New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain

Klein, Philipp; Johe, Patrick; Wagner, Annika; Jung, Sascha; Kühlborn, Jonas; Barthels, Fabian; Tenzer, Stefan; Distler, Ute; Waigel, Waldemar; Engels, Bernd; Hellmich, Ute A.; Opatz, Till; Schirmeister, Tanja

doi:10.3390/molecules25061451

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New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain

^1,†,

^2,†,

³,

^2,4,

¹,

²,

⁵

⁵

⁶,

⁶,

^3,7,

^1,*

and

Tanja Schirmeister

^2,*

Department of Chemistry, Organic Chemistry Section, Johannes Gutenberg-Universität, 55128 Mainz, Germany

Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-Universität, 55128 Main, Germany

Department of Chemistry, Biochemistry Section, Johannes Gutenberg-Universität, 55128 Mainz, Germany

⁴

Present address: Faculty of Chemistry and Chemical Biology, TU Dortmund University, 44227 Dortmund, Germany

⁵

Institute of Immunology, University Medical Center, Johannes Gutenberg-Universität Mainz, 55131 Mainz, Germany

⁶

Institute of Physical and Theoretical Chemistry, Universität Würzburg, 97074 Würzburg, Germany

⁷

Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt, 60323 Frankfurt, Germany

Authors to whom correspondence should be addressed.

^†

Both authors contributed equally.

Molecules 2020, 25(6), 1451; https://doi.org/10.3390/molecules25061451

Received: 11 February 2020 / Revised: 7 March 2020 / Accepted: 13 March 2020 / Published: 23 March 2020

(This article belongs to the Special Issue Covalent Inhibitors in Drug Discovery and Chemical Biology)

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Abstract

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the S_NAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (K_i = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the S_NAr reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation. View Full-Text

Keywords: cysteine protease; rhodesain; electrophilic (het)arene; nucleophilic aromatic substitution; Meisenheimer complex; π-complex; prodrug

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MDPI and ACS Style

Klein, P.; Johe, P.; Wagner, A.; Jung, S.; Kühlborn, J.; Barthels, F.; Tenzer, S.; Distler, U.; Waigel, W.; Engels, B.; Hellmich, U.A.; Opatz, T.; Schirmeister, T. New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain. Molecules 2020, 25, 1451. https://doi.org/10.3390/molecules25061451

AMA Style

Klein P, Johe P, Wagner A, Jung S, Kühlborn J, Barthels F, Tenzer S, Distler U, Waigel W, Engels B, Hellmich UA, Opatz T, Schirmeister T. New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain. Molecules. 2020; 25(6):1451. https://doi.org/10.3390/molecules25061451

Chicago/Turabian Style

Klein, Philipp, Patrick Johe, Annika Wagner, Sascha Jung, Jonas Kühlborn, Fabian Barthels, Stefan Tenzer, Ute Distler, Waldemar Waigel, Bernd Engels, Ute A. Hellmich, Till Opatz, and Tanja Schirmeister. 2020. "New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain" Molecules 25, no. 6: 1451. https://doi.org/10.3390/molecules25061451

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New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain

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