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CURATOR
A pinboard by
SUDHIR KUMAR PAL

PhD student, VIT University

PINBOARD SUMMARY

Structural and inhibition studies of LpxC

I am currently pursuing PhD at Centre for Bio-separation technology, Vellore Institute of technology, Tamil Nadu, India. The focus of my work is on structural and functional studies of UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) enzyme from Acinetobacter baumannii. This study will also incorporate the use of various biophysical techniques like DLS, CD, X-ray crystallography, and Cryo-EM, etc.

During my masters in Bioinformatics, I have been introduced and trained in molecular dynamics and various techniques in structural biology. I have done my master thesis on modelling of three dimensional structures of potent enzymes of Mycobacterium tuberculosis and their interaction with various drug candidates via molecular docking studies with the use of Phyre2, Docking Server and other concerned tools.

After completion of my Masters, I got an opportunity to work with Dr. Vijay Nema and gained better insight in Molecular Dynamics and Simulations of Proteins and Protein-ligand complexes using Gromacs and Charmm-gui.

During my research work at Masaryk University, Brno, Czech Republic under the supervision of Dr. Jozef Hritz, I gained detailed knowledge about molecular dynamics simulations and high-end techniques like SAXS and Cryo-Electron Microscopy. The main focus of my work was to elucidate three dimensional structure of Tyrosine Hydroxylase, mechanism of its activity and interaction with 14-3-3 proteins.

Furthermore, my research work at University of South Bohemia in Ceske Budejovice, Czech Republic under the guidance of Prof. Rudiger Ettrich helped me to gain better understanding of CryoEM generated model fitting and 3D structure generation of bio-molecules and protein X-rays crystallography.

Thus, I believe that training at HERCULES European School will provide me ample opportunities to learn advanced techniques and deepen up my knowledge in the field of structural biology. It is certain that experiences gained from this school will prove to be invaluable to me and my future endeavors to develop into a full-fledged independent researcher to give my best for the benefit of Science and mankind.

Besides getting enlightened from the theory, practicals and tutorials, the training will also open doors for close interaction with respective scientists which is very valuable in receiving great ideas and suggestions related to my queries about the subject.

24 ITEMS PINNED

Chlamydia spp. development is differentially altered by treatment with the LpxC inhibitor LPC-011.

Abstract: Chlamydia species are obligate intracellular bacteria that infect a broad range of mammalian hosts. Members of related genera are pathogens of a variety of vertebrate and invertebrate species. Despite the diversity of Chlamydia, all species contain an outer membrane lipooligosaccharide (LOS) that is comprised of a genus-conserved, and genus-defining, trisaccharide 3-deoxy-D-manno-oct-2-ulosonic acid Kdo region. Recent studies with lipopolysaccharide inhibitors demonstrate that LOS is important for the C. trachomatis developmental cycle during RB- > EB differentiation. Here, we explore the effects of one of these inhibitors, LPC-011, on the developmental cycle of five chlamydial species.Sensitivity to the drug varied in some of the species and was conserved between others. We observed that inhibition of LOS biosynthesis in some chlamydial species induced formation of aberrant reticulate bodies, while in other species, no change was observed to the reticulate body. However, loss of LOS production prevented completion of the chlamydial reproductive cycle in all species tested. In previous studies we found that C. trachomatis and C. caviae infection enhances MHC class I antigen presentation of a model self-peptide. We find that treatment with LPC-011 prevents enhanced host-peptide presentation induced by infection with all chlamydial-species tested.The data demonstrate that LOS synthesis is necessary for production of infectious progeny and inhibition of LOS synthesis induces aberrancy in certain chlamydial species, which has important implications for the use of LOS synthesis inhibitors as potential antibiotics.

Pub.: 26 Apr '17, Pinned: 05 Jan '18

In-vitro and in-vivo efficacy of LpxC inhibitor, CHIR-090, alone or combined with colistin against Pseudomonas aeruginosa biofilm.

Abstract: With the rapid spread of antimicrobial resistance in Gram-negative pathogens, biofilms-associated infections are increasingly harder to treat and combination therapy with colistin has become one of the most efficient therapeutic strategies. Our study aimed to evaluate the potential for synergy of colistin combined with CHIR-090, a potent LpxC inhibitor, against in-vitro and in-vivoPseudomonas aeruginosa biofilms. Four P. aeruginosa isolates with varying colistin susceptibility were chosen for evaluation. Tested isolates of P. aeruginosa exhibited MIC values ranging from 1 to 64 and 0.0625 to 0.5 μg/ml for colistin and CHIR-090, respectively. Against 24h static biofilms, MBEC values ranged from 256 to 512 and 8 to >128 μg/ml for colistin and CHIR-090, respectively. Interestingly, sub-inhibitory concentrations of CHIR-090 contributed to eradicate subpopulations of P. aeruginosa with highest colistin MIC values. Combination of colistin and CHIR-090 at sub-inhibitory concentrations demonstrated synergistic activity, both in-vivo and in-vitro, and prevented the formation of colistin tolerant sub-populations in in-vitro biofilms. In summary, our study highlights the in-vivo and in-vitro synergistic activity of colistin and CHIR-090 combination against both colistin-susceptible and non-susceptible P. aeruginosa biofilms. Further studies are warranted to investigate the clinical relevance of the combination of these two antimicrobials and outline the underlying mechanism for their synergistic action.

