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Current Enzyme Inhibition

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ISSN (Print): 1573-4080
ISSN (Online): 1875-6662

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Research Article

A Simplified Direct O2 Consumption-Based Assay to Test COX Inhibition

Author(s): Maria Grazia Perrone, Morena Miciaccia, Savina Ferorelli and Antonio Scilimati*

Volume 18, Issue 1, 2022

Published on: 25 March, 2022

Page: [10 - 18] Pages: 9

DOI: 10.2174/1573408018666220204104612

Price: $65

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Abstract

Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA), producing the hydroperoxide Prostaglandin G2 (PGG2), then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions is a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors.

Methods: We developed a new simplified and easy to be carried out protocol for O2 consumption measurement by using disrupted HEK293-derived adherent cells, stably transfected either with COX-1 or COX-2 genes, as a source of the COX enzymes. The Clark electrode is used to measure the O2 concentration variation during the enzyme-catalyzed reactions.

Results and Discussion: The novel assay was validated by determining the IC50 values of the known inhibitors such as indomethacin, ibuprofen, SC560, and celecoxib. Indomethacin and ibuprofen are two traditional non-steroidal anti-inflammatory drugs (tNSAIDs). SC560 is a commercially available reference compound used for COX-1 inhibition investigations. Celecoxib is a clinically used COXIBs. The assay was also applied to measure the kinetics and IC50 of mofezolac and P6. Mofezolac is the most potent selective COX-1 inhibitor, and active principle ingredient of Disopain® used to treat rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as a reference in in vitro and in vivo COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies.

Conclusion: The obtained results showed the suitability of the newly developed assay to measure COXs activity in the presence of inhibitors as well as the kinetics of the inhibition (i.e., Vmax and Km).

Keywords: Cyclooxygenases (COXs), Cyclooxygenase (COX) inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), in vitro assay, O2 consumption measurement, kinetics.

