Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.

Biology Centre CAS Institute of Parasitology University of South Bohemia Faculty of Science Branišovská 1160 31 37005 České Budějovice Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo n 2 16610 Prague Czech Republic

Institute of Pharmaceutical and Biomedical Sciences Johannes Gutenberg University of Mainz Staudingerweg 5 55128 Mainz Germany

Pharmaceutical Institute Department of Pharmaceutical and Medicinal Chemistry University of Bonn An der Immenburg 4 53121 Bonn Germany

Research Group Intestinal Microbiology German Institute of Human Nutrition Potsdam Rehbruecke Arthur Scheunert Allee 114 116 14558 Nuthetal Germany

Zobrazit více v PubMed

Pogorzelska A., Żołnowska B., Bartoszewski R., Biochimie 2018, 151, 85–106; PubMed

Pišlar A., Mitrović A., Sabotič J., Pečar Fonović U., Perišić Nanut M., Jakoš T., Senjor E., Kos J., PLoS Pathog. 2020, 16, e1009013. PubMed PMC

McCall L. I., McKerrow J. H., Trends Parasitol. 2014, 30, 342–349; PubMed

Ettari R., Previti S., Tamborini L., Cullia G., Grasso S., Zappalà M., Mini-Rev. Med. Chem. 2016, 16, 1374–1391; PubMed

Siqueira-Neto J. L., Debnath A., McCall L. I., Bernatchez J. A., Ndao M., Reed S. L., Rosenthal P. J., PLoS Neglected Trop. Dis. 2018, 12, e0006512. PubMed PMC

Johé P., Jaenicke E., Neuweiler H., Schirmeister T., Kersten C., Hellmich U. A., J. Biol. Chem. 2021, 296, 100565. PubMed PMC

Caffrey C. R., Hansell E., Lucas K. D., Brinen L. S., Alvarez Hernandez A., Cheng J., Gwaltney S. L., Roush W. R., Stierhof Y. D., Bogyo M., Steverding D., McKerrow J. H., Mol. Biochem. Parasitol. 2001, 118, 61–73. PubMed

Steverding D., Sexton D. W., Wang X., Gehrke S. S., Wagner G. K., Caffrey C. R., Int. J. Parasitol. 2012, 42, 481–488. PubMed

Kerr I. D., Wu P., Marion-Tsukamaki R., Mackey Z. B., Brinen L. S., PLoS Neglected Trop. Dis. 2010, 4, e701. PubMed PMC

Johé P., Jung S., Endres E., Kersten C., Zimmer C., Ye W., Sönnichsen C., Hellmich U. A., Sotriffer C., Schirmeister T., Neuweiler H., ACS Chem. Biol. 2021, 16, 661–670. PubMed

Schirmeister T., Schmitz J., Jung S., Schmenger T., Krauth-Siegel R. L., Gütschow M., Bioorg. Med. Chem. Lett. 2017, 27, 45–50; PubMed

Cianni L., Lemke C., Gilberg E., Feldmann C., Rosini F., dos Reis Rocho F., Ribeiro J. F., Tezuka D. Y., Lopes C. D., de Albuquerque S., Bajorath J., Laufer S., Leitão A., Gütschow M., Montanari C. A., PLoS Neglected Trop. Dis. 2020, 14, e0007755. PubMed PMC

Giroud M., Kuhn B., Saint-Auret S., Kuratli C., Martin R. E., Schuler F., Diederich F., Kaiser M., Brun R., Schirmeister T., Haap W., J. Med. Chem. 2018, 61, 3370–3388. PubMed

Previti S., Ettari R., Cosconati S., Amendola G., Chouchene K., Wagner A., Hellmich U. A., Ulrich K., Krauth-Siegel R. L., Wich P. R., Schmid I., Schirmeister T., Gut J., Rosenthal P. J., Grasso S., Zappalà M., J. Med. Chem. 2017, 60, 6911–6923. PubMed

