New insights on Ethambutol Targets in Mycobacterium tuberculosis

2019 ◽  
Vol 19 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Luciana D. Ghiraldi-Lopes ◽  
Paula A. Zanetti Campanerut-Sá ◽  
Geisa P. Caprini Evaristo ◽  
Jean E. Meneguello ◽  
Adriana Fiorini ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mechanism Of Action ◽  
Complex Mechanism ◽  
Electron Transfer Flavoprotein ◽  
Main Protein ◽  
Proteome Profile ◽  
Effective Drugs ◽  
Dihydrolipoyl Dehydrogenase

Background: In recent years, very few effective drugs against Mycobacterium tuberculosis have emerged, which motivates the research with drugs already used in the treatment of tuberculosis. Ethambutol is a bacteriostatic drug that affects cell wall integrity, but the effects of this drug on bacilli are not fully exploited. Objective: Based on the need to better investigate the complex mechanism of action of ethambutol, our study presented the proteome profile of M. tuberculosis after different times of ethambutol exposure, aiming to comprehend the dynamics of bacilli response to its effects. M. tuberculosis was exposed to ½ MIC of ethambutol at 24 and 48 hours. The proteins were identified by MALDI-TOF/TOF. Results: The main protein changes occurred in metabolic proteins as dihydrolipoyl dehydrogenase (Rv0462), glutamine synthetase1 (Rv2220), electron transfer flavoprotein subunit beta (Rv3029c) and adenosylhomocysteinase (Rv3248c). Conclusion: Considering the functions of these proteins, our results support that the intermediary metabolism and respiration were affected by ethambutol and this disturbance provided proteins that could be explored as additional targets for this drug.

scholarly journals Osmium–arene complexes with high potency towards Mycobacterium tuberculosis

Metallomics ◽  
2021 ◽  
Vol 13 (4) ◽  
Author(s):  
James P C Coverdale ◽  
Collette S Guy ◽  
Hannah E Bridgewater ◽  
Russell J Needham ◽  
Elizabeth Fullam ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Global Health ◽  
Mechanism Of Action ◽  
Health Problem ◽  
Normal Lung ◽  
Lung Cells ◽  
Redox Mechanism ◽  
Human Lung Cells ◽  
Half Sandwich ◽  
Global Health Problem

Abstract The treatment of tuberculosis (TB) poses a major challenge as frontline therapeutic agents become increasingly ineffective with the emergence and spread of drug-resistant strains of Mycobacterium tuberculosis (Mtb). To combat this global health problem, new antitubercular agents with novel modes of action are needed. We have screened a close family of 17 organometallic half-sandwich Os(II) complexes [(arene)Os(phenyl-azo/imino-pyridine)(Cl/I)]+Y– containing various arenes (p-cymene, biphenyl, or terphenyl), and NMe2, F, Cl, or Br phenyl or pyridyl substituents, for activity towards Mtb in comparison with normal human lung cells (MRC5). In general, complexes with a monodentate iodido ligand were more potent than chlorido complexes, and the five most potent iodido complexes (MIC 1.25–2.5 µM) have an electron-donating Me2N or OH substituent on the phenyl ring. As expected, the counter anion Y (PF6–, Cl–, I–) had little effect on the activity. The pattern of potency of the complexes towards Mtb is similar to that towards human cells, perhaps because in both cases intracellular thiols are likely to be involved in their activation and their redox mechanism of action. The most active complex against Mtb is the p-cymene Os(II) NMe2-phenyl-azopyridine iodido complex (2), a relatively inert complex that also exhibits potent activity towards cancer cells. The uptake of Os from complex 2 by Mtb is rapid and peaks after 6 h, with temperature-dependence studies suggesting a major role for active transport. Significance to Metallomics Antimicrobial resistance is a global health problem. New advances are urgently needed in the discovery of new antibiotics with novel mechanisms of action. Half-sandwich organometallic complexes offer a versatile platform for drug design. We show that with an appropriate choice of the arene, an N,N-chelated ligand, and monodentate ligand, half-sandwich organo–osmium(II) complexes can exhibit potent activity towards Mycobacterium tuberculosis (Mtb), the leading cause of death from a single infectious agent. The patterns of activity of the 17 azo- and imino-pyridine complexes studied here towards Mtb and normal lung cells suggest a common redox mechanism of action involving intracellular thiols.

1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis

2021 ◽  
Vol 64 (17) ◽  
pp. 12790-12807
Author(s):  
Lutete Peguy Khonde ◽  
Rudolf Müller ◽  
Grant A. Boyle ◽  
Virsinha Reddy ◽  
Aloysius T. Nchinda ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Wall Biosynthesis

scholarly journals Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

2020 ◽  
Vol 295 (36) ◽  
pp. 12618-12634
Author(s):  
H. Diessel Duan ◽  
Nishya Mohamed-Raseek ◽  
Anne-Frances Miller
Keyword(s):  
Electron Transfer ◽  
Density Functional ◽  
Protein Denaturation ◽  
Rhodopseudomonas Palustris ◽  
Density Functional Theory Calculations ◽  
Site Directed Mutagenesis ◽  
Functional Theory ◽  
Electron Transfer Flavoprotein ◽  
Midpoint Potential ◽  
Ct Complex

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

2003 ◽  
Vol 47 (1) ◽  
pp. 378-382 ◽  
Author(s):  
Michael S. Scherman ◽  
Katharine A. Winans ◽  
Richard J. Stern ◽  
Victoria Jones ◽  
Carolyn R. Bertozzi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targeting ◽  
Enzyme Inhibitor ◽  
Microtiter Plate ◽  
Biosynthetic Enzyme ◽  
Cell Wall Synthesis ◽  
Chemical Library ◽  
Microtiter Plate Assay ◽  
Plate Assay

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

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