scholarly journals Regioisomerization of Antimalarial Drug WR99210 Explains the Inactivity of a Commercial Stock

2020 ◽  
Vol 65 (1) ◽  
pp. e01385-20
Author(s):  
T. Parks Remcho ◽  
Sravanthi D. Guggilapu ◽  
Phillip Cruz ◽  
Glenn A. Nardone ◽  
Gavin Heffernan ◽  
...  
Keyword(s):  
Active Site ◽  
Antimalarial Drug ◽  
High Performance ◽  
Drug Candidate ◽  
Resonance Spectroscopy ◽  
Content Type ◽  
Computational Docking ◽  
Layer Chromatography ◽  
Commercial Stock ◽  
Selection Of

ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite’s dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.

scholarly journals Isomerization of antimalarial drug WR99210 explains its inactivity in a commercial stock

2020 ◽  
Author(s):  
T. Parks Remcho ◽  
Sravanthi D. Guggilapu ◽  
Phillip Cruz ◽  
Glenn A. Nardone ◽  
Gavin Heffernan ◽  
...  
Keyword(s):  
Active Site ◽  
Antimalarial Drug ◽  
High Performance ◽  
Mass Spectra ◽  
Drug Candidate ◽  
Resonance Spectroscopy ◽  
Computational Docking ◽  
Layer Chromatography ◽  
Commercial Stock ◽  
Selection Of

ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing human dhfr have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of 1 and 2; however, the compounds displayed differences by thin layer chromatography, reverse phase high performance liquid chromatography, and ultraviolet-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed 1 as WR99210 and 2 as an isomeric dihydrotriazine. Induced fit, computational docking models showed that 1 binds tightly and specifically in the P. falciparum DHFR active site whereas 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of isomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that can promote isomerization. Absorption spectroscopy may serve for assays of the unrearranged and rearranged triazines.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

Selection of selective laser sintering materials for different applications

2015 ◽  
Vol 21 (6) ◽  
pp. 630-648 ◽  
Author(s):  
Sunil Kumar Tiwari ◽  
Sarang Pande ◽  
Sanat Agrawal ◽  
Santosh M. Bobade
Keyword(s):  
Process Parameters ◽  
High Performance ◽  
Material Selection ◽  
Selective Laser Sintering ◽  
Laser Sintering ◽  
Optimal Process ◽  
Content Type ◽  
Volume Production ◽  
High Performance Application ◽  
Selection Of

Purpose – The purpose of this paper is to propose and evaluate the selection of materials for the selective laser sintering (SLS) process, which is used for low-volume production in the engineering (e.g. light weight machines, architectural modelling, high performance application, manufacturing of fuel cell, etc.), medical and many others (e.g. art and hobbies, etc.) with a keen focus on meeting customer requirements. Design/methodology/approach – The work starts with understanding the optimal process parameters, an appropriate consolidation mechanism to control microstructure, and selection of appropriate materials satisfying the property requirement for specific application area that leads to optimization of materials. Findings – Fabricating the parts using optimal process parameters, appropriate consolidation mechanism and selecting the appropriate material considering the property requirement of applications can improve part characteristics, increase acceptability, sustainability, life cycle and reliability of the SLS-fabricated parts. Originality/value – The newly proposed material selection system based on properties requirement of applications has been proven, especially in cases where non-experts or student need to select SLS process materials according to the property requirement of applications. The selection of materials based on property requirement of application may be used by practitioners from not only the engineering field, medical field and many others like art and hobbies but also academics who wish to select materials of SLS process for different applications.

scholarly journals Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

2013 ◽  
Vol 57 (6) ◽  
pp. 2603-2612 ◽  
Author(s):  
Narutoshi Uda ◽  
Yasuyuki Matoba ◽  
Takanori Kumagai ◽  
Kosuke Oda ◽  
Masafumi Noda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Homocysteine Thiolactone ◽  
Putative Gene ◽  
Content Type ◽  
Dna Fragment ◽  
Layer Chromatography

ABSTRACTWe have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic,d-cycloserine. The gene cluster is composed of 10 open reading frames, designateddcsAtodcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization ofO-ureidoserine. DcsD is similar toO-acetylserine sulfhydrylase, which generatesl-cysteine usingO-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generateO-ureido-l-serine usingO-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase convertingO-ureido-d-serine intod-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed inEscherichia coliand purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substratesO-acetyl-l-serine and hydroxyurea, synthesis ofd-cycloserine was successfully attained. Thesein vitrostudies yield the conclusion that DcsD and DcsG are necessary for the syntheses ofO-ureido-l-serine andd-cycloserine, respectively. DcsD was also able to catalyze the synthesis ofl-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclicd-amino acid analogs, such asd-homocysteine thiolactone.

