scholarly journals Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

2019 ◽  
Author(s):  
Yashwanth Ashok ◽  
Moona Miettinen ◽  
Danilo Kimio Hirabae de Oliveira ◽  
Mahlet Z. Tamirat ◽  
Katja Näreoja ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Whooping Cough ◽  
Binding Pocket ◽  
Antibiotic Development ◽  
Adp Ribosylation ◽  
Highly Active ◽  
Selective Approach ◽  
Dynamics Simulations ◽  
Adp Ribosyltransferase

ABSTRACTTargeted pathogen-selective approach to antibiotic development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5, 1:1:1:2:1) is a major virulence factor ofBordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify mg-levels of truncated but highly active recombinantB. pertussisPtxS1 fromEscherichia coliand anin vitrohigh throughput-compatible assay to quantify NAD+consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in thein vitroNAD+consumption assay that also were potent in an independentin vitroassay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove useful hit compounds in targetedB. pertussis-selective drug development.

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katharina Ernst ◽  
Ann-Katrin Mittler ◽  
Veronika Winkelmann ◽  
Carolin Kling ◽  
Nina Eberhardt ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Airway Epithelium ◽  
Whooping Cough ◽  
Apical Surface ◽  
Basal Cells ◽  
Pharmacological Chaperone ◽  
Infant Mouse ◽  
Novel Therapeutic Strategies

AbstractWhooping 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 α-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.

scholarly journals Evading the Proteasome: Absence of Lysine Residues Contributes to Pertussis Toxin Activity by Evasion of Proteasome Degradation

2007 ◽  
Vol 75 (6) ◽  
pp. 2946-2953 ◽  
Author(s):  
Zoë E. V. Worthington ◽  
Nicholas H. Carbonetti
Keyword(s):  
Pertussis Toxin ◽  
Intracellular Transport ◽  
Cellular Activity ◽  
Wild Type ◽  
Proteasome Degradation ◽  
Lysine Residues ◽  
Arginine Residues ◽  
A Subunit ◽  
Adp Ribosyltransferase

ABSTRACT Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.

scholarly journals Blocking bacterial naphthohydroquinone oxidation and ADP-ribosylation improves activity of rifamycins against Mycobacterium abscessus

2021 ◽  
Author(s):  
Uday S. Ganapathy ◽  
Tian Lan ◽  
Philipp Krastel ◽  
Marissa Lindman ◽  
Matthew D. Zimmerman ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Mycobacterium Abscessus ◽  
Bacterial Metabolism ◽  
Proof Of Concept ◽  
Intrinsic Resistance ◽  
Bacterial Oxidation ◽  
Adp Ribosylation ◽  
Dual Mechanism ◽  
Adp Ribosyltransferase

Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat M. abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti- M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against M. tuberculosis . Here, we asked i) why is rifabutin more active against M. abscessus than rifampicin, and ii) why is rifabutin’s anti- M. abscessus activity poorer than its anti-TB activity. Comparative analysis of naphthoquinone versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase Arr Mab like rifampicin, preventing it from achieving the nanomolar activity it displays against M. tuberculosis . Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule’s naphthoquinone core plus a modification that blocks ADP-ribosylation at its C23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively-resistant naphthoquinone core and C25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.

scholarly journals In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0246319
Author(s):  
Raphael J. Eberle ◽  
Danilo S. Olivier ◽  
Carolina C. Pacca ◽  
Clarita M. S. Avilla ◽  
Mauricio L. Nogueira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Chikungunya Virus ◽  
In Vitro Study ◽  
3D Models ◽  
Binding Pocket ◽  
Starting Point ◽  
Low Cytotoxicity ◽  
Inhibitory Potential ◽  
Dynamics Simulations

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

scholarly journals Characterization of Individual Human Antibodies That Bind Pertussis Toxin Stimulated by Acellular Immunization

2018 ◽  
Vol 86 (6) ◽  
pp. e00004-18 ◽  
Author(s):  
Edith Acquaye-Seedah ◽  
Elizabeth E. Reczek ◽  
Hugh H. Russell ◽  
Andrea M. DiVenere ◽  
Sara O. Sandman ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Protective Antigen ◽  
Whooping Cough ◽  
Structural Explanation ◽  
Booster Immunization ◽  
Human Antibodies ◽  
B Subunit ◽  
Individual Human

