterial activities with membrane lytic action. The illustration of desirable hydrophilic-lipophilic balance appeared in the dipeptide Trp-His[1-(3,5-di-tert-butylbenzyl)]-NHBn (13d) that produced the most promising antifungal activity with IC50 value of 2.10 µg/mL and MIC = 3.81 µg/mL against C. neoformans and antibacterial activity against E. faecalis and S. aureus with identical IC50 value of 4.40 µg/mL and MIC of 8.0 µg/mL. Peptide 13d did not exhibit cytotoxicity and hemolysis at the MIC value and above. This quintessence amphiphilicity was further corroborated by the mechanistic elucidations, which revealed that, peptide act by utilizing charge and hydrophobicity as the primary characteristic tools. Owing to their fundamental affinity, the negatively charged fungal membrane is enacted upon by the positively charged peptide, whereas the intrinsic hydrophobicity of the peptide allowed penetration into the lipophillic core of the fungal cell membrane. Consequently, the integrity of cell membrane is compromised leading to increased fluidity. The membrane eventually disintegrates thereby creating a hollow pore and appearance of a doughnut into the cell when visualized under SEM. The cell death mechanism and damage to the cell wall and intracellular organelles have been elucidated with the help of flow cytometry, TEM and CLSM studies.

Common garlic (Allium sativum L.) has potent Anti-Bacillus anthracis activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. AIM OF THE STUDY: Evaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. MATERIALS AND METHODS: Aqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. RESULTS: Garlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50 °C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. CONCLUSION: The Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L., while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas.

Design, synthesis and bio-evaluation of C-1 alkylated tetrahydro-ß-carboline derivatives as novel antifungal lead compounds.

The field of antifungal agent has become static and development of resistance by the pathogen as well as limited clinical efficacy of marketed drugs demand the constant development of new antifungals. The presence of hydrocarbon chain of specific length linked with various different heterocycles was found to be an important structural feature in various antifungal lead compounds. Based on the prominent antimicrobial activity of ß-carboline derivatives, a set of C1 alkylated tetrahydro-ß-carboline derivatives were proposed to be active against fungi. To validate and confirm the role of suitable alkyl chains linked to a ß-carboline scaffold, few related analogues having C1 aryl substituents were also synthesized in one step via classic Pictet-Spengler reaction. The synthesized library was evaluated for its antifungal activity against C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. glabrata, C. tropicalis and C. neoformans. One of the library members (compound 12c), with n-alkyl chain of eight carbons exhibited potent antifungal activity against C. glabrata and C. kefyr. The lead compound, being selectively toxic also demonstrated prominent synergy enhancing the potency of antifungal drugs up to 10-fold. The time kill kinetic studies confirmed the efficacy of compound 12c, where the results obtained were comparable to that of Amp B. FE-SEM analysis revealed the increased asymmetry, disintegration and roughness of cell surface which could be because of the possible interaction of compound 12c at membrane level or interference in cell wall structure. Apoptosis/necrosis detection assay confirmed the significant apoptotic activity in C. glabrata cells after 12c treatment which was responsible for the rapid killing of C. glabrata cells.

Design, synthesis, DFT, docking studies and ADME prediction of some new coumarinyl linked pyrazolylthiazoles: Potential standalone or adjuvant antimicrobial agents.

The control of antimicrobial resistance (AMR) seems to have come to a dead end. The major consequences of the use and abuse of antibacterial drugs are the development of resistant strains due to genetic mutability of both pathogenic and nonpathogenic microorganisms. We, herein, report the synthesis, characterization and biological activities of coumarin-thiazole-pyrazole (CTP) molecular hybrids with an effort to explore and overcome the increasing antimicrobial resistance. The compounds were characterized by analyzing their IR, Mass, 1H and13C NMR spectral data and elemental analysis. The in vitro antimicrobial activity of the synthesized compounds was investigated against various pathogenic strains; the results obtained were further explained with the help of DFT and molecular orbital calculations. Compound 1b and 1f displayed good antimicrobial activity and synergistic effects when used with kanamycin and amphotericin B. Furthermore, in vitro cytotoxicity of compounds 1b and 1f were studied against HeLa cells (cervical cancer cell) and Hek-293 cells. The results of molecular docking study were used to better rationalize the action and prediction of the binding modes of these compounds.

Antimicrobial activity of Bulbothrix setschwanensis (Zahlbr.) Hale lichen by cell wall disruption of Staphylococcus aureus and Cryptococcus neoformans.

