Structure-based drug repurposing to inhibit the DNA gyrase of Mycobacterium tuberculosis.

Drug repurposing is an alternative avenue for identifying new drugs to treat tuberculosis (TB). Despite the broad-range of anti-tubercular drugs, the emergence of multi-drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb) H37Rv, as well as the significant death toll globally, necessitates the development of new and effective drugs to treat TB. In this study, we have employed a drug repurposing approach to address this drug resistance problem by screening the drugbank database to identify novel inhibitors of the Mtb target enzyme, DNA gyrase. The compounds were screened against the ATPase domain of the gyrase B subunit (MtbGyrB47), and the docking results showed that echinacoside, doxorubicin, epirubicin, and idarubicin possess high binding affinities against MtbGyrB47. Comprehensive assessment using fluorescence spectroscopy, surface plasmon resonance spectroscopy (SPR), and circular dichroism (CD) titration studies revealed echinacoside as a potent binder of MtbGyrB47. Furthermore, ATPase, and DNA supercoiling assays exhibited an IC50 values of 2.1-4.7 µM for echinacoside, doxorubicin, epirubicin, and idarubicin. Among these compounds, the least MIC90 of 6.3 and 12 µM were observed for epirubicin and echinacoside, respectively, against Mtb. Our findings indicate that echinacoside and epirubicin targets mycobacterial DNA gyrase, inhibit its catalytic cycle, and retard mycobacterium growth. Further, these compounds exhibit potential scaffolds for optimizing novel anti-mycobacterial agents that can act on drug-resistant strains.

Specific keratinase derived designer peptides potently inhibit Aß aggregation resulting in reduced neuronal toxicity and apoptosis.

Compelling evidence implicates self-assembly of amyloid-ß (Aß1-42) peptides into soluble oligomers and fibrils as a major underlying event in Alzheimer's disease (AD) pathogenesis. Herein, we employed amyloid-degrading keratinase (kerA) enzyme as a key Aß1-42-binding scaffold to identify five keratinase-guided peptides (KgPs) capable of interacting with and altering amyloidogenic conversion of Aß1-42 The KgPs showed micromolar affinities with Aß1-42 and abolished its sigmoidal amyloidogenic transition, resulting in abrogation of fibrillogenesis. Comprehensive assessment using dynamic light scattering (DLS), atomic force microscopy (AFM) and Fourier-transform infrared (FTIR) spectroscopy showed that KgPs induced the formation of off-pathway oligomers comparatively larger than the native Aß1-42 oligomers but with a significantly reduced cross-ß signature. These off-pathway oligomers exhibited low immunoreactivity against oligomer-specific (A11) and fibril-specific (OC) antibodies and rescued neuronal cells from Aß1-42 oligomer toxicity as well as neuronal apoptosis. Structural analysis using molecular docking and molecular dynamics (MD) simulations showed two preferred KgP binding sites (Lys16-Phe20 and Leu28-Val39) on the NMR ensembles of monomeric and fibrillar Aß1-42, indicating an interruption of crucial hydrophobic and aromatic interactions. Overall, our results demonstrate a new approach for designing potential anti-amyloid molecules that could pave way for developing effective therapeutics against AD and other amyloid diseases.

Synergistic approaches unraveling regulation and aggregation of intrinsically disordered ß-amyloids implicated in Alzheimer's disease.

Alzheimer's disease is a severe brain illness that causes vast numbers of nerve cells in the brain to die, driven by the production and deposition of amyloid beta (Aß) peptides. Intrinsically disordered proteins (IDPs) generally lack stable structures and are abundant in nature. Aß peptide is a well-known IDP with a wide range of oligomeric forms. Dysfunctions in Aß lead to oligomerization, formation of fibrils, and neurodegenerative disorders or other forms of dementia. In this study, we used replica exchange molecular dynamics (REMD) to elucidate the roles of different osmolytes, particularly urea and trimethylamine N-oxide (TMAO), to study shifts in IDP populations. REMD samples the conformational space efficiently and at physiologically relevant temperatures, compared to conventional molecular dynamics that sample at a constant temperature. Urea is known to minimize the aggregation process, while TMAO is beneficial for its stabilizing action. The two osmolytes displayed characteristic effects on Aß peptides and resulted in progressive modulation of conformations. The present study underlines the hypothesis of "modulation of conformational ensembles" to explain the regulation and aggregation of IDPs.

Biotechnological applications and prospective market of microbial keratinases.

