In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive ½2 þ 2 e − reductions to release thioesterbound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e − reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.chain release | glycopeptidolipid | NAD(P)H | tyrosine-dependent oxidoreductase P olyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are multifunctional proteins that are known to produce a variety of complex natural products (1). While most of these natural products can be classified as secondary metabolites, in mycobacteria PKSs and NRPSs synthesize lipidic metabolites that are important for their survival and pathogenesis (2). The biosynthetic mechanism involves assembly-line repetitive condensation of specific monomeric units. During this process, the intermediates remain covalently tethered to the proteins through the thiol group of phosphopantetheine (ppant) moiety that is posttranslationally added onto the carrier domains (3). The ppantarm-bound substrate reaches out to the active centers of the various domains to facilitate successive catalytic steps. The chainreleasing domain then performs dual function of detaching the mature product and playing a role in determining the final structure of the metabolite.The most well-studied chain-releasing domains are thioesterases (TE) that hydrolyze the thioester bond to release linear as well as macrocyclic products (4). Recently, a new mechanism of chain release catalyzed by the reductase (R) domains has been identified. These domains utilize NAD(P)H as cofactor to reductively release the final product as aldehyde or alcohol (Fig. S1A) (5). Interestingly, 2 R domain homologues are shown to perform cofactor-independent Dieckmann's cyclization (referred to as R* domains) (6). Broadly, R domains show homology to the family of short-chain dehydrogenases/reductases (SDRs). The SDR family consists of tyrosine-dependent oxidoreductases that are known to share common sequ...

2-Butanone thiosemicarbazone ligand was prepared by condensation reaction between thiosemicarbazide and butanone. The ligand was characterized by H NMR,C NMR, FT-IR, mass spectrometry and UV spectroscopic studies. Docking studies were performed to study inhibitory action against topoisomerase II (Topo II) and ribonucleoside diphosphate reductase (RR) enzymes. Inhibition constants ( ) of the ligand were 437.87 and 327.4 μM for the two enzymes, respectively. The ligand was tested for its potential anticancer activity against two cancer cell lines MDA-MB-231 and A549 using MTT assay and was found to exhibit good activity at higher doses with an IC = 80 μM against human breast cancer cell line MDA-MB-231. On the other hand, no significant activity was obtained against the lung carcinoma cell line A549. Antibacterial activity of the ligand was tested against and using the disc diffusion method. Ligand did not exhibit any significant antibacterial activity. Four complexes of Co(III), Fe(II), Cu(II), and Zn(II) were prepared with the ligand and characterized by various spectroscopic studies. Low molar conductance values were obtained for all complexes displaying non-electrolyte nature except in Co(III) complex. As expected, complexation with metal ions significantly increased the cytotoxicity of the ligand against the tested cell lines viz. IC values of <20 μM for Co, Fe, and Zn complexes and approx. 80 μM against MDA cells versus IC value of <20 μM for Co and Cu complexes and that of 30 and 50 μM for Fe and Zn complexes, respectively, against A549 cells. The Cu complex was found to be active against and with MIC values in the range of 6-10 mg/mL. Other than Cu, only Co complex was found to possess antibacterial activity with MIC values of 5-10 mg/mL when tested against . Bioactivity score and Prediction of Activity Spectra for Substances (PASS) analysis also depicted the drug-like nature of ligand and complexes.

The steady rise in antimicrobial resistance poses a severe threat to global public health by hindering treatment of an escalating spectrum of infections. We have previously established the potent activity of α-MSH, a 13 residue antimicrobial peptide, against the opportunistic pathogen Staphylococcus aureus. Here, we sought to determine whether an increase in cationic charge in α-MSH could contribute towards improving its staphylocidal potential by increasing its interaction with anionic bacterial membranes. For this we designed novel α-MSH analogues by replacing polar uncharged residues with lysine and alanine. Similar to α-MSH, the designed peptides preserved turn/random coil conformation in artificial bacterial mimic 1,2-dimyristoyl-sn-glycero-3-phosphocholine:1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (7:3, w/w) vesicles and showed preferential insertion in the hydrophobic core of anionic membranes. Increased cationic charge resulted in considerable augmentation of antibacterial potency against MSSA and MRSA. With ~18-fold better binding than α-MSH to bacterial mimic vesicles, the most charged peptide KKK-MSH showed enhanced membrane permeabilization and depolarization activity against intact S. aureus. Scanning electron microscopy confirmed a membrane disruptive mode of action for KKK-MSH. Overall, increasing the cationic charge improved the staphylocidal activity of α-MSH without compromising its cell selectivity. The present study would help in designing more effective α-MSH-based peptides to combat clinically relevant staphylococcal infections.

