Gopal Singh Bisht

Recent approaches in design of peptidomimetics for antimicrobial drug discovery research.

Resistant pathogenic microbial strains are emerging at a rate that far exceeds the pace of new anti-infective drug development. In order to combat resistance development, there is pressing need to develop novel class of antibiotics having different mechanism of action in comparison to existing antibiotics. Antimicrobial peptides (AMPs) have been identified as ubiquitous components of innate immune system and widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from broad spectrum of activity to low propensity of resistance development. However, AMPs present several drawbacks that strongly limit their clinical applicability as ideal drug candidates such as; poor bioavailability, potential immunogenicity and high production cost. Thus, to overcome the limitations of native peptides, peptidomimetic becomes an important and promising approach. The different research groups worldwide engaged in antimicrobial drug discovery over the past decade have paid tremendous effort to design peptidomimetics. This review will focus on recent approaches in design of antimicrobial peptidomimetics their structure-activity relationship studies, mode of action, selectivity & toxicity.

Systematic Study of Non‐Natural Short Cationic Lipopeptides as Novel Broad‐Spectrum Antimicrobial Agents

We describe the design and synthesis of a new series of non‐natural short cationic lipopeptides (MW = 700) as antimicrobial agents. All of the synthesized lipopeptides were tested against a range of microbes such as Gram‐positive, Gram‐negative bacteria, fungi including methicillin‐resistant Staphylococcus aureus (MRSA) and methicillin‐resistant Staphylococcus epidermidis (MRSE). By systematic study of design template, we found that three ornithine residues conjugated with myristic acid are minimum requirement for a compound to be an antimicrobial agent. The most potent lipopeptide LP16 possesses broad‐spectrum antimicrobial activity and has MICs in the range of 1.5–6.25 μg/mL against Escherichia coli, S. aureus, Pseudomonas aeruginosa, Bacillus subtilis, and MRSE. All lipopeptides showed high selectivity toward microbial strains as compared to human red blood cells (HC50 > 250 μg/mL). Moreover, most potent lipopeptides (LP16 and LP23) did not induce drug resistance in S. aureus even after 15 rounds of passaging. In addition, a representative lipopeptide (LP16) showed tryptic stability for 24 h. These results suggest the potential of short cationic lipopeptides to boost the discovery of future antimicrobial therapeutics.

Antibacterial evaluation of structurally amphipathic, membrane active small cationic peptidomimetics: Synthesized by incorporating 3-amino benzoic acid as peptidomimetic element

A new series of small cationic peptidomimetics were synthesized by incorporating 3-amino benzoic acid (3-ABA) in a small structural framework with the objective to mimic essential properties of natural antimicrobial peptides (AMPs). The new design approach resulted into improvement of activity and selectivity in comparison to linear peptides and allowed us to better understand the influence of structural amphipathicity on biological activity. Lead peptidomimetics displayed antibacterial activities against resistant pathogens (MRSA & MRSE). A calcein dye leakage experiment revealed a membranolytic effect of 4g and 4l which was further confirmed by fluorescence microscopy. In addition, proteolytic stability and no sign of resistance development against Staphylococcus aureus and MRSA demonstrate their potential for further development as novel antimicrobial therapeutics.

Usefulness of Organic Acid Produced by Exiguobacterium sp. 12/1 on Neutralization of Alkaline Wastewater

The aim of this study was to investigate the role of organic acids produced by Exiguobacterium sp. strain 12/1 (DSM 21148) in neutralization of alkaline wastewater emanated from beverage industry. This bacterium is known to be able to grow in medium of pH as high as pH 12.0 and to neutralize alkaline industrial wastewater from pH 12.0 to pH 7.5. The initial investigation on the type of functional groups present in medium, carried out using FT-IR spectroscopy, revealed the presence of peaks corresponding to carbonyl group and hydroxyl group, suggesting the release of carboxylic acid or related metabolic product(s). The identification of specific carboxylic group, carried out using RP-HPLC, revealed the presence of a single peak in the culture supernatant with retention time most similar to formic acid. The concentration of acid produced on different carbon sources was studied as a function of time. Although acid was present in same final concentration, the rate of acid production was highest in case of medium supplemented with sucrose followed by fructose and glucose. The knowledge of metabolic products of the bacterium can be considered as a first step towards realization of its potential for large-scale bioremediation of alkaline wastewater from beverage industry.

Small cationic antimicrobial peptidomimetics: emerging candidate for the development of potential anti-infective agents.

