Hydrocarbon stapled temporin-L analogue as potential antibacterial and antiendotoxin agents with enhanced protease stability.

Antimicrobial resistance (AMR) is a serious global concern and a huge burden on the healthcare system. Antimicrobial peptides (AMPs) are considered as a solution of AMR due to their membrane-lytic and intracellular mode of action and therefore resistance development against AMPs is less frequent. One such AMPs, temporin-L (TL) is a 13-mer peptide reported as a potent and broad-spectrum antibacterial agent with significant immunomodulatory activity. However, TL is toxic to human erythrocytes at their antibacterial concentrations and therefore various analogues were synthesized with potent antimicrobial activity and lower hemolytic activity. In this work, we have selected a non-toxic engineered analogue of TL (eTL) and performed hydrocarbon stapling of amino acid residues at i to i + 4 positions at different part of sequence. The synthesized peptides were investigated against both the gram-positive and gram-negative bacteria as well as methicillin resistant S. aureus, its MIC was measured in the concentrations range of 0.9-15.2 µM. All analogues were found equal or better antibacterial as compared to parent peptide. Interestingly one analogue eTL [5-9] was found to be non-cytotoxic and stable in presence of the human serum. Mode of action studies revealed membrane depolarizing and disruptive mode of action with live MRSA. Further in vivo studies of antimicrobial against MRSA infection and anti-endotoxin activities in mice model revealed potential activity of the stapled peptide analogue. Overall, this reports on stapled analogue of the AMPs highlights an important strategy for the development of new antibacterial therapeutics against AMR.

Introduction of a ß-leucine residue instead of leucine9 and glycine10 residues in Temporin L for improved cell selectivity, stability and activity against planktonic and biofilm of methicillin resistant S. aureus.

Leucine and glycine residues, at the 9th and 10th positions of helical domain of naturally occurring antimicrobial peptide (AMP), Temporin L were substituted with an unnatural amino acid, ß-leucine (homovaline) to improve its serum protease stability, haemolytic/cytotoxic properties and reduce the size to some extent. The designed analogue, L9ßl-TL showed either equal or improved antimicrobial activity to TL against different microorganisms including the resistant strains. Interestingly, L9ßl-TL also exhibited lower haemolytic and cytotoxic activities against human red blood cells and 3T3 cells, respectively. Moreover, L9ßl-TL showed antibacterial activity in presence of 25% (v/v) human serum and showed resistance against proteolytic cleavage in presence of it that suggested the serum protease stability of the TL-analogue. L9ßl-TL exhibited un-ordered secondary structures in both bacterial and mammalian membrane mimetic lipid vesicles as compared to the helical structures of TL in these environments. However, tryptophan fluorescence studies demonstrated more selective interaction of L9ßl-TL with bacterial membrane mimetic lipid vesicles in comparison to non-selective interactions of TL with both kinds of lipid vesicles. Membrane depolarization studies with live MRSA and bacterial membrane-mimetic lipid vesicles suggested a membrane-disrupting mode of action of L9ßl-TL. L9ßl-TL showed faster bactericidal mechanism compared to TL against MRSA. Interestingly, L9ßl-TL was found as more potent than TL either in inhibiting biofilm formation or in eradicating the mature biofilm formed by MRSA. Overall, the present work demonstrates a simple and useful strategy to design of an analogue of TL, with minimal modifications while maintaining its antimicrobial activity with lesser toxicity and higher stability which could be attempted for other AMPs as well.

Report on Vincristine-Producing Endophytic Fungus Nigrospora zimmermanii from Leaves of Catharanthus roseus.