Ettari R., Previti S., Maiorana S., Amendola G., Wagner A., Cosconati S., Schirmeister T., Hellmich U. A., Zappalà M., J. Med. Chem. 2019, 62, 10617–10629; PubMed

Maiorana S., Ettari R., Previti S., Amendola G., Wagner A., Cosconati S., Hellmich U. A., Schirmeister T., Zappalà M., ChemMedChem 2020, 15, 1552–1561. PubMed

Dunny E., Doherty W., Evans P., Malthouse J. P. G., Nolan D., Knox A. J. S., J. Med. Chem. 2013, 56, 6638–6650; PubMed

Zhang H., Collins J., Nyamwihura R., Ware S., Kaiser M., Ogungbe I. V., Bioorg. Med. Chem. Lett. 2018, 28, 1647–1651. PubMed PMC

Zhang H., Collins J., Nyamwihura R., Crown O., Ajayi O., Ogungbe I. V., Bioorg. Med. Chem. Lett. 2020, 30, 127217. PubMed PMC

Breuning A., Degel B., Schulz F., Büchold C., Stempka M., U, Machon , Heppner S., Gelhaus C., Leippe M., Leyh M., Kisker C., Rath J., Stich A., Gut J., Rosenthal P. J., Schmuck C., Schirmeister T., J. Med. Chem. 2010, 53, 1951–1963. PubMed

Arafet K., González F. V., Moliner V., Chem. Eur. J. 2021, 27, 10142–10150. PubMed

Kerr I. D., Lee J. H., Farady C. J., Marion R., Rickert M., Sajid M., Pandey K. C., Caffrey C. R., Legac J., Hansell E., McKerrow J. H., Craik C. S., Rosenthal P. J., Brinen L. S., J. Biol. Chem. 2009, 284, 25697–25703. PubMed PMC

For a recent review, see Fang H., Peng B., Ong S. Y., Wu Q., Li L., Yao S. Q., Chem. Sci. 2021, 12, 8288–8310. PubMed PMC

For examples, see

Na Z., Li L., Uttamchandani M., Yao S. Q., Chem. Commun. 2012, 48, 7304–7306; PubMed

Frizler M., Yampolsky I. V., Baranov M. S., Stirnberg M., Gütschow M., Org. Biomol. Chem. 2013, 11, 5913–5921; PubMed

Sanman L. E., van der Linden W. A., Verdoes M., Bogyo M., Cell Chem. Biol. 2016, 23, 793–804; PubMed PMC

Pichler C. M., Krysiak J., Breinbauer R., Bioorg. Med. Chem. 2016, 24, 3291–3303; PubMed

Dana D., Garcia J., Bhuiyan A. I., Rathod P., Joo L., Novoa D. A., Paroly S., Fath K. R., Chang E. J., Pathak S. K., Bioorg. Chem. 2019, 85, 505–514; PubMed

van de Plassche M. A. T., O′Neill T. J., Seeholzer T., Turk B., Krappmann D., Verhelst S. H. L., J. Med. Chem. 2020, 63, 3996–4004; PubMed

Lemke C., Benýšek J., Brajtenbach D., Breuer C., Jílková A., Horn M., Buša M., Ulrychová L., Illies A., Kubatzky K. F., Bartz U., Mareš M., Gütschow M., J. Med. Chem. 2021, 64, 13793–13806; PubMed

Bhuiyan A. I., Rathod P., Ghoshal S., Dana D., Das T., Li G., Dickson A. A., Rafi F., Subramaniam G. S., Fath K. R., Paroly S., Chang E. J., Pathak S. K., Bioorg. Chem. 2021, 117, 105463. PubMed

Florent I., Lecaille F., Montagne J. J., Gauthier F., Schrével J., Lalmanach G., Biol. Chem. 2005, 386, 401–406; PubMed

Deu E., Leyva M. J., Albrow V. E., Rice M. J., Ellman J. A., Bogyo M., Chem. Biol. 2010, 17, 808–819; PubMed PMC