ABSTRACTDespite high vaccination rates, the incidence of whooping cough has steadily been increasing in developing countries for several decades. The current acellular pertussis (aP) vaccines all include the major protective antigen pertussis toxin (PTx) and are safer, but they appear to be less protective than infection or older, whole-cell vaccines. To better understand the attributes of individual antibodies stimulated by aP, we isolated plasmablast clones recognizing PTx after booster immunization of two donors. Five unique antibody sequences recognizing native PTx were recovered and expressed as recombinant human IgG1 antibodies. The antibodies all bind different epitopes on the PTx S1 subunit, B oligomer, or S1-B subunit interface, and just one clone neutralized PTx in anin vitroassay. To better understand the epitopes bound by the nonneutralizing S1-subunit antibodies, comprehensive mutagenesis with yeast display provided a detailed map of the epitope recognized by antibodies A8 and E12. Residue R76 is required for antibody A8 binding and is present on the S1 surface but is only partially exposed in the holotoxin, providing a structural explanation for A8's inability to neutralize holotoxin. The B-subunit-specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTxin vitroas well as in anin vivoleukocytosis assay. This is the first study, to our knowledge, to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of using these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning of protective immunity despite repeated aP immunization.

Induction of humoral immunity in response to immunization with recombinantMycobacterium bovisBCG expressing the S1 subunit ofBordetella pertussistoxin

2005 ◽  
Vol 51 (12) ◽  
pp. 1015-1020 ◽  
Author(s):  
Marco A Medeiros ◽  
Geraldo R.G Armôa ◽  
Odir A Dellagostin ◽  
Douglas McIntosh
Keyword(s):  
Immune Responses ◽  
Pertussis Toxin ◽  
Whooping Cough ◽  
Low Cost ◽  
Long Term Memory ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Specific Igg

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.Key words: recombinant BCG, humoral immune response, B. pertussis.

scholarly journals Independent and Coordinate Effects of ADP-Ribosyltransferase and GTPase-Activating Activities of Exoenzyme S on HT-29 Epithelial Cell Function

2001 ◽  
Vol 69 (9) ◽  
pp. 5318-5328 ◽  
Author(s):  
Jennifer E. Fraylick ◽  
Jeannine R. La Rocque ◽  
Timothy S. Vincent ◽  
Joan C. Olson
Keyword(s):  
Dna Synthesis ◽  
Cell Function ◽  
Double Mutation ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Cell Rounding ◽  
Inhibition Of Dna Synthesis ◽  
Adp Ribosyltransferase ◽  
Ht 29

ABSTRACT Type III-mediated translocation of exoenzyme S (ExoS) into HT-29 epithelial cells by Pseudomonas aeruginosa causes complex alterations in cell function, including inhibition of DNA synthesis, altered cytoskeletal structure, loss of readherence, microvillus effacement, and interruption of signal transduction. ExoS is a bifunctional protein having both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) functional domains. Comparisons of alterations in HT-29 cell function caused by P. aeruginosastrains that translocate ExoS having GAP or ADPRT mutations allowed the independent and coordinate functions of the two activities to be assessed. An E381A ADPRT mutation revealed that ExoS ADPRT activity was required for effects of ExoS on DNA synthesis and long-term cell rounding. Conversely, the R146A GAP mutation appeared to have little impact on the cellular effects of ExoS. While transient cell rounding was detected following exposure to the E381A mutant, this rounding was eliminated by an E379A-E381A ADPRT double mutation, implying that residual ADPRT activity, rather than GAP activity, was effecting transient cell rounding by the E381A mutant. To explore this possibility, E381A and R146A-E381A mutants were examined for their ability to ADP-ribosylate Ras in vitro or in vivo. While no ADP-ribosylation of Ras was detected by either mutant in vitro, both mutants were able to modify Ras when translocated by the bacteria, with the R146A-E381A mutant causing more efficient modification than the E381A mutant, in association with increased inhibition of DNA synthesis. Comparisons of Ras ADP-ribosylation by wild-type and E381A mutant ExoS by two-dimensional electrophoresis found the former to ADP-ribosylate Ras at two sites, while the latter modified Ras only once. These studies draw attention to the key role of ExoS ADPRT activity in causing the effects of bacterially translocated ExoS on DNA synthesis and cell rounding. In addition, the studies provide insight into the enhancement of ExoS ADPRT activity within the eukaryotic cell microenvironment and into possible modulatory roles that the GAP and ADPRT domains might have on the function of each other.

scholarly journals Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3710-3718 ◽  
Author(s):  
Ikuo Uchida ◽  
Ryoko Ishihara ◽  
Kiyoshi Tanaka ◽  
Eiji Hata ◽  
Sou-ichi Makino ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Salmonella Enterica ◽  
Phage Type ◽  
Chinese Hamster ◽  
Adp Ribosylation ◽  
Dependent Modification

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB – encoded by a prophage in S. Typhimurium DT104 – are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [32P]NAD and ArtA, and the label was released by HgCl2, which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA6Arg-Ala and ArtA115Glu-Ala, in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

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