Thermodynamics of complexes formation by ITC in methanol/water = 9/1 (v/v) solution: A case study

• Development of inhibitors acting against those viral enzymes operating via a cooperative two-metal ion mechanism, such as HIV integrase (IN), requires optimizing the binding affinity to the target.
• We have defined an experimental procedure for obtaining reliable thermodynamic data by ITC in methanol/water = 9/1 (v/v) as solvent.
• Formation heats in mixed solvent of the complexes formed by a ligand, model of Raltegravir, with Mg(II), Mn(II), Co(II) and Zn(II) are here reported.

Most enzymes that participate in the biochemistry of nucleic acids require divalent metal ion cofactors to promote activity. Development of potent inhibitors, acting against those viral enzymes operating via a cooperative two-metal ion mechanism, such as HIV integrase (IN) and RNase H, hepatitis C virus polymerase and influenza endonuclease, requires optimizing the binding affinity to the target, which is dictated by the binding free energy composed of both enthalpic and entropic contributions. They can be obtained by using isothermal titration microcalorimetry. We have defined an experimental procedure for obtaining reliable thermodynamic data in methanol/water = 9/1 0.1 M KCl as solvent, used to overcome solubility problems. In this way we have measured the heats of formation of the complexes formed by N-(4-fluorobenzyl)-5-hydroxy-2-isopropyl-1-methyl-6-oxo-1,6-dihydroxypyrimidine-4-carboxylate (HL, a model of Raltegravir, the antiretroviral drug produced by Merck & Co.), and a series of divalent metal ions of biological interest (Mg(II), Mn(II), Co(II) and Zn(II)), whose speciation was previously determined by potentiometry.

Integrase strand transfert inhibitors chelate the metal ions in the active site of HIV integrase.Figure optionsDownload as PowerPoint slide