Vincristine is an anti-cancer compound and one of the most crucial vinca alkaloids produced by the medicinal plant Catharanthus roseus (L.) G. Don. (Apocynaceae). This plant is home to hundreds of endophytic microbes, which produce a variety of bioactive secondary metabolites that are known for their medicinal properties. In this study, we focused on isolating an endophytic fungus that could increase the yield of vincristine under laboratory conditions as an alternative to plant-mediated extraction of vincristine. The endophytic fungus Nigrospora zimmermanii (Apiosporaceae) was isolated from Catharanthus roseus and it was found to be producing the anticancer compound vincristine. It was identified using high-performance thin-layer chromatography by matching the Rf value and spectral data with the vincristine standard and mass spectrometry data and the reference molecule from the PubChem database. The generation study of this microbe showed that the production of vincristine in the parent fungus was at its maximum, i.e., 5.344 µg/mL, while it was slightly reduced in subsequent generations. A colonization study was also performed and it showed that the fungus N. zimmermanii was able to re-infect the plant Catharanthus roseus after 20 days of inoculation. The colonization study showed that N. zimmernanii could infect the plant after isolation. This method is an efficient and easy way to obtain a high yield of vincristine, as compared to plant-mediated production.

Recognition and unfolding of human telomeric G-quadruplex by short peptide binding identified from the HRDC domain of BLM helicase.

Research in recent decades has revealed that the guanine (G)-quadruplex secondary structure in DNA modulates a variety of cellular events that are mostly related to serious diseases. Systems capable of regulating DNA G-quadruplex structures would therefore be useful for the modulation of various cellular events to produce biological effects. A high specificity for recognition of telomeric G-quadruplex has been observed for BLM helicase. We identified peptides from the HRDC domain of BLM using a molecular docking approach with various available solutions and crystal structures of human telomeres and recently created a peptide library. Herein, we tested one peptide (BLM HRDC peptide) from the library and examined its interaction with human telomeric variant-1 (HTPu-var-1) to understand the basis of G4-protein interactions. Our circular dichroism (CD) data showed that HTPu-var-1 folded into an anti-parallel G-quadruplex, and the CD intensity significantly decreased upon increasing the peptide concentration. There was a significant decrease in hypochromicity due to the formation of G-quadruplex-peptide complex at 295 nm, which indicated the unfolding of structure due to the decrease in stacking interactions. The fluorescence data showed quenching upon titrating the peptide with HTPu-var-1-G4. Electrophoretic mobility shift assay confirmed the unfolding of the G4 structure. Cell viability was significantly reduced in the presence of the BLM peptide, with IC50 values of 10.71 µM and 11.83 µM after 72 and 96 hours, respectively. These results confirmed that the selected peptide has the ability to bind to human telomeric G-quadruplex and unfold it. This is the first report in which a peptide was identified from the HRDC domain of the BLM G4-binding protein for the exploration of the G4-binding motif, which suggests a novel strategy to target G4 using natural key peptide segments.

Solid Self-Nano Emulsifying Nanoplatform Loaded with Tamoxifen and Resveratrol for Treatment of Breast Cancer.

The solid self-nanoemulsifying drug delivery system (s-SNEDDS) is a growing platform for the delivery of drugs via oral route. In the present work, tamoxifen (TAM) was loaded in SNEDDS with resveratrol (RES), which is a potent chemotherapeutic, antioxidant, anti-inflammatory and P-gp inhibitor for enhancing bioavailability and to obtain synergistic anti-cancer effect against breast cancer. SNEDDS were developed using capmul MCM as oil, Tween 80 as surfactant and transcutol-HP as co-surfactant and optimized by central composite rotatable design. Neusilin US2 concentration was optimized for adsorption of liquid SNEDDS to prepare s-SNEDDS. The developed formulation was characterized and investigated for various in vitro and cell line comparative studies. Optimized TAM-RES-s-SNEDDS showed spherical droplets of a size less than 200 nm. In all in vitro studies, TAM-RES-s-SNEDDS showed significantly improved (p ˂ 0.05) release and permeation across the dialysis membrane and intestinal lumen. Moreover, TAM-RES-s-SNEDDS possessed significantly greater therapeutic efficacy (p < 0.05) and better internalization on the MCF-7 cell line as compared to the conventional formulation. Additionally, oral bioavailability of TAM from SNEDDS was 1.63 folds significantly higher (p < 0.05) than that of combination suspension and 4.16 folds significantly higher (p < 0.05) than TAM suspension. Thus, findings suggest that TAM- RES-s-SNEDDS can be the future delivery system that potentially delivers both drugs to cancer cells for better treatment.

