Construction of lux-based promoter-reporter platforms in Mycobacterium bovis BCG for screening of drug repurposing small-molecule compounds as new anti-tuberculosis drugs

2021 ◽  
Author(s):  
Li Zhu ◽  
Annie Wing-tung Lee ◽  
Kelvin Ka-Lok WU ◽  
Peng GAO ◽  
Kingsley King-Gee Tam ◽  
...  
Keyword(s):  
Small Molecule ◽  
Drug Repurposing ◽  
Virulence Gene ◽  
Multidrug Resistant ◽  
Rapid Screening ◽  
Human Macrophage ◽  
Tuberculosis Complex ◽  
Low Cytotoxicity ◽  
Screening Platform

The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to construct a rapid screening system to identify novel anti-TB agents from a library of small-molecule compounds. In this study, a total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phoP, of M. tuberculosis complex using luminescence (lux)-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophage were determined. RNA-sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex. Six compounds displayed bactericidal activity against M. bovis BCG, in which Ebselen demonstrated the lowest cytotoxicity to macrophage and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex but down-regulated the expression of phoP specifically. Of them, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and could dysregulate the entire phoP-associated gene network, and thus identified as potential anti-virulence agents for M. tuberculosis. This study provides a rapid screening platform coupled with a systematic validation and eventually suggested one potential antibiotic and two anti-virulence agents for M. tuberculosis infections.

scholarly journals Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

2018 ◽  
Vol 11 (549) ◽  
pp. eaat7951 ◽  
Author(s):  
Daniel M. Foulkes ◽  
Dominic P. Byrne ◽  
Wayland Yeung ◽  
Safal Shrestha ◽  
Fiona P. Bailey ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Drug Repurposing ◽  
Protein Kinase Inhibitors ◽  
Cellular Mechanisms ◽  
Cellular Analysis

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

scholarly journals In Vitro Activities of Linezolid against Clinical Isolates of Mycobacterium tuberculosis Complex Isolated in Taiwan over 10 Years

2008 ◽  
Vol 52 (6) ◽  
pp. 2226-2227 ◽  
Author(s):  
Tsi-Shu Huang ◽  
Yung-Ching Liu ◽  
Cheng-Len Sy ◽  
Yao-Shen Chen ◽  
Hui-Zin Tu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
Baseline Period ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Tuberculosis Complex

ABSTRACT Significant increases in the MIC90s of linezolid in multidrug-resistant Mycobacterium tuberculosis isolates were seen between the baseline period of 2001 to 2003 (0.5 μg/ml) and 2004 (2 μg/ml). The MICs were 4 μg/ml in three strains. Both fluoroquinolones (except levofloxacin) and kanamycin were found to have statistically significant degrees of concordance with linezolid.

scholarly journals Radiation-induced PEGylated Ethambutol Has Low Antimycobacterial Activity in Vitro

2020 ◽  
Vol 11 (2) ◽  
pp. 8884-8894
Keyword(s):  
Antimycobacterial Activity ◽  
Multidrug Resistant ◽  
Biological Evaluation ◽  
Rapid Screening ◽  
Promising Tool ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Radiation Induced ◽  
Airborne Disease

Tuberculosis is an airborne disease caused by Mycobacterium tuberculosis and remains one of the leading causes of death worldwide. The rise in multidrug-resistant strains has prompted the search for novel strategies to produce tuberculostatic agents. This research is aimed at developing a derivative of ethambutol by gamma radiation-induced polymerization with polyethylene glycol (PEG). The synthesis was verified by Raman spectroscopy and UV–Vis spectrometry. The results show that PEG can be chemically bonded to ethambutol by amine and alcohol groups. In in vitro biological evaluation, PEGylated and neat ethambutol showed similar cell viabilities, while the modified drug lowered bacterial growth inhibitory activity. A mechanism for the polymerization is proposed. The particle size increased for PEGylated drugs concerning the starting polyether. Despite the low antimycobacterial activity in vitro, the product seems to be a promising tool for the rapid screening of hydrolase activity.

scholarly journals The Selective Class IIa Histone Deacetylase Inhibitor TMP195 Resensitizes ABCB1- and ABCG2-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs

2019 ◽  
Vol 21 (1) ◽  
pp. 238 ◽  
Author(s):  
Chung-Pu Wu ◽  
Sabrina Lusvarghi ◽  
Jyun-Cheng Wang ◽  
Sung-Han Hsiao ◽  
Yang-Hui Huang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Histone Deacetylase ◽  
Anticancer Drugs ◽  
Drug Transport ◽  
Drug Repurposing ◽  
Binding Pocket ◽  
Multidrug Resistant ◽  
Drug Induced ◽  
In Silico Docking

Multidrug resistance caused by the overexpression of the ATP-binding cassette (ABC) proteins in cancer cells remains one of the most difficult challenges faced by drug developers and clinical scientists. The emergence of multidrug-resistant cancers has driven efforts from researchers to develop innovative strategies to improve therapeutic outcomes. Based on the drug repurposing approach, we discovered an additional action of TMP195, a potent and selective inhibitor of class IIa histone deacetylase. We reveal that in vitro TMP195 treatment significantly enhances drug-induced apoptosis and sensitizes multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2 to anticancer drugs. We demonstrate that TMP195 inhibits the drug transport function, but not the protein expression of ABCB1 and ABCG2. The interaction between TMP195 with these transporters was supported by the TMP195-stimulated ATPase activity of ABCB1 and ABCG2, and by in silico docking analysis of TMP195 binding to the substrate-binding pocket of these transporters. Furthermore, we did not find clear evidence of TMP195 resistance conferred by ABCB1 or ABCG2, suggesting that these transporters are unlikely to play a significant role in the development of resistance to TMP195 in cancer patients.

scholarly journals Resveratrol Inhibits HCoV-229E and SARS-CoV-2 Coronavirus Replication In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 354
Author(s):  
Sébastien Pasquereau ◽  
Zeina Nehme ◽  
Sandy Haidar Ahmad ◽  
Fadoua Daouad ◽  
Jeanne Van Assche ◽  
...  
Keyword(s):  
Drug Repurposing ◽  
Effective Concentration ◽  
Selectivity Index ◽  
Viral Titer ◽  
Human Coronavirus ◽  
Candidate Drug ◽  
Drug Compounds ◽  
Low Cytotoxicity ◽  
Novel Coronavirus

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19.

scholarly journals Lung on a Chip Development from Off-Stoichiometry Thiol–Ene Polymer

Micromachines ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 546
Author(s):  
Roberts Rimsa ◽  
Artis Galvanovskis ◽  
Janis Plume ◽  
Felikss Rumnieks ◽  
Karlis Grindulis ◽  
...  
Keyword(s):  
Small Molecule ◽  
Light Transmission ◽  
Umbilical Vein ◽  
Drug Repurposing ◽  
In Vitro Models ◽  
Enzymatic Reactions ◽  
Promising Alternative ◽  
Prototype Development ◽  
Alternative Material

Current in vitro models have significant limitations for new respiratory disease research and rapid drug repurposing. Lung on a chip (LOAC) technology offers a potential solution to these problems. However, these devices typically are fabricated from polydimethylsiloxane (PDMS), which has small hydrophobic molecule absorption, which hinders the application of this technology in drug repurposing for respiratory diseases. Off-stoichiometry thiol–ene (OSTE) is a promising alternative material class to PDMS. Therefore, this study aimed to test OSTE as an alternative material for LOAC prototype development and compare it to PDMS. We tested OSTE material for light transmission, small molecule absorption, inhibition of enzymatic reactions, membrane particle, and fluorescent dye absorption. Next, we microfabricated LOAC devices from PDMS and OSTE, functionalized with human umbilical vein endothelial cell (HUVEC) and A549 cell lines, and analyzed them with immunofluorescence. We demonstrated that compared to PDMS, OSTE has similar absorption of membrane particles and effect on enzymatic reactions, significantly lower small molecule absorption, and lower light transmission. Consequently, the immunofluorescence of OSTE LOAC was significantly impaired by OSTE optical properties. In conclusion, OSTE is a promising material for LOAC, but optical issues should be addressed in future LOAC prototypes to benefit from the material properties.

