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

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.

Quantum Mechanics/Molecular Mechanics Studies of the Mechanism of Cysteine Proteases Inhibition by Dipeptidyl Nitroalkenes

“…As shown in Figure a, the quantum mechanics (QM) region, described with the AM1d semiempirical Hamiltonian, contains the whole inhibitor, the residue Cys25, and the imidazole ring of His159 (His163 in the case of the cathepsin L cysteine protease). The AM1d Hamiltonian has previously been employed in different computational studies of the mechanisms of catalysis and the inhibition of cysteine proteases . The rest of the system, the protein and water molecules, were described with the OPLS‐AA and TIP3P force fields, respectively.…”

“…On the basis of our results, we proposed that both irreversible inhibitors Cbz‐Tyr‐Ala‐CH 2 Cl and Cbz‐Phe‐Hph‐(S) are good candidates to develop an effective inhibitor of cruzain. Recently, Lameira and co‐workers studied the inhibition mechanism of cruzain by dipeptidyl nitriles using both isothermal titration calorimetry and the hybrid QM/MM approach . They concluded that the nucleophilic attack of Cys25 (cruzain numbering) and the proton transfer from His159 (cruzain numbering) take place in a single step and confirmed the reversible character of the inhibitor.…”

“…The inhibition mechanism of CPs by vinyl sulfones and other α,β‐unsaturated electron‐withdrawing groups proceeds by a Michael addition with an attack on the β‐carbon atom by the thiolate of the active‐site cysteine residue followed by protonation of the α‐carbon atom to form the thioether derivative (see Scheme ) . With regard to the cruzain cysteine protease, only a few inhibition mechanisms have been studied using computational tools that include protein environment effects . The inhibition mechanisms of cruzain by peptidyl halomethyl ketones (PHKs) and the dipeptidyl‐2,3‐epoxyketone Cbz‐Phe‐Hph‐(S) have been studied in our laboratory through quantum mechanics/molecular mechanics (QM/MM) simulations.…”

Ligand-induced conformational selection predicts the selectivity of cysteine protease inhibitors

“…Aspects of both their covalent and non-covalent interactions must be considered, but how to do so remains an open question. We have recently shown [32] that careful QM/MM calculations on the covalent binding of dipepdidyl nitrile ligands to cruzain can correlate well with experimental binding data, but the procedure is not suitable for high throughput use where the interactions of multiple ligands with multiple enzymes requires to be evaluated. Recently, Waldner et al [33] have shown that, for a panel of serine proteases, substrate specificity that could not be understood from an analysis of static (crystal) structures, was interpretable once the differential dynamics of the proteins was considered.…”

Atividade anti->i<Trypanosoma cruzi>/i< de agentes não-peptídicos como inibidores da cisteíno protease cruzaína

“…As cisteíno proteases são enzimas com função proteolítica, ou seja, que realizam a hidrólise de peptídeos, promovida por três resíduos de aminoácidos presentes no sítio ativo (Figura 2) O mecanismo catalítico mais comum para as cisteíno proteases, mostrado no Esquema 1, é consistente com a presença de um par iônico, formado após a transferência de um íon H + do resíduo de cisteína para o de histidina, levando a enzima a possuir um sítio ativo ionizado em sua forma livre (CysS − /HisH + ). A existência do par iônico em cisteíno proteases, como a papaína e catepsinas, foi confirmada experimentalmente e por modelos computacionais (28,29), sendo uma das características que diferem cisteíno proteases de serino proteases, que dependem da interação com o substrato para a ativação do grupo que dará início à hidrólise (30). O par iônico no sítio ativo explica a maior reatividade das cisteíno proteases frente a espécies eletrofílicas, quando comparadas a outras espécies nucleofílicas contendo enxofre, especialmente em meios mais ácidos, uma vez que o ânion tiolato é cerca de 10 10 vezes mais nucleofílico que o grupo tiol neutro (24,25,31).…”