EC Pharmacology and Toxicology

Breakdown of proteins into amino acids occurs in presence of Protease enzymes. In case of Serine proteases, serine acts as nucleophile amino acid. Serpines are known for ability to inhibit serine proteases. Serpins are involved in coagulation, inflammation and complement activation. Protease-activated receptors (PARs) are cleaved by serine. Protease inhibitors are known for anti-HIV action as anti-viral agents. This review summarizes types of proteases and serine proteases. Role of four PARs (1 to 4) are discussed in brief. It also highlights importance of serine protease inhibitors in inflammation. Various anti-viral agents such as Atazanavir, Darunavir, Fosamprenavir, indinavir, Lopinavir, Saquinavir, Nelfinavir, Ritonavir, Tipranavir approved by US-FDA are discussed in this review.

Keywords: HIV; Inflammation; PAR1; PAR2; Serine Protease

1. Mótyán János András., et al. “Research applications of proteolytic enzymes in molecular biology”. Biomolecules4 (2013): 923-942.
2. Oda Kohei. "New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases”. The Journal of Biochemistry 1 (2012): 13-25.
3. King John V., et al. “Molecular basis of substrate recognition and degradation by human presequence protease”. Structure 7 (2014): 996-1007.
4. Shen Yuequan., et al. “Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism”. Nature 7113 (2006): 870-874.
5. Versteeg Henri H and Wolfram Ruf. "Emerging insights in tissue factor-dependent signaling events”. Seminars in Thrombosis and Hemostasis1 (2006).
6. Rawlings Neil D., et al. “The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database”. Nucleic Acids ResearchD1 (2018): D624-D632.
7. Ovaere Petra., et al. “The emerging roles of serine protease cascades in the epidermis”. Trends in Biochemical Sciences 9 (2009): 453-463.
8. Evnin Luke B., et al. “Substrate specificity of trypsin investigated by using a genetic selection”. Proceedings of the National Academy of Sciences 17 (1990): 6659-6663.
9. Iván Gábor., et al. “Four spatial points that define enzyme families”. Biochemical and Biophysical Research Communications 4 (2009): 417-420.
10. Neurath H and GHf Dixon. "Structure and activation of trypsinogen and chymotrypsinogen”. Federation Proceedings 3 (1957).
11. Bode Wolfram and Peter Schwager. "The refined crystal structure of bovine β-trypsin at 1· 8 Å resolution: II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7· 0”. Journal of Molecular Biology 4 (1975): 693-717.
12. Renatus Martin., et al. “Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA”. The EMBO Journal 16 (1997): 4797-4805.
13. Hartley BS and BA Kilby. "The reaction of p-nitrophenyl esters with chymotrypsin and insulin”. Biochemical Journal 2 (1954): 288.
14. Bobofchak Kevin M., et al. “Energetic and Structural Consequences of Perturbing Gly-193 in the Oxyanion Hole of Serine Proteases”. Journal of Biological Chemistry 27 (2005): 25644-25650.
15. Soualmia Feryel and Chahrazade El Amri. "Serine protease inhibitors to treat inflammation: a patent review (2011-2016)”. Expert Opinion on Therapeutic Patents 2 (2018): 93-110.
16. Safavi Farinaz and Abdolmohamad Rostami. "Role of serine proteases in inflammation: Bowman–Birk protease inhibitor (BBI) as a potential therapy for autoimmune diseases”. Experimental and Molecular Pathology 3 (2012): 428-433.
17. Smith PK and JI Harper. "Serine proteases, their inhibitors and allergy”. Allergy12 (2006): 1441-1447.
18. Miranda E and DA Lomas. "Neuroserpin: a serpin to think about”. Cellular and Molecular Life Sciences CMLS 6 (2006): 709-722.
19. Wang Yingfei., et al. “Trypsin and trypsin-like proteases in the brain: proteolysis and cellular functions”. Cellular and Molecular Life Sciences 2 (2008): 237-252.
20. Ricagno Stefano., et al. “Human neuroserpin: structure and time-dependent inhibition”. Journal of Molecular Biology 1 (2009): 109-121.
21. Huntington James A., et al. “Structure of a serpin–protease complex shows inhibition by deformation”. Nature6806 (2000): 923-926.
22. Briand Christophe., et al. “Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease”. FEBS Letters 1 (2001): 18-22.
23. Almonte Antoine G and J David Sweatt. "Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior”. Brain Research 1407 (2011): 107-122.
24. Shigetomi Hiroshi., et al. “Anti-inflammatory actions of serine protease inhibitors containing the Kunitz domain”. Inflammation Research 9 (2010): 679-687.