Enhancement of vincristine under in vitro culture of Catharanthus roseus supplemented with Alternaria sesami endophytic fungal extract as a biotic elicitor / Mejora de vincristina bajo cultivo in vitro de Catharanthus roseus suplementado con extracto de hongos endófitos de Alternaria sésamo como un elicitor biótico

Vincristine, one of the major vinca alkaloid of Catharanthus roseus (L.) G. Don. (Apocynaceae), was enhanced under in vitro callus culture of C. roseus using fungal extract of an endophyte Alternaria sesami isolated from the surface-sterilized root cuttings of C. roseus. Vindoline, a precursor molecule for vincristine production, was detected for the first time in the fungal endophyte A. sesami which was used as a biotic elicitor in this study to enhance vincristine content in the C. roseus callus. It was identified using high-performance liquid chromatography and mass spectroscopy techniques by matching retention time and mass data with reference molecule. Supplementing the heat sterilized A. sesami endophytic fungal culture extract into the callus culture medium of C. roseus resulted in the enhancement of vincristine content in C. roseus callus by 21.717% after 105-day culture.(AU)

Spermine-Conjugated Short Proline-Rich Lipopeptides as Broad-Spectrum Intracellular Targeting Antibacterial Agents.

Toward the design of new proline-rich peptidomimetics, a short peptide segment, present in several proline-rich antimicrobial peptides (AMPs), was selected. Fatty acids of varying lengths and spermine were conjugated at the N- and C-terminals of the peptide, respectively. Spermine-conjugated lipopeptides, C10-PR-Spn and C12-PR-Spn, exhibited minimum inhibitory concentrations within 1.5-6.2 µM against the tested pathogens including resistant bacteria and insignificant hemolytic activity against human red blood cells up to 100 µM concentrations and demonstrated resistance against trypsin digestion. C10-PR-Spn and C12-PR-Spn showed synergistic antimicrobial activity against multidrug-resistant methicillin-resistant Staphylococcus aureus with several tested antibiotics. These lipopeptides did not permeabilize bacterial membrane-mimetic lipid vesicles or damage the Escherichia coli membrane like the nonmembrane-lytic AMP, buforin-II. The results suggested that C10-PR-Spn and C12-PR-Spn could interact with the 70S ribosome of E. coli and inhibit its protein synthesis. C10-PR-Spn and C12-PR-Spn demonstrated superior clearance of bacteria from the spleen, liver, and kidneys of mice, infected with S. aureus ATCC 25923 compared to levofloxacin.

Enhancement of vincristine under in vitro culture of Catharanthus roseus supplemented with Alternaria sesami endophytic fungal extract as a biotic elicitor.

Vincristine, one of the major vinca alkaloid of Catharanthus roseus (L.) G. Don. (Apocynaceae), was enhanced under in vitro callus culture of C. roseus using fungal extract of an endophyte Alternaria sesami isolated from the surface-sterilized root cuttings of C. roseus. Vindoline, a precursor molecule for vincristine production, was detected for the first time in the fungal endophyte A. sesami which was used as a biotic elicitor in this study to enhance vincristine content in the C. roseus callus. It was identified using high-performance liquid chromatography and mass spectroscopy techniques by matching retention time and mass data with reference molecule. Supplementing the heat sterilized A. sesami endophytic fungal culture extract into the callus culture medium of C. roseus resulted in the enhancement of vincristine content in C. roseus callus by 21.717% after 105-day culture.

Pharmacophore based drug design and synthesis of oxindole bearing hybrid as anticancer agents.

