scholarly journals Selective inhibition of PfATP6 by artemisinins and identification of new classes of inhibitors after expression in yeast

2021 ◽  
Author(s):  
Catherine Margaret Moore ◽  
Jigang Wang ◽  
Qingsong Lin ◽  
Pedro Eduardo Ferreira ◽  
Mitchell A Avery ◽  
...  
Keyword(s):  
Drug Targets ◽  
Screening Tool ◽  
Selective Inhibition ◽  
In Vitro Assays ◽  
Fitness Costs ◽  
Artemisinin Derivatives ◽  
Compound Screening ◽  
Screening Platform ◽  
Artemisinin Combination Therapies

Treatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work. Herein we report the successful development of an heterologous expression-based compound screening tool. Validated drug target P. falciparum calcium ATPase6 (PfATP6) and a mammalian ortholog (SERCA1a) were functionally expressed in yeast providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labelling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model. As previously hypothesised, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multi-targeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility.

Validating targets for antiparasite chemotherapy

Parasitology ◽  
1997 ◽  
Vol 114 (7) ◽  
pp. 31-44 ◽  
Author(s):  
C. C. WANG
Keyword(s):  
Drug Targets ◽  
Drug Transport ◽  
Gene Knockout ◽  
Specific Inhibitor ◽  
Selective Inhibition ◽  
Conclusive Evidence ◽  
Gene Encoding ◽  
Multiple Copies ◽  
Antiparasitic Agents

The enzymes and receptors in parasites that can be qualified as targets for antiparasite chemotherapy should perform essential functions in the parasites and demonstrate some feasibility for selective inhibition. They can be tentatively identified through detailed analysis of various aspects of metabolisms in the parasites or elucidation of the mechanisms of action among proven antiparasitic agents. Preliminary verifications of these putative targets can be indicated by in vitro antiparasite activity of an inhibitor of the target. However, before a major long-term effort to pursue in-depth structure-activity analysis of the target is to be committed for specific inhibitor design, further validations of the target are essential to insure that future studies are not misguided. One old-fashioned approach to validate a target in the pharmaceutical industry is by correlating target inhibitions with antiparasitic activities among large numbers of drug derivatives. The results are often indicative but hardly ever conclusive. Another method is by comparing the putative drug targets between the drug-sensitive and the drug-resistant parasites for potential discrepancies. Unfortunately, the latter often result from indirect causes, such as reduced drug transport, instead of an alteration of the drug target itself. The third experimental approach is by disrupting the gene encoding the putative target in parasite, which can provide the most conclusive evidence on whether the target plays an indispensible role in the parasite. But special conditions are needed for the gene knockout mutants to survive to exhibit their phenotypes and to allow genetic complementation studies for further verifications. Furthermore, gene knockout experiments are often difficult to perform on cells of multiple ploidy or genes of multiple copies, and are currently applicable only to a limited number of protozoan parasites. In the current article I have tried to take a cursory look at some eleven putative drug targets among various parasites, each supported by well-established antiparasitic agents identified as its inhibitors. I have also considered the evidence for validity of each of them and the potential means of further verifying their validity.

Innovative Methodology in the Discovery of Novel Drug Targets in the Free-Living Amoebae

2018 ◽  
Vol 20 (1) ◽  
pp. 60-69 ◽  
Author(s):  
Abdul Mannan Baig
Keyword(s):  
Drug Targets ◽  
Mortality Rates ◽  
In Vitro Assays ◽  
Cancer Drug ◽  
Cns Infection ◽  
Free Living ◽  
Drug Target Discovery ◽  
Novel Drug ◽  
Novel Drug Targets

Despite advances in drug discovery and modifications in the chemotherapeutic regimens, human infections caused by free-living amoebae (FLA) have high mortality rates (~95%). The FLA that cause fatal human cerebral infections include Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. Novel drug-target discovery remains the only viable option to tackle these central nervous system (CNS) infection in order to lower the mortality rates caused by the FLA. Of these FLA, N. fowleri causes primary amoebic meningoencephalitis (PAM), while the A. castellanii and B. Mandrillaris are known to cause granulomatous amoebic encephalitis (GAE). The infections caused by the FLA have been treated with drugs like Rifampin, Fluconazole, Amphotericin-B and Miltefosine. Miltefosine is an anti-leishmanial agent and an experimental anti-cancer drug. With only rare incidences of success, these drugs have remained unsuccessful to lower the mortality rates of the cerebral infection caused by FLA. Recently, with the help of bioinformatic computational tools and the discovered genomic data of the FLA, discovery of newer drug targets has become possible. These cellular targets are proteins that are either unique to the FLA or shared between the humans and these unicellular eukaryotes. The latter group of proteins has shown to be targets of some FDA approved drugs prescribed in non-infectious diseases. This review out-lines the bioinformatics methodologies that can be used in the discovery of such novel drug-targets, their chronicle by in-vitro assays done in the past and the translational value of such target discoveries in human diseases caused by FLA.

