Generic placeholder image

Current Enzyme Inhibition


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

Research Article

In Vitro Investigation of Potential Pepsin Inhibitors: New Perspectives for the Treatment of Gastroesophageal Reflux

Author(s): Luca Leoni, Valerio Damiani and Riccardo Salvio*

Volume 18, Issue 3, 2022

Published on: 26 April, 2022

Page: [162 - 171] Pages: 10

DOI: 10.2174/1573408018666220307121130

Price: $65


Background: In patients with Gastroesophageal Reflux Desease (GERD), the digestive enzyme pepsin can reach the esophagus and extraesophageal sites and cause damage with inflammation and other tedious symptoms.

Methods: In this work, a number of biocompatible, non-toxic, and hypoallergenic compounds were tested in vitro as pepsin inhibitors. The residual enzyme activity in the presence of the investigated compounds was measured through a convenient and reliable UV-vis method based on the cleavage of hemoglobin. This method is applicable even if the investigated additives are scarcely soluble in water and the test mixtures are dispersions rather than solutions.

Results: A few negatively charged saccharides showed the highest effect among the investigated compounds. The inhibitory activity of pepstatin and lovastatin was also tested with the same method in a wide range of concentrations. These compounds turned out to be effective even if present in extremely low amounts. A docking/molecular dynamic investigation providesuseful insights into the binding site and the mechanism of action of pepstatin as an inactivating agent toward pepsin.

Conclusion: In particular, the computational study indicates that the binding with this compound significantly increases the mobility of the active site residues and prevents them from cooperating in the reactive event.

Keywords: Anionic polysaccharides, pepstatin, alginates, molecular dynamics, pepsin, gastroesophageal reflux, enzyme inhibition.

