scholarly journals Synthesis and Selection of De Novo Proteins That Bind and Impede Cellular Functions of an Essential Mycobacterial Protein

2006 ◽  
Vol 73 (4) ◽  
pp. 1320-1331 ◽  
Author(s):  
Alka Rao ◽  
Geeta Ram ◽  
Adesh Kumar Saini ◽  
Reena Vohra ◽  
Krishan Kumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
De Novo ◽  
Cell Damage ◽  
Vitro Assay ◽  
De Novo Proteins ◽  
Protein Pool ◽  
Protein Libraries ◽  
Selection Of

ABSTRACT Recent advances in nonrational and part-rational approaches to de novo peptide/protein design have shown increasing potential for development of novel peptides and proteins of therapeutic use. We demonstrated earlier the usefulness of one such approach recently developed by us, called “codon shuffling,” in creating stand-alone de novo protein libraries from which bioactive proteins could be isolated. Here, we report the synthesis and selection of codon-shuffled de novo proteins that bind to a selected Mycobacterium tuberculosis protein target, the histone-like protein HupB, believed to be essential for mycobacterial growth. Using a versatile bacterial two-hybrid system that entailed utilization of HupB and various codon-shuffled protein libraries as bait and prey, respectively, we were able to identify proteins that bound strongly to HupB. The observed interaction was also confirmed using an in vitro assay. One of the protein binders was expressed in Mycobacterium smegmatis and was shown to appreciably affect growth in the exponential phase, a period wherein HupB is selectively expressed. Furthermore, the transcription profile of hupB gene showed a significant reduction in the transcript quantity in mycobacterial strains expressing the protein binder. Electron microscopy of the affected mycobacteria elaborated on the extent of cell damage and hinted towards a cell division malfunction. It is our belief that a closer inspection of the obtained de novo proteins may bring about the generation of small-molecule analogs, peptidomimetics, or indeed the proteins themselves as realistic leads for drug candidates. Furthermore, our strategy is adaptable for large-scale targeting of the essential protein pool of Mycobacterium tuberculosis and other pathogens.

Elaboration of Novel TTK1 Inhibitory Leads via QSAR-Guided Selection of Crystallographic Pharmacophores Followed By In vitro Assay

2020 ◽  
Vol 16 ◽  
Author(s):  
Mahmoud A. Al-Sha'er ◽  
Mutasem O. Taha
Keyword(s):  
Standard Error ◽  
Qsar Model ◽  
Qsar Modeling ◽  
Vitro Assay ◽  
Physicochemical Descriptors ◽  
Qsar Models ◽  
Ic50 Values ◽  
Pharmacophore Hypotheses ◽  
Selection Of

Introduction: Tyrosine threonine kinase (TTK1) is a key regulator of chromosome segregation. TTK targeting received recent concern for the enhancement of possible anticancer therapies. Objective: In this regard we employed our well-known method of QSAR-guided selection of best crystallographic pharmacophore(s) to discover considerable binding interactions that anchore inhibitors into TTK1 binding site. Method:Sixtyone TTK1 crystallographic complexes were used to extract 315 pharmacophore hypotheses. QSAR modeling was subsequently used to choose a single crystallographic pharmacophore that when combined with other physicochemical descriptors elucidates bioactivity discrepancy within a list of 55 miscellaneous inhibitors. Results: The best QSAR model was robust and predictive (r2(55) = 0.75, r2LOO = 0.72 , r2press against external testing list of 12 compounds = 0.67), Standard error of estimate (training set) (S)= 0.63 , Standard error of estimate (testing set)(Stest) = 0.62. The resulting pharmacophore and QSAR models were used to scan the National Cancer Institute (NCI) database for new TTK1 inhibitors. Conclusion: Five hits confirmed significant TTK1 inhibitory profiles with IC50 values ranging between 11.7 and 76.6 micM.

scholarly journals De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Rashmi Ravindran Nair ◽  
Kishor Jakkala ◽  
Atul Pradhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hydroxyl Radical ◽  
Prolonged Exposure ◽  
De Novo ◽  
Random Mutagenesis ◽  
Content Type ◽  
Genome Wide ◽  
Resistant Mutants ◽  
Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

scholarly journals Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

2018 ◽  
Vol 115 (39) ◽  
pp. 9779-9784 ◽  
Author(s):  
Sangeeta Tiwari ◽  
Andries J. van Tonder ◽  
Catherine Vilchèze ◽  
Vitor Mendes ◽  
Sherine E. Thomas ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
De Novo ◽  
Target Space ◽  
Biosynthesis Pathway ◽  
Arginine Biosynthesis ◽  
Arginine Deprivation