In the present study, antimicrobial activity of a common Himalayan lichen viz. Bulbothrix setschwanensis (Zahlbr.) Hale extract in three common solvents (acetone, chloroform and methanol) was evaluated against six bacterial and seven fungal clinical strains. The acetone extract showed promising antimicrobial activity against S. aureus (1.56 mg/mL) and C. neoformans (6.25 mg/mL). Further, GC-MS analysis revealed 2,3-bis(2-methylpentanoyloxy)propyl 2-methylpentanoate and Ethyl 2-[(2R,3R,4aR,8aS)-3-hydroxy-2,3,4,4a,6,7,8,8a-octahydropyrano [3,2-b]pyran-2-yl]acetate as the predominant compounds. The combination of acetone extract with antibacterial drugs [kanamycin (KAN), rifampicin (RIF)] and antifungal drugs [amphotericin B (Amp B) and fluconazole (FLC)] showed lysis of S. aureus and C. neoformans at non-inhibitory concentration (FICI values were 0.31 for KAN, 0.18 for RIF, 0.37 for Amp B and 0.30 for FLC, respectively). Notably, the acetone extract confirmed cell wall damage of both S. aureus and C. neoformans cells and was clearly visualized under scanning electron microscopy (SEM), flow cytometry and confocal microscopy. Besides this, the three extracts also have less significant cytotoxic activity at MIC concentrations against mammalian cells (HEK-293 and HeLa). This study for the first time suggests that the chemical compounds present in the acetone extract of B. setschwanensis could be used against S. aureus and C. neoformans infections.

Synthesis, stability and mechanistic studies of potent anticryptococcal hexapeptides.

The growing incidents of cryptococcosis in immuno-compromised patients have created a need for novel drug therapies capable of eradicating the disease. The peptide-based drug therapy offers many advantages over the traditional therapeutic agents, which has been exploited in the present study by synthesizing a series of hexapeptides that exhibits promising activity against a panel of Gram-negative and Gram-positive bacteria and various pathogenic fungal strains; the most exemplary activity was observed against Cryptococcus neoformans. The peptides 3, 24, 32 and 36 displayed potent anticryptococcal activity (IC50 = 0.4-0.46 µg/mL, MIC = 0.63-1.25 µg/mL, MFC = 0.63-1.25 µg/mL), and stability under proteolytic conditions. Besides this, several other peptides displayed promising inhibition of pathogenic bacteria. The prominent ones include peptides 18-20, and 26 that exhibited IC50 values ranged between 2.1 and 3.6 µg/mL, MICs of 5-20 µg/mL and MBCs of 10-20 µg/mL against Staphylococcus aureus and methicillin-resistant S. aureus. The detailed mechanistic study on selected peptides demonstrated absolute selectivity towards the bacterial membranes and fungal cells by causing perturbations in the cell membranes, confirmed by the scanning electron microscopy and transmission electron microscopy studies.

Facile synthesis, structural evaluation, antimicrobial activity and synergistic effects of novel imidazo[1,2-a]pyridine based organoselenium compounds.

A simple and efficient method has been described to synthesize the hitherto unknown imidazo[1,2-a]pyridine selenides (5a-l) by reaction of 2-chloroimidazo [1,2-a]pyridines with aryl/heteroaryl selenols, generated in situ by reduction of various diselenides with hypophosphorous acid. The crystal structures of 3-nitro-2-(phenylselanyl)-imidazo[1,2-a]pyridine (5a), 2-(mesitylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5d) and 3-nitro-2-(pyridin-2-ylselanyl)-imidazo[1,2-a]pyridine (5e) were confirmed by X-ray crystallography and the DFT calculations were performed to determine various structural parameters which were correlated with the X-ray crystal structures. The synthesized compounds were subjected to antimicrobial evaluation and it was found that compounds 5a and 5j were active against gram negative bacterium Escherichia coli whereas compound 5e was active against different fungal strains. Time kill assay was performed to understand the microbial activity of synthesized organoselenium compounds and the toxicity of these compounds was evaluated against human cell lines. Synergistic effects of active compounds 5a and 5e were tested with existing antibiotic drugs which exhibited that the antibiotic combination with synthesized organoselenium compounds efficiently enhanced the antimicrobial activity.

Chemosensitization of multidrug resistant Candida albicans by the oxathiolone fused chalcone derivatives.

Three structurally related oxathiolone fused chalcone derivatives appeared effective chemosensitizers, able to restore in part sensitivity to fluconazole of multidrug-resistant C. albicans strains. Compound 21 effectively chemosensitized cells resistant due to the overexpression of the MDR1 gene, compound 6 reduced resistance of cells overexpressing the ABC-type drug transporters CDR1/CDR2 and derivative 18 partially reversed fluconazole resistance mediated by both types of yeast drug efflux pumps. The observed effect of sensitization of resistant strains of Candida albicans to fluconazole activity in the presence of active compounds most likely resulted from inhibition of the pump-mediated efflux, as was revealed by the results of studies involving the fluorescent probes, Nile Red, Rhodamine 6G and diS-C3(3).