Anil Kumar Chhillar

Functional aspects of the solution structure and dynamics of PAF – a highly‐stable antifungal protein from Penicillium chrysogenum

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G‐protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five β‐strands forming two orthogonally packed β‐sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S–S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site‐directed mutagenesis, we demonstrate that the highly‐conserved and positively‐charged lysine‐rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent‐exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

Investigations toward new lead compounds from medicinally important plants

Extensive phytochemical investigations on 30 Piper species growing in India and other medicinal plants have revealed the presence of a large number of novel compounds belonging to different classes. The antiviral activity of several lignans and neolignans belonging to different structural types has been evaluated against six different viral strains. Further, the effects of ethanol, chloroform, and hexane extracts of Piper longum and Piper galiatum on TNF-α induced expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells have been studied, a novel aromatic ester was isolated from the most active extract of P. longum. A potential antifungal compound having implications in treating aspergillosis was isolated from an important Indian medicinal plant, Datura metel.

Quantitative analysis of catechins in Saraca asoca and correlation with antimicrobial activity

Herbal medicines are highly complex and have unknown mechanisms in diseases treatment. Saraca asoca (Roxb.), De. Wild has been recommended to treat gynecological disorders and used in several commercial polyherbal formulations. In present study, efforts have been made to explore antimicrobial activity and its co-relation with the distributions of catechins in the organs of S. asoca using targeted MS/MS. Eight extracts (cold and hot water) from four different organs of S. asoca and two drugs were prepared and antimicrobial activity was assessed by microbroth dilution assay. Quantitative and qualitative analysis of catechins in crude extracts was done by using targeted and auto-MS/MS and correlated with antimicrobial activity. (+)-Catechin and (+)-epicatechin and their biosynthesis related compound were found to be up-regulated in regenerated bark and leaves extracts. (−)-Epigallocatechin was found to be significantly higher in bark water extract as compared to others but showed low antimicrobial activity. Result showed down-regulation of (−)-epigallocatechin and up-regulation of (+)-catechin and (+)-epicatechin in the regenerated bark and leaves of S. asoca. It might be the contributing factor in the antimicrobial activity of regenerated bark and leaves of the plant. The concentration of (+)-epicatechin in processed drugs (Ashokarishta) from Baidyanath was found to be seven times higher than that of Dabur Pvt. Ltd., but no antimicrobial activity was observed, indicating the variations among the plant based drugs. This will be helpful in rational use of S. asoca parts. Furthermore, the analytical method developed is sensitive, repeatable and reliable; therefore, it is suitable for quality control of herbal drugs.

Analysis Toward Innovative Herbal Antibacterial & Antifungal Drugs

The antimicrobial activities of four medicinal plants Argemona mexicana, Achyranthes aspera, Catharanthus roseus, and Syzygium cumini were evaluated against Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Proteus vulgaris, Bacillus subtilis, Salmonella typhi and three Aspergillus species. Extracts from Achyranthes aspera and Catharanthus roseus showed the highest antimicrobial potential (MIC 0.375-0.750 mg/ml) while extract from Argemona mexicana and Syzygium cumini, showed less activity. In disc diffusion assay, only eight out of twenty extracts showed antimicrobial activity at a concentration of 25.0 μg/ disc. The GCMS investigation reveals the existence of 2-bornanone; 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; hexadecanoic acid, methyl ester and hexatriacontane in water extract fraction of C. roseus. The present research article provides a review of some medicinal plants incorporating antimicrobial drugs, together with recent advances in emerging therapeutics in clinical development and related patents for exploitation of herbal medicine.

Combination Therapy: The Propitious Rationale for Drug Development

Therapeutic options for many infections are extremely limited and at crisis point. We run the risk of entering a second pre-antibiotic era. There had been no miracle drug for the patients infected by resistant microbial pathogens. Most of the very few new drugs under development have problems with their toxicity, or pharmacokinetics and pharmacodynamics. We are already decades behind in the discovery, characterization and development of new antimicrobials. In that scenario, we could not imagine surviving without newer and effective antimicrobial agents. Bacteria have been the champions of evolution and are still evolving continuously, where they pose serious challenges for humans. Along with the crisis of evolving resistance, the condition is made worst by the meager drug pipeline for new antimicrobials. Despite ongoing efforts only 2 new antibiotics (Telavancin in 2009 and Ceftaroline fosamil in 2010) have been approved since 2009 pipeline status report of Infectious Disease Society of America (IDSA). Recent approval of new combination based antiviral drugs such as Stribild (combination of four drugs for HIV treatment) and Menhibrix (combination vaccine to prevent meningococcal disease and Haemophilus influenzae type b in children) proves that combination therapy is still the most promising approach to combat the ever evolving pathogens. Combination therapy involves the drug repurposing and regrouping of the existing antimicrobial agents to provide a synergistic approach for management of infectious diseases. This review article is an effort to highlight the challenges in new drug development and potential of combination drug therapy to deal with them.

