Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents

Smith, Roxanne P.; Paxman, Jason J.; Scanlon, M.J.; Heras, Begoña

doi:10.3390/molecules21070811

Cited by 55 publications

(79 citation statements)

References 80 publications

(109 reference statements)

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“…Upon addition of 3 µM of oxidized DsbA to the magnetic tweezers experiment, a shift in the folding probability is observed (Fig 2A). At an equilibrium force of 8.9 pN, the protein L polyprotein hops between its 5th, 6th, and 7th folded state in the presence of oxidized DsbA ( A peptide antagonist blocks the chaperone activities of DsbA -DsbA has emerged as an attractive therapeutic target due to its vital role in the maturation of bacterial virulence factors, including toxins, adhesins, and secretion machinery (17,35). In principle, inhibition of DsbA could lessen the severity of bacterial infections without a selective bactericidal activity that would foster resistance.…”

Section: Single Molecule Measurement Of Protein Folding By Magnetic Tmentioning

confidence: 99%

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DsbA is a redox-switchable mechanical chaperone

Eckels¹,

Chaudhuri²,

Chakraborty³

et al. 2018

Preprint

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Force is a crucial protein denaturant in cells, occurring during processes including translation, degradation, and translocation. In bacteria, many toxins and virulence factors pass through a translocon pore as unfolded polypeptides en route to periplasmic and extracellular compartments.DsbA is a ubiquitous bacterial oxidoreductase that associates with substrates during and after translocation, yet its involvement in protein folding and translocation remains an open question.Here, using magnetic tweezers-based single molecule force spectroscopy, we characterize a mechanical chaperone activity of DsbA on a cysteine-free domain from the protein L superantigen. Interaction of DsbA with unfolded protein L substrates prominently increases the fraction of time that protein L spends in its native folded state. This chaperone activity is tuned by the oxidation state of DsbA; oxidized DsbA is a strong promoter of folding, but the effect is weakened by reduction of the catalytic CXXC motif. We further localize the chaperone binding site of DsbA using a seven residue peptide which effectively blocks the chaperone activity. We calculated that DsbA assisted folding of proteins in the periplasm generates enough mechanical work to decrease the ATP consumption needed for periplasmic translocation by up to 33%. In turn, pharmacologic inhibition of this chaperone activity may open up a new class of antivirulence agents.

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Section: Single Molecule Measurement Of Protein Folding By Magnetic Tmentioning

confidence: 99%

“…Notably, DsbA is an ongoing target for drug development due to its direct relevance in urinary tract infections, bacteremia, and biological weapons development (13)(14)(15)(16). To date, a set of small molecules, peptide compounds have been developed in one direction that block the oxidoreductase activity of this enzyme (17).…”

Section: Introductionmentioning

confidence: 99%

DsbA is a redox-switchable mechanical chaperone

Eckels¹,

Chaudhuri²,

Chakraborty³

et al. 2018

Preprint

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“…[121,122]). In humans, blood coagulation involves the activity of the enzyme vitamin K epoxide reductase (VKOR), which is inhibited by the anticoagulant drug warfarin (Coumadin).…”

Section: Dsb Enzymes As Novel Antimicrobial Targetsmentioning

confidence: 99%

From Biology to Biotechnology: Disulfide Bond Formation in <i>Escherichia coli</i>

Landgraf¹,

Ren²,

Masuch³

et al. 2017

≪i>Escherichia Coli

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Disulfide bonds formed between a pair of oxidized cysteines are important to the structural integrity and proper folding of many proteins. Accordingly, Nature has evolved several systems for the genesis and maintenance of such bonds. Beginning with the discovery of protein disulfide isomerase, which provided the first evidence for enzyme-catalyzed disulfide-bond formation, many years of research have resulted in the explication of the complex network of electron transport pathways needed for this process. Herein, we take a historical approach in describing the elucidation of disulfide-bond formation in E. coli. We frame this topic in the context of genome sequencing eras. The first section describes the discovery of eukaryotic protein disulfide isomerase and the subsequent research that followed from the early 1960s to the early 1990s, a time period we have named the pre-genomic sequencing era. The second section details the renaissance in research on disulfide-bond formation in the periplasm of prokaryotes, fueled by bacterial genetic screens and the development of genomic sequencing technology. Accordingly, we have named this section the genomic sequencing era, which ranges from the early 1990s to approximately 2010. The final section outlines the use of bacterial genetic screens to select for new oxidoreductase enzymes and their potential uses in biotechnological and pharmaceutical applications. This era we have dubbed the post-genomic sequencing era, and we envision it to represent the future of research on oxidative folding.

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“…Intramolecular disulfide bonds are often essential for the native folding and subsequent function of multiple secreted or surface proteins, including fimbriae, flagellar motor, secretion systems, and secreted toxins (13, 16). Given that many of these proteins are bona fide virulence factors or form integral components of machinery for virulence factor assembly, this makes DsbA and DsbB ideal targets for the development of antivirulence drugs (13, 16, 20). Recently, several classes of small molecule inhibitors of DsbA, as well as inhibitors of its cognate DsbB, have been reported, primarily through screening campaigns involving biophysical and/or biochemical assays (12, 21–25).…”

Section: Introductionmentioning
confidence: 99%

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics
Verderosa
¹
,
Dhouib
²
,
Hong
³

et al. 2020
Preprint
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21Antibiotics are failing fast, and the development pipeline is alarmingly dry. New drug research 22 and development is being urged by world health officials, with new antibacterials against 23 multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, 24 which are inhibitors of bacterial pathogenicity factors, are a class of promising antibacterials, 25 however, their development is often stifled by lack of standardised preclinical testing akin to 26 what guides antibiotic development. The lack of established target-specific microbiological 27 assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors 28 is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead 29 optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays 30 developed for the identification and early preclinical evaluation of DsbA inhibitors. Inhibitors 31 of DsbA block bacterial oxidative protein folding and were previously identified by biophysical 32 and biochemical assays. Here we use existing Escherichia coli DsbA inhibitors and 33 uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that a combination of a 34 cell-based AssT sulfotransferase assay and the UPEC motility assay, modified for a higher 35 throughput format, can provide a robust and target-specific platform for the evaluation of DsbA 36 inhibitors. Our pipeline could also be used in fragment and compound screening for the 37 identification of new DsbA inhibitor classes or hits with a broad spectrum of activity. In 38 conclusion, the establishment of accurate, high-throughput microbiological assays for 39 antivirulence drug identification and early preclinical development, is a significant first step 40 towards their translation into effective therapeutics. 41 42 3Importance: 43 The safety net of last resort antibiotics is quickly vanishing as bacteria become increasingly 44 resistant to most available drugs. If no action is taken, we will likely enter a post-antibiotic era, 45 where common infections and minor injuries are once again lethal. The paucity in new 46 antibiotic discovery of the past decades has compounded the problem of increasing antibiotic 47 resistance, to the point that it now constitutes a global health crisis that demands global action. 48There is currently an urgent need for new antibacterial drugs with new targets and modes of 49 action. To address this, research and development efforts into antivirulence drugs, such as 50 DsbA inhibitors, have been ramping up globally. However, the development of microbiological 51 assays as tools for effectively identifying and evaluating antivirulence drugs is lagging behind. 52Here, we present a high-throughput cell-based screening and evaluation pipeline, which could 53 significantly advance development of DsbA inhibitor as antivirulence therapeutics. 54 55 4Introduction: 56 In 2014 the World Health Organisation (WHO) released a statement declaring an...

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Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents

Cited by 55 publications

References 80 publications

DsbA is a redox-switchable mechanical chaperone

DsbA is a redox-switchable mechanical chaperone

From Biology to Biotechnology: Disulfide Bond Formation in <i>Escherichia coli</i>

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics

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