LMH001, a novel Nox2 inhibitor specifically designed to block phosphorylated p47phox binding to p22phox has great potential to treat oxidative stress-related human diseases

Jian-Mei Li, Daniel Meijles, Maziah M Ghazaly, Brendan Howlin. University of Surrey, Guildford, UK

Oxidative stress (or oxidative damage) due to the activation of an O2.--generating enzyme, Nox2, has been recognized as a fundamental common mechanism underlying the pathogenesis of many diseases such as hypertension, atherosclerosis, inflammation, type 2 diabetes and ageing-related health deterioration. However, specific inhibitor to Nox2 is missing, and common anti-oxidant therapies are not effective. Researches from our group and others have discovered that the binding of phosphorylated p47phox to p22phox is a key step of Nox2 activation. We have successfully designed novel inhibitors (named LMH) using the pharmacophore generated by computer modeling and protein:protein docking to block the interaction of phosphorylated p47phox with p22phox.

The master compound (LMH001, MW: 290.27) was synthesised, dissolved initially in DMSO (0.5M) and further diluted in PBS before use. Toxicity was tested both in vitro and in vivo. LMH001 at a dose up to 10µM for 24 h showed no cytotoxicity to human hepatocyte cells (HepG2 cells), human pulmonary microvascular endothelial cells (HPMEC) and primary mouse bone marrow hematopoietic stem cells (BMHSC) as examined by both MTT and trypan blue exclusion assays. Intra-peritoneal injection of LMH001 in C57BL/6J mice (5 month old, 3mg/kg/day for 2 weeks) had no toxic effect on BMHSC proliferation. LMH does not affect neutrophil oxidative response to challenge because the peripheral blood mononuclear cells (PBMC) isolated from LMH-treated mice had similar levels of O2.- production (detected by lucigenin-chemiluminescence) as compared to PBMC isolated from vehicle-treated mice in response to fMLP or phorbol 12-myristate 13-acetate (PMA) stimulation. The therapeutic potential of LMH was examined on cell culture models of human diseased conditions where oxidative stress is involved. LMH001 (5µM, 24h) under physiological conditions had no significant effect on basal O2.- production and cell proliferation. However, LMH001 significantly inhibited (1) PMA-induced O2.- production by human neutrophils (HL60 cells) (IC50≤3µM); (2) TNFα (100U/ml), or angiotensin II (AngII, 100nM), or high levels of glucose (40mM)-induced endothelial O2.- production (IC50≤2µM); and (3) Epithelial cell growth factor (10ng/ml)-induced O2.- production and cell proliferation by human lung cancer cells (A549 cells) (IC50≤2µM) (all p<0.01). Compared to conventional Nox2 inhibitors: diphenyleneiodonium (20µM) and apocynin (20µM), LMH001 (5µM) showed significantly higher inhibition on TNFα or AngII-induced endothelial O2.- production. In conclusion, LMH is a novel Nox2 inhibitor specifically designed to block phosphorylated p47phox interaction with p22phox. It is non-toxic and inhibits effectively Nox2-derived oxidative stress in cells under diseased conditions. LMH has great therapeutic potential to treat oxidative stress-related human diseases.

All studies were performed in accordance with protocols approved by the Home Office under the Animals (Scientific Procedures) Act 1986 UK.