Graphical Abstract
Durazzo M, Lupi G, Cicerchia F. et al. Extra-esophageal presentation of gastroesophageal reflux disease: 2020 update. J Clin Med 2020; 9(8): 2559.
[] [PMID: 32784573]
Vakil N, van Zanten SV. P. Kahrilas J, Dent R Jones. The montreal definition and classification of gastroesophageal reflux disease. Am J Gastroenterol 2006; 101: 1900-20.
[] [PMID: 16928254]
Nirwan JS, Hasan SS, Babar ZU, Conway BR, Ghori MU. Global prevalence and risk factors of Gastro-Oesophageal Reflux Disease (GORD): Systematic review with meta-analysis. Sci Rep 2020; 10(1): 5814.
[] [PMID: 32242117]
Tytgat GN, McColl K, Tack J. et al. New algorithm for the treatment of gastro-oesophageal reflux disease. Aliment Pharmacol Ther 2008; 27(3): 249-56.
[] [PMID: 17973975]
Varela JE, Hinojosa MW, Nguyen NT. Laparoscopic fundoplication compared with laparoscopic gastric bypass in morbidly obese patients with gastroesophageal reflux disease. Surg Obes Relat Dis 2009; 5(2): 139-43.
[] [PMID: 18996768]
Katz PO, Gerson LB, Vela MF. Guidelines for the diagnosis and management of gastroesophageal reflux disease. Am J Gastroenterol 2013; 108(3): 308-28.
[] [PMID: 23419381]
Scarpignato C. Advances in drug therapy of gastroesophageal reflux disease. Basel: Krager 1992.
Samloff IM. Pepsinogens, pepsins, and pepsin inhibitors. Gastroenterology 1971; 60(4): 586-604.
[] [PMID: 4324336]
Wald M, Rehbein H, Beermann C, Bußmann B, Schwarz K. Purification and characterization of pepsinogen and pepsin from the stomach of rainbow trout (Oncorhynchus mykiss). Eur Food Res Technol 2016; 242: 1925-35.
Bardhan KD, Strugala V, Dettmar PW. Reflux revisited: advancing the role of pepsin. Int J Otolaryngol 2012; 2012: 646901.
[] [PMID: 22242022]
Johnston N, Wells CW, Samuels TL, Blumin JH. Pepsin in nonacidic refluxate can damage hypopharyngeal epithelial cells. Ann Otol Rhinol Laryngol 2009; 118(9): 677-85.
[] [PMID: 19810610]
Farrell S, McMaster C, Gibson D, Shields MD, McCallion WA. Pepsin in bronchoalveolar lavage fluid: a specific and sensitive method of diagnosing gastro-oesophageal reflux-related pulmonary aspiration. J Pediatr Surg 2006; 41(2): 289-93.
[] [PMID: 16481237]
Saber H, Ghanei M. Extra-esophageal manifestations of gastroesophageal reflux disease: Controversies between epidemiology and clicnic. Open Respir Med J 2012; 6: 121-6.
[] [PMID: 23166570]
Kung YM, Hsu WH, Wu MC. et al. Recent advances in the pharmacological management of gastroesophageal reflux disease. Dig Dis Sci 2017; 62(12): 3298-316.
[] [PMID: 29110162]
Magliulo G, Plateroti R, Plateroti AM. Gastroesophageal reflux disease and the presence of pepsin in the tears. Med Hypotheses 2013; 80(2): 129-30.
[] [PMID: 23218443]
Eto T, Tompkins RK. Inhibition of pepsin activity by ursodeoxycholic acids and chenodeoxycholic acids. Am J Surg 1985; 150(5): 564-7.
[] [PMID: 3933371]
Eto T, Tompkins RK. Further studies on the inhibition of pepsin by bile salts. Ann Surg 1986; 203(1): 8-12.
[] [PMID: 3079997]
Sunderland AM, Dettmar PW, Pearson JP. Alginates inhibit pepsin activity in vitro; a justification for their use in gastro-oesophageal reflux disease (gord). Gastroenterology 2000; 118(4): A21.
Strugala V, Kennington EJ, Campbell RJ, Skjåk-Braek G, Dettmar PW. Inhibition of pepsin activity by alginates in vitro and the effect of epimerization. Int J Pharm 2005; 304(1-2): 40-50.
[] [PMID: 16139974]
Strugala V, Avis J, Jolliffe IG, Johnstone LM, Dettmar PW. The role of an alginate suspension on pepsin and bile acids - key aggressors in the gastric refluxate. Does this have implications for the treatment of gastro-oesophageal reflux disease? J Pharm Pharmacol 2009; 61(8): 1021-8.
[] [PMID: 19703345]
Chater PI, Wilcox MD, Brownlee IA, Pearson JP. Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin. Carbohydr Polym 2015; 131: 142-51.