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

Examining P-gp efflux kinetics guided by the BDDCS – Rational selection of in vitro assay designs and mathematical models

2019 ◽  
Vol 132 ◽  
pp. 132-141 ◽  
Author(s):  
Julia Riede ◽  
Ken-Ichi Umehara ◽  
Patrick Schweigler ◽  
Felix Huth ◽  
Hilmar Schiller ◽  
...  
Keyword(s):  
Mathematical Models ◽  
In Vitro Assay ◽  
Rational Selection ◽  
Vitro Assay ◽  
Efflux Kinetics ◽  
Selection Of

scholarly journals Second-Generation Recombination-Based In Vivo Expression Technology for Large-Scale Screening for Vibrio cholerae Genes Induced during Infection of the Mouse Small Intestine

2005 ◽  
Vol 73 (2) ◽  
pp. 972-980 ◽  
Author(s):  
C. G. Osorio ◽  
J. A. Crawford ◽  
J. Michalski ◽  
H. Martinez-Wilson ◽  
J. B. Kaper ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Large Scale ◽  
Screening Method ◽  
Mouse Small Intestine ◽  
In Vivo Expression ◽  
Induced Genes ◽  
El Tor ◽  
Selection Of

ABSTRACT We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.

scholarly journals Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence in Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Parthasarathi Ajitkumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Prolonged Exposure ◽  
De Novo ◽  
Efflux Pump ◽  
Persister Cells ◽  
Efflux Pump Inhibitor ◽  
Antibiotic Resistant ◽  
Mechanical Removal ◽  
Derivatives Of

ABSTRACTPersisters constitute a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. Multiple mechanisms have been suggested for bacterial persistence against antibiotics. With mycobacteria being no exception to this behaviour, we had reported the de novo emergence of genetically antibiotic-resistant Mycobacterium tuberculosis from persister cells upon prolonged exposure to microbicidal concentrations of the anti-tuberculosis drugs, rifampicin and moxifloxacin. Here, we present evidence for reduced permeability to rifampicin as a mechanism for persistence of Mycobacterium tuberculosis in vitro. We observed that rifampicin persistent M. tuberculosis cells developed a thick outer layer (TOL) capsule. The TOL restricted the entry of fluorochrome-conjugated rifampicin, 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), which retained only 2.5% of its original bactericidal activity, but high levels of permeability, on actively growing mid-log phase cells. Gentle mechanical removal of TOL significantly enhanced 5-FAM-rifampicin entry into the persister cells. The level of 5-FAM-rifampicin in the persister cells was not affected by the pre-incubation of the cells with verapamil, a drug efflux pump inhibitor, ruling out the involvement of efflux pumps in the reduced intracellular concentration of 5-FAM-rifampicin. GC-MS analysis of TOL showed the presence of ∼7-fold, ∼5-fold and ∼2- fold higher levels of α-D-glucopyranoside, 1,2,5-linked-mannitol, and 3,4-linked mannose, respectively, among ∼2-fold higher levels of derivatives of several other types of sugars such as arabinose and galactose. Taken together, the present study reveals that rifampicin-persistent M. tuberculosis cells develop TOL that enables the bacilli to restrict entry of rifampicin and thereby remain tolerant to the antibiotic in vitro.

EFFECTISAN FOR DISINFECTION OF OBJECTS OF VETERINARY SUPERVISION

2018 ◽  
Vol 1 (2) ◽  
pp. 36-41
Author(s):  
D. I. Udavliev ◽  
◽  
A. M. Abdullaeva ◽  
S. S. Shikhov ◽  
N. E. Vanner ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Quality Control ◽  
Mycobacterium Tuberculosis ◽  
Consumption Rate ◽  
High Antimicrobial Activity ◽  
E Coli ◽  
Selection Of Cultures ◽  
Selection Of ◽  
Test Cultures

