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Design, Synthesis and Trypanocidal Activity of Covalent Reversible Inhibitors of the Enzyme Cruzain. Molecular Hybridization

Grant number: 15/14304-7
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): March 01, 2016
Effective date (End): April 30, 2018
Field of knowledge:Physical Sciences and Mathematics - Chemistry
Principal Investigator:Carlos Alberto Montanari
Grantee:Daniela de Vita
Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
Associated research grant:13/18009-4 - Molecular design, synthesis and trypanocidal activity of cruzain reversible covalent inhibitors, AP.TEM


Cruzain, the main Trypanosoma cruzi cysteine protease, is an enzyme essential for the life cycle of the parasite and has been used as a viable target to search for enzyme inhibitor drug candidates. The K777 peptidemimetic compound inhibits cruzain in nanomolar concentrations and acts through a mechanism of irreversible inhibition. Analogs or derivatives of the K777 usually contain electrophilic functional groups known as "warheads" that can bind covalently to the active site of the cruzain via nucleophilic attack promoted by the catalytic cysteine. Although this mechanism has been extensively studied, few other studies have explored the influence of the nature of the covalent bond in the process of reversible covalent inhibition in cruzain. The Medicinal Chemistry Group of the IQSC/USP began studying this mechanism through the FAPESP 2011/01893-3 and 2011/20572-3 projects, with huge success, through the use of nitriles as warheads to form reversible covalent binding of dipeptidyl-nitriles with cruzain. The results of such research yielded in the approval of a thematic project by FAPESP (2013/18009-4), which is in course since May 2014, effective for 5 years. A similar set of prototypical inhibitors to cathepsin K (which has been used as target in the pharmaceutical industry) was selected for containing a chemical skeleton susceptible to optimization processes. All the synthesized compounds in the NEQUIMED group are active against cruzain at concentrations of low-micromolar to sub-micromolar, being the most potent compound Nequimed409 (Neq0409) that inhibited the enzyme with IC50 equal to 1.89 ± 0.11 µM (pIC50 = 5.7). Furthermore, also by the first time, these compounds showed trypanocidal activity against the infective form trypomastigote/amastigotes Tulahuen lacZ strain at concentrations less than 50 mM, wherein the prototype compound Neq0409 is a trypanocidal agent (dependent concentration-response) with an EC50 of 2.7 ± 0.3 mM (pEC50 = 5.6) against the amastigote form of the same strain. The Neq0409 is more potent than the drug benznidazole (BZ), which was used as control (pEC50 = 4.6) and shows cytotoxicity on mouse spleen > 500 mM (comparable value to that of the BZ is > 500 mM). The dipeptidyl-nitriles studied exhibit characteristics of lead compounds that can be optimized for drug candidates: T. cruzi pEC50 > 5 (pEC50 (Neq0409) = 5.6) with SI > 50 (ratio SI = IC50 (cyto)/IC50 (T. cruzi) = 185, BZ = 20.6); PFI < 8 (PFINeq0409 = 3.7); # rings Ar < 5 MW and < 500 Da. In this work, studies will be carried out to (i) synthesize Neq0409 analogues/derivatives; (ii) obtaining structure-activity relationship (SAR) with the aim of identifying the chemical-biologic reasons for the search of new drug candidates; (iii) determining the potency against cruzain. The synthesis of dipeptidyl-nitriles will be based on previous results already obtained in the group and will consist of the preparation of compounds of the non-basic inhibitors type of cruzain with 1-aminocyclopropane-1-carbonitrile group as warhead.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
SILVA, DANIEL G.; RIBEIRO, JEAN F. R.; DE VITA, DANIELA; CIANNI, LORENZO; FRANCO, CAIO HADDAD; FREITAS-JUNIOR, LUCIO H.; MORAES, CAROLINA BORSOI; ROCHA, JOSMAR R.; BURTOLOSO, ANTONIO C. B.; KENNY, PETER W.; et al. A comparative study of warheads for design of cysteine protease inhibitors. Bioorganic & Medicinal Chemistry Letters, v. 27, n. 22, p. 5031-5035, . (13/18009-4, 13/01128-0, 16/10362-5, 15/14304-7)

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