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1.
Future Med Chem ; 13(1): 25-43, 2021 01.
Article in English | MEDLINE | ID: mdl-33289603

ABSTRACT

Aim: Compounds that block enzyme activity can kill pathogens and help develop effective and safe drugs for Chagas disease and leishmaniasis. Materials & methods: A library of nonpeptidic nitrile-based compounds was synthesized and had their inhibitory affinity tested against cruzain, Leishmania mexicana cysteine protease B and cathepsin L. Isothermal titration calorimetry experiments and molecular simulations were performed for selected compounds to obtain thermodynamic fingerprints and identify main interactions and putative modes of binding with cruzain. Results: The derivatives provided increased affinity against all enzymes compared with the lead, and thermodynamic and computational studies showed improved thermodynamic properties and a possible different mode of binding. Conclusion: Our studies culminated in 1b, a compound 60-fold more potent in cruzain than its lead that also showed entropic and enthalpic contributions favorable to Gibbs binding energy.


Subject(s)
Chagas Disease/drug therapy , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Fluorine/chemistry , Leishmaniasis/drug therapy , Nitriles/chemical synthesis , Cathepsin L/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Drug Discovery , Humans , Leishmania mexicana/metabolism , Models, Molecular , Nitriles/pharmacology , Protozoan Proteins/metabolism , Structure-Activity Relationship , Thermodynamics
2.
Bioorg Med Chem ; 28(22): 115743, 2020 11 15.
Article in English | MEDLINE | ID: mdl-33038787

ABSTRACT

Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.


Subject(s)
Aza Compounds/pharmacology , Cathepsin B/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Leishmania mexicana/drug effects , Trypanocidal Agents/pharmacology , Aza Compounds/chemical synthesis , Aza Compounds/chemistry , Cathepsin B/metabolism , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dipeptides/pharmacology , Dose-Response Relationship, Drug , Leishmania mexicana/enzymology , Molecular Dynamics Simulation , Molecular Structure , Nitriles/chemical synthesis , Nitriles/chemistry , Nitriles/pharmacology , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism , Structure-Activity Relationship , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/chemistry
3.
Bioorg Chem ; 101: 104039, 2020 08.
Article in English | MEDLINE | ID: mdl-32629285

ABSTRACT

Cysteine proteases (CPs) are involved in a myriad of actions that include not only protein degradation, but also play an essential biological role in infectious and systemic diseases such as cancer. CPs also act as biomarkers and can be reached by active-based probes for diagnostic and mechanistic purposes that are critical in health and disease. In this paper, we present the modulation of a CP panel of parasites and mammals (Trypanosoma cruzi cruzain, LmCPB, CatK, CatL and CatS), whose inhibition by nitrile peptidomimetics allowed the identification of specificity and selectivity for a given CP. The activity cliffs identified at the CP inhibition level are useful for retrieving trends through multiple structure-activity relationships. For two of the cruzain inhibitors (10g and 4e), both enthalpy and entropy are favourable to Gibbs binding energy, thus overcoming enthalpy-entropy compensation (EEC). Group contribution of individual molecular modification through changes in enthalpy and entropy results in a separate partition on the relative differences of Gibbs binding energy (ΔΔG). Overall, this study highlights the role of CPs in polypharmacology and multi-target screening, which represents an imperative trend in the actual drug discovery effort.


Subject(s)
Cysteine Proteases/chemistry , Animals , Mammals , Molecular Dynamics Simulation , Molecular Structure , Structure-Activity Relationship
4.
PLoS Negl Trop Dis ; 14(3): e0007755, 2020 03.
Article in English | MEDLINE | ID: mdl-32163418

ABSTRACT

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC50 value of ca. 4.0 µM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.


Subject(s)
Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/pharmacology , Leishmania mexicana/enzymology , Nitriles/pharmacology , Protozoan Proteins/antagonists & inhibitors , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/enzymology , Cysteine Endopeptidases , Cysteine Proteases/metabolism , Humans
5.
J Chem Inf Model ; 60(3): 1666-1677, 2020 03 23.
Article in English | MEDLINE | ID: mdl-32126170

ABSTRACT

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi (T. cruzi) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor (Neq0682) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682. The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to -26.7 and -5.9 kcal mol-1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the ΔG of the reaction is very negative for the process involving K777, consequently, the covalent adduct cannot revert to a noncovalent protein-ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases.