The article provides information about the effectiveness of the drug is Effectisan in the laboratory. As a result of the conducted researches it is established that the tested preparation possesses a certain high antimicrobial activity. The drug (in vitro) is effective at concentrations from 0.2 to 1.0% against E. coli (pcs 1257), St. aureus (pcs 209-P), Mycobacterium B-5, Bac. cereus (pcs 96). With wet disinfection at a concentration of 0.5% and a consumption rate of 350 ml/m2, the drug has a pronounced disinfection activity against surfaces made of various materials, the quality control of disinfection in which is carried out by the presence or absence of growth in the washouts of test cultures E. coli, S. aureus. At a concentration of 2.0% and the rate of 350 ml/m2 Effectisan has a pronounced disinfectant activity during processing objects and for quality control of disinfection by selection of cultures of Mycobacterium tuberculosis (pcs-5).

scholarly journals Evaluation of an In Vitro Assay for Gamma Interferon Production in Response to Mycobacterium tuberculosis Infections

2004 ◽  
Vol 11 (6) ◽  
pp. 1089-1093 ◽  
Author(s):  
Edward W. Taggart ◽  
Harry R. Hill ◽  
Roland G. Ruegner ◽  
Thomas B. Martins ◽  
Christine M. Litwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Negative Control ◽  
Gamma Interferon ◽  
Low Risk ◽  
Interferon Response ◽  
Vitro Assay ◽  
Negative Case ◽  
Group 2 ◽  
Group 1

ABSTRACT The tuberculin skin test (TST) is the “gold standard” for detecting infection with Mycobacterium tuberculosis. We compared the TST using purified protein derivative to the QuantiFERON-TB test (QFT). Two groups were examined. Group 1 individuals (n = 66) (low risk) were at low risk for exposure to M. tuberculosis and were not Mycobacterium bovis BCG vaccinated. Group 2 (n = 29) include individuals who were likely to have been exposed to a high prevalence of M. tuberculosis infections and were BCG vaccinated. Group 1 individuals were given a TST. Group 2 individuals were not given a TST because of possible adverse reactions. A 10- to 15-mm indurated area 48 h after TST was considered positive. A positive QFT result was defined as a significant gamma interferon response to M. tuberculosis antigen, Mycobacterium avium antigen, and a nonspecific mitogen stimulus and no response in the negative control. In group 1, 60 of 66 individuals (90.9%) were negative by both methods, and 1 person was positive by both methods. There was one QFT-negative, TST-positive case, one QFT-positive, TST-negative case, and three conditional QFT-positive, TST-negative cases. In group 2, 12 of 29 (41.4%) were positive by QFT and considered likely to be TST positive because of prior BCG vaccination. QFT testing in our low-risk group resulted in an agreement of 96.8%, a sensitivity of 50%, and a specificity of 98.4% compared with TST results. QFT testing with TST in low-risk groups can aid in the detection of latent M. tuberculosis infections.

Evaluation of an In Vitro Bioassay for the Detection of Purified Ricin and Castor Bean in Beverages and Liquid Food Matrices

2007 ◽  
Vol 70 (10) ◽  
pp. 2377-2382 ◽  
Author(s):  
JENNIFER L. BRZEZINSKI ◽  
DAVID L. CRAFT
Keyword(s):  
Castor Bean ◽  
Colorimetric Assay ◽  
Cell Damage ◽  
Jurkat Cells ◽  
Biologically Active ◽  
Detection Methods ◽  
Mouse Bioassay ◽  
Animal Testing ◽  
Vitro Assay

The potential use of ricin as a biological weapon in food highlights the necessity for the development of food-specific detection methods. Current methods for the detection of ricin consist of various immunoassays, which detect only one subunit of the ricin toxin and therefore may not be indicative of a biologically active molecule. An in vivo assay, such as a mouse bioassay, can indicate the biological activity of the toxin; however, this method is not feasible for laboratories that do not have animal testing facilities. The purpose of this study was to develop an in vitro assay for the detection of biologically active ricin in beverages and liquid foods. Acidic and high-protein beverages were spiked with either purified ricin or ground castor beans and added to cultured human Jurkat cells. After an overnight incubation, the supernatant was tested for lactate dehydrogenase (LDH) activity with a colorimetric assay. LDH was released from the cytosol upon cell damage and was positively correlated with cell death. Ricin was detectable in all the matrices tested, with a sensitivity of 10 to 100 pg/ml. Biologically active ricin was detectable in all the matrices incubated with ground castor bean material. This method provides a confirmatory way to detect biologically active ricin that can be utilized by laboratories lacking animal facilities.

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