Pub.: 04 May '17, Pinned: 05 Jan '18

MolProbity: More and better reference data for improved all-atom structure validation.

Abstract: This paper describes the current update on macromolecular model validation services that are provided at the MolProbity website, emphasizing changes and additions since the previous review in 2010. There have been many infrastructure improvements, including rewrite of previous Java utilities to now use existing or newly written Python utilities in the open-source CCTBX portion of the Phenix software system. This improves long-term maintainability and enhances the thorough integration of MolProbity-style validation within Phenix. There is now a complete MolProbity mirror site at http://molprobity.manchester.ac.uk. GitHub serves our open-source code, reference datasets, and the resulting multi-dimensional distributions that define most validation criteria. Coordinate output after Asn/Gln/His "flip" correction is now more idealized, since the post-refinement step has apparently often been skipped in the past. Two distinct sets of heavy-atom-to-hydrogen distances and accompanying van der Waals radii have been researched and improved in accuracy, one for the electron-cloud-center positions suitable for X-ray crystallography and one for nuclear positions. New validations include messages at input about problem-causing format irregularities, updates of Ramachandran and rotamer criteria from the million quality-filtered residues in a new reference dataset, the CaBLAM Cα-CO virtual-angle analysis of backbone and secondary structure for cryoEM or low-resolution X-ray, and flagging of the very rare cis-nonProline and twisted peptides which have recently been greatly overused. Due to wide application of MolProbity validation and corrections by the research community, in Phenix, and at the worldwide Protein Data Bank, newly deposited structures have continued to improve greatly as measured by MolProbity's unique all-atom clashscore. This article is protected by copyright. All rights reserved.

Pub.: 27 Oct '17, Pinned: 02 Jan '18

The recognition of LpxC inhibitors as potential antibiotics could revolutionise the management of sepsis in veterinary patients if their unknown biological properties are widely evaluated in suitable animal models

Abstract: Publication date: Available online 2 December 2014 Source:International Journal of Veterinary Science and Medicine Author(s): Saul Chemonges Current studies continue to show that UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetyl glucosamine deacetylase (LpxC) inhibitors such as pyridone methylsulfone hydroxymate 1 and 2a (LpxC-2) downregulate the lethal effects of sepsis initiated by multi-drug resistant Gram-negative bacteria (MDRGNB) by curtailing lipopolysaccharide (LPS) synthesis in murine models. Sepsis initiated by MDRGNB is a leading cause of shock and systemic inflammatory response syndrome (SIRS) in intensive care unit (ICU) patients. To date however, the biological effects of LpxC-2 and related molecules in companion and production animals remain largely unexplored in vivo and are therefore unknown. Such studies would be greatly informative in the expectation of LpxC-2 progressing to human clinical trials. Mechanistic studies to interrogate this novel antibiotic candidate in realistic and clinically applicable large animal models of veterinary importance are sorely lacking. To be relevant, the physiology of the chosen animal models should closely match that of humans such as ovine or porcine, or even better, non-human primate based studies, as they are more genetically similar to humans than murine models. If discovered to have subtle or negligible side effects, LpxC-2 could have a future role in the treatment and management of MDRGNB-induced infections that lead to sepsis in both animals and humans. More research is indicated on LpxC-2 use in many veterinary species, as data remains scarce.

Pub.: 08 Dec '14, Pinned: 02 Jan '18

3D-QSAR, Molecular Docking and Molecular Dynamics Simulation of Pseudomonas aeruginosa LpxC Inhibitors.

Abstract: As an important target for the development of novel antibiotics, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) has been widely studied. Pyridone methylsulfone hydroxamate (PMH) compounds can effectively inhibit the catalytic activity of LpxC. In this work, the three-dimensional quantitative structure-activity relationships of PMH inhibitors were explored and models with good predictive ability were established using comparative molecular field analysis and comparative molecular similarity index analysis methods. The effect of PMH inhibitors' electrostatic potential on the inhibitory ability of Pseudomonas aeruginosa LpxC (PaLpxC) is revealed at the molecular level via molecular electrostatic potential analyses. Then, two molecular dynamics simulations for the PaLpxC and PaLpxC-inhibitor systems were also performed respectively to investigate the key residues of PaLpxC hydrolase binding to water molecules. The results indicate that orderly alternative water molecules can form stable hydrogen bonds with M62, E77, T190, and H264 in the catalytic center, and tetracoordinate to zinc ion along with H78, H237, and D241. It was found that the conformational transition space of PaLpxC changes after association with PMH inhibitors through free energy landscape and cluster analyses. Finally, a possible inhibitory mechanism of PMH inhibitors was proposed, based on our molecular simulation. This paper will provide a theoretical basis for the molecular design of LpxC-targeting antibiotics.

Pub.: 10 May '17, Pinned: 02 Jan '18

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

Abstract: The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

Pub.: 27 Jul '17, Pinned: 02 Jan '18

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

Abstract: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India.A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations.The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding.Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Pub.: 20 Aug '17, Pinned: 02 Jan '18