Anne Minnich

Molecular Characterization of Antigen‐Induced Lung Inflammation in a Murine Model of Asthma

Abstract: Asthma is one of the foremost contributors to morbidity and mortality in industrialized countries. Our objective was to characterize the acute response to allergen and to identify potentially novel molecular targets for pharmacological intervention in asthma. We therefore designed a study to identify genes whose regulation was altered following ovalbumin (OVA) challenge in the presence and absence of treatment with glucocorticoids in BALB/c mice. RNA was isolated from lungs for gene profiling from 8‐week‐old sensitized mice, 3 and 18 hours post OVA challenge on days 1, 4, and 7 of aerosol challenge. Taqman (real time RT‐PCR) analysis of marker genes indicative of Th2 (IL‐4, IL‐13), eosinophil (RANTES, eotaxin), Th1/macrophage (IFNγ) and epithelial cell (MUC5AC) phenotypes were used to characterize responses to allergen challenge. Histological evaluation of lungs from additional challenged animals revealed inflammatory infiltrates on days 4 and 7, but not on day 1 post challenge. We postulate that expression of IL‐4, IL‐13 and other genes by OVA at day 1 probably reflects activation of resident cells, whereas the fivefold increase in the number of regulated genes at day 7 reflects the contribution of recruited cells. Of the regulated genes, only a subset was counter‐regulated by dexamethasone treatment. Although regulated genes included genes in many protein families, herein we report regulation of two proteases whose role in response to OVA challenge has not been characterized. This model will be used to generate disease hypotheses for which may play an important role in initiating disease pathology in this model.

A conformationally constrained inhibitor with an enhanced potency for β-tryptase and stability against semicarbazide-sensitive amine oxidase (SSAO).

A novel β-tryptase inhibitor with a basic benzylamine P1 group, a piperidine-amide linker, and a substituted indole P4 group was discovered. A substitution at 4-indole position was introduced to constrain the conformational flexibility of the inhibitor to the bioactive conformation exhibited by X-ray structures so that entropic penalty was decreased. More importantly, this constrained conformation limited the accessibility of this molecule to anti-targets, especially SSAO, so that an enhanced metabolic profile was achieved.

A β-tryptase inhibitor with a tropanylamide scaffold to improve in vitro stability and to lower hERG channel binding affinity.

Tropanylamide was investigated as a possible scaffold for β-tryptase inhibitors with a basic benzylamine P1 group and a substituted thiophene P4 group. Comparing to piperidinylamide, the tropanylamide scaffold is much more rigid, which presents less opportunity for the inhibitor to bind with off-target proteins, such as cytochrome P450, SSAO, and hERG potassium channel. The proposed binding mode was further confirmed by an in-house X-ray structure through co-crystallization.

Structure-based design, synthesis, and profiling of a β-tryptase inhibitor with a spiro-piperidineamide scaffold, benzylamine P1 group, and a substituted indole P4 group

A β-tryptase inhibitor with a basic benzylamine P1 group, a substituted indole P4 group, and a spiro-piperidineamide scaffold linker was designed, synthesized, and tested for its β-tryptase potency and ADMET properties. Comparing to its non-spiro analogs, the inhibitor with a spiro-piperidineamide scaffold demonstrated superior metabolic stability in human liver microsome and against semicarbazide-sensitive amine oxidase (SSAO).