Among the leukodystrophies, X-linked adrenoleukodystrophy (X-ALD) is the most prevalent, with an allele frequency of about 1:17,000. All males with mutation in their X-ALD gene, ABCD1, have elevated tissue levels of saturated very long-chain fatty acids (VLCFA) beginning in utero. Untreated, elevated VLCFA levels are maintained throughout life, irrespective of whether the patient is presymptomatic or affected by the fatal demyelinating cerebral phenotype. Current therapies, including Lorenzo's oil, bone marrow transplantation, or ex vivo gene therapy, are beneficial to a subset of patients. Notably, each of these treatment modalities is associated with reducing the patient's VLCFA levels. Unfortunately these therapies also have undesirable risks and/or side effects. Thus, there is a need for newer, more effective treatments. No small molecule drugs are currently available and none are currently in clinical trials in the U.S. We developed a medium-throughput screening assay in 96-well plate format for assessing the effect of small molecule inhibitors on VLCFA levels in transformed X-ALD skin fibroblasts. A preliminary screen of 1040 FDA-approved drugs from the Spectrum collection (MicroSource) identified 6 candidate drugs. Two of these proved effective at lowering VLCFA levels >40% in at least 3 primary skin fibroblast cultures from different X-ALD patients; these are undergoing preclinical testing in X-ALD mice. Based on the number of hits in the preliminary screen, we hypothesize that there exists a significant number of additional compounds that could lower VLCFA levels in X-ALD fibroblasts and that would be candidates for further evaluation. To assess this, we propose:
Aim 1) To screen chemical libraries for additional candidate drugs for X-ALD, and Aim 2) To perform secondary level evaluation of candidate drugs for X-ALD identified in Aim 1.
In Aim 1, transformed X-ALD fibroblasts will be seeded into 96-well plates; drugs (10 M in DMSO) or vehicle alone will be added and cells cultured for 72 hrs, washed, solubilized, extracted with methanol, and VLCFA quantitated by tandem mass spectrometry. The specific VLCFA-containing lipid measured will be C26:0 lysophosphatidyl choline (C26- LPC). We will assess additional approved drugs from the Spectrum collection, as well as >600 pure natural products in the Spectrum collection, and a subset of the 50,000+ molecule CNS- Set (ChemBridge) library, which includes small molecule drug-like compounds more likely to pass the blood brain barrier. We estimate that 8,000-10,000 compounds can be screened in this project.
In Aim 2, molecules that lower C26-LPC in transformed X-ALD fibroblasts by >50% will be verified in transformed and primary X-ALD fibroblasts at concentrations ranging from 1 nM to 10 M. Both C26-LPC and total lipid VLCFA levels will be measured. Based on our preliminary findings, we expect that this approach will identify additional drugs that could ultimately prove to be therapeutically effective in X-ALD.
All patients with the fatal neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) have elevated plasma and tissue levels of very long-chain fatty acids (VL CFA), and all therapies that benefit at least some patients are associated with lowering VLCFA levels. All current therapies also have undesirable risks and/or side effects, including death, and no drug treatment is available. We developed a drug screening method which, in preliminary studies, identified 2 drug candidates (out of 1040 tested) that are currently undergoing further evaluation, and propose in this study to screen 8,000-10,000 additional drugs for their ability to benefit X- ALD patients by lowering VLCFA levels.
