[6] K.-H. Lin, E.A. Nalivaika, K.L. Prachanronarong, N.K. Yilmaz,
The continuous enzymatic assays were perfoArmCedCinEPblTacEk D96 MANUSCRIPT
C.A. Schiffer, Dengue Protease Substrate Recognition: Binding of
the Prime Side, ACS Infectious Diseases, 2 (2016) 734-743.
[7] M.A.M. Behnam, D. Graf, R. Bartenschlager, D.P. Zlotos, C.D.
Klein, Discovery of Nanomolar Dengue and West Nile Virus
Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue,
Journal of Medicinal Chemistry, 58 (2015) 9354-9370.
[8] A.K. Timiri, B.N. Sinha, V. Jayaprakash, Progress and prospects
on DENV protease inhibitors, European Journal of Medicinal
Chemistry, 117 (2016) 125-143.
well V-bottom plates (Greiner Bio-One, Germany) on a BMG
Labtech Fluostar OPTIMA Microtiter fluorescence plate reader
using excitation and emission wavelengths of 320 and 405 nm,
respectively. The enzymatic activity was determined as slope per
second (relative fluorescence units per second) and monitored for
15 min. Percentage inhibition was calculated relative to positive
controls (without the inhibitor). All experiments were performed
in triplicate and averaged.
[9] D. Ekonomiuk, X.-C. Su, K. Ozawa, C. Bodenreider, S.P. Lim,
G. Otting, D. Huang, A. Caflisch, Flaviviral Protease Inhibitors
Identified by Fragment-Based Library Docking into a Structure
Generated by Molecular Dynamics, Journal of Medicinal Chemistry,
52 (2009) 4860-4868.
[10] C. Steuer, C. Gege, W. Fischl, K.H. Heinonen, R.
Bartenschlager, C.D. Klein, Synthesis and biological evaluation of α-
ketoamides as inhibitors of the Dengue virus protease with antiviral
activity in cell-culture, Bioorganic & Medicinal Chemistry, 19
(2011) 4067-4074.
[11] C. Nitsche, C. Steuer, C.D. Klein, Arylcyanoacrylamides as
inhibitors of the Dengue and West Nile virus proteases, Bioorganic
& Medicinal Chemistry, 19 (2011) 7318-7337.
[12] T. Mendgen, C. Steuer, C.D. Klein, Privileged Scaffolds or
Promiscuous Binders: A Comparative Study on Rhodanines and
Related Heterocycles in Medicinal Chemistry, Journal of Medicinal
Chemistry, 55 (2012) 743-753.
[13] G. Bönner, H. Babst, W. Kaufmann, In vivo effects of camostat
mesilate on plasma kallikrein, plasma kininase II and renal kallikrein
of man, Arzneimittelforschung, 37 (1987) 535-537.
[14] T. Knehans, A. Schüller, D.N. Doan, K. Nacro, J. Hill, P.
Güntert, M.S. Madhusudhan, T. Weil, S.G. Vasudevan, Structure-
guided fragment-based in silico drug design of dengue protease
inhibitors, Journal of Computer-Aided Molecular Design, 25 (2011)
263-274.
[15] M. Hosoya, S. Matsuyama, M. Baba, H. Suzuki, S. Shigeta,
Effects of protease inhibitors on replication of various myxoviruses,
Antimicrobial Agents and Chemotherapy, 36 (1992) 1432-1436.
[16] M.K. Ramjee, I.M.J. Henderson, S.B. McLoughlin, A. Padova,
The Kinetic and Structural Characterization of the Reaction of
Nafamostat with Bovine Pancreatic Trypsin, Thrombosis Research,
98 (2000) 559-569.
[17] S.A. Beyler, L.J.D. Zaneveld, Antifertility activity of
systemically administered proteinase (acrosin) inhibitors,
Contraception, 26 (1982) 137-146.
ESI-MS Analysis of Covalent Inhibitor Binding
The DENV protease (DENVP) and the DENV protease
mutant (M S135A) (final concentration: 3 µM) were incubated
with the test compounds (final concentration: 100 µM, from 10
mM stock solutions in DMSO) in assay-buffer (50 mM Tris-HCl
pH 9, ethylene glycol (10% v/v), and 0.0016% Brij 58) at room
temperature. Untreated enzymes served as controls by replacing
the volume of the stock solution with pure DMSO. The
incubation volume (200 µL) was stored in an HPLC vial. After
chosen incubation times a volume of 20 µL was analyzed by flow
injection analysis using an Agilent 1200 series HPLC device
coupled to an ESI-MS instrument (micrOTOF-QII, Bruker
Daltonik, Bremen) operating in positive ionization mode.