Dual TK inhibitors have shown significant clinical effects against many tumors, but with unmanageable side effects. Design approach and selectivity of these inhibitors plays substantial role in their potency and side-effects. Understanding the homology of binding sites in targeted receptors, and involvement of signaling proteins after the inhibition might help in producing less toxic but effective inhibitors. Herein, we designed benzylideneindolon-2-one derivatives based on homology modeling in binding sites of VEGFR-2 and EGFR receptors as dual- inhibitor potent anticancer compounds with high selectivity. The benzylideneindolon-2-one derivatives were found to possess conformational switch in form of oxindole, substituted at 2-benzimidazole. Within synthesized compounds, 5b was found most active in in-vitro enzyme inhibition assay against VEGFR-2 and EGFR with highest IC50 value of 6.81 ± 2.55 and 13.04 ± 4.07 nM, respectively. Interestingly, cytotoxicity studies revealed selective toxicity of compound 5b against proliferation of A-431 cell lines (over expressed VEGFR-2 and EGFR) with GI50 value of 0.9 ± 0.66 µM. However, the compounds showed mild to moderate activity in all other cancer cell line in the range of 0.2-100 µM. Further mode of action studies by flow cytometry and western blot on A-431 indicated that they work via apoptosis at S- phase following Bcl/Bax pathway, and cell migration via MMP9. 5b not only suppressed tumor growth but also improved vandetanib associated with weight loss toxicity. Moreover, 5b was found safer than sunitinib and erlotinib with LD50 of 500 mg/kg body weight. These results propose 5b as potential anti-tumor drug with safer profile of conventional inhibitors of VEGFR-2 and EGFR for solid tumors.

Cell-penetrating peptide conjugates of indole-3-acetic acid-based DNA primase/Gyrase inhibitors as potent anti-tubercular agents against planktonic and biofilm culture of Mycobacterium smegmatis.

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that caused 1.5 million fatalities globally in 2018. New strains of Mtb resistant to all known classes of antibiotics pose a global healthcare problem. In this work, we have conjugated novel indole-3-acetic acid-based DNA primase/gyrase inhibitor with cell-penetrating peptide via cleavable and non-cleavable bonds. For non-cleavable linkage, inhibitor was conjugated with peptide via an amide bond to the N-terminus, whereas a cleavable linkage was obtained by conjugating the inhibitor through a disulfide bond. We performed the conjugation of the inhibitor either directly on a solid surface or by using solution-phase chemistry. M. smegmatis (non-pathogenic model of Mtb) was used to determine the minimal inhibitory concentration (MIC) of the synthetic conjugates. Conjugates were found more active as compared to free inhibitor molecules. Strikingly, the conjugate also impairs the development of biofilm, showing a therapeutic potential against infections caused by both planktonic and sessile forms of mycobacterium species.

Biomarker Analysis Based Chemoprofiling of Polyherbal Ayurvedic Formulation Containing Vitis vinifera L. by Validated UPLC-MS/MS Method.

BACKGROUND: Drakshasava is one of the commercial Ayurvedic medicines from India, prepared from grapes and spices. It is believed to address health imbalances and claimed to be beneficial for weakness, bleeding disorders, and various inflammatory diseases. It has been reported to possess pharmacological activities such as diuretic, cardioprotective, and antimicrobial. Being a polyherbal mixture, it faces challenges in its standardization and quality control. OBJECTIVE: The aim of the present study is to develop a validated UPLC-MS/MS method for simultaneous quantification of 10 polyphenolic biomarkers in Drakshasava. It explores the effect of Vitis vinifera L. and additional herbs on fermentation with respect to bioactive compounds through the successive addition method. METHODS: The MS methods were optimized in multiple-reaction monitoring (MRM) mode with ESI while chromatographic separation was achieved on an Acquity UPLC BEH C18 column using both isocratic and gradient elution in water and acetonitrile containing 0.1% formic acid. RESULTS: The developed method was validated as per ICH-Q2B guidelines and found to be within the assay variability limits. Gallic acid was found to be the most abundant marker in all the samples followed by resveratrol. The content of all the markers has been found to be increased significantly post-fermentation, compared to decoction except kaempferol. The successive addition of prashpeka drvya (minor herbs) in the formulation showed variability at different stages with respect to the selected markers and did not exhibit major changes in the chemical profiling of the final product. CONCLUSION: The developed method was found to be rapid, accurate, reliable, and highly sensitive for the simultaneous quantification of selected biomarkers in Drakshasava. The research is the first chemometric report on the standardization of Drakshasava by validated UPLC-MS/MS method. It may prove to be a useful tool for the development of new phytopharmaceutical drugs and further quality control of other polyherbal formulations.