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[1]
Garavito RM, DeWitt DL. The cyclooxygenase isoforms: Structural insights into the conversion of arachidonic acid to prostaglandins. Biochim Biophys Acta 1999; 1441(2-3): 278-87.
[http://dx.doi.org/10.1016/S1388-1981(99)00147-X] [PMID: 10570255]
[2]
Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: The biology of prostaglandin synthesis and inhibition. Pharmacol Rev 2004; 56(3): 387-437.
[http://dx.doi.org/10.1124/pr.56.3.3] [PMID: 15317910]
[3]
Blobaum AL, Marnett LJ. Structural and functional basis of cyclooxygenase inhibition. J Med Chem 2007; 50(7): 1425-41.
[http://dx.doi.org/10.1021/jm0613166] [PMID: 17341061]
[4]
Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol 2011; 31(5): 986-1000.
[http://dx.doi.org/10.1161/ATVBAHA.110.207449] [PMID: 21508345]
[5]
Perrone MG, Miciaccia M, Vitale P, et al. An attempt to chemically state the cross-talk between monomers of COX homodimers by double/hybrid inhibitors mofezolac-spacer-mofezolac and mofezolac-spacer-arachidonic acid. Eur J Med Chem 2021; 209: 112919.
[http://dx.doi.org/10.1016/j.ejmech.2020.112919] [PMID: 33129592]
[6]
Kurumbail RG, Kiefer JR, Marnett LJ. Cyclooxygenase enzymes: Catalysis and inhibition. Curr Opin Struct Biol 2001; 11(6): 752-60.
[http://dx.doi.org/10.1016/S0959-440X(01)00277-9] [PMID: 11751058]
[7]
Aïd S, Bosetti F. Targeting cyclooxygenases-1 and -2 in neuroinflammation: Therapeutic implications. Biochimie 2011; 93(1): 46-51.
[http://dx.doi.org/10.1016/j.biochi.2010.09.009] [PMID: 20868723]
[8]
Choi S-H, Aid S, Bosetti F. The distinct roles of cyclooxygenase-1 and -2 in neuroinflammation: Implications for translational research. Trends Pharmacol Sci 2009; 30(4): 174-81.
[http://dx.doi.org/10.1016/j.tips.2009.01.002] [PMID: 19269697]
[9]
Brenneis C, Coste O, Altenrath K, et al. Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation. J Biol Chem 2011; 286(3): 2331-42.
[http://dx.doi.org/10.1074/jbc.M110.157362] [PMID: 21075851]
[10]
Perrone MG, Centonze A, Miciaccia M, Ferorelli S, Scilimati A. Cyclooxygenase inhibition safety and efficacy in inflammation-based psychiatric disorders. Molecules 2020; 25(22): 5388.
[http://dx.doi.org/10.3390/molecules25225388] [PMID: 33217958]
[11]
Gupta RA, Tejada LV, Tong BJ, et al. Cyclooxygenase-1 is overexpressed and promotes angiogenic growth factor production in ovarian cancer. Cancer Res 2003; 63(5): 906-11.
[PMID: 12615701]
[12]
Scilimati A, Ferorelli S, Iaselli MC, et al. Targeting COX-1 by mofezolac-based fluorescent probes for ovarian cancer detection. Eur J Med Chem 2019; 179: 16-25.
[http://dx.doi.org/10.1016/j.ejmech.2019.06.039] [PMID: 31229884]
[13]
Perrone MG, Luisi O, De Grassi A, Ferorelli S, Cormio G, Scilimati A. Translational theragnosis of ovarian cancer: where do we stand? Curr Med Chem 2020; 27(34): 5675-715.
[http://dx.doi.org/10.2174/0929867326666190816232330] [PMID: 31419925]
[14]
Casalino G, Coluccia M, Pati ML, et al. Intelligent microarray data analysis through non-negative Matrix factorization to study human multiple myeloma cell lines. ApplSciences (Switzerland) 2019; 9(24): 5552.
[15]
Khan R, Sharma M, Kumar L, Husain SA, Sharma A. Interrelationship and expression profiling of cyclooxygenase and angiogenic factors in Indian patients with multiple myeloma. Ann Hematol 2013; 92(1): 101-9.
[http://dx.doi.org/10.1007/s00277-012-1572-5] [PMID: 22971811]
[16]
Scilimati A, Miciaccia M, Pati ML, et al. Patient bone marrow aspiration to explore the cyclooxygenases (COXs) involvement in multiple myeloma. J Cancer Res Ther Oncol 2021; 9: 1-19.
[http://dx.doi.org/10.17303/jcrto.2021.9.103]
[17]
Rostevanov IS, Boyko M, Ferorelli S, et al. Inhibition of cyclooxygenase-1 does not reduce mortality in post-ischemic stroke rats. Neurosci Lett 2020; 737: 135296.
[http://dx.doi.org/10.1016/j.neulet.2020.135296] [PMID: 32777346]
[18]
Kitamura T, Kawamori T, Uchiya N, et al. Inhibitory effects of mofezolac, a cyclooxygenase-1 selective inhibitor, on intestinal carcinogenesis. Carcinogenesis 2002; 23(9): 1463-6.