25. Molehin Adebayo J., et al. “McMANUS. "Serine protease inhibitors of parasitic helminths”. Parasitology6 (2012): 681-695.
26. Barry Michele and R Chris Bleackley. "Cytotoxic T lymphocytes: all roads lead to death”. Nature Reviews Immunology 6 (2002): 401-409.
27. Neurath H and GHf Dixon. "Structure and activation of trypsinogen and chymotrypsinogen”. Federation Proceedings 3 (1957).
28. Di Cera E. “Thrombin”. Molecular Aspects of Medicine 29(2008): 203-254.
29. Fehlhammer Heinz., et al. “Crystal structure of bovine trypsinogen at 1· 8 Å resolution: II. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin”. Journal of Molecular Biology 4 (1977): 415-438.
30. Venekei István., et al. “Attempts to convert chymotrypsin to trypsin”. FEBS Letters 2 (1996): 143-147.
31. Hung Su-Hwi and Lizbeth Hedstrom. "Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity”. Protein Engineering 8 (1998): 669-673.
32. Jelinek Balázs., et al. “Ala226 to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin‐like protease”. Protein Engineering Design and Selection 2 (2004): 127-131.
33. Bah Alaji., et al. “Rapid kinetics of Na+ binding to thrombin”. Journal of Biological Chemistry 52 (2006): 40049-40056.
34. Kirby Anthony J and Florian Hollfelder. "Enzymes under the nanoscope”. Nature7218 (2008): 45-47.
35. Sigala Paul A., et al. “Testing geometrical discrimination within an enzyme active site: Constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole”. Journal of the American Chemical Society 41 (2008): 13696-13708.
36. Pineda Agustin O., et al. “Crystal structure of thrombin in a self-inhibited conformation”. Journal of Biological Chemistry 43 (2006): 32922-32928.
37. Reiling K Kinkead., et al. “Structure of human pro-chymase: a model for the activating transition of granule-associated proteases”. Biochemistry 9 (2003): 2616-2624.
38. Fersht Alan R. "Conformational equilibria in α-and δ-chymotrypsin: the energetics and importance of the salt bridge”. Journal of Molecular Biology 2 (1972): 497-509.
39. Fersht Alan R and Yolanda Requena. "Equilibrium and rate constants for the interconversion of two conformations of α-chymotrypsin: the existence of a catalytically inactive conformation at neutral pH”. Journal of Molecular Biology 2 (1971): 279-290.
40. Gandhi Prafull S., et al. “Structural identification of the pathway of long-range communication in an allosteric enzyme”. Proceedings of the National Academy of Sciences 6 (2008): 1832-1837.
41. Rohr Kerstin B., et al. “X-ray structures of free and leupeptin-complexed human αI-tryptase mutants: indication for an α→ β-tryptase transition”. Journal of Molecular Biology 1 (2006): 195-209.
42. Krojer Tobias., et al. “Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine”. Nature 6879 (2002): 455-459.
43. Jing Hua., et al. “Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity”. Journal of Molecular Biology 5 (1998): 1061-1081.
44. Coughlin Shaun R. "Thrombin signalling and protease-activated receptors”. Nature 6801 (2000): 258-264.
45. Ramachandran R and MD Hollenberg. "Proteinases and signalling: pathophysiological and therapeutic implications via PARs and more”. British Journal of Pharmacology S1 (2008): S263-S282.
46. Noorbakhsh Farshid., et al. “Proteinase-activated receptors in the nervous system Nature reviews”. Neuroscience12 (2003): 981-990.
47. Luo Weibo., et al. “Protease-activated receptors in the brain: receptor expression, activation, and functions in neurodegeneration and neuroprotection”. Brain Research Reviews 2 (2007): 331-345.
48. Sokolova Elena and Georg Reiser. "Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: localization, expression and participation in neurodegenerative diseases”. Thrombosis and Haemostasis 10 (2008): 576-581.
49. Weinstein JR., et al. “Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA”. Journal of Neuroscience 4 (1995): 2906-2919.
50. Junge Candice E., et al. “Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes”. Experimental Neurology 1 (2004): 94-103.
51. Gómez-Gonzalo Marta., et al. “An excitatory loop with astrocytes contributes to drive neurons to seizure threshold”. PLoS Biology 4 (2010): e1000352.
52. Tsirka Stella E., et al. “Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator”. Nature6547 (1995): 340-344.
53. Lee C Justin., et al. “Astrocytic control of synaptic NMDA receptors”. The Journal of Physiology 3 (2007): 1057-1081.
54. Nicole Olivier., et al. “Activation of protease-activated receptor-1 triggers astrogliosis after brain injury”. Journal of Neuroscience 17 (2005): 4319-4329.
55. Hamill Cecily E., et al. “Exacerbation of dopaminergic terminal damage in a mouse model of Parkinson's disease by the G-protein-coupled receptor protease-activated receptor 1”. Molecular Pharmacology 3 (2007): 653-664.
56. Nagai Taku., et al. “The rewards of nicotine: regulation by tissue plasminogen activator–plasmin system through protease activated receptor-1”. Journal of Neuroscience 47 (2006): 12374-12383.
57. Ito Mina., et al. “Possible involvement of protease‐activated receptor‐1 in the regulation of morphine‐induced dopamine release and hyperlocomotion by the tissue plasminogen activator‐plasmin system”. Journal of Neurochemistry 5 (2007): 1392-1399.
58. Gingrich Melissa B., et al. “Potentiation of NMDA receptor function by the serine protease thrombin”. Journal of Neuroscience 12 (2000): 4582-4595.
59. Maggio Nicola., et al. “Thrombin induces long-term potentiation of reactivity to afferent stimulation and facilitates epileptic seizures in rat hippocampal slices: toward understanding the functional consequences of cerebrovascular insults”. Journal of Neuroscience 3 (2008): 732-736.
60. Connolly Andrew J., et al. “Role of the thrombin receptor in development and evidence for a second receptor”. Nature6582 (1996): 516-519.
61. Almonte AG., et al. “Program No. 715.9 2009 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience; 2009. Protease-activated receptor-1 (PAR1) function modulates hippocampal synaptic plasticity (2009).
62. Bushell Trevor. "The emergence of proteinase‐activated receptor‐2 as a novel target for the treatment of inflammation‐related CNS disorders”. The Journal of Physiology 1 (2007): 7-16.
63. Lohman Rink-Jan., et al. “Protease-activated receptor-2 regulates trypsin expression in the brain and protects against seizures and epileptogenesis”. Neurobiology of Disease 1 (2008): 84-93.
64. Sorensen Brit B., et al. “Antiapoptotic effect of coagulation factor VIIa”. Blood5 (2003): 1708-1715.
65. McCoy Kelly L., et al. “PAR1 and PAR2 couple to overlapping and distinct sets of G proteins and linked signaling pathways to differentially regulate cell physiology”. Molecular Pharmacology 6 (2010): 1005-1015.
66. Jin Guang., et al. “Deficiency of PAR-2 gene increases acute focal ischemic brain injury”. Journal of Cerebral Blood Flow and Metabolism 3 (2005): 302-313.
67. Greenwood Sam M and Trevor J Bushell. "Astrocytic activation and an inhibition of MAP kinases are required for proteinase‐activated receptor‐2‐mediated protection from neurotoxicity”. Journal of Neurochemistry 6 (2010): 1471-1480.
68. Lohman Rink-Jan., et al. “A regulatory role for protease-activated receptor-2 in motivational learning in rats”. Neurobiology of Learning and Memory 3 (2009): 301-309.
69. Gan J., et al. “Indirect modulation of neuronal excitability and synaptic transmission in the hippocampus by activation of proteinase‐activated receptor‐2”. British Journal of Pharmacology 5 (2011): 984-994.
70. Striggow Frank., et al. “Four different types of protease‐activated receptors are widely expressed in the brain and are up‐regulated in hippocampus by severe ischemia”. European Journal of Neuroscience 4 (2001): 595-608.
71. Macfarlane Scott R., et al. “Proteinase-activated receptors”. Pharmacological Reviews 2 (2001): 245-282.
72. Henrich-Noack Petra., et al. “Focal ischemia induces expression of protease-activated receptor1 (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra”. Brain Research1 (2006): 232-241.
73. Henrich‐Noack P., et al. “Cellular expression pattern of the protease‐activated receptor 4 in the hippocampus in naïve rats and after global ischaemia”. Journal of Neuroscience Research 4 (2010): 850-857.
74. Mao Yingying., et al. “Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice”. Journal of Cerebral Blood Flow and Metabolism 5 (2010): 1044-1052.
75. Rizk Elsie., et al. “Alteplase for the treatment of midline catheter occlusions: a retrospective, single-cohort descriptive study”. British Journal of Nursing 14 (2022): S6-S16.
76. Wander Gurpreet Singh and Shibba Takkar Chhabra. "Critical analysis of various drugs used for thrombolytic therapy in acute myocardial infarction”. Med Update 23 (2013): 109-116.
77. Melandri Giovanni., et al. “Review of tenecteplase (TNKase) in the treatment of acute myocardial infarction”. Vascular Health and Risk Management 5 (2009): 249.