Recent advances and prospects in gemcitabine drug delivery systems.

Cancer is a community health hazard which progress at a fatal rate in various countries across the globe. An agent used for chemotherapy should exhibit ideal properties to be an effective anticancer medicine. The chemotherapeutic medicines used for treatment of various cancers are, gemcitabine, paclitaxel, etoposide, methotrexate, cisplatin, doxorubicin and 5-fluorouracil. However, many of these agents present nonspecific systemic toxicity that prevents their treatment efficiency. Of all, gemcitabine has shown to be an active agent against colon, pancreatic, colon, ovarian, breast, head and neck and lung cancers in amalgamation with various anticancer agents. Gemcitabine is considered a gold-standard and the first FDA approved agent used as a monotherapy in management of advanced pancreatic cancers. However due to its poor pharmacokinetics, there is need of newer drug delivery system for efficient action. Nanotechnology has shown to be an emerging trend in field of medicine in providing novel modalities for cancer treatment. Various nanocarriers have the potential to deliver the drug at the desired site to obtain information about diagnosis and treatment of cancer. This review highlights on various nanocarriers like polymeric nanoparticles, solid lipid nanoparticles, mesoporous silica nanoparticles, magnetic nanoparticles, micelles, liposomes, dendrimers, gold nanoparticles and combination approaches for delivery of gemcitabine for cancer therapy. The co-encapsulation and concurrent delivery of Gem with other anticancer agents can enhance drug action at the cancer site with reduced side effects.

HPLC-UV and spectrofluorimetric methods for simultaneous estimation of fluticasone furoate and vilanterol in rabbit plasma: A pharmacokinetic study.

Fluticasone furoate (FF) and vilanterol trifenatate (VT) is a widely prescribed combination in the management of asthma and chronic obstructive pulmonary disease. In the present study, two quantitative methods based on HPLC-UV and spectrofluorimetric analysis had been developed and validated for simultaneous estimation of FF and VT in rabbit plasma using baclomethasone as internal standard (ISTD). Analytes and ISTD were separated from plasma using simple step of protein precipitation with acetonitrile. Chromatographic separation was achieved on Spherisorb S5 ODS2 (250 mm × 4.6 mm, 5.0 µm) column using mobile phase that constitute acetonitrile-0.01% glacial acetic acid in water (70:30, v/v) and then detected on a UV detector at 235 nm wavelength. Spectrofluorimetric detection was performed using absorption/emission wavelength (λabs/em) of 286/352 nm and 362/407 nm for FF and VT, respectively. For both analytes, linearity ranged from 4-200 ng/mL to 10-200 ng/mL using HPLC-UV and spectrofluorimetric method, respectively. Methods were validated as per FDA recommendations. Statistical analysis revealed that these detection methods are statistically insignificant difference and can be used interchangeably without any bias. Further, these methods were applied in pharmacokinetic study for simultaneous estimation of FF and VT in rabbit plasma.

Synthesis of stable benzimidazole derivatives bearing pyrazole as anticancer and EGFR receptor inhibitors.

