Towards selective inhibitors of adenylyl cyclase toxin from Bordetella pertussis

doi:10.1016/j.tim.2012.04.002

Trends in Microbiology

Volume 20, Issue 7, July 2012, Pages 343-351

https://doi.org/10.1016/j.tim.2012.04.002 Get rights and content

Whooping cough is a very important medical problem that requires novel approaches for treatment. The disease is caused by Bordetella pertussis, with the calmodulin (CaM)-activated adenylyl cyclase (AC) toxin (also known as CyaA) being a major virulence factor. Hence, CyaA inhibitors could constitute novel therapeutics, but it has been difficult to develop potent drugs with high selectivity over mammalian membranous ACs (mACs). Recent studies have shown that bis-anthraniloyl-substituted nucleoside 5′-triphosphates are potent and selective CyaA inhibitors. In addition, the interaction of CyaA with CaM is very different from the interaction of membranous mAC1 with CaM. Accordingly, compounds that interfere with the CyaA–CaM interaction may constitute a novel class of drugs against whooping cough.

Section snippets

CyaA as major virulence factor of Bordetella pertussis

Whooping cough is caused by Bordetella pertussis and remains a major health problem 1, 2, 3. According to the World Health Statistics 2011 report by the World Health Organization, more than 100 000 whooping cough causes were recorded in 2009, but many cases, specifically in developing countries, go undetected due to poorly functioning reporting systems 4, 5, 6, 7. Despite widespread vaccination in the developed countries, the number of whooping cough outbreaks in various sociocultural settings

Towards CyaA inhibitors targeting the catalytic site

The first AC inhibitors available were the P-site inhibitors. P-site inhibitors constitute nucleoside 3′-(rather than 5′)-mono-, di-, tri-, or tetraphosphates. P-site inhibitors probably bind to the catalytic site of mACs in a specific conformation. The type of inhibition is non-competitive with respect to the substrate ATP, but competitive versus the products cAMP and pyrophosphate [18]. However, most P-site inhibitors such as 2′-d-3′-AMP (Figure 1) are nearly inactive at CyaA [19].

Towards CyaA inhibitors targeting the CyaA–CaM interface

Several hydrophobic compounds inhibit the interaction between CaM and target proteins by binding to CaM 27, 28. The most potent CaM inhibitor known so far is calmidazolium (Figure 1) [28]. Calmidazolium binds to CaM with a K_D of ∼2 nM and inhibits CyaA-induced cAMP accumulation in intact cells with an IC₅₀ of 2.5 μM [29], in other words a potency that is ∼1000-fold lower than expected from the binding affinity for CaM. These data were the first hint that calmidazolium inhibits CyaA by a mechanism

Systematic exploration of the catalytic site of CyaA as target for inhibitors

The elucidation of the crystal structure of the catalytic site of CyaA in complex with PMEApp and of the catalytic core of mAC with various nucleotides, together with the availability of systematically varied compound libraries and sophisticated molecular modeling techniques, allowed rigorous analysis of CyaA in comparison to mACs 13, 22, 35, 36, 37, 38, 39, 40, 41.

Major goals in the development of CyaA inhibitors are high potency together with high selectivity relative to mACs.

Molecular analysis of the CyaA–CaM interaction site and recent studies with CaM antagonists

In contrast to the catalytic site, so far no rational inhibitor design has been applied with respect to the CyaA–CaM interaction site. However, substantial biophysical and biochemical work has been performed providing an excellent basis for pharmacological studies. The analysis of the crystal structure of the CyaA–CaM complex revealed that CyaA interacts with the C-terminal half of CaM (C-CaM) via four different regions, with 49 amino acids of CyaA and 41 amino acids of CaM being involved [13].

Unresolved questions and future directions

The first potent and selective CyaA inhibitors have now been obtained, but these compounds have still to be considered as pharmacological tools rather than as drug candidates. Specifically, (M)ANT-nucleotides can bind to numerous proteins [48] and, to this end, bis-Cl-ANT-ATP has only been examined with three of the nine mAC isoforms [41]. It is likely that bis-Cl-ANT-ATP also binds to other nucleotide-binding proteins, for example protein kinases, resulting in off-target effects. A

Concluding remarks

In conclusion, we have provided evidence for the notion that inhibition of the catalytic activity of CyaA is a worthwhile pharmacological approach to treat whooping cough. Early studies suggested that CyaA is an intractable pharmacological target such that, overall, relatively few data have been published so far. However, the limited number of available studies suggests that CyaA is much more amendable to pharmacological manipulation than previously appreciated. Both the catalytic site and the