78. Capitanescu Cristian., et al. “Molecular processes in the streptokinase thrombolytic therapy”. Journal of Enzyme Inhibition and Medicinal Chemistry 6 (2016): 1411-1414.
79. Lindsberg Perttu J and Valeria Caso. "Desmoteplase after ischemic stroke in patients with occlusion or high-grade stenosis in major cerebral arteries”. Stroke3 (2016): 901-903.
80. Macheroux Peter., et al. “L‐Amino‐acid oxidase from the Malayan pit viper Calloselasma rhodostoma: Comparative sequence analysis and characterization of active and inactive forms of the enzyme”. European Journal of Biochemistry 6 (2001): 1679-1686.
81. Becker Stephen. "Atazanavir: improving the HIV protease inhibitor class”. Expert Review of Anti-Infective Therapy 3 (2003): 403-413.
82. Squires Kathleen., et al. “Comparison of once-daily atazanavir with efavirenz, each in combination with fixed-dose zidovudine and lamivudine, as initial therapy for patients infected with HIV”. JAIDS Journal of Acquired Immune Deficiency Syndromes 5 (2004): 1011-1019.
83. Murphy Robert L., et al. “Dose-ranging, randomized, clinical trial of atazanavir with lamivudine and stavudine in antiretroviral-naive subjects: 48-week results”. Aids18 (2003): 2603-2614.
84. Noor Mustafa A., et al. “The effects of HIV protease inhibitors atazanavir and lopinavir/ritonavir on insulin sensitivity in HIV-seronegative healthy adults”. Aids 16 (2004): 2137-2144.
85. Dauchy Frédéric-Antoine., et al. “Increased risk of abnormal proximal renal tubular function with HIV infection and antiretroviral therapy”. Kidney International 3 (2011): 302-309.
86. Lefebvre Eric and Celia A Schiffer. "Resilience to resistance of HIV-1 protease inhibitors: profile of darunavir”. AIDS Reviews 3 (2008): 131.
87. Tie Yunfeng., et al. “High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains”. Journal of Molecular Biology 2 (2004): 341-352.
88. Brower Evan T., et al. “Inhibition of HIV‐2 protease by HIV‐1 protease inhibitors in clinical use”. Chemical Biology and Drug Design 4 (2008): 298-305.
89. Bartlett JG. "Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents”. The Department of Health and Human Services Panel on Antiretroviral Guidelines for Adult and Adolescents (2008): 42-43.
90. Molina Jean-Michel., et al. “Safety and efficacy of darunavir (TMC114) with low-dose ritonavir in treatment-experienced patients: 24-week results of POWER 3”. JAIDS Journal of Acquired Immune Deficiency Syndromes 1 (2007): 24-31.
91. Eron Jr Joseph., et al. “The KLEAN study of fosamprenavir-ritonavir versus lopinavir-ritonavir, each in combination with abacavir-lamivudine, for initial treatment of HIV infection over 48 weeks: a randomised non-inferiority trial”. The Lancet 9534 (2006): 476-482.
92. Gathe Jr., et al. “Long-term (120-week) antiviral efficacy and tolerability of fosamprenavir/ritonavir once daily in therapy-naive patients with HIV-1 infection: an uncontrolled, open-label, single-arm follow-on study”. Clinical Therapeutics 5 (2006): 745-754.
93. González de Requena Daniel., et al. “Indinavir plasma concentrations and resistance mutations in patients experiencing early virological failure”. AIDS Research and Human Retroviruses 6 (2003): 457-459.
94. Sham Hing L., et al. “ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease”. Antimicrobial Agents and Chemotherapy 12 (1998): 3218-3224.
95. Max Blake and Renslow Sherer. "Management of the adverse effects of antiretroviral therapy and medication adherence”. Clinical Infectious Diseases 2 (2000): S96-S116.
96. Kempf Dale J., et al. “Pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus protease by coadministration with ritonavir”. Antimicrobial Agents and Chemotherapy 3 (1997): 654-660.
97. US Food and Drug Administration. Roche, Fortovase® (saquinavir) Silver Spring, MD: US Food and Drug Administration (2003).
98. Weller Ian VD and IG Williams. "Antiretroviral drugs”. British Medical Journal 7299 (2001): 1410-1412.
99. Cameron DW., et al. “Ritonavir and saquinavir combination therapy for the treatment of HIV infection”. AIDS 2 (1999): 213-224.
100. Orman Jennifer S and Caroline M Perry. “Tipranavir: a review of its use in the management of HIV infection”. Drugs 10 (2008): 1435-1463.