Information on how small molecules bind to the target enzyme has the potential to impact immensely on how medicinal chemists go about antiparasitic drug discovery. In this review, for the first time, we intend to make an assessment of the structural aspects of trypanothione reductase as drug target, and its complexes with several reversible drugs from the Protein Data Bank (PDB). We attempt to reveal the mechanism of these interactions by careful accounting of the X-ray structures and their possible roles in biological activity to treat Trypanosomatidae diseases. We focus on some of the outstanding findings from structures that are relevant to anti-trypanocidal drug discovery. We also review new interesting compounds that have appeared in the literature based on these X-ray structures.

The insulin−protamine interaction is at the core of the mode of action in many insulin formulations (Zn + insulin + protamine) and to treat diabetes, in which protamine is added to the stable form of hexameric insulin (Zn−insulin). However, due to the unavailability of quantitative data and a high-resolution structure, the binding mechanism of the insulin− protamine complex remains unknown. In this study, it was observed that Zn−insulin experiences destabilization as observed by the loss of secondary structure in circular dichroism (CD), and reduction in thermal stability in melting study, upon protamine binding. In isothermal titration calorimetry (ITC), it was found that the interactions were mostly enthalpically driven. This is in line with the positive ΔC m value (+880 cal mol −1 ), indicating the role of hydrophilic interactions in the complex formation, with the exposure of hydrophobic residues to the solvent, which was firmly supported by the 8-anilino-1-naphthalene sulfonate (ANS) binding study. The stoichiometry (N) value in ITC suggests the multiple insulin molecules binding to the protamine chain, which is consistent with the picture of the condensation of insulin in the presence of protamine. Atomic force microscopy (AFM) suggested the formation of a heterogeneous Zn−insulin−protamine complex. In fluorescence, Zn−insulin experiences strong Tyr quenching, suggesting that the location of the protamine-binding site is near Tyr, which is also supported by the molecular docking study. Since Tyr is critical in the stabilization of insulin self-assembly, its interaction with protamine may impair insulin's self-association ability and thermodynamic stability while at the same time promoting its flexible conformation desired for better biological activity.

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There is a successful use of protamine-insulin formulation (Zn+insulin+protamine) to treat diabetes in which protamine is added to the stable form of hexameric insulin (Zn-insulin). The biophysical characterization of Zn-insulin, which can dissociate to form biologically active monomers, is well reported. However, its interaction with protamine, which is at the core of the mode of action in many pharmaceutical formulations, is unresolved. Through biophysical characterization, we have tried to dissect the interactions driving the Zn-insulin-protamine complexation. Based on the thermal melting study, it was found that protamine indulges in the destabilization of Zn-insulin. Fluorescence results revealed that Zn-insulin experiences Tyr quenching in the presence of protamine, undergoing a significant conformational change. As shown by the molecular docking study, protamine disturbs the H-bonding network at the dimer interface by binding to the amino acid residues involved in the dimer stabilization. It may result in the freeying of B-chain, introducing conformational fluctuations in the insulin. This is well supported by the loss of helical content seen in circular dichroism. Further, the insulin-protamine complex formation was strongly dominated by hydrogen bonding and a few hydrophobic contacts. The endothermic heat and positive entropy observed in isothermal titration calorimetry in the dissociation of Zn-insulin-protamine is a reflection of that. Finally, the ANS binding study proposed the adaption of a flexible conformation by the Zn-insulin-protamine complex containing exposed hydrophobic residues, a potential arrangement for successful receptor binding.