scholarly journals Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases

2020 ◽  
Vol 26 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Sarah Wazir ◽  
Mirko M. Maksimainen ◽  
Heli I. Alanen ◽  
Albert Galera-Prat ◽  
Lari Lehtiö
Keyword(s):  
Drug Targets ◽  
Microtiter Plate ◽  
Post Translational Modification ◽  
Screening Assay ◽  
Fluorescent Compound ◽  
Vitro Assay ◽  
Adp Ribosylation ◽  
Compound Screening ◽  
A Chain

ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of β-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases.

scholarly journals Evaluation of Gene Expression and Phenotypic Profiling Data as Quantitative Descriptors for Predicting Drug Targets and Mechanisms of Action

2019 ◽  
Author(s):  
Maris Lapins ◽  
Ola Spjuth
Keyword(s):  
Gene Expression ◽  
Drug Discovery ◽  
Drug Targets ◽  
Chemical Structure ◽  
Predictive Accuracy ◽  
Mechanisms Of Action ◽  
In Vitro Assays ◽  
Using Data ◽  
Drug Leads

AbstractProfiling drug leads by means of in silico and in vitro assays as well as omics is widely used in drug discovery for safety and efficacy predictions. In this study, we evaluate the performance of machine learning models trained on data from gene expression and phenotypic profiling assays, with models trained on chemical structure descriptors, for prediction of various drug mechanisms of action and target proteins. Models for several hundred mechanisms of actions and targets were trained using data on 1484 compounds characterized in both gene expression using L1000 profiles, and phenotypic profiling with cell painting assay. The results indicate that the accuracy of the three profiling technologies varies for different endpoints, and indicate a clear potential synergistic effect if these methods are combined. We also study the effect of predictive accuracy of data from different cell lines for L1000 profiles, showing that the choice of cell line has a non-negligible effect on the predictive accuracy. The results strengthen the idea of integrated approaches for predicting drug targets and mechanisms of action in preclinical drug discovery.

scholarly journals Antimycobacterial, Enzyme Inhibition, and Molecular Interaction Studies of Psoromic Acid in Mycobacterium tuberculosis: Efficacy and Safety Investigations

2018 ◽  
Vol 7 (8) ◽  
pp. 226 ◽  
Author(s):  
Sherif Hassan ◽  
Miroslava Šudomová ◽  
Kateřina Berchová-Bímová ◽  
Shanmugaraj Gowrishankar ◽  
Kannan Rengasamy
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Active Sites ◽  
In Silico Analysis ◽  
Inhibitory Effect ◽  
In Vitro Assays ◽  
Standard Drug ◽  
Antituberculosis Therapy ◽  
Ic50 Value

The current study explores the antimycobacterial efficacy of lichen-derived psoromic acid (PA) against clinical strains of Mycobacterium tuberculosis (M.tb). Additionally, the inhibitory efficacy of PA against two critical enzymes associated with M.tb, namely, UDP-galactopyranose mutase (UGM) and arylamine-N-acetyltransferase (TBNAT), as drug targets for antituberculosis therapy were determined. PA showed a profound inhibitory effect towards all the M.tb strains tested, with minimum inhibitory concentrations (MICs) ranging between 3.2 and 4.1 µM, and selectivity indices (SIs) ranging between 18.3 and 23.4. On the other hand, the standard drug isoniazid (INH) displayed comparably high MIC values (varying from 5.4 to 5.8 µM) as well as low SI values (13.0–13.9). Interestingly, PA did not exhibit any cytotoxic effects on a human liver hepatocellular carcinoma cell line even at the highest concentration tested (75 µM). PA demonstrated remarkable suppressing propensity against UGM compared to standard uridine-5'-diphosphate (UDP), with 85.8 and 99.3% of inhibition, respectively. In addition, PA also exerted phenomenal inhibitory efficacy (half maximal inhibitory concentration (IC50) value = 8.7 µM, and 77.4% inhibition) against TBNAT compared with standard INH (IC50 value = 6.2 µM and 96.3% inhibition). Furthermore, in silico analysis validated the outcomes of in vitro assays, as the molecular interactions of PA with the active sites of UGM and TBNAT were unveiled using molecular docking and structure–activity relationship studies. Concomitantly, our findings present PA as an effective and safe natural drug plausible for use in controlling tuberculosis infections.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal
Keyword(s):  
In Silico ◽  
Docking Studies ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Chronic Obstructive ◽  
Lipoxygenase Inhibitors ◽  
Lipoxygenase Inhibition ◽  
In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

