Cysteine proteases
are important targets for the discovery of novel
therapeutics for many human diseases. From parasitic diseases to cancer,
cysteine proteases follow a common mechanism, the formation of an
encounter complex with subsequent nucleophilic reactivity of the catalytic
cysteine thiol group toward the carbonyl carbon of a peptide bond
or an electrophilic group of an inhibitor. Modulation of target enzymes
occurs preferably by covalent modification, which imposes challenges
in balancing cross-reactivity and selectivity. Given the resurgence
of irreversible covalent inhibitors, can they impair off-target effects
or are reversible covalent inhibitors a better route to selectivity?
This Perspective addresses how small molecule inhibitors may achieve
selectivity for different cathepsins, cruzain, rhodesain, and falcipain-2.
We discuss target- and ligand-based designs emphasizing repurposing
inhibitors from one cysteine protease to others.
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.
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.
The free energy perturbation using the covalent and noncovalent states can predict the binding affinity of covalent halogenated dipeptidyl nitrile inhibitors of the human Cathepsin L (hCatL).
In this paper, the selective interactions of synthetic derivatives of two natural compounds, berberine and palmatine, with DNA G-quadruplex structures were reported. In particular, the previous works on this subject concerning berberine were further presented and discussed, whereas the results concerning palmatine are presented here for the first time. In detail, these palmatine derivatives were developed by inserting seven different small peptide basic chains, giving several new compounds that have never been reported before. The preliminary studies of the interactions of these compounds with various G-quadruplex-forming sequences were carried out by means of various structural and biochemical techniques, which showed that the presence of suitable side chains is very useful for improving the interaction of the ligands with G-quadruplex structures. Thus, these new palmatine derivatives might act as potential anticancer drugs.
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