Molecular Insights on Pertussis Toxin: Structure, Function, and Inhibition

ABSTRACT The rise of antibiotic-resistant pathogenic bacteria and the increasing understanding of the beneficial role of the bacterial microbiota, disrupted by antibiotics, prompt the development of new anti-bacterial therapies. One such approach targets bacterial virulence factors such as exotoxins, which are often the disease-causing virulence factor, making them appealing drug development targets. Detailed molecular understanding of exotoxins is crucial for efficient drug development efforts. This study focuses on pertussis toxin (PT), a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. PT, an ADP-ribosyltransferase (ART) toxin, disrupts cellular signaling by transferring ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to inhibitory α-subunits of G proteins of the host cell, leading to variety of systemic pathologies. The aim of the study was to identify small molecules inhibiting the ART activity of PT, and to obtain atomic resolution structural insights of the binding poses of the inhibitors as well as of the ART activity of PT. An in vitro high-throughput multiwell assay to screen small molecules inhibiting the ART activity of PT was developed. Two compounds, effective at low micromolar levels in vitro, with one also potent in living cells, were identified. No binding poses for the compounds were obtained with X-ray crystallography. However, crystal structures of PT in complex with NAD+, its hydrolysis products ADP-ribose and nicotinamide, NAD+ analog PJ34 (ART inhibitor), and a novel NAD+ analog formed upon crystallization with 3-aminobenzamide (ART inhibitor) and NAD+, were obtained. These structures provide novel insights into pre- and post-NAD+ hydrolysis steps of the ART activity of PT and provide a rational basis to develop ART inhibitors as well as the biocatalytic use of PT to produce the novel NAD+ analog for biochemical and structural experiments with NAD+ binding proteins. In conclusion, this study identified small molecules inhibiting the ART activity of PT and obtained atomic resolution structural insights of the ART activity, as well as binding poses of the small molecules in PT, known to inhibit other ARTs. These findings could aid in the rational drug design approaches and development of PT-specific small-molecule inhibitors. KEYWORDS:whooping cough, pertussis toxin, ADP-ribosyltransferase, inhibitor, drug developmentTIIVISTELMÄ Antibioottiresistentit bakteerit sekä ymmärrys antibioottien haitallisesta vaikutuksesta suotuisaan mikrobistoon korostavat tarvetta kehittää vaihtoehtoisia hoitomuotoja bakteeritaudeille. Yksi keino on bakteerien virulenssitekijöiden, kuten eksotoksiinien, kohdentaminen. Eksotoksiineilla on usein keskeinen osuus taudin aiheuttamisessa, mikä tekee niistä hyviä lääkekehityskohteita. Eksotoksiinien yksityiskohtainen ymmärtäminen on tärkeää uusien hoitomuotojen kehittämisessä. Tämä tutkimus keskittyy pertussistoksiiniin (PT), joka on hinkuyskää aiheuttavan Bordetella pertussis -bakteerin tärkeä virulenssitekijä. PT on ADP-ribosyyli-transferaasi (ART), joka häiritsee soluviestintää siirtämällä ADP-riboosin nikotiiniadeniinidinukleotidilta (NAD+) isäntäsolun G-proteiinin inhibitoriseen α-alayksikköön, johtaen moniin systeemisiin häiriöihin. Tavoitteena oli löytää pienmolekyylisiä yhdisteitä, jotka estävät PT:n ART-toimintaa, ja tutkia atomitasolla PT:n rakennetta yhdisteiden sitoutumisen sekä ART-toiminnan kannalta. Tässä työssä kehitettiin erittäin tehokas PT:n toimintaa estävien pienmolekyylisten yhdisteiden in vitro -seulontamenetelmä, jolla löydettiin kaksi PT:n aktiivisuutta estävää pienmolekyylistä yhdistettä. Ne osoittautuivat tehokkaiksi matalina pitoisuuksina in vitro, ja toinen on tehokas myös elävissä soluissa. Näille yhdisteille ei saatu kiderakennetta, mutta rakenteet saatiin PT:lle yhdessä NAD+, sen hydrolyysituotteiden ADP-riboosin ja nikotiininamidin, NAD+ analogi PJ34:n (ART-inhibiittori) sekä uuden NAD+ analogin kanssa, joka muodostui kiteytettäessä 3-aminobentsamidia (ART-inhibiittori) ja NAD+:ia. Nämä rakenteet antavat uutta tietoa PT:n ART-toiminnasta NAD+-hydrolyysin eri vaiheissa ja luovat pohjaa PT:n ART-toimintaa estävien yhdisteiden rationaaliselle lääkesuunnittelulle sekä PT:n käytölle biokatalyyttisena alustana uuden NAD+ analogin tuottamiseen biokemiallisiin ja rakenteellisiin kokeisiin NAD+:ia sitovien proteiinien kanssa. Yhteenvetona tämä väitöstyö löysi pienmolekyylisiä yhdisteitä, jotka estävät PT:n toimintaa ja tarjoaa atomitason rakenteellista tietoa PT:n ART-toiminnasta sekä sitoutumisesta tunnettujen ART-entsyymejä inhiboivien pienmolekyylisten yhdisteiden kanssa. Tämä tieto voi auttaa sekä rationaalisen lääkesuunnittelun menetelmien että PT:lle suunnattuja pienimolekyylisten yhdisteiden kehittämisessä. AVAINSANAT: hinkuyskä, pertussistoksiini, ADP-ribosyylitransferaasi, inhibiittori, lääkehehity

