The aggregation of the peptide amyloid-beta (Abeta) to form amyloid plaques is a key event in Alzheimer's disease. It has been shown that CuII can bind to soluble Abeta and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the CuII binding to Abeta16 and Abeta28, models of the soluble Abeta. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these CuII-Abeta complexes: (1) ITC showed two CuII binding sites, with an apparent Kd of 10(-7) and 10(-5) M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to CuII in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the CuII in the low-affinity binding site in Abeta28 but not in Abeta16; (5) 1H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to CuII in the high-affinity binding site; (6) the pKa of 11.3 of tyrosine in position 10 was not influenced by the binding of 2 equivalents of CuII.

Historically the early stages of drug discovery have been based on finding the highest affinity compounds that bind to the target of interest, with little consideration for the forces driving the binding event. The association constant (K a ) can be defined by the equation DG = ÀRTln K a , with DG = DHÀTDS. To fully describe K a it would therefore be beneficial to characterize both of the thermodynamic terms (DH and DS) that drive this affinity for binding. The importance of separating affinity into its thermodynamic components is emphasized by the ubiquitous "enthalpy/entropy compensation effect", where large changes in DH and DS tend to be of similar but opposite signs and there is no net change in affinity, despite potentially very different binding mode.[1] It has been proposed by Freire, [2] and Ward & Holdgate [3] that it is advantageous, in terms of both potency and selectivity, to start from an enthalpically-driven lead. It can also be argued that choosing compounds with different binding modes increases the variety of chemical substrate for optimization, therefore reducing the risk of all the compounds encountering the same side effects. These points emphasize the need to measure thermodynamic signatures of lead compounds as early in the drug discovery process as possible.The only method that directly measures the thermodynamics of a binding event in solution is isothermal titration calorimetry (ITC).[4] Even though ITC can give a full thermodynamic signature (DG obs , DH obs , DS obs and K B, obs ) from a single experiment, the full utilization of the technique for lead optimization has been hampered by technical limitations requiring substantial quantities of reagents. In addition, data have frequently been collected from optimized, but varied, experimental conditions for a particular system, and without appropriate controls the interpretation of results between studies is difficult. Here, we demonstrate that with recent advances in ITC technology [5] and comparing subtly modified ligands against the same target, under identical conditions, and with X-ray data support, thermodynamic measurements can provide medicinal chemists with another differentiator in their quest to discover the best lead compounds. Moreover, these data are informative to medicinal chemists as they are applicable to situations where a less complete biophysical analysis is possible.We chose human carbonic anhydrase (hCA II) as a favorable system for this investigation as there is already a wealth of both 3D structures and calorimetric data available, which has established this protein as the leading model system. [6][7][8][9][10][11] Additionally, the protein binds benzene sulfonamides (BSAs) with a 1:1 stoichiometry and does not undergo gross conformational changes upon binding, providing an essentially thermodynamically closed system that will therefore not complicate interpretation of the binding thermodynamics. The binding of BSA to hCA II is driven mainly through four H bonds from the sulfonamide, two H bonds to the Zn co-...

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Isothermal titration calorimetry (ITC) is a biophysical technique that allows a thermodynamic characterization of an interactive system. It is a free in solution technique that requires no labeling, using heat as signal. ITC allows simultaneous determination of affinity K a, stoichiometry n, enthalpy change ΔH and calculation of free energy change ΔG and entropy change ΔS in one single experiment. It is the only technique that allows direct enthalpy change measurement. By accessing the enthalpy change, we get a step closer in estimating the driving forces that characterize the interaction of a protein with a ligand, information much needed in the drug discovery process.