High-Throughput Screening to Identify Inhibitors of the Type I Interferon-Major Histocompatibility Complex Class I Pathway in Skeletal Muscle.

Travis B Kinder,Patricia K Dranchak,James Inglese

doi:10.1021/acschembio.0c00343

Travis B Kinder, Patricia K Dranchak + Show 1 more

Open Access

https://doi.org/10.1021/acschembio.0c00343

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Abstract

Immunosuppressants used to treat autoimmunity are often not curative and have many side effects. Our purpose was to identify therapeutics for autoimmunity of the skeletal muscle termed idiopathic inflammatory myopathies (myositis). Recent evidence shows that the pro-inflammatory type I interferons (IFN) and a downstream product major histocompatibility complex (MHC) class I are pathogenic in myositis. We conducted quantitative high-throughput screening on >4500 compounds, including all approved drugs, through a series of cell-based assays to identify those that inhibit the type I IFN-MHC class I pathway in muscle precursor cells (myoblasts). The primary screen utilized CRISPR/Cas9 genome-engineered human myoblasts containing a pro-luminescent reporter HiBit fused to the C-terminus of endogenous MHC class I. Active compounds were counter-screened for cytotoxicity and validated by MHC class I immunofluorescence, Western blot, and RT-qPCR. Actives included Janus kinase inhibitors, with the most potent being ruxolitinib, and epigenetic/transcriptional modulators like histone deacetylase inhibitors and the hypoxia-inducible factor 1 inhibitor echinomycin. Testing in animal models and clinical trials is necessary to translate these therapies to myositis patients. These robust assay technologies can be further utilized to interrogate the basic mechanisms of the type I IFN-MHC class I pathway, identify novel molecular probes, and elucidate possible environmental triggers that may lead to myositis.

Highlights

Autoimmunity is the result of a break in immune self-tolerance, where the body’s adaptive immune system mistakenly attacks host tissues
Clone AB-7 was chosen for quantitative high throughput screening (qHTS) due to its superior signal-to-background ratio of 32.5 and Z’-factor of 0.89 (Supplementary Table 1), a parameter to assess HTS assays’ dynamic range that takes into account the difference between positive and negative controls and the variability of each (Zhang et al, 1999)
To expand the arsenal of therapeutics that might be effective for managing myositis, a high throughput screening campaign was performed to identify compounds that inhibit the type I IFN – major histocompatibility complex (MHC) class I pathway in skeletal muscle

Summary

Introduction

Autoimmunity is the result of a break in immune self-tolerance, where the body’s adaptive immune system mistakenly attacks host tissues. The idiopathic inflammatory myopathies, collectively referred to as myositis, is a group of systemic autoimmune diseases that has characteristics of autoinflammation, with the common feature of skeletal muscle inflammation and weakness often accompanied by autoantibodies and pathologies of the skin, lungs, or vasculature (Dalakas, 2015). Their causes are not fully understood but thought to be a combination of genetic predisposition and environmental triggers (Miller et al, 2018). These therapeutics can reduce disease burden in many patients, they have serious side effects and often do not completely eliminate muscle inflammation or restore muscle function (Lundberg et al, 2000)

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