Rapid increase in the emergence and spread of microbes resistant to conventionally used antibiotics has become a major threat to global health care. Antimicrobial peptides (AMPs) are considered as a potential source of novel antibiotics because of their numerous advantages such as broad-spectrum activity, lower tendency to induce resistance, immunomodulatory response and unique mode of action. However, AMPs have several drawbacks such as; susceptibility to protease degradation, toxicity and high costs of manufacturing. Therefore, extensive research efforts are underway to explore the therapeutic potential of these fascinating natural compounds. This review highlights the potential of small cationic antimicrobial peptidomimetics (SCAMPs; M.W. ≅ 700 Da) as new generation antibiotics. In particular, we focused on recently identified small active pharmacophore from bulky templates of native AMPs, β-peptides, and lipopeptides. In addition, various design strategies recently undertaken to improve the physicochemical properties (proteolytic stability & plasma protein binding) of small cationic peptides have also been discussed.

Design and synthesis of cell selective α/β-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 β-amino acids. Sequence A-12 was protease susceptible whereas its α/β-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-/β-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.

Engineering Yeast as Cellular Factory

Yeast is used as an attractive host for metabolic engineering. It is widely used in industries for the production of enzymes, fine chemicals, alcoholic beverages, baker’s yeast, pharmaceutical products, biofuels, recombinant proteins, etc. Yeast can be grown easily as it requires simple growth factors such as carbon, vitamins, and salts which makes it economic for large-scale production. Among different types of yeast like Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Kluyveromyces lactis, and Saccharomyces cerevisiae, S. cerevisiae is most commonly used. Yeast can be genetically modified to produce chemicals (glycerol, propanediol, organic acids, etc.) and fatty acid derivatives (fatty acid ester, fatty alkanes, and fatty alcohols). Yeast engineering for the production of biofuel has made the production of biofuel affordable and eco-friendly. Yeast has the ability to perform posttranslational modification and, therefore, is widely employed for producing human recombinant proteins. Generally modifications of enzyme activity are employed in yeast engineering to obtain desired product. Yeast engineering can be achieved by either introducing a new pathway in yeast or by altering the native pathway. In this chapter we have presented general considerations along with different strategies developed for the synthesis of various products using engineered yeast.

Nanomaterial in Diverse Biological Applications

Nanotechnology has revolutionized the world ranging from simple food packaging articles to medicinal therapeutics. Nano-sized objects have found application in most of the fields in sciences. Nanotechnology has been applied to create molecules possessing dimensions according to the type of application desired. Pharmaceutical industry has shown an immense interest in nanotechnology to improve pharmacologic and therapeutic properties of conventional drugs. Pharmaceutical formulations based on nanotechnology have improved the therapy strategies to treat simple as well as complex diseases. Selective targeting potential of these formulations not only deliver drug at active sites, but they also protect healthy cells from harmful effects of active drug. Nanotechnology has provided us with a lot of advantages, but along with these advantages, certain nanomaterials have been found to be toxic to human health as well as the environment. Nanotechnology-based products must be checked for their safety and toxicity profiles before launching them in market. The study of safety aspects and toxicity profile of nanomaterials is emerging as a new area of research. This chapter focuses on pharmacological applications, theranostic potential, safety aspects, and risk factors of various nanomaterial-based products. Along with this, it highlights the role of nanomaterials in bioremediation and controlling pollution.

Factories for Antibody Generation

Antibodies are gathering increasing importance as diagnostic tools and as therapeutic agents. The major limiting factor is their low quality and productivity. Progress in exploiting molecular biology tools has provided platforms for developing recombinant antibodies. The major focus is on (1) performing complicated engineering by simpler techniques, (2) building up binders and fusing to diverse effectors and tags, (3) manufacturing the constructs economically using microbes, and (4) retaining stable material clonality for higher reproducibility. Among the most regularly used expression systems used for generating artificial antibody fragments are microalgae, bacteria, and mammalian cells. Each expression system has its own merits and demerits; however, mammalian cell expression systems have quite a few advantages: for recombinant protein production is their ability to induce proper folding of the protein, posttranslational changes, and generating the product—factors necessary for proper biological activity.

In Vitro and In Vivo Evaluation of Small Cationic Abiotic Lipopeptides as Novel Antifungal Agents

We investigated the antifungal potential of short lipopeptides against clinical fungal isolates with an objective to evaluate their clinical feasibility. All tested lipopeptides exhibit good antifungal activity with negligible difference between the MICs against susceptible and drug‐resistant clinical fungal isolates. The MTT assay results revealed the lower cytotoxicity of lipopeptides toward mammalian cells (NRK‐52E). In particular, LP24 displayed highest potency against most of the tested fungal isolates with MICs in the range of 1.5–4.5 μg/mL. Calcein dye leakage experiments with model membrane suggested the membrane‐active mode of action for LP24. Extending our work from model membranes to intact Aspergillus fumigatus in scanning electron micrographs, we could visualize surface perturbation caused by LP24. LP24 (5 mg/kg) significantly reduces the A. fumigatus burden among the various organs of infected animals, and 70% of the infected mice survived when observed for 28 days. This study underscores the potential of small cationic abiotic lipopeptides to develop into the next‐generation antimicrobial therapy.