Column chromatography was performed as follows: column:
Reprosil-Pur ODS-3, Dr. Maisch GmbH, Germany, 3 µm,
50 x 2 mm; method: eluent A, water (+0.1% formic acid); eluent
B, acetonitrile (+0.1% formic acid); flow rate, 0.3 mL/min;
gradient, 10% B (0 min), 95% B (4 min), 95% B (8 min), 10% B
(0 min) and 10% B (5-7 min). Instrument calibration and
external mass calibration were performed with ESI Tuning mix
(Fluka) calibration standard at the end of each analysis run. Mass
spectra of analytes with multiple, variable charges were
deconvoluted using the Maximum Entropy Deconvolution
algorithm (Compass DataAnalysis Version 4.0 SP4, Bruker
Daltonik).
Acknowledgements
We thank Heiko Rudy for measuring ESI high resolution mass
spectra and Natascha Stefan for measuring routine protease
assays. The project was sponsored by the Deutsche
Forschungsgemeinschaft,
KL-1356/3-2.
Tonko
Dražić
acknowledges support by an EU Marie-Curie grant (LIsTEN
748447).
[18] M. Bartolini, V. Cavrini, V. Andrisano, Characterization of
reversible and pseudo-irreversible acetylcholinesterase inhibitors by
means of an immobilized enzyme reactor, Journal of
Chromatography A, 1144 (2007) 102-110.
References and notes
[19] R.A. Bauer, Covalent inhibitors in drug discovery: from
accidental discoveries to avoided liabilities and designed therapies,
Drug Discovery Today, 20 (2015) 1061-1073.
[20] X. Koh-Stenta, J. Joy, S.F. Wang, P.Z. Kwek, J.L.K. Wee, K.F.
Wan, S. Gayen, A.S. Chen, C. Kang, M.A. Lee, A. Poulsen, S.G.
Vasudevan, J. Hill, K. Nacro, Identification of covalent active site
inhibitors of dengue virus protease, Drug Design, Development and
Therapy, 9 (2015) 6389-6399.
[21] C. Jöst, C. Nitsche, T. Scholz, L. Roux, C.D. Klein, Promiscuity
and Selectivity in Covalent Enzyme Inhibition: A Systematic Study
of Electrophilic Fragments, Journal of Medicinal Chemistry, 57
(2014) 7590-7599.
[1] M.G. Guzman, S.B. Halstead, H. Artsob, P. Buchy, J. Farrar, D.J.
Gubler, E. Hunsperger, A. Kroeger, H.S. Margolis, E. Martínez,
M.B. Nathan, J.L. Pelegrino, C. Simmons, S. Yoksan, R.W. Peeling,
Dengue: a continuing global threat, Nature Reviews Microbiology, 8
(2010) 7-16.
[2] S. Bhatt, P.W. Gething, O.J. Brady, J.P. Messina, A.W. Farlow,
C.L. Moyes, J.M. Drake, J.S. Brownstein, A.G. Hoen, O. Sankoh,
M.F. Myers, D.B. George, T. Jaenisch, G.R.W. Wint, C.P. Simmons,
T.W. Scott, J.J. Farrar, S.I. Hay, The global distribution and burden
of dengue, Nature, 496 (2013) 504-507.
[3] V. Boldescu, M.A.M. Behnam, N. Vasilakis, C.D. Klein, Broad-
spectrum agents for flaviviral infections: dengue, Zika and beyond,
Nature Reviews Drug Discovery, 16 (2017) 565-586.
[4] C. Nitsche, S. Holloway, T. Schirmeister, C.D. Klein,
Biochemistry and Medicinal Chemistry of the Dengue Virus
Protease, Chemical Reviews, 114 (2014) 11348-11381.
[5] L. Cregar-Hernandez, G.-S. Jiao, A.T. Johnson, A.T. Lehrer,
T.A.S. Wong, S.A. Margosiak, Small Molecule Pan-dengue and
West Nile Virus NS3 Protease Inhibitors, Antiviral chemistry &
chemotherapy, 21 (2011) 209-218.
Supplementary Material
Supplementary material is provided as a separate electronic
file.