Yang P. Y., Wang M., Li L., Wu H., He C. Y., Yao S. Q., Chem. Eur. J. 2012, 18, 6528–6541. PubMed

Arastu-Kapur S., Ponder E. L., Fonović U. P., Yeoh S., Yuan F., Fonović M., Grainger M., Phillips C. I., Powers J. C., Bogyo M., Nat. Chem. Biol. 2008, 4, 203–213; PubMed

Yang P. Y., Wang M., He C. Y., Yao S. Q., Chem. Commun. 2012, 48, 835–837; PubMed

Choy J. W., Bryant C., Calvet C. M., Doyle P. S., Gunatilleke S. S., Leung S. S. F., Ang K. K. H., Chen S., Gut J., Oses-Prieto J. A., Johnston J. B., Arkin M. R., Burlingame A. L., Taunton J., Jacobson M. P., McKerrow J. M., Podust L. M., Renslo A. R., Beilstein J. Org. Chem. 2013, 9, 15–25; PubMed PMC

Tan M. S. Y., Davison D., Sanchez M. I., Anderson B. M., Howell S., Snijders A., Edgington-Mitchell L. E., Deu E., PLoS One 15 2020, e0227341. PubMed PMC

For examples, see

Trippier P. C., ChemMedChem 2013, 8, 190–201; PubMed

Chakrabarty S., Kahler J. P., van de Plassche M. A., Vanhoutte R., Verhelst S. H. L., Curr. Top. Microbiol. Immunol. 2019, 420, 253–281; PubMed

Beard H. A., Korovesis D., Chen S., Verhelst S. H. L., Mol. Omics 2021, 17, 197–209. PubMed

Jaishankar P., Hansell E., Zhao D. M., Doyle P. S., McKerrow J. H., Renslo A. R., Bioorg. Med. Chem. Lett. 2008, 18, 624–628; PubMed

Jung S., Fuchs N., Johe P., Wagner A., Diehl E., Yuliani T., Zimmer C., Barthels F., Zimmermann R. A., Klein P., Waigel W., Meyr J., Opatz T., Tenzer S., Distler U., Räder H. J., Kersten C., Engels B., Hellmich U. A., Klein J., Schirmeister T., J. Med. Chem. 2021, 64, 12322–12358. PubMed

Breidenbach J., Bartz U., Gütschow M., Biochim. Biophys. Acta Proteins Proteom. 2020, 1868, 140445. PubMed PMC

Kada G., Falk H., Gruber H. J., Biochim. Biophys. Acta 1999, 1427, 33–43. PubMed

Steverding D., Caffrey C. R., Mol. Biochem. Parasitol. 2021, 245, 111395. PubMed

Palmer J. T., Rasnick D., Klaus J. L., Brömme D., J. Med. Chem. 1995, 38, 3193–3196; PubMed

Roush W. R., Gwaltney S. L., Cheng J., Scheidt K. A., McKerrow J. H., Hansell E., J. Am. Chem. Soc. 1998, 120, 10994–10995;

Chen Y. T., Brinen L. S., Kerr I. D., Hansell E., Doyle P. S., McKerrow J. H., Roush W. R., PLoS Neglected Trop. Dis. 2010, 4, e825. PubMed PMC

Häußler D., Schulz-Fincke A. C., Beckmann A. M., Keils A., Gilberg E., Mangold M., Bajorath J., Stirnberg M., Steinmetzer T., Gütschow M., Chem. Eur. J. 2017, 23, 5205–5209. PubMed

Frizler M., Lohr F., Lülsdorff M., Gütschow M., Chem. Eur. J. 2011, 17, 11419–11423. PubMed

Mertens M. D., Schmitz J., Horn M., Furtmann N., Bajorath J., Mareš M., Gütschow M., ChemBioChem 2014, 15, 955–959. PubMed

Hirumi H., Hirumi K., J. Parasitol. 1989, 75, 985–989. PubMed