Discovery of novel small molecule inhibitors of cardiac hypertrophy using high throughput, high content imaging

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Highlights

  • High throughput screening with primary cardiomyocytes

  • Phenotypic screening with primary cardiomyocytes

  • Small molecular library screening with primary cardiomyocytes

  • Novel inhibitors of cardiac hypertrophy

Abstract

Chronic cardiac hypertrophy is maladaptive and contributes to the pathogenesis of heart failure. The objective of this study was to identify small molecule inhibitors of pathological cardiomyocyte hypertrophy. High content screening was performed with primary neonatal rat ventricular myocytes (NRVMs) cultured on 96-well plates and treated with a library of 3241 distinct small molecules. Non-toxic hit compounds that blocked hypertrophy in response to phenylephrine (PE) and phorbol myristate acetate (PMA) were identified based on their ability to reduce cell size and inhibit expression of atrial natriuretic factor (ANF), which is a biomarker of pathological cardiac hypertrophy. Many of the hit compounds are existing drugs that have not previously been evaluated for benefit in the setting of cardiovascular disease. One such compound, the anti-malarial drug artesunate, blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). These findings demonstrate that phenotypic screening with primary cardiomyocytes can be used to discover anti-hypertrophic lead compounds for heart failure drug discovery. Using annotated libraries of compounds with known selectivity profiles, this screening methodology also facilitates chemical biological dissection of signaling networks that control pathological growth of the heart.

Introduction

The power of small molecule high throughput screening (HTS) in finding leads for drug discovery is well established [1]. HTS has historically been performed in industry and has largely focused on identifying modulators of distinct biochemical targets using simple in vitro assays or engineered reporter cell lines [2]. The standard approach has been to screen for modulators of a single target. Recently, phenotype-based screening has emerged as a more information-rich alternative to traditional target-based screening [3]. Phenotypic screening attempts to incorporate as much relevant biological information as possible and eliminate toxic hits, or hits with undesirable mechanisms-of-action, at an early stage of the discovery process. As such, phenotype-based screens have the potential to significantly lower the otherwise high attrition rates of lead compounds in the path of optimization and development into new drugs.

Heart failure affects 6 million people in the US alone, with 500,000 new diagnoses annually and a 5-year mortality rate of 42%, exceeding that of many cancers [4]. Cardiac hypertrophy is a hallmark of heart failure. Long-term suppression of cardiac hypertrophy is associated with reduced morbidity and mortality, and thus there is intense interest in developing novel therapeutics to target this growth response [5], [6], [7]. Current treatment of heart failure involves the use of drugs that inhibit signaling pathways triggered by cell surface receptors, such as the angiotensin receptor and the β-adrenergic receptor [8]. However, given the multitude of redundant signaling pathways capable of promoting pathological cardiac hypertrophy, it is hypothesized that increased efficacy will be obtained with therapies that target distal nodal points in the hypertrophic response. Phenotypic screening methods provide a unique opportunity to identify and target such nodal points [9].

Here, we describe results of an HTS campaign designed to discover small molecule inhibitors of pathological cardiomyocyte hypertrophy. This phenotypic screen employed primary neonatal rat cardiomyocytes and a high content screening platform, which enabled simultaneous image-based quantification of effects of compounds on cardiomyocyte cell area, biomarker expression, and viability. The results illustrate the power of phenotypic screening as a means to identify leads for heart failure drug discovery, and to yield compounds that can be employed as chemical biological probes to uncover nodal effectors of pathological cardiac hypertrophy.

Section snippets

Chemical libraries

The NIH Clinical Collections I and II (446 and 281 compounds, respectively) were obtained from Evotec US (South San Francisco, CA). The Spectrum Collection (2320 compounds) was purchased from Microsource (Gaylordsville, CT). The Kinase Inhibitor Library (194 compounds) was obtained from SelleckChem (Houston, TX). All compounds were supplied as 10 mM stock solutions in DMSO.

Cardiomyocyte cell culture and compound treatment

Neonatal rat ventricular myocytes (NRVMs) were isolated from the hearts of 1–3 day-old Sprague Dawley rats (Charles River),

Hypertrophy high throughput assay design and validation

In order to develop an assay to screen chemical libraries for anti-hypertrophic compounds, a cell-based high content imaging approach was developed (Fig. 1A). Freshly isolated neonatal rat ventricular myocytes (NRVMs) were plated on gelatin-coated 96-well plates with optically clear bottoms. Subsequently, NRVMs were treated with phenylephrine (PE, 10 μM), or the diacylglycerol mimetic phorbol 12-myristate 13-acetate (PMA, 50 nM). PE elicits cardiomyocyte hypertrophy by stimulating the α1

Discussion

We discovered multiple small molecule inhibitors of cardiomyocyte hypertrophy by employing primary cardiomyocytes and high throughput, high content imaging. The findings establish the feasibility of using phenotypic screens to initiate heart failure drug discovery programs. Furthermore, since the protein targets of many of the small molecule hits are known, this work has uncovered the potential involvement of novel effectors that control pathological cardiac growth.

Based on current guidelines,

Conclusions

Drug discovery typically involves the identification of potential drug targets and validation of a role for these targets in disease. Target discovery and validation often takes many years, thereby delaying the start of small molecule screening efforts, chemical optimization of hit compounds, and in vivo testing to confirm that manipulation of the target will provide therapeutic benefit without unacceptable side effects. The work presented here provides an example of an alternative approach, in

Acknowledgements

We thank B. Ferguson for NRVM preparation, R. Fickes and J. Spiltoir for compound administration, and M. Bristow for valuable discussions. This work was supported by grants from the Butcher Foundation and a Bioscience Discovery Evaluation Grant from the University of Colorado Denver Technology Transfer Office (CU3167H). T.A.M. was supported by the NIH (HL116848, HL127240 and AG043822) and the American Heart Association (Grant-in-Aid, 14510001). M.S.S. was funded by T32 training grants and an

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