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 mug/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.

Background:: Computational or in silico studies are undertaken to assess the drug like properties of lead compounds. These studies help in fast prediction of relevant properties. Objective: : Through this review, an effort is made to encapsulate some of the important parameters which should be met by a compound for it to be considered as a potential drug candidate along with an overview of automated softwares which can be used for making various predictions. Methods:: Drug uptake, its absorption, evacuation and associated hazardous effects are important factors for consideration in drug designing and should be known in early stages of drug development. Several important physicochemical properties like molecular weight, polar surface area (PSA), molecular flexibility etc. have to be taken into consideration in drug designing. Toxicological assessment is another important aspect of drug discovery which predicts the safety and adverse effects of a drug. Results: : Additionally, bioactivity scores of probable drug leads against various human receptors can also be predicted to evaluate the probability of them to act as a potential drug candidate. The in vivo biological targets of a molecule can also be efficiently predicted by molecular docking studies. Conclusion:: Some important software like iGEMDOCK, AutoDock, OSIRIS property explorer, Molinspiration, MetaPrint2D, admetSAR and their working methodology and principle of working have been summarized in this review.

Escalating multidrug resistance and highly evolved virulence mechanisms have aggravated the clinical menace of methicillin-resistant Staphylococcus aureus (MRSA) infections. Towards development of economically viable staphylocidal agents here we report eight structurally novel tryptophan-arginine template based peptidomimetics. Out of the designed molecules, three lipopeptidomimetics (S-6, S-7 and S-8) containing 12-amino dodecanoic acid exhibited cell selectivity and good to potent activity against clinically relevant pathogens MRSA, methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (MIC: 1.4-22.7 μg/mL). Mechanistically, the active peptidomimetics dissipated membrane potential and caused massive permeabilization on MRSA concomitant with loss of viability. Against stationary phase MRSA under nutrient-depleted conditions, active peptidomimetics S-7 and S-8 achieved > 6 log reduction in viability upon 24 h incubation while both S-7 (at 226 μg/mL) and S-8 (at 28 μg/mL) also destroyed 48 h mature MRSA biofilm causing significant decrease in viability (p < 0.05). Encouragingly, most active peptidomimetic S-8 maintained efficacy against MRSA in presence of serum/plasma while exhibiting no increase in MIC over 17 serial passages at sub-MIC concentrations implying resistance development to be less likely. Therefore, we envisage that the current template warrants further optimization towards the development of cell selective peptidomimetics for the treatment of device associated MRSA infections.Pan-resistant microbial pathogens refractory to current clinical antibiotics are spreading at an unprecedented rate 1 . Among human pathogens, methicillin-resistant Staphylococcus aureus (MRSA) is a high priority clinical threat 2 . Multidrug resistance and expression of multiple virulence factors have further exacerbated the clinical severity of MRSA infections in clinics as well as community settings 3 . Moreover, with the growing use of medical implants in clinics, almost 65-80% MRSA infections in vivo have been reported to be associated with biofilm formation. Biofilms are large agglomerations of bacterial cells encased in a self-produced matrix that exhibit substantial recalcitrance to antibiotic treatment 4 (10 to 1000-fold more antibiotic concentration is required to eradicate biofilms). The recalcitrance of biofilms is multifactorial involving high localized inoculum, poor penetration of antibiotics to the core of biofilms, slow-growing nutrient-depleted bacterial populations and presence of persister cells 5,6 . Additionally, matrix formation allows the creation of a microenvironment which promotes resistance and tolerance development in microbes along with providing protection from host immune surveillance 7 . Therefore, a lot of efforts are being directed towards optimization of novel molecules/strategies to eradicate clinically relevant biofilms [8][9][10] . Recently, the consensus is emerging that unlike single target capturing antibiotics; membrane disruptive, dual target...

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