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Synthesis and evaluation of trypanocidal activity of potential reversible covalent inhibitors of cruzain enzyme

Grant number: 16/07946-5
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): May 01, 2016
Effective date (End): November 30, 2019
Field of knowledge:Physical Sciences and Mathematics - Chemistry - Organic Chemistry
Principal Investigator:Carlos Alberto Montanari
Grantee:Lorenzo Cianni
Home 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
Associated scholarship(s):17/17386-0 - Molecular design and synthesis of new cruzain inhibitors with trypanocidal activity, BE.EP.DD


The cruzain, major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite's life cycle and it has been used as a viable target for searching new enzyme inhibitors candidate as pharmaceutical drugs. The peptide-mimetic compound K777 inhibits cruzain at nanomolar concentrations, and acts by irreversible inhibition mechanism. Analogues or derivatives of K777 generally contain electrophilic functional groups known as "warheads" that can bind covalently to the active site of cruzain via nucleophilic attack promoted by the catalytic cysteine. Although this mechanism has been extensively studied, just few other studies have explored the reversible inhibition of cruzain process by covalent bond. The Medicinal Chemistry Group IQSC / USP (NEQUIMED / IQSC / USP) began to study the mechanism through FAPESP 2011 / 01893-3 and 2011 / 20572-3 projects with great success by using the nitriles as warheads to form reversible covalent bond of dipeptidyl nitriles compounds with cysteine proteases. Results of this research were used to the thematic project by FAPESP (2013 / 18009-4) which is in force from May 2014 to May 2019.A series of similar inhibitors to cathepsin K (which has been used as a target in the pharmaceutical industry) was selected for containing a potential structure for an optimization process. All the synthesized compounds in NEQUIMED are active against cruzain in low-micromolar to sub-micromolar concentrations, where the most potent compound is Nequimed409 (Neq0409) that inhibits the enzyme with IC50 equal to 1.89 ± 0.11 uM (pIC50 = 5.7). Furthermore, also for the first time, these compounds showed trypanocidal activity against the form infective trypomastigote / amastigote of the Tulahuen lacZ strains in concentration less than 50 uM, indeed the compound Neq0409 is a trypanocidal agent (dependence on concentration response) with a IC50 of 2.7 ± 0.3 uM (pIC50 = 5.6) against the amastigote form of the same strain.Neq 409 is more potent than the drug benznidazole (BZ) which was used as control (pIC50 = 4.6) and presents cytotoxicity in mice spleen> 500 uM (comparable to the value of BZ is> 500 uM). The dipeptidyl nitriles studied compounds have characteristics of matrices that can be optimized for drug candidates: T. cruzi pIC50> 5 (pIC50 (Neq409) = 5.6) with SI> 10 (IC50 = SI reason (I quote) / IC50 ( T. cruzi) = 185, BZ = 20.6); PFI <8 (PFINeq409 = 3.7); # rings Ar<5 and MW <500 Da. Recently, in a preliminary process to optimize Neq0409, the molecular structures of Neq0565 and Neq0566 were established using methods in silico developing in NEQUIMED / IQSC / USP, and then synthesized by the group of Prof. Paula Gomes at the University of Porto. In preliminary studies of trypanocidal activity, Neq0565 was activated against the amastigote forms of T. cruzi, Tulahuen strain (²-galactosidase) with pIC50 of 4.9 and SI >> 50 (IC50 = SI dose / IC50 (T. cruzi), while its enantiomer (Neq0566) was not active. In this project, studies will be conducted: (i) synthesis and characterization of analogs / derivatives of Neq0565 (ii) evaluation of the potency of inhibition against cruzain (iii) study in silico of structural factors important to establish a strong ligand-enzyme interaction (studies (Q) SARs) for future structural modifications propose leading to more potent and selective inhibitors, (iv) evaluation of the trypanocidal activity for the most potent compounds against cruzain. (AU)

Articles published in other media outlets (1 total):
UOL: Pesquisa amplia leque de substâncias promissoras contra doença de Chagas (09/May/2020)

Scientific publications (4)
(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)
CIANNI, LORENZO; ROCHO, FERNANDA DOS REIS; ROSINI, FABIANA; BONATTO, VINICIUS; RIBEIRO, JEAN F. R.; LAMEIRA, JERONIMO; LEITAO, ANDREI; SHAMIM, ANWAR; MONTANARI, CARLOS A. Optimization strategy of single-digit nanomolar cross-class inhibitors of mammalian and protozoa cysteine proteases. BIOORGANIC CHEMISTRY, v. 101, AUG 2020. Web of Science Citations: 0.
CIANNI, LORENZO; LEMKE, CARINA; GILBERG, ERIK; FELDMANN, CHRISTIAN; ROSINI, FABIANA; ROCHO, FERNANDA DOS REIS; RIBEIRO, JEAN F. R.; TEZUKA, DAIANE Y.; LOPES, CARLA D.; DE ALBUQUERQUE, SERGIO; BAJORATH, JUERGEN; LAUFER, STEFAN; LEITAO, ANDREI; GUETSCHOW, MICHAEL; MONTANARI, CARLOS A. Mapping the S1 and S1' subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents. PLoS Neglected Tropical Diseases, v. 14, n. 3 MAR 2020. Web of Science Citations: 0.
CIANNI, LORENZO; FELDMANN, CHRISTIAN WOLFGANG; GILBERG, ERIK; GUETSCHOW, MICHAEL; JULIANO, LUIZ; LEITAO, ANDREI; BAJORATH, JUERGEN; MONTANARI, CARLOS A. Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity?. Journal of Medicinal Chemistry, v. 62, n. 23, p. 10497-10525, DEC 12 2019. Web of Science Citations: 1.
GOMES, JULIANA C.; CIANNI, LORENZO; RIBEIRO, JEAN; ROCHO, FERNANDA DOS REIS; MARTINS SILVA, SAMELYN DA COSTA; JATAI BATISTA, PEDRO HENRIQUE; MORAES, CAROLINA BORSOI; FRANCO, CAIO HADDAD; FREITAS-JUNIOR, LUCIO H. G.; KENNY, PETER W.; LEITAO, ANDREI; BURTOLOSO, ANTONIO C. B.; DE VITA, DANIELA; MONTANARI, CARLOS A. Synthesis and structure-activity relationship of nitrile-based cruzain inhibitors incorporating a trifluoroethylamine-based P2 amide replacement. Bioorganic & Medicinal Chemistry, v. 27, n. 22 NOV 15 2019. Web of Science Citations: 0.

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