An Exploration of Therapeutic Applications for Dinitrosyl Iron Complexes (DNIC's)

Abstract

The therapeutic nature and mechanisms of in vitro nitric oxide (NO) release from dinitrosyl iron complexes (DNICs) are explored herein. First, the ideal primary coordination environment for the sustained liberation of NO while limiting the toxicity related to iron and NO was investigated. Dimeric RRE-type {Fe(NO)2}9 complexes, SPhRRE [(µ-SPh)Fe(NO)2]2 and TGTA-RRE, [(µ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-β-d-glucose tetraacetate), were found to deliver NO with the lowest effect on cell toxicity (i.e., highest IC50) with TGTA-RRE delivering a higher concentration of NO to the cytosol of SMCs. Monomeric DNICs with bulky N-heterocyclic carbenes (NHC), namely 1,3-bis(2,4,6-trimethylphenyl)imidazolidene (IMes), have IC50’s of ~7 µM, but didn’t release NO into SMCs. The reduced, mononuclear {Fe(NO)2}10 neocuproine-based DNIC increased intracellular NO. Given the efficacy of TGTA-RRE, instead of redesigning entirely new DNICs, the release rate of NO was tuned with the addition of biomolecules histidine and glutathione. From the Griess assay and X-band EPR data, decomposition of the histidine-cleaved dimer, [(TGTA)(NHis)Fe(NO)2], generated Fe(III) and increased the NO release rate compared to the TGTA-RRE precursor. In contrast, increasing concentrations of glutathione generated the stable [(TGTA)(GS)Fe(NO)2]- and depressed the NO release rate. This work provides insight into tuning NO release beyond the design of DNICs, through the incubation with biomolecules. A structure activity relationship between thiolate identity and expected protease inhibition was investigated in silico and in vitro via AutoDock 4.2.6 (AD4) and FRET protease assays respectively. AD4 was validated for coordinatively unsaturated DNIC binding using a crystal structure of a protein-bound DNIC, PDB – 1ZGN (calculation RMSD = 1.77). The dimeric DNICs TGTA-RRE and TG-RRE, [(µ-S-TG)Fe(NO)2]2 (TG = 1-thio-β-d-glucose), were identified as leads via the in silico study. Computations suggest inhibition at the catalytic Cys145 of SC2Mpro. In vitro studies indicate inhibition of protease activity upon TGTA-RRE treatment, with an IC50 of 38 µM for TGTA-RRE and 33 µM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions as well as validating the in silico leads in vitro.