Acknowledgments

We would like to acknowledge the collaboration with Drs Jens Geduhn, Andreas Gille, Martin Göttle, Burkhard König, Carolin Lübker, Gerald H. Lushington, Mark Richter, Dominik Schuler, Phillip Steindel, Yuequan Shen, and Wei-Jen Tang on the CyaA project. We also thank Drs Detlef Neumann and Carolin Lübker for critical reading of the manuscript. Thanks are also due to the reviewers for their constructive critique. This work was supported by grants of the Deutsche Forschungsgemeinschaft (GRK 760,

References (53)

F. Berger
Investigation on a pertussis outbreak in a military school: risk factors and approach to vaccine efficacy
Vaccine
(2010)
D. Ladant et al.
Bordetella pertussis adenylate cyclase: a toxin with multiple talents
Trends Microbiol.
(1999)
R. Shrivastava et al.
Virulence factor secretion and translocation by Bordetella species
Curr. Opin. Microbiol.
(2009)
W.J. Tang et al.
The adenylyl cyclase activity of anthrax edema factor
Mol. Aspects Med.
(2009)
R. Seifert
Inhibitors of membranous adenylyl cyclases
Trends Pharmacol. Sci.
(2012)
C.W. Dessauer
The interactions of adenylate cyclases with P-site inhibitors
Trends Pharmacol. Sci.
(1999)
R.A. Johnson et al.
Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and ‘P’-site agonists
J. Biol. Chem.
(1990)
S. Soelaiman
Structure-based inhibitor discovery against adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough
J. Biol. Chem.
(2003)
C. Pinto
Structure–activity relationships for the interactions of 2′- and 3′-(O)-(N-methyl)anthraniloyl-substituted purine and pyrimidine nucleotides with mammalian adenylyl cyclases
Biochem. Pharmacol.
(2011)
M.. Erdorf
Impact of divalent cations on regulation of adenylyl cyclase isoforms by forskolin analogs
Biochem. Pharmacol.
(2011)

I. Marzouqi

Development of improved vaccines against whooping cough: current status

Hum. Vaccin.

(2010)

Cited by (15)

Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases
2021, European Journal of Medicinal Chemistry
Citation Excerpt :
The most extensively studied bacterial adenylate cyclase toxins are adenylate cyclase toxin from Bordetella pertussis (ACT) and edema factor from Bacillus anthracis (EF) [6–8]. It has been speculated, that inhibition of bacterial adenylate cyclase toxins could be exploited as potential targets for prophylactic or therapeutic strategies to combat infectious diseases [9,10]. Furthermore, selective modulation of mammalian ACs’ activity represents also a promising strategy for the development of new agents to treat various human neurological and inflammatory diseases [11,12].
A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC₅₀ as low as 37 nM) and B. anthracis edema factor (IC₅₀ as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.
Bordetella protein toxins
2015, The Comprehensive Sourcebook of Bacterial Protein Toxins
Pathogenic Bordetella species produce a number of bone fide toxins, which are believed to play a major role in the pathogenesis of these bacteria. The most well characterized Bordetella species are Bordetella pertussis, the etiologic agent of whooping cough, Bordetella parapertussis, responsible for a milder whooping-cough-like syndrome, and Bordetella bronchiseptica, causing kennel cough and swinal rhinitis. All three species produce the protein toxins adenylate cyclase and dermonecrotic toxin. However, only B. pertussis produces pertussis toxin, although the genes coding for pertussis toxin are present in the genomes of most B. parapertussis and B. bronchiseptica isolates. This chapter focuses on these major protein toxins and reviews current knowledge on their biogenesis, mode of action, involvement in pathogenesis and, where appropriate, biotechnological applications.
From canonical to non-canonical cyclic nucleotides as second messengers: Pharmacological implications
2015, Pharmacology and Therapeutics
Citation Excerpt :
Many bacteria exploit signal transduction mechanisms in mammalian cells to corrupt host defense and facilitate bacteria survival. Accordingly, often, bacterial toxins mimic or, more accurately, exaggerate the effects of second messengers (Muanprasat & Chatsudthipong, 2013; Seifert & Dove, 2012; Seifert & Dove, 2013). One can also use pharmacological activators and inhibitors of generators and/or effectors to delineate specific signal transduction pathways.
This review summarizes our knowledge on the non-canonical cyclic nucleotides cCMP, cUMP, cIMP, cXMP and cTMP. We place the field into a historic context and discuss unresolved questions and future directions of research. We discuss the implications of non-canonical cyclic nucleotides for experimental and clinical pharmacology, focusing on bacterial infections, cardiovascular and neuropsychiatric disorders and reproduction medicine. The canonical cyclic purine nucleotides cAMP and cGMP fulfill the criteria of second messengers. (i) cAMP and cGMP are synthesized by specific generators, i.e. adenylyl and guanylyl cyclases, respectively. (ii) cAMP and cGMP activate specific effector proteins, e.g. protein kinases. (iii) cAMP and cGMP exert specific biological effects. (iv) The biological effects of cAMP and cGMP are terminated by phosphodiesterases and export. The effects of cAMP and cGMP are mimicked by (v) membrane-permeable cyclic nucleotide analogs and (vi) bacterial toxins. For decades, the existence and relevance of cCMP and cUMP have been controversial. Modern mass-spectrometric methods have unequivocally demonstrated the existence of cCMP and cUMP in mammalian cells. For both, cCMP and cUMP, the criteria for second messenger molecules are now fulfilled as well. There are specific patterns by which nucleotidyl cyclases generate cNMPs and how they are degraded and exported, resulting in unique cNMP signatures in biological systems. cNMP signaling systems, specifically at the level of soluble guanylyl cyclase, soluble adenylyl cyclase and ExoY from Pseudomonas aeruginosa are more promiscuous than previously appreciated. cUMP and cCMP are evolutionary new molecules, probably reflecting an adaption to signaling requirements in higher organisms.
Allosteric activation of Bordetella pertussis Adenylyl cyclase by calmodulin: Molecular dynamics and mutagenesis studies
2014, Journal of Biological Chemistry
Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg³³⁸, Asn³⁴⁷, and Asp³⁶⁰) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn³⁴⁷; ACm1A), two (Arg³³⁸ and Asp³⁶⁰; ACm2A), or three residues (Arg³³⁸, Asn³⁴⁷, and Asp³⁶⁰; ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3A·C-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.
ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation
2014, Biochemical and Biophysical Research Communications
Citation Excerpt :
EF from Bacillus anthracis is an AC that is taken up into host cells via PA and is activated by calmodulin as well [13]. CyaA contributes to the pathogenesis of whooping cough [14], and EF contributes to the pathogenesis of anthrax disease [15]. ExoY is a type III secretion protein from Pseudomonas aeruginosa that possesses structural similarity with CyaA and EF in the catalytic domain and generates cAMP, too [16].
In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.
Inhibitors of Bacillus anthracis edema factor
2013, Pharmacology and Therapeutics
Citation Excerpt :
Accordingly, EF inhibitors could be valuable drugs in the therapy of anthrax disease (Hicks et al., 2011a; Sweeney et al., 2011). Along the same line, CyaA inhibitors could be used for the treatment of whooping cough (Seifert & Dove, 2012). This review focuses on EF inhibitors.
Edema factor (EF) is a calmodulin (CaM)-activated adenylyl cyclase (AC) toxin from Bacillus anthracis that contributes to anthrax pathogenesis. Anthrax is an important medical problem, but treatment of B. anthracis infections is still unsatisfying. Thus, selective EF inhibitors could be valuable drugs in the treatment of anthrax infection, most importantly shock. The catalytic site of EF, the EF/CaM interaction site and allosteric sites constitute potential drug targets. To this end, most efforts have been directed towards targeting the catalytic site. A major challenge in the field is to obtain compounds with high selectivity for AC toxins relative to mammalian membranous ACs (mACs). 3′-(N-methyl)anthraniloyl-2′-deoxyadenosine-5′-triphosphate is the most potent EF inhibitor known so far (K_i, 10 nM), but selectivity relative to mACs needs to be improved (currently ~5–50-fold, depending on the specific mAC isoform considered). AC toxin inhibitors can be identified in virtual screening studies based on available EF crystal structures and examined in cellular test systems or at the level of purified toxin using classic radioisotopic or non-radioactive fluorescence assays. Binding of certain MANT-nucleotides to AC toxins elicits large direct fluorescence- or fluorescence resonance energy transfer signals upon interaction with CaM, and these signals can be used to identify toxin inhibitors in competition binding studies. Collectively, potent EF inhibitors are available, but before they can be used clinically, selectivity against mACs must be improved. However, several methodological approaches, complementing each other, are now available to direct the development of potent, selective, orally applicable and clinically useful EF inhibitors.

View all citing articles on Scopus

View full text

Trends in Microbiology

ReviewTowards selective inhibitors of adenylyl cyclase toxin from Bordetella pertussis

Section snippets

CyaA as major virulence factor of Bordetella pertussis

Towards CyaA inhibitors targeting the catalytic site

Towards CyaA inhibitors targeting the CyaA–CaM interface

Systematic exploration of the catalytic site of CyaA as target for inhibitors

Molecular analysis of the CyaA–CaM interaction site and recent studies with CaM antagonists

Unresolved questions and future directions

Concluding remarks

Acknowledgments

Vaccine

Trends Microbiol.

Curr. Opin. Microbiol.

Mol. Aspects Med.

Trends Pharmacol. Sci.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

Chem. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Biochem. Pharmacol.

Methods Enzymol.

J. Biol. Chem.

Anal. Biochem.

Recent developments in pertussis

Lancet

Development of improved vaccines against whooping cough: current status

Hum. Vaccin.

Review
Towards selective inhibitors of adenylyl cyclase toxin from Bordetella pertussis