Repurposing of auranofin against bacterial infections: An In silico and In vitro study

2020 ◽  
Vol 16 ◽  
Author(s):  
Nidhi Sharma ◽  
Arti Singh ◽  
Ruchika Sharma ◽  
Anoop Kumar
Keyword(s):  
Broad Spectrum ◽  
In Silico ◽  
Antibacterial Agent ◽  
Bacterial Infections ◽  
In Vitro Assays ◽  
Infection Model ◽  
Synergistic Activity ◽  
Bacterial Strains ◽  
Molecular Docking Study

Aim: The aim of the study was to find out the role of auranofin as a promising broad spectrum antibacterial agent. Methods: In-vitro assays (Percentage growth retardation, Bacterial growth kinetics, Biofilm formation assay) and In-silico study (Molegro virtual docker (MVD) version 6.0 and Molecular operating environment (MOE) version 2008.10 software). Results: The in vitro assays have shown that auranofin has good antibacterial activity against Gram positive and Gram negative bacterial strains. Further, auranofin has shown synergistic activity in combination with ampicillin against S. aureus and B. subtilis whereas in combination with neomycin has just shown additive effect against E. coli, P. aeruginosa and B. pumilus. In vivo results have revealed that auranofin alone and in combination with standard drugs significantly decreased the bioburden in zebrafish infection model as compared to control. The molecular docking study have shown good interaction of auranofin with penicillin binding protein (2Y2M), topoisomerase (3TTZ), UDP-3-O-[3- hydroxymyristoyl] N-acetylglucosaminedeacetylase (3UHM), cell adhesion protein (4QRK), β-lactamase (5CTN) and arylsulphatase (1HDH) enzyme as that of reference ligand which indicate multimodal mechanism of action of auranofin. Finally, MTT assay has shown non-cytotoxic effect of auranofin. Conclusion: In conclusion, auranofin in combination with existing antibiotics could be developed as a broad spectrum antibacterial agent; however, further studies are required to confirm its safety and efficacy. This study provides possibility of use of auranofin apart from its established therapeutic indication in combination with existing antibiotics to tackle the problem of resistance.

Current Status and Future Perspective for Research on Medicinal Plants with Anticancerous Activity and Minimum Cytotoxic Value

2019 ◽  
Vol 20 (12) ◽  
pp. 1227-1243
Author(s):  
Hina Qamar ◽  
Sumbul Rehman ◽  
D.K. Chauhan
Keyword(s):  
Side Effects ◽  
Medicinal Plants ◽  
Anticancer Agents ◽  
Drug Targets ◽  
Psidium Guajava ◽  
Current Status ◽  
Future Perspective ◽  
Wide Range

Cancer is the second leading cause of morbidity and mortality worldwide. Although chemotherapy and radiotherapy enhance the survival rate of cancerous patients but they have several acute toxic effects. Therefore, there is a need to search for new anticancer agents having better efficacy and lesser side effects. In this regard, herbal treatment is found to be a safe method for treating and preventing cancer. Here, an attempt has been made to screen some less explored medicinal plants like Ammania baccifera, Asclepias curassavica, Azadarichta indica, Butea monosperma, Croton tiglium, Hedera nepalensis, Jatropha curcas, Momordica charantia, Moringa oleifera, Psidium guajava, etc. having potent anticancer activity with minimum cytotoxic value (IC50 >3μM) and lesser or negligible toxicity. They are rich in active phytochemicals with a wide range of drug targets. In this study, these medicinal plants were evaluated for dose-dependent cytotoxicological studies via in vitro MTT assay and in vivo tumor models along with some more plants which are reported to have IC50 value in the range of 0.019-0.528 mg/ml. The findings indicate that these plants inhibit tumor growth by their antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic molecular targets. They are widely used because of their easy availability, affordable price and having no or sometimes minimal side effects. This review provides a baseline for the discovery of anticancer drugs from medicinal plants having minimum cytotoxic value with minimal side effects and establishment of their analogues for the welfare of mankind.

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