School of Medicine and Health Sciences Poster Presentations

Discovery of Novel Drugs to Alleviate Muscle Weakness in Myositis

Poster Number

14

Document Type

Poster

Publication Date

3-2016

Abstract

Idiopathic inflammatory myopathies (myositis) often exhibit refractory muscle weakness, which persists despite elimination of infiltrating inflammatory cells with immunosuppressive drugs. Myositis patients and our mouse model of myositis have been reported to acquire a deficiency of the metabolic enzyme AMP deaminase 1 (AMPD1), which catalyzes the rate-limiting step of the purine nucleotide cycle. In humans, a congenital AMPD1 deficiency due to a loss-of-function allele is associated with easy fatigability, weakness, and cramping. We hypothesize that an acquired AMPD1 deficiency contributes to muscle weakness in myositis, so we sought to find drugs that increased expression of AMPD1 to alleviate this refractory weakness. We created a coincidental reporter HEK293 cell line that used the AMPD1 promoter to drive the expression of a bicistronic transcript for the renilla and firefly luciferase genes intervened by a ribosomal skipping sequence, designed to reduce false positives by assessing the readout from two non-homologous reporters. The resulting cell line was utilized for quantitative high throughput screening (qHTS) of 3 libraries, totaling 4194 compounds at 7 to 11 dilutions each. Actives were then validated in cultured myotubes by RT-qPCR and an enzymatic activity assay of endogenous AMPD1. Regulation of the coincidental reporter in HEK293 cells was similar to that in muscle cells and the qHTS assay conditions were optimized for 1536 well-plates (Z’>0.5). The commercially available LOPAC1280 and FDA approved libraries identified microtubule polymerization inhibitors, such as podophyllotoxin and colchicine, as the most active compounds. Podophyllotoxin was verified to up-regulate endogenous AMPD1 mRNA and increase enzymatic activity in cultured myotubes. Additionally, qHTS of a 50 compound library of aza-podophyllotoxin analogues identified novel actives that are being pursued as lead compounds. Most of the active drugs identified by qHTS were microtubule polymerization inhibitors, suggesting a mechanistic link between microtubule polymerization and AMPD1 transcription. Active compounds identified by these assays will undergo preclinical testing in our mouse model of myositis for their ability to up-regulate AMPD1 and alleviate muscle weakness.

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Presented at: GW Research Days 2016

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Discovery of Novel Drugs to Alleviate Muscle Weakness in Myositis

Idiopathic inflammatory myopathies (myositis) often exhibit refractory muscle weakness, which persists despite elimination of infiltrating inflammatory cells with immunosuppressive drugs. Myositis patients and our mouse model of myositis have been reported to acquire a deficiency of the metabolic enzyme AMP deaminase 1 (AMPD1), which catalyzes the rate-limiting step of the purine nucleotide cycle. In humans, a congenital AMPD1 deficiency due to a loss-of-function allele is associated with easy fatigability, weakness, and cramping. We hypothesize that an acquired AMPD1 deficiency contributes to muscle weakness in myositis, so we sought to find drugs that increased expression of AMPD1 to alleviate this refractory weakness. We created a coincidental reporter HEK293 cell line that used the AMPD1 promoter to drive the expression of a bicistronic transcript for the renilla and firefly luciferase genes intervened by a ribosomal skipping sequence, designed to reduce false positives by assessing the readout from two non-homologous reporters. The resulting cell line was utilized for quantitative high throughput screening (qHTS) of 3 libraries, totaling 4194 compounds at 7 to 11 dilutions each. Actives were then validated in cultured myotubes by RT-qPCR and an enzymatic activity assay of endogenous AMPD1. Regulation of the coincidental reporter in HEK293 cells was similar to that in muscle cells and the qHTS assay conditions were optimized for 1536 well-plates (Z’>0.5). The commercially available LOPAC1280 and FDA approved libraries identified microtubule polymerization inhibitors, such as podophyllotoxin and colchicine, as the most active compounds. Podophyllotoxin was verified to up-regulate endogenous AMPD1 mRNA and increase enzymatic activity in cultured myotubes. Additionally, qHTS of a 50 compound library of aza-podophyllotoxin analogues identified novel actives that are being pursued as lead compounds. Most of the active drugs identified by qHTS were microtubule polymerization inhibitors, suggesting a mechanistic link between microtubule polymerization and AMPD1 transcription. Active compounds identified by these assays will undergo preclinical testing in our mouse model of myositis for their ability to up-regulate AMPD1 and alleviate muscle weakness.