Understanding the structure-activity relationship between metal-substituted polyoxometalate complexes and biomolecules

Unraveling the three-dimensional structure and function of proteins remains of utmost importance as they serve as important drug targets. Their large size and structural complexity however complicate their structural investigations by existing experimental methods. In order to facilitate the study of proteins, most biochemical procedures require the selective hydrolysis of proteins into smaller and more manageable fragments. However, the high kinetic stability of the peptide bond, with a half-life ranging from 350 to 600 years at physiological pH, makes this a challenging procedure. This partially explains the fact that there are only a small number of commercially available selective proteases. Two main classes of commercially available proteases exist. These include proteolytical enzymes, of which trypsin is the most well known, and chemical reagents such as cyanogen bromide (CNBr). However, their various shortcomings make the search for efficient and selective artificial proteases indispensable. During the past decades, the development of metal-based artificial proteases has become an increasingly important area of research, with special focus on developing cleavage methods that are selective. To overcome the limitations of currently existing proteases, our research group recently proposed ‘‘polyoxometalates’’, also referred to as ‘‘POMs’’, as a novel class of artificial proteases. These negatively charged metal-oxygen clusters electrostatically interact with the positively charged surface patches of proteins. More importantly, incorporation of a strong Lewis acidic metal ion into the POM framework generates a metal-substituted POM, which has been proven to induce hydrolytic cleavage of proteins in a regioselective manner. This PhD thesis consists of several research projects which were conducted to get a better overall understanding on i) the biological activity of metal-substituted POMs and ii) the structure-activity relationships between metal-substituted POMs and biomolecules (proteins in particular). First of all, the factors influencing the reactivity and selectivity of a Zr-substituted Wells-Dawson POM towards an oligopeptide were explored. The specific type and order of amino acid residues together with the flexible nature of the polypeptide chain play an important role in the observed selectivity of hydrolysis. Additionally, a Zr-substituted Keggin POM was successfully cocrystallized with hen egg-white lysozyme (HEWL). The crystal structure exclusively showed the presence of a monomeric 1:1 Keggin species and three binding sites at the interface of the negatively charged POM surface and the positively charged regions of HEWL. Moreover, the hydrolytic cleavage of the globular protein, cytochrome c, was investigated by different metal-substituted POMs. It was clearly shown that the nature of the imbedded Lewis-acid metal ion played a crucial role in tuning the reactivity and selectivity during the hydrolytic process. Furthermore, it was demonstrated for the first time that a metal-substituted POM was compatible to act as a metalloprotease in the presence of a zwitterionic surfactant. Human serum albumin (HSA) was successfully hydrolyzed by a Zr-substituted Wells-Dawson POM in the presence of a zwitterionic surfactant. This result is a promising step forward towards the potential application of metal-substituted POMs as a class of artificial proteases for membrane proteins. Also, a comparison of the reactivity of the isostructural and strong Lewis acidic Zr(IV)- and Hf(IV)-substituted Wells-Dawson POMs towards the glycoprotein, ovalbumin, revealed small differences in terms of hydrolytic selectivity. Furthermore, we demonstrated that several Zr(IV)-, Hf(IV)- and Ce(IV)-based POMs are able to inhibit Taq DNA polymerase and DNA polymerase I, Klenow fragment. As DNA polymerases are important therapeutic targets, this POM-induced enzyme inhibition may be an important step forward in the search for novel therapeutic approaches.nrpages: 186status: accepte