Keratinases are well-recognized enzymes with the unique ability to attack highly cross-linked, recalcitrant structural proteins such as keratin. Their potential in environmental clean-up of huge amount of feather waste has been well established since long. Today, they have gained importance in various other biotechnological and pharmaceutical applications. However, commercial availability of keratinases is still limited. Hence, to attract entrepreneurs, investors and enzyme industries it is utmost important to explicitly present the market potential of keratinases through detailed account of its application sectors. Here, the application areas have been divided into three parts: the first one is dealing with the area of exclusive applications, the second emphasizes protease dominated sectors where keratinases would prove better substitutes, and the third deals with upcoming newer areas which still await practical documentation. An account of benefits of keratinase usage, existing market size, and available commercial sources and products has also been presented.

Thermostable keratinase from Bacillus pumilus KS12: production, chitin crosslinking and degradation of Sup35NM aggregates.

Production of thermostable keratinase from Bacillus pumilus KS12 was enhanced up to seven fold by statistical methods. The enzyme was partially purified by ultrafiltration followed by thermal precipitation with purity of 3.2-fold and recovery of 89%. Keratinase was immobilized using covalent method by crosslinking 2 mg protein (688 U/mg) onto 1g chitin activated with 2.5% (v/v) glutaraldehyde for 60 min. Its comparative biochemical studies with that of free keratinase revealed the shift in optimum pH with increased stability towards pH from 9.0 to 10.0 and temperature. Also, it showed statistically significant improved hydrolysis of a number of soluble and insoluble substrates in comparison to free keratinase. Owing to improved catalytic efficiency of immobilized keratinase, its potential for degradation of Sup35NM was evaluated, where 100 µg of enzyme could degrade 60 µg Sup35NM after 60 min at pH 7.0 and 37°C.

A hydrolytic γ-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation.

γ-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with γ-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni(2+)-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t (1/2) of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Ptγ-GT shared 37 % sequence identity and 53 % homology with an extremophile γ-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.

Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: altered substrate specificity and thermostability.

Pro-sequences were swapped in cis between keratinases from Bacillus licheniformis (Ker BL) and Bacillus pumilus (Ker BP) to construct Ker ProBP-BL and Ker ProBL-BP, respectively. Expression of these keratinases was carried out constitutively by E. coli HB101-pEZZ18 system. They were characterized with respect to their parent enzymes, Ker BL and Ker BP, respectively. Ker ProBP-BL became more thermostable with a t(1/2) of 45 min at 80°C contrary to Ker BL which was not stable beyond 60°C. Similarly, the activity of Ker ProBP-BL on keratin and casein substrate, i.e. K:C ratio increased to 1.2 in comparison to 0.1 for Ker BL. Hydrolysis of insulin B-chain revealed that the cleavage sites increased to six from four in case of Ker ProBP-BL in comparison to Ker BL. However, cleavage sites decreased from seven to four in case of Ker ProBL-BP in comparison to the parent keratinase, Ker BP. Likewise, Ker ProBL-BP revealed altered pH and temperature kinetics with optima at pH 10 and 60°C in comparison to Ker BP which had optima at pH 9 and 70°C. It also cleaved soluble substrates with better efficiency in comparison to Ker BP with K:C ratio of 1.6. Pro-sequence mediated conformational changes were also observed in trans and were almost similar to the features acquired by the chimeras constructed in cis by swapping the pro-sequence region.

Biochemical Characterization of a Thiol-Activated, Oxidation Stable Keratinase from Bacillus pumilus KS12.

An extracellular keratinase from Bacillus pumilus KS12 was purified by DEAE ion exchange chromatography. It was a 45 kDa monomer as determined by SDS PAGE analysis. It was found to be an alkaline, serine protease with pH and temperature optima of 10 and 60°C, respectively. It was thiol activated with two- and eight-fold enhancement in presence of 10 mM DTT and ß-mercaptoethanol, respectively. In addition, its activity was stimulated in the presence of various surfactants, detergents, and oxidizing agents where a nearly 2- to 3-fold enhancement was observed in presence of H(2)O(2) and NaHClO(3). It hydrolyzed broad range of complex substrates including feather keratin, haemoglobin, fibrin, casein,and α-keratin. Analysis of amidolytic activity revealed that it efficiently cleaved phenylalanine → leucine → alanine- p-nitroanilides. It also cleaved insulin B chain between Val(2)- Asn(3), Leu(6)-Cys(7) and His(10)-Leu(11) residues.