Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin

Ashok, Yashwanth; Miettinen, Moona; Oliveira, Danilo Kimio Hirabae de; Tamirat, Mahlet Z.; Näreoja, Katja; Tiwari, Avlokita; Hottiger, Michael O.; Johnson, Mark S.; Lehtiö, L.; Pulliainen, Arto T.

doi:10.1021/acsinfecdis.9b00412

Cited by 32 publications

(37 citation statements)

References 54 publications

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“…No ADP-ribose signal was detected between 35 and 55 kDa markers if the reaction conditions lacked NAD + . As previously reported, 22 rPtxS1-wt and to lesser extent rPtxS1-Q127D/E129D had already become auto-ADP-ribosylated in the E. coli expression host as seen by the appearance of an ADP-ribose signal between 15 and 25 kDa markers. Based on these results, we concluded that the rPtxS1-wt ADP-ribosylated the rGαi target in the studied concentrations in the presence of NAD + .…”

Section: Resultssupporting

confidence: 81%

“…Of note, rPtxS1 is an ADP-ribosyltransferase, which consumes NAD + also in the absence of its Gαi substrate, although to a much lower quantities. 22 In all assays, the NAD + concentration was fixed to 400 nM to ensure that the assay functionality is in the linear range determined by the manufacturer. The enzyme reaction was performed using 1 μM of substrates (PC10, PC15, and rGαi) and 200 nM of rPtxS1-wt.…”

Section: Resultsmentioning

confidence: 99%

“…Expression and purification of recombinant PtxS1 (wt and Q127D/E129D mutant) and Gαi proteins, here after called rPtxS1-wt, rPtxS1-Q127D/E129D and rGαi, have been described previously. 22 For rHIS-GST-Af1521, the expression plasmid pNH-HIS-GST-Af1521 was transformed into BL21(DE3) and selected o/n at 37 °C on Luria–Bertani (LB) agar with appropriate antibiotics (kanamycin, 50 μg/mL). Next day, a single colony was transferred into LB media with appropriate antibiotics and cultured at 37 °C, 250 rpm, o/n.…”

Section: Methodsmentioning

confidence: 99%

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Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

et al. 2020

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Post-translational modifications (PTMs) are one of the most important regulatory mechanisms in cells, and they play key roles in cell signaling both in health and disease. PTM catalyzing enzymes have become significant drug targets, and therefore, tremendous interest has been focused on the development of broad-scale assays to monitor several different PTMs with a single detection platform. Most of the current methodologies suffer from low throughput or rely on antibody recognition, increasing the assay costs, and decreasing the multifunctionality of the assay. Thus, we have developed a sensitive time-resolved Förster resonance energy transfer (TR-FRET) detection method for PTMs of cysteine residues using a single-peptide approach performed in a 384-well format. In the developed assay, the enzyme-specific biotinylated substrate peptide is post-translationally modified at the cysteine residue, preventing the subsequent thiol coupling with a reactive AlexaFluor 680 acceptor dye. In the absence of enzymatic activity, increase in the TR-FRET signal between the biotin-bound Eu(III)-labeled streptavidin donor and the cysteine-coupled AlexaFluor 680 acceptor dye is observed. We demonstrate the detection concept with cysteine modifying S-nitrosylation and ADP-ribosylation reactions using a chemical nitric oxide donor S-nitrosoglutathione and enzymatic ADP-ribosyltransferase PtxS1-subunit of pertussis toxin, respectively. As a proof of concept, three peptide substrates derived from the small GTPase K-Ras and the inhibitory α-subunit of the heterotrimeric G-protein Gαi showed expected functionality in both chemical and enzymatic assays. Measurements yielded signal-to-background ratios of 28.7, 33.0, and 8.7 between the modified and the nonmodified substrates for the three peptides in the S-nitrosylation assay, 5.8 in the NAD + hydrolysis assay, and 6.8 in the enzymatic ADP-ribosyltransferase inhibitor dose–response assay. The developed antibody-free assay for cysteine-modifying enzymes provides a detection platform with low nanomolar peptide substrate consumption, and the assay is potentially applicable to investigate various cysteine-modifying enzymes in a high throughput compatible format.

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

et al. 2020

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“…Recombinant Gαi and recombinant PTS1 were expressed and purified as described earlier [ 46 ]. The used peptides α-defensin-1 and -5 as well as β-defensin-1 were purchased from PeptaNova (Sandhausen, Germany).…”

Section: Methodsmentioning

confidence: 99%

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Kling

Pulliainen

Barth

et al. 2021

Toxins

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Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not β-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.

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“…The Eu-probe was purified as described before, [31] and the concentration was determined using the DELFIA technique and a commercial EuCl 3 standard from PerkinElmer Life and Analytical Sciences, Wallac (Turku, Finland). The protein substrates, recombinant pertussis toxin (PTX), G protein alpha I subunit (Gαi) [33], human Ras GTPase-activating protein 1 (p120GAP) [34], wild type Kirsten RAt Sarcoma virus (KRAS, 2-188), human son of sevenless SOS1 catalytic domain (SOS cat , 564-1048) [35], and eukaryotic initiation factor 4A1 (eIF4A1) [32] were kind gifts from our collaborators. Malate dehydrogenase (MDH) was purchased from Roche (Basel, Switzerland).…”

Section: Materials Instrumentation and Assay Buffersmentioning

confidence: 99%

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

Vuorinen

Valtonen

Hassan

et al. 2021

IJMS

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Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.

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Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin

Cited by 32 publications

References 54 publications

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

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