[] [PMID: 26256170]
Yue Y, Sun Y, Yan X, Liu J, Zhao S, Zhang J. Evaluation of the binding of perfluorinated compound to pepsin: Spectroscopic analysis and molecular docking. Chemosphere 2016; 161: 475-81.
[] [PMID: 27459159]
Ikeda K, Kusano T. In vitro inhibition of digestive enzymes by Indigestible Polysaccharides. Cereal Chem 1983; 60(4): 260-3.
Minekus M, Alminger M, Alvito P. et al. A standardised static in vitro digestion method suitable for food - an international consensus. Food Funct 2014; 5(6): 1113-24.
[] [PMID: 24803111]
Workman RJ, Burkitt DW. Pepsin inhibition by a high specific activity radioiodinated derivative of pepstatin. Arch Biochem Biophys 1979; 194(1): 157-64.
[] [PMID: 375833]
Umezawa H, Aoyagi T, Morishima H, Matsuzaki M, Hamada M, Takekuchi T. Pepstatin, a new pepsin inhibitor produced by Actinomy-cetes. J Antibiot (Tokyo) 1970; 23(5): 259-62.
[] [PMID: 4912600]
Kunimoto S, Aoyagi T, Morishima H, Takeuchi T, Umezawa H. Mechanism of inhibition of pepsin by pepstatin. J Antibiot (Tokyo) 1972; 25(4): 251-5.
[] [PMID: 4559274]
Fujinaga M, Chernaia MM, Tarasova NI, Mosimann SC, James MNG. Crystal structure of human pepsin and its complex with pepstatin. Protein Sci 1995; 4(5): 960-72.
[] [PMID: 7663352]
Salvio R, Cacciapaglia R, Mandolini L, Sansone F, Casnati A. Diguanidinocalix[4]arenes as effective and selective catalysts of the cleavage of diribonucleoside monophosphates. RSC Advances 2014; 4(65): 34412-6.
Salvio R, Moliterno M, Caramelli D. et al. Kinetic resolution of phosphoric diester by Cinchona alkaloid derivatives provided with a guan-idinium unit. Catal Sci Technol 2016; 6(7): 2280-8.
Salvio R, Volpi S, Cacciapaglia R, Sansone F, Mandolini L, Casnati A. Phosphoryl transfer processes promoted by a trifunctional ca-lix[4]arene inspired by dna topoisomerase I. J Org Chem 2016; 81(19): 9012-9.
[] [PMID: 27579493]
Salvio R, Volpi S, Cacciapaglia R, Sansone F, Mandolini L, Casnati A. Upper rim bifunctional cone-calix[4]arenes based on a ligated metal ion and a guanidinium unit as DNAase and RNAase mimics. J Org Chem 2016; 81(11): 4728-35.
[] [PMID: 27135962]
Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010; 31(2): 455-61.
[] [PMID: 19499576]
Hess B, Kutzner C, van der Spoel D, Lindahl E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular sim-ulation. J Chem Theory Comput 2008; 4(3): 435-47.
[] [PMID: 26620784]
Pronk S, Páll S, Schulz R. et al. GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 2013; 29(7): 845-54.
[] [PMID: 23407358]
Schmid N, Eichenberger AP, Choutko A. et al. Definition and testing of the GROMOS force-field versions 54A7 and 54B7. Eur Biophys J 2011; 40(7): 843-56.
[] [PMID: 21533652]
Darden T, York D, Pedersen L. Particle mesh Ewald: AnN⋅log(N) method for Ewald sums in large systems. J Chem Phys 1993; 98: 10089-92.
Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys 2007; 126(1): 014101.
[] [PMID: 17212484]
Segreto GE, Alba J, Salvio R, D’Abramo M. DNA cleavage by endonuclease I-DmoI: A QM/MM study and comparison with experimental data provide indications on the environmental effects. Theor Chem Acc 2020; 139(3): 1-7.
Salvio R, D’Abramo M. Conformational mobility and efficiency in supramolecular catalysis. A computational approach to evaluate the performances of enzyme mimics. Eur J Org Chem 2020; 37: 6004-11.
Heller W, Pangonis WJ. Theoretical investigations on the light scattering of colloidal spheres. I. The specific turbidity. J Chem Phys 1957; 26: 498-506.
Villalva DG, Giansanti L, Mauceri A, Ceccacci F, Mancini G. Influence of the state of phase of lipid bilayer on the exposure of glucose residues on the surface of liposomes. Colloids Surf B Biointerfaces 2017; 159: 557-63.
[] [PMID: 28850920]
