Interactions of azole-based inhibitors with human heme oxygenase.

Human heme oxygenase-1 (hHO-1) plays a crucial role in human physiology because of its ability to metabolize free heme. The heme degradation products, biliverdin and bilirubin, were shown to have protective antioxidant properties in cells. In the context of cancer, hHO-1 function grants cancer cells defense from standard chemotherapy treatments, leading to the development of azole-based inhibitors that target hHO-1 for potential anticancer therapy. This work reports experimental and theoretical characterization of interactions between three azole-based inhibitors and the active site of hHO-1. It was found that all three compounds have Kd values within the µM order. The electronic absorption and resonance Raman (rR) spectra indicated that they bind to the ferric heme and coordinate through a nitrogen atom. rR measurements revealed varying effects of inhibitors on the geometry of heme vinyl groups in the ferric form of hHO-1. Changes in peripheral group orientation are known to affect heme redox potential, and consequently can reflect the inhibitory properties of studied azoles. The subsequent docking studies showed that inhibitors with lower Kd values are located close to two vinyl groups, while the compound with higher Kd is situated near only one, consistent with the rR studies. Finally, the rR studies of the CO adducts showed that the inhibitors bind to the heme in a reversible manner. Altogether, the combination of ligand binding studies, UV-Vis and rR spectroscopies, as well as computational approach revealed an importance of the steric hindrance imposed by the inhibitor's side chain.

Structural and biophysical characterization of the Burkholderia pseudomallei IspF inhibitor L-tryptophan hydroxamate.

The enzyme 2-methylerythritol 2,4-cyclodiphosphate synthase, IspF, is essential for the biosynthesis of isoprenoids in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The development of inhibitors that target IspF may lead to novel classes of anti-infective agents or herbicides. Enantiomers of tryptophan hydroxamate were synthesized and evaluated for binding to Burkholderia pseudomallei (Bp) IspF. The L-isomer possessed the highest potency, binding BpIspF with a KD of 36 µM and inhibited BpIspF activity 55% at 120 µM. The high-resolution crystal structure of the L-tryptophan hydroxamate (3)/BpIspF complex revealed a non-traditional mode of hydroxamate binding where the ligand interacts with the active site zinc ion through the primary amine. In addition, two hydrogen bonds are formed with active site groups, and the indole group is buried within the hydrophobic pocket composed of side chains from the 60 s/70 s loop. Along with the co-crystal structure, STD NMR studies suggest the methylene group and indole ring are potential positions for optimization to enhance binding potency.

Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase.

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore's disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR.