Small-molecule enzyme inhibitors utilizing active-site metal chelation: Prolyl 4-hydroxylase and microbial ribonucleases

Metal cofactors play a variety of structural and reactive roles in enzymes. Their versatility makes them both a viable target for binding small-molecule inhibitors, and an excellent vehicle for specific inhibition in the active sites of both metalloenzymes and non-metalloenzymes. This dissertation describes the development of novel inhibitors by exploiting the diverse roles that metals play in enzymic systems.; The iron-dependent enzyme prolyl 4-hydroxylase (P4H) catalyzes the post-translational modification of proline residues to form 4-hydroxyproline, an essential residue for collagen stability. Chapter Two describes the development of a screen for bioavailable inhibitors of P4H using the nematode, Caenorhabditis elegans. Using this assay, an epoxy ketone was discovered that specifically inhibits the P4H enzyme in vivo. To characterize this interaction, a simple in vitro assay for P4H was used to demonstrate that the epoxy ketone binds in the enzymic active site, presumably with the oxygens of the ketone and epoxy groups as ligands of the endogenous iron. There, the epoxy group undergoes nucleophilic attack by an enzymic residue to inactivate the enzyme irreversibly. The ketone of the inhibitor provides a functional group of unique reactivity that can be utilized in proteomics applications.; Chapter Three describes the inhibition of a ribonuclease by a small-molecule in a complex with an exogenous metal. The inhibition of ribonuclease Bi by 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is enhanced by 30-fold in the presence of Zn2+. Thus, an N-hydroxyurea nucleotide can recruit Zn2+ to inhibit the enzymatic activity of a ribonuclease. This result engenders a general strategy for the inhibition of non-metalloenzymes by metal complexes.; Chapter Four describes the basis for the competitive inhibition of ribonuclease Sa (RNase Sa) by 3'-N-hydroxyurea-3 '-deoxythymidine 5'-phosphate. Inhibition is enhanced by nearly 10-fold in the presence of Zn2+, which could coordinate to the N-hydroxyurea group along with enzymic residues. The data indicate that an N-hydroxyurea nucleotide can recruit Zn2+ to inhibit the enzymatic activity of RNase Sa, and suggest that the carboxylate of Glu54 is a ligand for that Zn 2+. These results extend the generality of zinc(II) complexes with an N-hydroxyurea nucleotide as inhibitors of ribonucleases.