The interaction between ZnO nanoparticles (NPs) and lysozyme has been studied using calorimetric as well as spectrophotometric techniques, and interpreted in terms of the three-dimensional structure. The circular dichroism spectroscopic data show an increase in alpha-helical content on interaction with ZnO NPs. Glutaraldehyde cross-linking studies indicate that the monomeric form occurs to a greater extent than the dimer when lysozyme is conjugated with ZnO NPs. The enthalpy-driven binding between lysozyme and ZnO possibly involves the region encompassing the active site in the molecule, which is also the site for the dimer formation in a homologous structure. The enzyme retains high fraction of its native structure with negligible effect on its activity upon attachment to NPs. Compared to the free protein, lysozyme-ZnO conjugates are more stable in the presence of chaotropic agents (guanidine hydrochloride and urea) and also at elevated temperatures. The possible site of binding of NP to lysozyme has been proposed to explain these observations. The stability and the retention of a higher level of activity in the presence of the denaturing agent of the NP-conjugated protein may find useful applications in biotechnology ranging from diagnostic to drug delivery.

Although curcumin is known for its anticarcinogenic properties, the exact mechanism of its action or the identity of the target receptor is not completely understood. Studies on a series of curcumin analogues, synthesized to investigate their tubulin binding affinities and tubulin self-assembly inhibition, showed that: (i) curcumin acts as a bifunctional ligand, (ii) analogues with substitution at the diketone and acetylation of the terminal phenolic groups of curcumin are less effective, (iii) a benzylidiene derivative, compound 7, is more effective than curcumin in inhibiting tubulin self-assembly. Cell-based studies also showed compound 7 to be more effective than curcumin. Using fluorescence spectroscopy we show that curcumin binds tubulin 32 Å away from the colchicine-binding site. Docking studies also suggests that the curcumin-binding site to be close to the vinblastine-binding site. Structure-activity studies suggest that the tridented nature of compound 7 is responsible for its higher affinity for tubulin compared to curcumin.

The discovery of several sulfonamide drugs paved the way toward the synthesis of 6 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide, E7010) and 7 (N-(3-fluoro-4-methoxyphenyl)pentafluorobenzenesulfonamide, T138067), both of which inhibit tubulin polymerization and are under clinical development. A series of diarylsulfonamides containing an indole scaffold was also found to have antimitotic properties, but their mode of interactions with tubulin has remained unidentified so far. In this study, we demonstrate that these sulfonamide drugs bind to the colchicine site of tubulin in a reversible manner. They quenched intrinsic tryptophan fluorescence of tubulin presumably due to drug-induced conformational changes in the protein, but were unable to modulate GTPase activity of tubulin in contrast to colchicine that enhances the same enzymatic activity. Further investigation using isothermal titration calorimetry (ITC) revealed that 5 (N-(5-chloro-7-indolyl)-4-methoxybenzenesulfonamide) afforded a large positive value of heat capacity change (DeltaC(p)() = +264 cal mol(-1) K(-1)) on binding to tubulin, suggesting a substantial conformational transition in the protein along with partial enthalpy-entropy compensation. On the other hand, the 2-chloro regioisomer 2 gave a large negative value of DeltaC(p)() (-589 cal mol(-1) K(-1)) along with complete enthalpy-entropy compensation. This thermodynamic profile was thought to be attributable to a prominent contribution of van der Waals interaction and hydrogen bonding between specific groups in the drug-tubulin complex. These results indicate that a mere alteration in the position of a single substituent chlorine on the indole scaffold has a great influence on the drug-tubulin binding thermodynamics.

Curcumin has shown promising therapeutic utilities for many diseases, including cancer; however, its clinical application is severely limited because of its poor stability under physiological conditions. Here we find that curcumin also loses its activity instantaneously in a reducing environment. Curcumin can exist in solution as a tautomeric mixture of keto and enol forms, and the enol form was found to be responsible for the rapid degradation of the compound. To increase the stability of curcumin, several analogues were synthesized in which the diketone moiety of curcumin was replaced by isoxazole (compound 2) and pyrazole (compound 3) groups. Isoxazole and pyrazole curcumins were found to be extremely stable at physiological pH, in addition to reducing atmosphere, and they can kill cancer cells under serum-depleted condition. Using molecular modeling, we found that both compounds 2 and 3 could dock to the same site of tubulin as the parent molecule, curcumin. Interestingly, compounds 2 and 3 also show better free radical scavenging activity than curcumin. Altogether, these results strongly suggest that compounds 2 and 3 could be good replacements for curcumin in future drug development.

Tubulin, an α,β heterodimer, has four distinct ligand binding sites (for paclitaxel, peloruside/laulimalide, vinca, and colchicine). The site where colchicine binds is a promising drug target for arresting cell division and has been observed to accommodate compounds that are structurally diverse but possess comparable affinity. This investigation, using two such structurally different ligands as probes (one being colchicine itself and another, TN16), aims to provide insight into the origin of this diverse acceptability to provide a better perspective for the design of novel therapeutic molecules. Thermodynamic measurements reveal interesting interplay between entropy and enthalpy. Although both these parameters are favourable for TN16 binding (ΔH < 0, ΔS > 0), but the magnitude of entropy has the determining role for colchicine binding as its enthalpic component is destabilizing (ΔH > 0, ΔS > 0). Molecular dynamics simulation provides atomistic insight into the mechanism, pointing to the inherent flexibility of the binding pocket that can drastically change its shape depending on the ligand that it accepts. Simulation shows that in the complexed states both the ligands have freedom to move within the binding pocket; colchicine can switch its interactions like a "flying trapeze", whereas TN16 rocks like a "swing cradle", both benefiting entropically, although in two different ways. Additionally, the experimental results with respect to the role of solvation entropy correlate well with the computed difference in the hydration: water molecules associated with the ligands are released upon complexation. The complementary role of van der Waals packing versus flexibility controls the entropy-enthalpy modulations. This analysis provides lessons for the design of new ligands that should balance between the "better fit" and "flexibility"', instead of focusing only on the receptor-ligand interactions.