A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

Steven S An,John M Peltier,Kwangmi Ahn,Sudhir Sahasrabudhe,Jeffrey J Fredberg,Thomas I Zarembinski,Peter S Askovich,Moritz Von Rechenberg

doi:10.1186/1465-9921-12-8

Abstract

BackgroundA newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.MethodsUsing a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.ResultsAmong 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.ConclusionsThis staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

Highlights

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins
One of the major protein substrates for protein kinase (PKA) is the small heat shock protein 20 (HSP20) [10,11,12], and phosphorylation of HSP20 is linked to relaxation of both airway and vascular smooth muscle [10,11,12,13,14,15]
Using Surface plasmon resonance (SPR)-based evaluation of protein interactions, we found that phosphorylated form of HSP20 (pHSP20) exhibited the highest binding affinity for the g isoform of 14-3-3 proteins (Figure 1B)

Summary

Introduction

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. The primary pathway by which b2-agonists modulate ASM contraction is through activation of adenylyl cyclase, One of the major protein substrates for PKA is the small heat shock protein 20 (HSP20) [10,11,12], and phosphorylation of HSP20 is linked to relaxation of both airway and vascular smooth muscle [10,11,12,13,14,15]. Mounting evidence points to the molecular interaction between pHSP20 and a class of 14-3-3 proteins as a critical determinant of cofilin-mediated disruption of actin stress fibers and smooth muscle relaxation [15,19,23]

Methods

Results

Conclusion