2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion

Sayer, James R; Wallden, K.; Pesnot, Thomas; Campbell, Frederick; Gane, Paul J.; Simone, Michela I.; Koss, Hans; Buelens, Floris; Boyle, Timothy P.; Selwood, David L.; Waksman, Gabriel; Tabor, Alethea B.

doi:10.1016/j.bmc.2014.09.036

Cited by 31 publications

(30 citation statements)

References 42 publications

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“…Such a large conformational spectrum might be the reason why it has been difficult to design VirB11-targetting drugs. [18][19][20][21] The novel insights we now provide might prove decisive in progressing these efforts to a successful conclusion.…”

Section: Resultsmentioning

confidence: 96%

“…Therefore, our study reveals an unprecedented level of conformational complexity, which might be exploited functionally at many different levels. Such a large conformational spectrum might be the reason why it has been difficult to design VirB11‐targetting drugs . The novel insights we now provide might prove decisive in progressing these efforts to a successful conclusion.…”

Section: Resultsmentioning

confidence: 98%

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X‐ray crystal structures of the type IVb secretion system DotB ATPases

Prevost

Waksman

2018

Protein Science

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Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilizes a Type IVb secretion (T4bS) system termed “dot/icm” to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS‐like N‐terminal domain coupled to a RecA‐like C‐terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and β, with an αβαβαβ configuration. The existence of α and β conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααβααβ configuration. The two conformers co‐exist regardless of the nucleotide‐bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug design to develop more specific antibiotics to tackle Legionnaire's disease.PDB Code(s): http://firstglance.jmol.org/fg.htm?mol=Will; http://firstglance.jmol.org/fg.htm?mol=be; http://firstglance.jmol.org/fg.htm?mol=provided

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 98%

X‐ray crystal structures of the type IVb secretion system DotB ATPases

Prevost

Waksman

2018

Protein Science

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“…We here used a similar unbiased DSF-based screen to identify fragments that bind Cagα without specifically targeting its ATPase active site. Competitive inhibitors of Cagα ATPase activity are already available [29,30], and whereas these molecules may have potential for development into antivirulence drugs, the possibility that they bind other ATPases in bacteria or in host cells remains a concern.…”

Section: Discussionmentioning

confidence: 99%

“…High-throughput small molecule screening and chemical synthesis led to the identification of inhibitors of the ATPase activity of Cagα that likely bind at the ATPase active site, but structural information on their binding site is not available [29,30]. Whereas the isolation of competitive inhibitors of the ATPase activity of VirB11 homologs is interesting, there are concerns about the specificity of these molecules since they may also inhibit other ATPases in bacteria or in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Fragment-based screening identifies inhibitors of the ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

Arya

Oudouhou

Casu

et al. 2018

Preprint

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Type IV secretion systems are membrane-bound multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system is encoded by the cag pathogenicity island that encodes 27 Cag proteins and most of these are essential for bacterial virulence. We here present our work on the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein during thermal denaturation suggesting that they bind Cagα. Several of these molecules affect binding of ADP and four of them inhibit the ATPase enzyme activity of Cagα.Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the ATPase active site. Cross-linking analysis suggests that the active molecules change the conformation of the protein and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Analysis by X-ray crystallography reveals that molecule 1G2 binds at the interface between Cagα subunits. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins and the identified molecules have potential for the development into antivirulence drugs. Author summaryWe here report the results of a small-molecule screening approach to identify inhibitors of an essential virulence factor from the gastrointestinal pathogen Helicobacter pylori. We identifed novel chemical entities that bind to the Cagα protein and inhibit its function. We discovered a novel inhibitor binding site that disrupts the hexameric quaternary structure and inhibits ATPase enzyme activity of Cagα. Based on the structural information on the binding site, these molecules could be developed into high-affinity inhibitors that may have potential as anti-virulence drugs. This approach represents a generally applicable strategy for the inhibition of bacterial virulence factors for which structural information is available that could be applied to target similar proteins from many bacterial pathogens.

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“…Cagα can also be regulated by HP1451, a protein unique to H. pylori that forms a complex with the hexamer and reduces the ATP hydrolysis activity of Cagα (Hare et al 2007) although the conservation and importance of this regulatory mechanism is still not known. Finally, VirB11 was found as a potent target for ATPase inhibitors (Sayer et al 2014) in order to block CagA secretion (Hilleringmann et al 2006)[LT4].…”

Section: Virb11mentioning

confidence: 99%

Structural and Molecular Biology of Type IV Secretion Systems

Bergé

Waksman

Terradot

2017

Current Topics in Microbiology and Immunology

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Type IV secretion systems (T4SSs) are nanomachines that Gram-negative, Gram-positive bacteria, and some archaea use to transport macromolecules across their membranes into bacterial or eukaryotic host targets or into the extracellular milieu. They are the most versatile secretion systems, being able to deliver both proteins and nucleoprotein complexes into targeted cells. By mediating conjugation and/or competence, T4SSs play important roles in determining bacterial genome plasticity and diversity; they also play a pivotal role in the spread of antibiotic resistance within bacterial populations. T4SSs are also used by human pathogens such as Legionella pneumophila, Bordetella pertussis, Brucella sp., or Helicobacter pylori to sustain infection. Since they are essential virulence factors for these important pathogens, T4SSs might represent attractive targets for vaccines and therapeutics. The best-characterized conjugative T4SSs of Gram-negative bacteria are composed of twelve components that are conserved across many T4SSs. In this chapter, we will review our current structural knowledge on the T4SSs by describing the structures of the individual components and how they assemble into large macromolecular assemblies. With the combined efforts of X-ray crystallography, nuclear magnetic resonance (NMR), and more recently electron microscopy, structural biology of the T4SS has made spectacular progress during the past fifteen years and has unraveled the properties of unique proteins and complexes that assemble dynamically in a highly sophisticated manner.

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2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion

Abstract: Graphical abstract

Cited by 31 publications

References 42 publications

X‐ray crystal structures of the type IVb secretion system DotB ATPases

X‐ray crystal structures of the type IVb secretion system DotB ATPases

Fragment-based screening identifies inhibitors of the ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

Structural and Molecular Biology of Type IV Secretion Systems

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