[http://dx.doi.org/10.1093/carcin/23.9.1463] [PMID: 12189188]
[19]
Howe JR, Skryabin BV, Belcher SM, Zerillo CA, Schmauss C. The responsiveness of a tetracycline-sensitive expression system differs in different cell lines. J Biol Chem 1995; 270(23): 14168-74.
[http://dx.doi.org/10.1074/jbc.270.23.14168] [PMID: 7775477]
[20]
Abdelazeem AH, El-Saadi MT, Safi El-Din AG, Omar HA, El-Moghazy SM. Design, synthesis and analgesic/anti-inflammatory evaluation of novel diarylthiazole and diarylimidazole derivatives towards selective COX-1 inhibitors with better gastric profile. Bioorg Med Chem 2017; 25(2): 665-76.
[http://dx.doi.org/10.1016/j.bmc.2016.11.037] [PMID: 27916468]
[21]
Miciaccia M, Belviso BD, Iaselli M, et al. Three-dimensional structure of human cyclooxygenase (hCOX)-1. Sci Rep 2021; 11(1): 4312.
[http://dx.doi.org/10.1038/s41598-021-83438-z] [PMID: 33619313]
[22]
Takahashi E, Yamaoka Y. Simple and inexpensive technique for measuring oxygen consumption rate in adherent cultured cells. J Physiol Sci 2017; 67(6): 731-7.
[http://dx.doi.org/10.1007/s12576-017-0563-7] [PMID: 28785888]
[23]
Diepart C, Verrax J, Calderon PB, Feron O, Jordan BF, Gallez B. Comparison of methods for measuring oxygen consumption in tumor cells in vitro. Anal Biochem 2010; 396(2): 250-6.
[http://dx.doi.org/10.1016/j.ab.2009.09.029] [PMID: 19766582]
[24]
Kulmacz RJ, Lands WE. Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents. J Biol Chem 1985; 260(23): 12572-8.
[http://dx.doi.org/10.1016/S0021-9258(17)38909-3] [PMID: 3930499]
[25]
Callan OH, So OY, Swinney DC. The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J Biol Chem 1996; 271(7): 3548-54.
[http://dx.doi.org/10.1074/jbc.271.7.3548] [PMID: 8631960]
[26]
Pairet M, Engelhardt G. Distinct isoforms (COX-1 and COX-2) of cyclooxygenase: possible physiological and therapeutic implications. Fundam Clin Pharmacol 1996; 10(1): 1-17.
[http://dx.doi.org/10.1111/j.1472-8206.1996.tb00144.x] [PMID: 8900495]
[27]
Walker MC, Kurumbail RG, Kiefer JR, et al. A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors. Biochem J 2001; 357(Pt 3): 709-18.
[http://dx.doi.org/10.1042/bj3570709] [PMID: 11463341]
[28]
Gierse JK, Koboldt CM, Walker MC, Seibert K, Isakson PC. Kinetic basis for selective inhibition of cyclo-oxygenases. Biochem J 1999; 339(Pt 3): 607-14.
[http://dx.doi.org/10.1042/bj3390607] [PMID: 10215599]
[29]
Walenga RW, Wall SF, Setty BN, Stuart MJ. Time-dependent inhibition of platelet cyclooxygenase by indomethacin is slowly reversible. Prostaglandins 1986; 31(4): 625-37.
[http://dx.doi.org/10.1016/0090-6980(86)90170-X] [PMID: 3088675]
[30]
Llorens O, Perez JJ, Palomer A, Mauleon D. Differential binding mode of diverse cyclooxygenase inhibitors. J Mol Graph Model 2002; 20(5): 359-71.
[http://dx.doi.org/10.1016/S1093-3263(01)00135-8] [PMID: 11885959]
[31]
Selinsky BS, Gupta K, Sharkey CT, Loll PJ. Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations. Biochemistry 2001; 40(17): 5172-80.
[http://dx.doi.org/10.1021/bi010045s] [PMID: 11318639]
[32]
Llorens O, Perez JJ, Palomer A, Mauleon D. Structural basis of the dynamic mechanism of ligand binding to cyclooxygenase. Bioorg Med Chem Lett 1999; 9(19): 2779-84.
[http://dx.doi.org/10.1016/S0960-894X(99)00481-3] [PMID: 10522690]
[33]
Gupta K, Kaub CJ, Carey KN, Casillas EG, Selinsky BS, Loll PJ. Manipulation of kinetic profiles in 2-aryl propionic acid cyclooxygenase inhibitors. Bioorg Med Chem Lett 2004; 14(3): 667-71.
[http://dx.doi.org/10.1016/j.bmcl.2003.11.034] [PMID: 14741265]
[34]
Rome LH, Lands WE. Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs. Proc Natl Acad Sci USA 1975; 72(12): 4863-5.
[http://dx.doi.org/10.1073/pnas.72.12.4863] [PMID: 1061075]
[35]
Morrison JF, Walsh CT. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 1988; 61: 201-301.
[http://dx.doi.org/10.1002/9780470123072.ch5] [PMID: 3281418]

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