Functional high-throughput contractility assay suite to develop therapeutics for asthma andhypertension

SUMMARY / ABSTRACT Asthmatic bronchoconstriction and hypertensive vasoconstriction are extremely common disease states in which excessive contractile cellular forces directly contribute to the pathophysiology. Existing treatments for these diseases, which affect 25 million and 75 million Americans, respectively, have severe side-effects, become desensitized over prolonged use, or lack efficacy altogether. In particular, LABAs used in asthma management carry a “black-box” warning, and 15-20% of hypertensive patients require andgt;3 drugs to control blood pressure. Despite understanding the role of cellular force in these scenarios, drug developers have lacked the drug discovery tools that directly target this critically important phenotype. Instead, many new drug development efforts continue to focus on known pathways. Clearly, there is a significant clinical unmet need in treating resistant asthma and hypertension, and there are large associated (andgt;$20B) markets worldwide. Specifically, there is need to develop new classes of drugs with molecular mechanisms of action that are orthogonal to existing therapies that promote smooth muscle cell relaxation causing bronchodilation or vasodilation. Forcyte Biotechnologies is an early-stage bio-pharmaceutical company based in Los Angeles that is leveraging a UCLA-borne microtechnology known as FLECS – a high-throughput screening (HTS) platform that measures contractility of single-cells in a 384-wellplate format – to identify and bring to market new compound classes that act on force-generating pathways within cells. This is the first and only reported assay that obtains functional force generation data for single cells, at HTS scales. Our initial programs will focus on treatment resistant asthma and hypertension, but can extend to other diseases associated with abnormal cellular force. In this proposal, we will focus on the natural progression of our platform, seeking to first design and implement workflows to automate the end-to-end execution the assay to enable throughputs of 10K cmpds/day and we will automate the multiplexing strategies we established feasibility for during phase 1. We will also build on our phase 1 research to develop the first truly high-throughput quantitative tissue-level (“tissuoid”) contractility assay (in a 384-wellplate format) to supplement our single-cell assays and enable for the first time, the engineering of treatment-resistant contractile disease models on-chip, completing our commercial assay suite. Finally, we will perform a large-scale validation screen of a small molecule library to obtain key performance metrics of our assay workflow, and to validate the strength of our technique. The products and services enabled by this research are novel and will address urgent needs for both pharmaceutical companies seeking to bolster their pipelines, and researchers working on early drug discovery.In the proposed work, we will continue development of the first high-throughput single-cell contractility screening platform, based on fluorescently-labeled elastomeric contractible surfaces (“FLECS”), as a means for bolstering the pharmaceutical pipeline for asthma and hypertension therapeutics. FLECS currently integrates with 384-wellplate formats to facilitate high-throughput screening of compounds that act on cellular contractility. This proposal aims to implement automation workflows to maximize our screening throughput potential, ensure reproducibility, and provide a walk-away assay solution to pharma end-users. We will also use our assay development expertise and phase 1 data to establish a companion tissue-level “tissuoid” contractility assay to perform quantitative follow-up on hits and enable the engineering of treatment-resistant contractile disease models on-chip. Following this development. we will expect a large validation screen of a small molecule library. These goals will allow Forcyte to provide novel research tools and validated normal engineered disease state models to pharmaceutical clients to screen thousands of compounds for novel diabetes therapies