Exotoxin-Targeted Drug Modalities as Antibiotic Alternatives

The paradigm of antivirulence therapy dictates that bacterial pathogens are specifically disarmed but not killed by neutralizing their virulence factors. Clearance of the invading pathogen by the immune system is promoted. As compared to antibiotics, the pathogen-selective antivirulence drugs hold promise to minimize collateral damage to the beneficial microbiome. Also, selective pressure for resistance is expected to be lower because bacterial viability is not directly affected. Antivirulence drugs are being developed for stand-alone prophylactic and therapeutic treatments but also for combinatorial use with antibiotics. This Review focuses on drug modalities that target bacterial exotoxins after the secretion or release-upon-lysis. Exotoxins have a significant and sometimes the primary role as the disease-causing virulence factor, and thereby they are attractive targets for drug development. We describe the key pre-clinical and clinical trial data that have led to the approval of currently used exotoxin-targeted drugs, namely the monoclonal antibodies bezlotoxumab (toxin B/TcdB, Clostridioides difficile), raxibacumab (anthrax toxin, Bacillus anthracis), and obiltoxaximab (anthrax toxin, Bacillus anthracis), but also to challenges with some of the promising leads. We also highlight the recent developments in pre-clinical research sector to develop exotoxin-targeted drug modalities, i.e., monoclonal antibodies, antibody fragments, antibody mimetics, receptor analogs, neutralizing scaffolds, dominant-negative mutants, and small molecules. We describe how these exotoxin-targeted drug modalities work with high-resolution structural knowledge and highlight their advantages and disadvantages as antibiotic alternatives

Crystal structures of pertussis toxin with NAD(+) and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (G alpha i) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre-and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum (ER), where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the ER into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G-protein coupled receptor (GPCR) signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide (3AB) and NAD+, which we name benzamide amino adenine dinucleotide (BaAD). These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small molecule inhibitors

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein alpha -subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough

Persistent T cell-mediated immune responses against Omicron variants after the third COVID-19 mRNA vaccine dose

IntroductionThe prime-boost COVID-19 mRNA vaccination strategy has proven to be effective against severe COVID-19 disease and death. However, concerns have been raised due to decreasing neutralizing antibody levels after COVID-19 vaccination and due to the emergence of new immuno-evasive SARS-CoV-2 variants that may require additional booster vaccinations.MethodsIn this study, we analyzed the humoral and cell-mediated immune responses against the Omicron BA.1 and BA.2 subvariants in Finnish healthcare workers (HCWs) vaccinated with three doses of COVID-19 mRNA vaccines. We used enzyme immunoassay and microneutralization test to analyze the levels of SARS-CoV-2 specific IgG antibodies in the sera of the vaccinees and the in vitro neutralization capacity of the sera. Activation induced marker assay together with flow cytometry and extracellular cytokine analysis was used to determine responses in SARS-CoV-2 spike protein stimulated PBMCs.ResultsHere we show that within the HCWs, the third mRNA vaccine dose recalls both humoral and T cell-mediated immune responses and induces high levels of neutralizing antibodies against Omicron BA.1 and BA.2 variants. Three weeks after the third vaccine dose, SARS-CoV-2 wild type spike protein-specific CD4+ and CD8+ T cells are observed in 82% and 71% of HCWs, respectively, and the T cells cross-recognize both Omicron BA.1 and BA.2 spike peptides. Although the levels of neutralizing antibodies against Omicron BA.1 and BA.2 decline 2.5 to 3.8-fold three months after the third dose, memory CD4+ T cell responses are maintained for at least eight months post the second dose and three months post the third vaccine dose.DiscussionWe show that after the administration of the third mRNA vaccine dose the levels of both humoral and cell-mediated immune responses are effectively activated, and the levels of the spike-specific antibodies are further elevated compared to the levels after the second vaccine dose. Even though at three months after the third vaccine dose antibody levels in sera decrease at a similar rate as after the second vaccine dose, the levels of spike-specific CD4+ and CD8+ T cells remain relatively stable. Additionally, the T cells retain efficiency in cross-recognizing spike protein peptide pools derived from Omicron BA.1 and BA.2 subvariants. Altogether our results suggest durable cellmediated immunity and protection against SARS-CoV-2

Opioid-Induced Bowel Dysfunction in Patients Undergoing Spine Surgery: Comparison of Oxycodone and Oxycodone-Naloxone Treatment

<p><strong>Article full text</strong></p> <p><br> The full text of this article can be found <a href="https://link.springer.com/article/10.1007/s12325-016-0456-9"><b>here</b>.</a><br> <br> <strong>Provide enhanced digital features for this article</strong><br> If you are an author of this publication and would like to provide additional enhanced digital features for your article then please contact <u>[email protected]</u>.<br> <br> The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.<br> <br> Other enhanced features include, but are not limited to:<br> • Slide decks<br> • Videos and animations<br> • Audio abstracts<br> • Audio slides<u></u></p