Lucidi M, Marsan M, Pudda F. et al. Geometrical-optics approach to measure the optical density of bacterial cultures using a LED-based photometer. Biomed Opt Express 2019; 10(11): 5600-10.
[] [PMID: 31799033]
Savelli C, Salvio R. Guanidine-based polymer brushes grafted onto silica nanoparticles as efficient artificial phosphodiesterases. Chemistry 2015; 21(15): 5856-63.
[] [PMID: 25735267]
Piper DW, Fenton BH. pH stability and activity curves of pepsin with special reference to their clinical importance. Gut 1965; 6(5): 506-8.
[] [PMID: 4158734]
Samloff IM, O’Dell C. Inhibition of peptic activity by sucralfate. Am J Med 1985; 79(2): 15-8.
[] [PMID: 3929601]
Li Z, Wang M, Wang F. et al. gamma-Cyclodextrin: A review on enzymatic production and applications. Appl Microbiol Biotechnol 2007; 77(2): 245-55.
[] [PMID: 17891389]
Gramage-Doria R, Armspach D, Matt D. Metallated cavitands (calixarenes, resorcinarenes, cyclodextrins) with internal coordination sites. Coord Chem Rev 2013; 257(3-4): 776-816.
Raynal M, Ballester P, Vidal-Ferran A, van Leeuwen PW. Supramolecular catalysis. Part 2: Artificial enzyme mimics. Chem Soc Rev 2014; 43(5): 1734-87.
[] [PMID: 24365792]
Cacciapaglia R, Di Stefano S, Mandolini L, Salvio R. Reactivity of carbonyl and phosphoryl groups at calixarenes. Supramol Chem 2013; 25(9-11): 537-54.
Bjerre J, Rousseau C, Marinescu L, Bols M. Artificial enzymes, “chemzymes”: Current state and perspectives. Appl Microbiol Biotechnol 2008; 81(1): 1-11.
[] [PMID: 18787819]
Aachmann FL, Otzen DE, Larsen KL, Wimmer R. Structural background of cyclodextrin-protein interactions. Protein Eng 2003; 16(12): 905-12.
[] [PMID: 14983070]
Santamaría VJ, Rozo TG, Barreto CB. Characterization of a κ-Carrageenan Hydrogel and its Evaluation as a Coating Material for Fertilizers. J Polym Environ 2019; 27: 774-83.
Zhang J, Li Q, Jiang X. et al. Effect of sulfated polysaccharides on the digestion of DNA by pepsin under simulated gastric juice in vitro. Food Funct 2020; 11(2): 1790-7.
[] [PMID: 32053124]
Haynes MW. Handbook of Chemistry and Physics. Boca Raton, London and, New York: CHR Press, Taylor & Francis Group 2016.
Aryee FNA, Nickerson MT. Effect of pH, biopolymer mixing ratio and salts on the formation and stability of electrostatic complexes formed within mixtures of lentil protein isolate and anionic polysaccharides (κ-carrageenan and gellan gum). Int J Food Sci Technol 2014; 49(1): 65-71.
Marciniszyn J Jr, Hartsuck JA, Tang J. Mode of inhibition of acid proteases by pepstatin. J Biol Chem 1976; 251(22): 7088-94.
[] [PMID: 993206]
Goswami S, Vidyarthi AS, Bhunia B, Mandal T. A review on lovastatin and its production. J Biomol Tech 2012; 4(1): 581-7.
Smith I, Schmidt R, Halm EA, Mansi IA. Do statins increase the risk of esophageal conditions? Findings from four propensity score-matched analyses. Clin Drug Investig 2018; 38(2): 135-46.
[] [PMID: 29081029]
Wijarnpreecha K, Panjawatanan P, Leelasinjaroen L, Ungprasert P. Statins and gastroesophageal reflux disease: A meta-analysis. J Postgrad Med 2019; 65(4): 207-11.
[] [PMID: 31603078]
Khoury T, Mari A, Amara H. et al. Impact of chronic statins use on the development of esophagitis in patients with gastroesophageal re-flux disease. Can J Gastroenterol Hepatol 2019; 2019: 6415757.
[] [PMID: 30854351]
Andreeva NS, Rumsh LD. Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalyt-ic site of pepsin-like enzymes. Protein Sci 2001; 10(12): 2439-50.
[] [PMID: 11714911]
Campos LA, Sancho J. The active site of pepsin is formed in the intermediate conformation dominant at mildly acidic pH. FEBS Lett 2003; 538(1-3): 89-95.
[] [PMID: 12633859]
Davies DR. The structure and function of the aspartic proteinases. Annu Rev Biophys Biophys Chem 1990; 19(1): 189-215.
[] [PMID: 2194475]

Rights & Permissions Print Export Cite as
© 2023 Bentham Science Publishers | Privacy Policy