Characterisation of Na/K-ATPase, its isoforms, and the inotropic response to ouabain in isolated failing human hearts

Objective: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. Methods: For contractility studies, a total of 57 trabeculae were isolated from two non- failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three α isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. Results: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p≪0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol·g−1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three α isoforms were expressed at the mRNA level in both normal and failing hearts. Conclusions: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition. Cardiovascular Research 1993;27:2229-223

Fluorescence Lifetime-based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro

Fluorescence lifetime- (FLT-) based assays have developed to highly attractive tools in drug discovery. All recently published examples of FLT based assays describe essentially their use for monitoring enzyme-mediated peptide modifications such as proteolytic cleavage or phosphorylation/de-phosphorylation. Here we report the development of competitive binding assays as a novel, inhibitor-centric assay principle employing the fluorescence lifetime of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details on the rationale of the design and the synthesis of probes, i.e., active-site directed low-molecule weight (lmw) inhibitors conjugated to PT14, are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer- (TR-FRET-) based competitive binding assays

Design, synthesis and pre-clinical characterization of selective Factor D inhibitors targeting the alternative complement pathway

Complement Factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis type II (MPGNII) and paroxysmal nocturnal hemoglobinuria (PNH). Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide (LPS)-induced AP activation model, as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD

The discovery of 4-((2S,4S)-4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic acid (LNP023), a Factor B inhibitor, specifically designed to be applicable to treating a diverse array of complement mediated diseases

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS) and various glomerular diseases. The serine protease Factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP pathway selective, orally bioavailable inhibitors has not been reported previously. Herein we describe our efforts to identify FB inhibitors by leveraging insights from several X-ray co-crystal structures. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse indications