Correction to: Stabilized Human Cystatin C, Variant L47C/G69C, is a Better Reporter than the Wild-type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases.

The original publication of this article contained a number of grammatical errors. Unfortunately, an incorrect version of the file that did not include some final language editing was inadvertently published online. The original article has been corrected.

Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases.

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M-1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10-100 µM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.

In Silico Study of the Resistance to Organophosphorus Pesticides Associated with Point Mutations in Acetylcholinesterase of Lepidoptera: B. mandarina, B. mori, C. auricilius, C. suppressalis, C. pomonella, H. armígera, P. xylostella, S. frugiperda, and S. litura.

An in silico analysis of the interaction between the complex-ligands of nine acetylcholinesterase (AChE) structures of Lepidopteran organisms and 43 organophosphorus (OPs) pesticides with previous resistance reports was carried out. To predict the potential resistance by structural modifications in Lepidoptera insects, due to proposed point mutations in AChE, a broad analysis was performed using computational tools, such as homology modeling and molecular docking. Two relevant findings were revealed: (1) Docking results give a configuration of the most probable spatial orientation of two interacting molecules (AChE enzyme and OP pesticide) and (2) a predicted ΔGb. The mutations evaluated in the form 1 acetylcholinesterase (AChE-1) and form 2 acetylcholinesterase (AChE-2) structures of enzymes do not affect in any way (there is no regularity of change or significant deviations) the values of the binding energy (ΔGb) recorded in the AChE-OPs complexes. However, the mutations analyzed in AChE are associated with a structural modification that causes an inadequate interaction to complete the phosphorylation of the enzyme.

Structure-Based Virtual Screening and In Vitro Evaluation of New Trypanosoma cruzi Cruzain Inhibitors.

Chagas disease (CD), or American trypanosomiasis, causes more than 10,000 deaths per year in the Americas. Current medical therapy for CD has low efficacy in the chronic phase of the disease and serious adverse effects; therefore, it is necessary to search for new pharmacological treatments. In this work, the ZINC15 database was filtered using the N-acylhydrazone moiety and a subsequent structure-based virtual screening was performed using the cruzain enzyme of Trypanosoma cruzi to predict new potential cruzain inhibitors. After a rational selection process, four compounds, Z2 (ZINC9873043), Z3 (ZINC9870651), Z5 (ZINC9715287), and Z6 (ZINC9861447), were chosen to evaluate their in vitro trypanocidal activity and enzyme inhibition. Compound Z5 showed the best trypanocidal activity against epimatigote (IC50 = 36.26 ± 9.9 µM) and trypomastigote (IC50 = 166.21 ± 14.5 µM and 185.1 ± 8.5 µM on NINOA and INC-5 strains, respectively) forms of Trypanosoma cruzi. In addition, Z5 showed a better inhibitory effect on Trypanosoma cruzi proteases than S1 (STK552090, 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]-indol-4-amine), a known cruzain inhibitor. This study encourages the use of computational tools for the rational search for trypanocidal drugs.

In Silico Analysis of Homologous Heterodimers of Cruzipain-Chagasin from Structural Models Built by Homology.

The present study gives an overview of the binding energetics of the homologous heterodimers of cruzipain-chagasin based on the binding energy (ΔGb) prediction obtained with FoldX. This analysis involves a total of 70 homologous models of the cruzipain-chagasin complex which were constructed by homology from the combinatory variation of nine papain-like cysteine peptidase structures and seven cysteine protease inhibitor structures (as chagasin-like and cystatin-like inhibitors). Only 32 systems have been evaluated experimentally, ΔGbexperimental values previously reported. Therefore, the result of the multiple analysis in terms of the thermodynamic parameters, are shown as relative energy |ΔΔG| = |ΔGbfrom FoldX - ΔGbexperimental|. Nine models were identified that recorded |ΔΔG| < 1.3, five models to 2.8 > |ΔΔG| > 1.3 and the other 18 models, values of |ΔΔG| > 2.8. The energetic analysis of the contribution of ΔH and ΔS to ΔGb to the 14-molecular model presents a ΔGb mostly ΔH-driven at neutral pH and at an ionic strength (I) of 0.15 M. The dependence of ΔGb(I,pH) at 298 K to the cruzipain-chagasin complex predicts a linear dependence of ΔGb(I). The computational protocol allowed the identification and prediction of thermodynamics binding energy parameters for cruzipain-chagasin-like heterodimers.

Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain.

Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain- cystatin affinity.