Experimental study and computational modelling of cruzain cysteine protease inhibition by dipeptidyl nitriles

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

Determination of thermodynamic signature of cruzain cysteine protease inhibitors

O reconhecimento bimolecular pode ocorrer por interveniência de diferentes interações não-covalentes: ligação hidrogênio, forças de van der Waals e hidrofóbicas, interações π-π, eletrostáticas. Através da ITC (calorimetria de titulação isotérmica), a mudança de calor quando um composto interage com uma proteína alvo permite a determinação precisa da afinidade, bem como as forças que dirigem o processo. A titulação na presença de ligantes fornece informações sobre as forças que controlam a interação e permite a identificação dos complexos intermoleculares que são relevantes para o planejamento baseado em estrutura. A ITC é a única técnica que mede diretamente a entalpia de interação (ΔH). Os sistemas bioquímicos exibem caracteristicamente compensação entalpia-entropia (EEC) em que o aumento da entalpia é compensado por uma penalidade entrópica, reduzindo a magnitude da mudança na afinidade. Ao caracterizar as relações estrutura-atividade (SAR), a maioria dos grupos envolvidos na interação pode ser observada como contribuindo para ΔH, mas não para a afinidade. Assim, os valores de ΔH podem destacar uma possível descontinuidade na SAR, de modo que os dados estruturais experimentais provavelmente serão particularmente valiosos no planejamento molecular. Neste trabalho, tivemos como objetivo usar a ITC para determinar a assinatura termodinâmica de inibidores da cisteíno protease cruzaína com o principal objetivo de avaliar eventuais descontinuidades na SAR. Além disso, a calorimetria diferencial de varredura (DSC) foi empregada para avaliar as mudanças na temperatura de transição da proteína (Tm) que ocorre quando da interação do ligante. Enquanto a ITC permite avaliar a contribuição do ligante para o complexo, a DSC permite a correlação da termodinâmica que impulsiona a interação com as alterações conformacionais na macromolécula. As duas técnicas foram utilizadas para o estudo de moléculas biotivas contra o Trypanosoma cruzi, causador da doença de Chagas, que ainda é uma doença negligenciada.Bimolecular recognition can occur via distinct non-covalent interactions: hydrogen bonding, van der Waals and hydrophobic forces, π-π, electrostatic interactions. Through ITC (isothermal titration calorimetry), the heat change when a compound interacts with a target protein allows accurate determination of the affinity as well as the forces driving the process. Titration in the presence of ligands provides information about the forces that control the interaction and allows the identification of intermolecular complexes that are relevant for structure-based design. ITC is the only technique that directly measures the enthalpy of interaction (ΔH). Biochemical systems characteristically exhibit enthalpy-entropy compensation (EEC) in which the increase in enthalpy is offset by an entropic penalty, reducing the magnitude of the change in affinity. When characterizing the structure-activity relationships (SAR), most of the groups involved in the interaction can be observed as contributing to ΔH, but not to affinity. so the experimental structural data are likely to be particularly valuable in molecular design. In this work, we aimed to use the ITC to determine the thermodynamic signature of cysteine protease cruzi inhibitors with the main objective of evaluating eventual discontinuities in the SAR. In addition, differential scanning calorimetry (DSC) was employed to assess changes in protein transition temperature (Tm) that occur upon ligand interaction. While the ITC allows the assessment of the ligand\'s contribution to the complex, the DSC allows the correlation of the thermodynamics that drives the interaction with the conformational changes in the macromolecule. Both techniques were used to study bioactive molecules against Trypanosoma cruzi, which causes Chagas disease, which is still a neglected disease

Experimentos didáticos envolvendo radiação microondas Microwave-assisted experiments for undergraduate courses

<abstract language="eng">Theoretical and practical aspects of the use of microwave-assisted strategies in chemistry are introduced for students using simple and safe experiments employing a domestic oven. Three procedures are proposed for evaluating the distribution of microwave radiation inside the microwave oven cavity: (1) variation of the volume of marshmallows; (2) drying of filter paper wetted with Co(II) solution, and (3) variation of water temperature, after microwave-assisted heating. These experiments establish the position with the highest incidence of microwave radiation in the oven cavity, which was chosen for the synthesis of salicylic acid acetate. This synthesis was performed in 5 min of heating and the yield was around 85%. All experiments can be carried out in a 4 h lab-session using low-cost instrumentation

Leveraging the cruzain S3 subsite to increase affinity for reversible covalent inhibitors

Cruzain is themajor 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 un-substituted 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.G. The inclusion of the chlorine atom in the P3-meta position results in the highest reduction of the detrimental entropic contribution observed in P3

Assessment of the cruzain cysteine protease reversible and Irreversible covalent inhibition mechanism

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

Crystal structure of Leishmania mexicana cysteine protease B in complex with a high-affinity azadipeptide nitrile inhibitor

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

Molecular Design, Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors.

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