Martin Bechem

Potent and Selective Human Neutrophil Elastase Inhibitors with Novel Equatorial Ring Topology: In Vivo Efficacy of the Polar Pyrimidopyridazine Bay-8040 in a Pulmonary Arterial Hypertension Rat Model.

Human neutrophil elastase (HNE) is a key driver of inflammation in many cardiopulmonary and systemic inflammatory and autoimmune conditions. Overshooting high HNE activity is the consequence of a disrupted protease–antiprotease balance. Accordingly, there has been an intensive search for potent and selective HNE inhibitors with suitable pharmacokinetics that would allowing oral administration in patients. Based on the chemical probe BAY‐678 and the clinical candidate BAY 85‐8501 we explored further ring topologies along the equator of the parent pyrimidinone lead series. Novel ring systems were annulated in the east, yielding imidazolo‐, triazolo‐, and tetrazolopyrimidines in order to ensure additional inhibitor–HNE contacts beyond the S1 and the S2 pocket of HNE. The western annulation of pyridazines led to the polar pyrimidopyridazine BAY‐8040, which combines excellent potency and selectivity with a promising pharmacokinetic profile. In vivo efficacy with regard to decreasing cardiac remodeling and amelioration of cardiac function was shown in a monocrotaline‐induced rat model for pulmonary arterial hypertension. This demonstrated in vivo proof of concept in animals.

A luminescence-based assay for sensitive nitric oxide detection

Nitric oxide (NO) plays an important role in the protection against the onset and progression of various cardiovascular diseases, including hypertension and atherosclerosis, which are associated with an apparently reduced NO bioavailability. Therefore, the NO/cGMP signaling pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based and highly sensitive nitric oxide reporter assay which is suitable for ultra-high-throughput screening. In our coculture system, endothelial nitric oxide synthase (eNOS) mediated NO generation is monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx through the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor [1]. Using this NO reporter assay, a fully automated highthroughput screening campaign for stimulators of NO synthesis was performed. The coculture system reflects most aspects of the natural NO/cGMP signaling pathway. Namely, Ca2+-dependent and Ca2+-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability, as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP pathway. References 1. Wunder F, Stasch JP, Hutter J, Alonso-Alija C, Huser J, Lohrmann E: A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the NO/cGMP pathway. Anal Biochem 2005, 339:104-112. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007