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One cannot rule them all: Are bacterial toxins-antitoxins druggable?

Chan WT, Balsa D, Espinosa M - FEMS Microbiol. Rev. (2015)

Bottom Line: The result is a cessation of cell growth or even death.Appropriate fragments could disrupt the T:A interfaces leading to the release of the targeted TA pair.Possible ways of delivery and formulation of Tas are also discussed.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28006-Madrid, Spain.

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Detection of disruption of TA protein complex by i-PPI via BRET assays. The BRET assay technology is based on the efficient resonance energy transfer between a bioluminescent donor moiety and a fluorescent acceptor moiety. The bioluminescent Renilla luciferase (RLUC) that was fused with toxin protein catalyses the coelenterazine substrate to coelenteramide with concomitant light emission at 480 nm. When the acceptor enhanced yellow fluorescent protein (EYFP)-antitoxin is in close proximity to RLUC, EYFP will absorb the energy emitted by the RLUC/coelenterazine reaction and emit yellow light that can be measured at 530 nm. If the interactions between the toxin and antitoxin proteins are disrupted by i-PPI, RLUC and EYFP will be too far apart for resonance energy transfer and only the blue-emitting spectrum of RLUC will be detected.
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fig3: Detection of disruption of TA protein complex by i-PPI via BRET assays. The BRET assay technology is based on the efficient resonance energy transfer between a bioluminescent donor moiety and a fluorescent acceptor moiety. The bioluminescent Renilla luciferase (RLUC) that was fused with toxin protein catalyses the coelenterazine substrate to coelenteramide with concomitant light emission at 480 nm. When the acceptor enhanced yellow fluorescent protein (EYFP)-antitoxin is in close proximity to RLUC, EYFP will absorb the energy emitted by the RLUC/coelenterazine reaction and emit yellow light that can be measured at 530 nm. If the interactions between the toxin and antitoxin proteins are disrupted by i-PPI, RLUC and EYFP will be too far apart for resonance energy transfer and only the blue-emitting spectrum of RLUC will be detected.

Mentions: A specific bioluminiscence resonance energy transfer (BRET) assay (Xu, Piston and Johnson 1999) to test libraries of peptides that could disrupt the T:A interfaces was developed (Nieto et al., 2006). This tool is an in vivo assay, amenable for high-throughput scaling up, and with clear advantages over fluorescence resonance energy transfer (FRET)-based assays because BRET is not susceptible to photo-bleaching, and thus yields more reliable results. BRET has been proven to be a constructive tool to identify potential hits for T:A interruption (Fig. 3) (Lioy et al., 2010). In this study, several libraries encompassing various millions of peptides were used in attempt to disrupt the Epsilon–Zeta TA protein complex. Two positive 17 amino-acid-long hits were found in two sublibraries; however, no new records on further progresses have been reported (Lioy et al., 2010). It also remains unknown whether the peptides were binding to the antitoxin or to the toxin. Besides disruption of the interfaces of both proteins, it is important to ensure that the binding of the peptides, if to the toxin, does not hinder the catalytic activity of the toxin, so that the released toxin can still act on its target to kill the pathogens. As shown in another study, peptides designed based on antitoxin sequences were able to disrupt the T:A interface, but the toxicity of the toxin was also reduced (Agarwal et al., 2010). We suggest that peptides designed based on toxin sequence to bind to antitoxin would be a better approach. Employments of peptides of sizes ranging from 6 to 60 amino acids to tackle disruption of PPIs have important advantages such as high specificity and potentially lower failure rates after its inclusion into the drug pipeline of clinical trials. However, biological molecules have shown several shortcomings, such as poor oral availability and high costs of bioprocessing.


One cannot rule them all: Are bacterial toxins-antitoxins druggable?

Chan WT, Balsa D, Espinosa M - FEMS Microbiol. Rev. (2015)

Detection of disruption of TA protein complex by i-PPI via BRET assays. The BRET assay technology is based on the efficient resonance energy transfer between a bioluminescent donor moiety and a fluorescent acceptor moiety. The bioluminescent Renilla luciferase (RLUC) that was fused with toxin protein catalyses the coelenterazine substrate to coelenteramide with concomitant light emission at 480 nm. When the acceptor enhanced yellow fluorescent protein (EYFP)-antitoxin is in close proximity to RLUC, EYFP will absorb the energy emitted by the RLUC/coelenterazine reaction and emit yellow light that can be measured at 530 nm. If the interactions between the toxin and antitoxin proteins are disrupted by i-PPI, RLUC and EYFP will be too far apart for resonance energy transfer and only the blue-emitting spectrum of RLUC will be detected.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487406&req=5

fig3: Detection of disruption of TA protein complex by i-PPI via BRET assays. The BRET assay technology is based on the efficient resonance energy transfer between a bioluminescent donor moiety and a fluorescent acceptor moiety. The bioluminescent Renilla luciferase (RLUC) that was fused with toxin protein catalyses the coelenterazine substrate to coelenteramide with concomitant light emission at 480 nm. When the acceptor enhanced yellow fluorescent protein (EYFP)-antitoxin is in close proximity to RLUC, EYFP will absorb the energy emitted by the RLUC/coelenterazine reaction and emit yellow light that can be measured at 530 nm. If the interactions between the toxin and antitoxin proteins are disrupted by i-PPI, RLUC and EYFP will be too far apart for resonance energy transfer and only the blue-emitting spectrum of RLUC will be detected.
Mentions: A specific bioluminiscence resonance energy transfer (BRET) assay (Xu, Piston and Johnson 1999) to test libraries of peptides that could disrupt the T:A interfaces was developed (Nieto et al., 2006). This tool is an in vivo assay, amenable for high-throughput scaling up, and with clear advantages over fluorescence resonance energy transfer (FRET)-based assays because BRET is not susceptible to photo-bleaching, and thus yields more reliable results. BRET has been proven to be a constructive tool to identify potential hits for T:A interruption (Fig. 3) (Lioy et al., 2010). In this study, several libraries encompassing various millions of peptides were used in attempt to disrupt the Epsilon–Zeta TA protein complex. Two positive 17 amino-acid-long hits were found in two sublibraries; however, no new records on further progresses have been reported (Lioy et al., 2010). It also remains unknown whether the peptides were binding to the antitoxin or to the toxin. Besides disruption of the interfaces of both proteins, it is important to ensure that the binding of the peptides, if to the toxin, does not hinder the catalytic activity of the toxin, so that the released toxin can still act on its target to kill the pathogens. As shown in another study, peptides designed based on antitoxin sequences were able to disrupt the T:A interface, but the toxicity of the toxin was also reduced (Agarwal et al., 2010). We suggest that peptides designed based on toxin sequence to bind to antitoxin would be a better approach. Employments of peptides of sizes ranging from 6 to 60 amino acids to tackle disruption of PPIs have important advantages such as high specificity and potentially lower failure rates after its inclusion into the drug pipeline of clinical trials. However, biological molecules have shown several shortcomings, such as poor oral availability and high costs of bioprocessing.

Bottom Line: The result is a cessation of cell growth or even death.Appropriate fragments could disrupt the T:A interfaces leading to the release of the targeted TA pair.Possible ways of delivery and formulation of Tas are also discussed.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28006-Madrid, Spain.

Show MeSH