scholarly journals A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

2014 ◽  
Vol 59 (1) ◽  
pp. 356-364 ◽  
Author(s):  
Wesley Wu ◽  
Zachary Herrera ◽  
Danny Ebert ◽  
Katie Baska ◽  
Seok H. Cho ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Drug Targets ◽  
Specific Activity ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Rescue ◽  
Growth Inhibitory ◽  
Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

scholarly journals Metabolic Profiling of Saponin-Rich Ophiopogon japonicus Roots Based on 1H NMR and HPTLC Platforms

Planta Medica ◽  
2019 ◽  
Vol 85 (11/12) ◽  
pp. 917-924 ◽  
Author(s):  
Yanhui Ge ◽  
Xiaojia Chen ◽  
Dejan Gođevac ◽  
Paula C. P. Bueno ◽  
Luis F. Salomé Abarca ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Nuclear Magnetic Resonance Spectroscopy ◽  
High Performance ◽  
Resonance Spectroscopy ◽  
Layer Chromatography ◽  
Nuclear Magnetic

AbstractIdeally, metabolomics should deal with all the metabolites that are found within cells and biological systems. The most common technologies for metabolomics include mass spectrometry, and in most cases, hyphenated to chromatographic separations (liquid chromatography- or gas chromatography-mass spectrometry) and nuclear magnetic resonance spectroscopy. However, limitations such as low sensitivity and highly congested spectra in nuclear magnetic resonance spectroscopy and relatively low signal reproducibility in mass spectrometry impede the progression of these techniques from being universal metabolomics tools. These disadvantages are more notorious in studies of certain plant secondary metabolites, such as saponins, which are difficult to analyse, but have a great biological importance in organisms. In this study, high-performance thin-layer chromatography was used as a supplementary tool for metabolomics. A method consisting of coupling 1H nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography was applied to distinguish between Ophiopogon japonicus roots that were collected from two growth locations and were of different ages. The results allowed the root samples from the two growth locations to be clearly distinguished. The difficulties encountered in the identification of the marker compounds by 1H nuclear magnetic resonance spectroscopy was overcome using high-performance thin-layer chromatography to separate and isolate the compounds. The saponins, ophiojaponin C or ophiopogonin D, were found to be marker metabolites in the root samples and proved to be greatly influenced by plant growth location, but barely by age variation. The procedure used in this study is fully described with the purpose of making a valuable contribution to the quality control of saponin-rich herbal drugs using high-performance thin-layer chromatography as a supplementary analytical tool for metabolomics research.

A Review article on Analytical Method Development for the combination of Azelnidipine and Telmisartan

2021 ◽  
pp. 227-234
Author(s):  
Nirma Chavda ◽  
Suresh Kumar
Keyword(s):  
Liquid Chromatography ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Method Development ◽  
Ultra Performance Liquid Chromatography ◽  
Resonance Spectroscopy ◽  
Stability Indicating ◽  
Stability Indicating Method ◽  
Layer Chromatography

The literature survey explains that there is not any stability indicating method reportedly for combination of Azelnidipine and Telmisartan till date. Validation and development of stability indicating analytical methods is possible as per ICH Guidelines. There are several of Spectroscopic methods such as Ultraviolet Spectroscopy, Mass spectroscopy, infrared spectroscopy, Nuclear magnetic resonance spectroscopy and Chromatographic methods such as High performance liquid chromatography, Thin layer Chromatography, High Performance thin layer chromatography, Gas chromatography and Ultra performance liquid chromatography etc. used for stability indicating method development and validation.

Identification of ß-carotene producing bacteria from terrestrial habitats of Jaipur, Rajasthan

2019 ◽  
Vol 48 (4) ◽  
pp. 329-336
Author(s):  
Ankit Chokriwal ◽  
Bjorn John Stephen ◽  
Abhijeet Singh
Keyword(s):  
High Performance ◽  
Design Methodology ◽  
Biochemical Characterization ◽  
Dpph Assay ◽  
Content Type ◽  
Isolation And Purification ◽  
Ic50 Value ◽  
Terrestrial Habitats ◽  
Layer Chromatography

Purpose Carotenoids are pigments that have significant value as colorants and antioxidants in pharmaceutical, food, textile and cosmetic industries. Owing to its high demand, this study aims to identify ß-carotene producing bacteria from different terrestrial habitats of Jaipur region. Design/methodology/approach In this paper, standard isolation and purification process was used, followed by colony morphology and biochemical characterization of ß-carotene producing bacteria. ß-carotene concentration was determined quantitatively using spectrophotometric method. Findings Out of 43 isolates, 21 isolates showed peak range between 400 and 500 nm confirming the presence of carotenoids. Only one bacteria SAN-A has capacity to produce ß-carotene confirmed by the thin layer chromatography and high performance liquid chromatography (HPLC) with a yield of 1.68 mg/l. The 2, 2-Diphenyl-2-picrylhydrazil (DPPH) assay showed an IC50 value of 4.0 mg/ml. Originality/value The present study revealed the presence of ß-carotene producing bacteria in the soil of different terrestrial habitat of Jaipur region which can be exploited as an economical source for ß-carotene production.

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