Combinatorial effect of TIMP-1 and α1AT gene polymorphisms on development of chronic obstructive pulmonary disease.

OBJECTIVE To study the role of α(1)AT and TIMP-1 gene polymorphisms in development of COPD. DESIGN AND METHODS Blood samples from total 408 subjects (217 COPD patients and 191 controls) were used for genotyping and estimating biolevels of α(1)AT, TIMP-1 and inflammatory cytokines. Data was analyzed to determine the role of interaction of TIMP-1 and α(1)AT genes; and interplay between various genotypes and biolevels of α(1)AT, TIMP-1 and inflammatory cytokines in development of COPD. RESULTS Significantly low levels of α(1)AT and TIMP-1 were observed in COPD patients as compared to controls (P = 0.001), where as the inflammatory cytokines were found to be increased in patients. PIM3 allele of α(1)AT gene in COPD patients was found to be associated with low levels of α(1)AT (P = 0.001), the effect being more pronounced when PIM3 combined with rs6609533 of TIMP-1 gene (P = 0.0001). Combination of genotypes rs6609533 of TIMP-1 and PIM3 of α(1)AT containing the risk alleles was over-represented in patients (P = 0.005). CONCLUSION The SNP rs6609533 of TIMP-1 gene interacted with PIM3 of α(1)AT to make a possible risk combination for development of COPD.

Differential expression of Aspergillus fumigatus protein in response to treatment with a novel antifungal compound, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate.

A dihydropyridine derivative, diethyl 4‐(4‐methoxyphenyl)‐2,6‐dimethyl‐1,4‐dihydropyridin‐3,5‐dicarboxylate (2e), having potent antifungal activity against pathogenic species of Aspergillus was investigated for its possible molecular mechanism of action. The SDS‐PAGE coupled with nano‐high‐performance liquid chromatography–tandem mass spectrometry was used directly to assess both absolute abundance and differential expression of proteins in the secretory phases of Aspergillus fumigatus under the influence of 2e. It was observed that the compound inhibited the expression of two proteins of 60.99 and 79.77 kDa. Both of these secretory proteins that were inhibited by 2e, were analysed further by matrix assisted laser desorption ionization time‐of‐flight mass spectrometry. The 60.99‐ and 79.77‐kDa proteins were identified as probable retroelement pol polyprotein and elongation factor G respectively. These targeted proteins could be the products of potentially virulence‐related genes of A. fumigatus which may unravel the mode of action of 2e and pathobiology of A. fumigatus.

Investigations on anti-Aspergillus properties of bacterial products.

Aims:  To investigate the anti‐Aspergillus properties of bacterial products.

Genomics of Chronic Obstructive Pulmonary Disease (COPD); Exploring the SNPs of Protease-Antiprotease Pathway

The COPD has been an important respiratory condition that affects people worldwide and its incidence has been alarming. The increasing incidence of this disorder has been attributed to global industrialization and environmental pollution. Although the exposures to environmental pollutants and smoking have been important triggers, the genetic component of individuals has been shown to be important for development and progression of COPD. Recent literature reported that protease-antiprotease imbalance to be important in etiopathogenesis of COPD. The enzymes namely neutrophil elastase and matrix metalloprotienases are considered to be foremost proteolytic molecules released by neutrophils and macrophages during inflammatory events in COPD. Normally, the lungs remain protected from the destructive effect of these two antiproteases by α1-antitrypsin (α1AT) and tissue inhibitors of metalloproteinases (TIMPs) respectively. In this review, we are trying to highlight the work by various research groups in exploring the SNPs of various genes of inflammatory pathways and the protease-antiprotease pathway, which may have some degree of association with COPD.

Siderophores; iron scavengers: the novel & promising targets for pathogen specific antifungal therapy

ABSTRACT Introduction: The recent emergence of resistance, toxicity paradigm and limited efficacy of conventional antifungal drugs necessitate the identification of de novo targets in fungal metabolism. One of the most critical physiological processes during in vivo pathogenesis is maintenance of iron homeostasis. The most life threatening opportunistic human fungal pathogens like Aspergillus, Candida and Cryptococcus exploit the siderophore mediated iron uptake mechanism either for survival, virulence, propagation or resistance to oxidative stress envisaged in vivo during infection. Areas covered: In this review, we will highlight the metabolic pathways; specifically siderophore biosynthesis, uptake and utilisation, triggered in the fungal pathogens in iron starving conditions and the various putative targets viable in these pathways to be recruited as novel therapeutic antidotes either via biosynthetic enzymes catalytic site inhibitors or as drug conjugates through trojan horse approach and further role in the development of fungal specific reliable diagnostic markers. Expert opinion: Siderophores are the weapons released by a pathogen to conquer the battle for iron acquisition. Hence, the fungal siderophore biosynthetic pathways along with their uptake and utilisation mechanisms represent an ideal target for pathogen specific, host friendly therapeutic strategy which would block the proliferation of parasite without causing any harm to the mammalian host.