Identification of drug modulators targeting gene-dosage disease CMT1A

Abstract

The structural integrity of myelin formed by Schwann cells in the peripheral nervous system (PNS) is required for proper nerve conduction and is dependent on adequate expression of myelin genes including peripheral myelin protein 22 (PMP22). Consequently, excess PMP22 resulting from its genetic duplication and overexpression has been directly associated with the peripheral neuropathy called Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent type of CMT. Here, in an attempt to identify transcriptional inhibitors with therapeutic value toward CMT1A, we developed a cross-validating pair of orthogonal reporter assays, firefly luciferase (FLuc) and β-lactamase (βLac), capable of recapitulating PMP22 expression, utilizing the intronic regulatory element of the human PMP22 gene. Each compound from a collection of approximately 3,000 approved drugs was tested at multiple titration points to achieve a pharmacological end point in a 1536-well plate quantitative high-throughput screen (qHTS) format. In conjunction with an independent counter-screen for cytotoxicity, the design of our orthogonal screen platform effectively contributed to selection and prioritization of active compounds, among which three drugs (fenretinide, olvanil, and bortezomib) exhibited marked reduction of endogenous Pmp22 mRNA and protein. Overall, the findings of this study provide a strategic approach to assay development for gene-dosage diseases such as CMT1A.

Figure 1. Assay design and validation

(A) A schematic diagram of reporter constructs derived from the intronic element of the human PMP22 locus. Dimeric Sox10 binding site and Egr2 binding sites are represented by ovals and boxes, respectively. (B) siRNA-induced depletion of Sox10 leads to downregulation of both the Fluc and βLac reporters without affecting cell viability. The reporter (FLuc or βLac) expressing S16 cells were treated with Sox10-siRNA or NT (non-targeting)-siRNA as a control for 48hr. Whereas the FLuc cells were subjected to luciferase assay (i) for FLuc expression or CellTiter-Glo assay (ii) for viability, the βLac cells were processed for βLac expression (iii) and viability (iv) measured at 460nm and 535nm, respectively. Error bars indicate the SD of replicates (n=144 for the FLuc cells, n=192 for the βLac cells).

Figure 2. qHTS profiles and active compound selection

3D plots from qHTS in the FLuc assay (A), the βLac assay (fluorescence at 460nm), and CellTiter-Glo assay. CRCs are colored according to curve classification defined in Supplementary Figure 1; light blue (class 1), green (class 2), dark blue (class 3), gray (class 4), and magenta (stimulatory). (B) Potency distribution of compounds identified in the FLuc assay. Gray bars indicate the total number of actives identified, blue: concordant actives in the FLuc and βLac assays, red: concordant actives with no significant cytotoxicity. Bottom panel: potency distribution of compounds identified in the βLac assay. Gray bars indicate the total number of actives identified, blue: concordant actives in the FLuc and βLac assays, red: concordant actives with no significant cytotoxicity. (C) Activity plots of the 9 active compounds in FLuc (open circle), βLac (solid circle), and CellTiter-Glo (solid triangle) assays. (D) Potency correlation of the 9 actives (solid circles) from the qHTS assays was indicated by the experimental R² (a solid line) versus the ideal R² of 1 (a dotted line). Open circles represent compounds that are active in both FLuc and βLac (460nm) assays but cytotoxic in the CellTiter-Glo assay. The data from these screens have been deposited into PubChem Summary AID # xxxx.

Figure 3. Transcription of endogenous Pmp22 is negatively modulated by actives from qHTS

The FLuc-expressing S16 cells were treated with either DMSO as a control or each compound indicated at 10µM for 24hr and then subjected to quantitative reverse transcriptase-PCR (RT-qPCR) with primers specific to endogenous Pmp22 or FLuc (A). Similar results were observed with Pmp22 and βLac in the βLac-expressing S16 cells (data not shown). Primary rat Schwann cells were cultured and treated with each drug as described in the Supporting Information before subjected to RT-qPCR for Pmp22 expression analysis (B). Data were first normalized to beta-actin (ActB) used as an endogenous loading control across samples and then plotted relative to the untreated sample set to 1 for each target gene. Error bars indicate the SD of three replicates.

Figure 4. Expression analysis of Pmp22 mRNA transcript variants and protein

The S16 cells were treated with increasing concentrations of each drug for 24hr and then subjected to either RT-qPCR with primers specific to Pmp22-1a and -b. (A) or Western blot (B). For mRNA analysis, data were first normalized to ActB used as a loading control across samples and then plotted relative to the untreated sample set to 1 for each target. Error bars indicate the SD of three replicates. For protein analysis, fold change was calculated as a ratio of Pmp22 to β-actin (a loading control) normalized to that of the untreated sample set to 1. Higher concentrations of bortezomib were also tested along with other proteasome inhibitors (Supplementary Figure 7). Vehicles used were ethanol for fenretinide and DMSO for olvanil and bortezomib.

Figure 5. Dynamic changes in expression of myelination regulators

The S16 cells were treated with each drug at 10uM for 24hr and then applied to RT-qPCR targeting major myelin genes (A) and their regulators (B). Data were first normalized to ActB used as a loading control across samples and then plotted relative to the untreated sample set to 1 for each target gene. Lines are drawn to compare a fold-decrease of Pmp22 with that of the other myelin genes by each drug (A). Error bars indicate the SD of three replicates.