scholarly journals Broad Spectrum Antibacterial Ocotillol-Type Derivatives: Semi-Synthesis, Structure-Activity Relationship, and Membrane-Active Mode of Action

2021 ◽  
Author(s):  
Xianming Zeng ◽  
Ziyi Zhang ◽  
Yunyun Zhou ◽  
Shengyu Zhang ◽  
Zhiwen Zhou
Keyword(s):  
Antibacterial Activity ◽  
Antibacterial Agents ◽  
Bacterial Resistance ◽  
Synergistic Effects ◽  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Hek 293 ◽  
Low Cytotoxicity ◽  
New Compounds

A series of 3-amino substituted ocotillol-type derivatives were designed and synthesized for the first time. The in vitro antibacterial activity tests showed that some of the new compounds exhibited excellent antibacterial activity. Compound 13d, which was the most active one, displayed particularly strong antibacterial activity against S. aureus, B. subtilis, MRSA (methicillin-resistant S. aureus) and E. coli with minimum inhibitory concentration (MIC) values of 1-4 μg mL-1. Further research also suggested that 13d showed low cytotoxicity to human normal cells HEK-293 and L02, strong synergistic effects with kanamycin or chloramphenicol and a broad antibacterial spectrum including against multidrug-resistant strains. This active molecule 13d also induced bacterial resistance more slowly than norfloxacin and colistin. Furthermore, the research results demonstrated that this type of compounds could disperse the established bacterial biofilms, thus suppressing or delaying the development of drug resistance. Mechanism studies have shown that compound 13d could damage the integrity of cell membranes, which in turn facilitated the antibacterial agents binding to deoxyribonucleic acid (DNA), leading to cell death. Therefore, these results indicated that the membrane active ocotillol-type derivatives are a promising class of antibacterial agents to fight against super bacteria and deserve further attention.

scholarly journals Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms

2020 ◽  
Author(s):  
Ana C. Puhl ◽  
Ethan James Fritch ◽  
Thomas R. Lane ◽  
Longping V. Tse ◽  
Boyd L. Yount ◽  
...  
Keyword(s):  
Cell Lines ◽  
Small Molecule ◽  
Ebola Virus ◽  
Early Stage ◽  
Drug Repurposing ◽  
Marburg Virus ◽  
Small Molecule Drugs ◽  
Speed Up

AbstractSARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola virus and demonstrated activity against SARS-CoV-2 in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in vitro in HeLa cells and of mouse adapted Ebola virus in mouse in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with Kd values of 339 nM and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 hence justifying in vivo evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.

scholarly journals Synergy between Circular Bacteriocin AS-48 and Ethambutol against Mycobacterium tuberculosis

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Clara Aguilar-Pérez ◽  
Begoña Gracia ◽  
Liliana Rodrigues ◽  
Asunción Vitoria ◽  
Rubén Cebrián ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Antimycobacterial Activity ◽  
Multidrug Resistant ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Antituberculosis Therapy ◽  
Circular Bacteriocin ◽  
Low Cytotoxicity

ABSTRACT The increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains and the very few drugs available for treatment are promoting the discovery and development of new molecules that could help in the control of this disease. Bacteriocin AS-48 is an antibacterial peptide produced by Enterococcus faecalis and is active against several Gram-positive bacteria. We have found that AS-48 was active against Mycobacterium tuberculosis, including H37Rv and other reference and clinical strains, and also against some nontuberculous clinical mycobacterial species. The combination of AS-48 with either lysozyme or ethambutol (commonly used in the treatment of drug-susceptible tuberculosis) increased the antituberculosis action of AS-48, showing a synergic interaction. Under these conditions, AS-48 exhibits a MIC close to some MICs of the first-line antituberculosis agents. The inhibitory activity of AS-48 and its synergistic combination with ethambutol were also observed on M. tuberculosis-infected macrophages. Finally, AS-48 did not show any cytotoxicity against THP-1, MHS, and J774.2 macrophage cell lines at concentrations close to its MIC. In summary, bacteriocin AS-48 has interesting antimycobacterial activity in vitro and low cytotoxicity, so further studies in vivo will contribute to its development as a potential additional drug for antituberculosis therapy.

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