Subject(s)
Cysteine Proteases , Trypanosoma cruzi , Cysteine Endopeptidases , Cysteine Proteinase Inhibitors/pharmacology , Protozoan Proteins
6.
Phys Chem Chem Phys ; 20(37): 24317-24328, 2018 Sep 26.
Article in English | MEDLINE | ID: mdl-30211406

ABSTRACT

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases.


Subject(s)
Cysteine Endopeptidases/chemistry , Cysteine Proteases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Nitriles/chemical synthesis , Protozoan Proteins/chemistry , Trypanocidal Agents/chemistry , Trypanosoma cruzi/enzymology , Drug Design , Molecular Dynamics Simulation , Protein Binding , Quantum Theory , Thermodynamics
7.
Bioorg Chem ; 79: 285-292, 2018 09.
Article in English | MEDLINE | ID: mdl-29783099

ABSTRACT

Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease. Reversible covalent cruzain inhibitors can block the steps of cell differentiation in the parasite and kill the organism. To this end, the description of how inhibitors modified at the P2/P3 positions lead to analogs with greater cruzain affinity to the S2/S3 subsites is of fundamental importance. Albeit many efforts are being employed in the characterization of the interaction processes with S2 subsite, little is known about the cruzain S3 subsite. In this work, we show a brief but consistent study to identify favorable substitutions in P3 of dipeptidyl nitriles that increase cruzain affinity. Using molecular dynamics simulations, we have identified some dipeptidyl nitrile analogs with modifications at P3 position that had higher cruzain inhibition than the original unsubstituted compound. A matched molecular pair analysis shows the importance of including a chlorine atom in the P3-meta position. The modifications implemented in P3 are confirmed when profiling the thermodynamic parameters via isothermal titration calorimetry. The classical enthalpy-entropy compensation phenomenon, in which enthalpy changes are counterbalanced by entropy results in a small modification of ΔG. The inclusion of the chlorine atom in the P3-meta position results in the highest reduction of the detrimental entropic contribution observed in P3.


Subject(s)
Cysteine Proteinase Inhibitors/pharmacology , Protozoan Proteins/antagonists & inhibitors , Trypanosoma cruzi/enzymology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Molecular Dynamics Simulation , Molecular Structure , Protozoan Proteins/metabolism , Structure-Activity Relationship
8.
PLoS Negl Trop Dis ; 9(7): e0003916, 2015.
Article in English | MEDLINE | ID: mdl-26173110

ABSTRACT

A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A Ki value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC50 value of 28 µM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.


Subject(s)
Chagas Disease/parasitology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Binding Sites , Chagas Disease/drug therapy , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Drug Design , Enzyme Inhibitors/chemistry , Humans , Kinetics , Nitriles/chemistry , Nitriles/pharmacology , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Structure-Activity Relationship , Trypanocidal Agents/chemistry , Trypanosoma cruzi/enzymology
9.
Talanta ; 73(5): 845-9, 2007 Oct 31.
Article in English | MEDLINE | ID: mdl-19073110

ABSTRACT

The behavior of selenium in thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) was studied and the developed procedure was applied for selenium determination in biological materials after microwave-assisted sample digestion. A sample volume of 600muL was introduced into the hot metallic Ni tube at a flow rate of 0.4mLmin(-1) using water as carrier. The limit of detection obtained for Se was 8.7mugL(-1) (3s(blank)/slope, n=10), which is 95-fold better than that typically obtained using FAAS. The applicability of the TS-FF-AAS procedure was evaluated for selenium determination in biological materials. Certified reference materials of pig kidney (BCR 186) and mussel (GBW 08571) were analyzed and a t-test had not shown any statistically significant difference at a 95% confidence level between determined and certified values for both materials. The procedure was successfully applied for determination of Se in pig kidney and shellfish. It was demonstrated that TS-FF-AAS improved the performance of FAAS (flame atomic absorption spectrometry) for determination of Se.

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