A new series of benzimidazole linked pyrazole derivatives were synthesized by cyclocondensation reaction through one-pot multicomponent reaction in absolute ethanol. All the synthesized compounds were tested for their in vitro anticancer activities on five human cancer cell lines including MCF-7, HaCaT, MDA-MB231, A549 and HepG2. EGFR receptor inhibitory activities were carried out for all the compounds. Majority of the compounds showed potent antiproliferative activity against the tested cancer cell lines. Compound 5a showed the most effective activity against the lungs cancer cell lines (IC50 = 2.2 µM) and EGFR binding (IC50 = 0.97 µM) affinity as compared to other members of the series. Compound 5a inhibited growth of A549 cancer cells by inducing a strong G2/M phase arrest. In addition, same compound inhibited growth of A549 cancer cells by inducing apoptosis. In molecular docking studies compound 5a was bound to the active pocket of the EGFR (PDB 1M17) with five key hydrogen bonds and two π-π interaction with binding energies ΔG = -34.581 Kcal/mol.

Design and synthesis of cell selective α/ß-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus.

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or ß-amino acids. Sequence A-12 was protease susceptible whereas its α/ß-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2-32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/ß-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.

Design, synthesis, docking and QSAR study of substituted benzimidazole linked oxadiazole as cytotoxic agents, EGFR and erbB2 receptor inhibitors.

The synthesis of benzimidazole linked oxadiazole derivatives designed as potential EGFR and erbB2 receptor inhibitors with anticancer and apoptotic activity were studied. Compounds 7a specifically inhibit EGFR and erbB2 receptor at 0.081 and 0.098 µM concentration. Some of the compounds showed strong, broad-spectrum antiproliferative activitiy when tested against five human cancer cell lines. Compounds 7a and 7n were more cytotoxic than 5-fluorouracil against MCF-7 cancer cell, with IC50 values of 5.0 and 2.55 µM whereas, only 7a led to cell cycle arrest at G2/M phase accompanied by an increase in apoptosis. Compounds 7a and 7n showed normal architecture of myofibrils in cardiomyopathy study whereas only compound 7a showed nearly equal biochemical parameters (SGOT and SGPT) when compared to control. Molecular docking & 3D-QSAR studies were used to establish interactions of 7a and 7n within the active site of enzyme for ATP binding site of kinase domain.

N-terminally modified linear and branched spermine backbone dipeptidomimetics against planktonic and sessile methicillin-resistant Staphylococcus aureus.

Toward the discovery of useful therapeutic molecules, we report the design and synthesis of a focused library of new ultrashort N-terminally modified dipeptidomimetics, with or without modifications in the spermine backbone leading to linear (series 1) or branched (series 2) tryptophans, as antimicrobial agents. Eight peptidomimetics in the library showed good antibacterial activity (MICs of 1.77 to 14.2 µg/ml) against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis bacterial strains. Tryptophan fluorescence measurements on artificial bacterial or mammalian mimic membranes and assessment of the MRSA potential depolarization ability of the designed compounds revealed membrane interactions dependent on tryptophan positioning and N-terminal tagging. Among active peptidomimetics, compounds 1c and 1d were found to be nonhemolytic, displaying rapid bactericidal activity (at 4× MIC) against exponentially growing MRSA. Further, scanning electron microscopy of peptidomimetic 1c- and 1d-treated MRSA showed morphological changes with damage to cell walls, defining a membrane-active mode of action. Moreover, peptidomimetics 1c and 1d did not induce significant drug resistance in MRSA even after 17 passages. We also investigated the activity of these molecules against MRSA biofilms. At sub-MIC levels (∼2 to 4 µg/ml), both peptidomimetics inhibited biofilm formation. At concentrations higher than the MIC (35 to 140 µg/ml), peptidomimetics 1c and 1d significantly reduced the metabolic activity and biomass of mature (24-h) MRSA biofilms. These results were corroborated by confocal laser scanning microscopy (live/dead assay). The in vitro protease stability and lower cytotoxicity of peptidomimetics against peripheral blood mononuclear cells (PBMCs) support them being novel staphylocidal peptidomimetics. In conclusion, this study provides two peptidomimetics as potential leads for treatment of staphylococcal infections under planktonic and sessile conditions.