Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors

Abstract

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Double-headed nanosystems for oral drug delivery

“…21 Tris(2-aminoethyl) amine (TREN), a flexible C 3 symmetric linker, has been extensively utilized in co-ordination chemistry as tri-or tetradentate ligand for various metal ion interactions and sensor based applications. [22][23][24][25] It has been reported that the flexibility of TREN assisted in self-assembly of the collagen mimetic scaffold due to effective adjusting of the three peptide chains to form triple helical packing. 26 The TREN binding site in the artificial L-serine derivative based metallocatalyts has been reported to act as allosteric regulation sites due to conformation change upon metal binding.…”

Examining the Scope and Thermodynamics of Assembly in Nesting Complexes Comprising Molecular Baskets and TPA Ligands

“…Likewise, C 3symmetric and complementary guests (Figure 1) contain positively charged ammoniums 2 3+ , neutral hydrogen atoms 3, or negatively charged carboxylates 4 3− at the periphery; in order to estimate the overall charge of each ligand at pH = 7.2, we took into account pK a values of protonated TPA (pK a < 6.28), 15 peripheral carboxylic acids (pK a < 5), and ammoniums (pK a ≈ 9.0). 10 For trigonal bipyramidal Zn(II) coordination compounds, 16 however, the pK a of a complexed water molecule 17 is assumed to be approximately 8. 10 Incremental addition of Zn−3 2+ to basket 1 3− in 10 mM phosphate buffer at pH = 7.2 was monitored by 1 H NMR spectroscopy (Figure 2A); the results of DOSY NMR measurements of basket aggregates in this solvent.…”