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Molecular design, synthesis and trypanocidal activity of cruzain reversible covalent inhibitors

Grant number: 16/04096-0
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): May 01, 2016
Effective date (End): September 30, 2016
Field of knowledge:Physical Sciences and Mathematics - Chemistry - Organic Chemistry
Principal Investigator:Antonio Carlos Bender Burtoloso
Grantee:João Henrique Carvalho Batista
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 cysteine protease of Trypanosoma cruzi, is an essential enzyme for the life cycle of the parasite and has been used as a viable target for searching for enzyme inhibitors as drug candidates. The peptide mimetic compound K11777 inhibits cruzain at nanomolar concentrations, and acts by an irreversible inhibition mechanism. Analogs or derivatives K11777 usually contain electrophilic functional groups known as "warheads" that can bind covalently to the active site of cruzain via nucleophilic attack promoted by 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 IQSC / USP began studying this mechanism through FAPESP 2011/01893-3 and 2011/20572-3 projects, with great success using nitriles as warheads to form covalent-reversible dipeptidyl nitriles with cysteine proteases. Inhibitors similar to the prototypical inhibitors of cathepsin K (which has been used in the pharmaceutical industry as a target) were selected to contain a skeleton susceptible to chemical optimization processes. All the synthesised NEQUIMED compounds were active against cruzain in the range of low- to sub-micromolar concentrations. The most potent compound, Nequimed409 (Neq409), inhibited the enzyme with IC50 of 1.89 ± 0.11 µM (pIC50 = 5.7). These compounds showed trypanocidal activity against the trypomastigote infective form Tulahuen lacZ strain at concentrations less than 50 µM. The prototype compound Neq409, potent against cruzain is also a trypanocidal agent (concentration-dependent response) with IC50 of 2.7 ± 0.3 µM (pIC50 = 5.6). Neq409 is 9 times more potent than the drug benznidazole (BZ), which was used as control (pIC50 = 4.6) and has minimal cytotoxicity in mice spleen (> 500 µM, comparable to the BZ which is > 500 µM). The dipeptidyl nitriles exhibit characteristics of leads that can be optimized for drug candidates: T. cruzi pIC50 > 5 (pIC50 (Neq409) = 5.6) with SI > 10 (SI ratio = IC50(cyto)/IC50(T. cruzi) = 185, BZ = 20.6); PFI < 8 (PFINeq409 = 3.7); # Ar rings < 5 and MW 500 < Da. The Aims of this proposal are (i) determine the in silico docking poses with molecular dynamics simulation and calculation of the free energy of interaction (MM-PBSA) and to obtain structure-activity relationships (SAR) to identify new drug candidates; (ii) synthesise Neq409 analogues/derivatives with three different objectives: (1) optimising the potency of dipeptidyl nitriles as cruzain nanomolar inhibitors (2) identification of new warheads as alternatives to nitrile and (3) substitution of the amide group by heterocycles to tailor the ligands to have less peptidic character and reduced hydrolytic instability; (iii) determine the potency against cruzain; (iv) obtaining X-ray data of the crystallographic structure of ligand-cruzain co-crystal; (v) evaluating of trypanocidal activity in vitro and in vivo; (vi) evaluating of cytotoxic activity and (vii) determining the in vivo pharmacokinetic profile. (AU)

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