Single amino acid mutations in p97 AAA ATPase (also known as VCP, CDC48, TER ATPase) cause autosomal dominant human disorders embracing inclusion body myopathy, Paget's disease of bone, frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS) among others. The most common p97 mutation (R155H) accounts for half the incidence of this rare, progressively debilitating, currently untreatable, fatal disorder. Strong data from IBMPFD/ALS human patients and mouse models suggests that targeting the disease-mutations of p97 will lead to a treatment. Since wild type (WT) p97 and its many ubiquitin-binding cofactors regulate the functions of protein substrates, WT p97?s essential functions must remain intact for human health. Previous studies demonstrated that R155H-p97 has differential sensitivity to inhibitors, higher basal ATPase activity, and is differentially regulated by interacting proteins. This suggests that mutant-selective small molecules can be identified which will inhibit R155H-p97, without effects on wild type. The goal of this project is to identify those mutant-selective small molecule candidates for drug development to treat or prevent IBMPFD/ALS pathology.
Three specific aims are proposed to achieve our goal.
AIM 1 : Identify compounds that inhibit R155H, the most common p97 mutant causing IBMPFD/ALS. Conditions for high-throughput screening were developed and validated during a R155H-p97 pilot screen with the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection of 2,400 approved drugs. This project will carry out a quantitative high-throughput screening (qHTS) of 500,000 diverse compounds for inhibitors of R155H-p97 in the presence of p47 (one of the major cofactors of p97) in collaboration with the NCATS. The compounds will be ranked according to selectivity followed by potency.
AIM 2 : Test the compounds identified in AIM 1 in a cascade of biochemical and cell-based assays to ensure that the compounds will have the same effect inside as well as outside cells. The compounds will be characterized in established and validated cell-based assays to monitor the expression of seven potential IBMPFD/ALS disease biomarkers. Finally AIM 3: Use the lead compounds that emerge from Aim 2 to evaluate whether they correct disease in fibroblasts obtained from diseased and healthy mice and both human subjects with IBMPFD/ALS and healthy individuals. This data along with evaluation of Blood, Brain Barrier permeability should identify best candidates for further therapy development.
This proposal is relevant to public health because mutations in the macromolecule known as p97 ATPase cause progressive degenerative diseases including Paget?s disease and ALS. The protein quality control and other tasks of normal p97 are critical for health. This project will identify, and then characterize small molecules which correct mutated p97 activity without damaging the normal p97 functions, potentially leading to new drugs to treat IBMPFD/ALS.
