Diverse Chemical Scaffolds Enhance Oligodendrocyte Formation by Inhibiting CYP51, TM7SF2, or EBP

Abstract

Small molecules that promote oligodendrocyte formation have been identified in "drug repurposing" screens to nominate candidate therapeutics for diseases in which myelin is lost, including multiple sclerosis. We recently reported that many such molecules enhance oligodendrocyte formation not by their canonical targets but by inhibiting a narrow range of enzymes in cholesterol biosynthesis. Here we identify enhancers of oligodendrocyte formation obtained by screening a structurally diverse library of 10,000 small molecules. Identification of the cellular targets of these validated hits revealed a majority inhibited the cholesterol biosynthesis enzymes CYP51, TM7SF2, or EBP. In addition, evaluation of analogs led to identification of CW3388, a potent EBP-inhibiting enhancer of oligodendrocyte formation poised for further optimization.

Keywords: cholesterol; glial biology; high-content imaging; multiple sclerosis; oligodendrocyte; phenotypic screening; remyelination; sterol signaling; target identification.

Figure 1.

Most validated hits inhibit CYP51 or EBP and accumulate 8,9-unsaturated sterols in OPCs. A. Schematic representation of screening strategy. B. Dot-scattered plot of percentage of MBP+ oligodendrocytes generated after 72 h of treatment. Retested molecules are green. C. Heat-map representing percentage of MBP+ oligodendrocytes generated in two derivations of OPCs after treatment with top hits. D. Structure of CYP51 or EBP inhibitors identified. See Figure S1 for remaining hits. E, F GC-MS-based quantification of lanosterol (E) and zymostenol (F) after treatment with indicated molecules. In experiments C, E and F, n = 2 replicates per condition. All treatments 10 μM. See also Figure S1.

Figure 2.

CW9956 and CW9009 enhance oligodendrocyte formation by inhibiting EBP and causing zymostenol accumulation. A. Cholesterol biosynthesis pathway in brief. B. Structure of CW9009 and CW9956. C, D. Percentage of MBP+ oligodendrocytes generated following treatment with CW9009 and CW9956. E. Representative images following treatments at 5 μM; scale bar, 100 μM. F. Zymostenol levels in OPC-5 after treatment with CW9956. G. EBP enzymatic activity in a biochemical assay; treatments, 10 μM. H. Percentage of MBP+ oligodendrocytes following treatment with the indicated small molecules or combinations of small molecules (Ro 48–8071, 11 nM, ketoconazole 2.5 μM, CW9956, 1 μM). P = 0.0076 for a combination of Ro 48–8071 and CW9956 in comparison to CW9956; P = 0.6509 for the combination of ketoconazole and CW9956 in comparison to ketoconazole; n. s. = non-significant, two-tailed Student’s t-test. Experiments in C-G are representative of at least two independent experiments. In C, D and H: n = 4 wells per condition except DMSO and ketoconazole n = 16 in C, D. n = 2 replicates per condition in panels F, G. Throughout, OPC-5 derivation used unless noted. Data in panels C, D and H are presented as mean ± standard deviation (s.d.). See also Figure S2 and Data S1.

Figure 3.

Evaluation of structural analogs of CW9956 and CW9009. A. Heatmap representing percentage of MBP+ oligodendrocytes following treatment with 76 structural analogs of CW9009/CW9956. Structures of molecules labeled in bold are given in panels B or E; for all analogs, see Data S1. B, E. Molecules discussed in this figure. C, D. Percentage of MBP+ oligodendrocytes generated following treatment with the indicated molecules. n = 4 wells per condition except DMSO, n = 8. F. Percentage of MBP+ oligodendrocytes generated following treatment with CW1143 or CW8438. n = 4 wells per condition. P = 0.0003 for CW1143 in comparison to DMSO; P = <0.0005 for CW1143 in comparison to CW8438; two-tailed Student’s t-test, 95% confidence interval. G-J. Quantification of zymostenol (G, I), 14-dehydrozymostenol (H) and 7-dehydrocholesterol (J) after treatment with the indicated analogs. n = 2 replicates per condition. Each analog was tested at 1 μM in H-J. K. Representative images following CW1143 or CW8438 treatment (1 μM), scale bar, 100 μM. Green labels indicate enhancers of oligodendrocyte formation; red labels indicate molecules with no effect. Data in experiments C-K are representative of two or more independent experiments. Data in panels C, D and F are represented as mean ± s.d.. See also Figures S3–4 and Data S2 and S3.

Figure 4.

EBP inhibitors promote wrapping of microfibers but lack the potent off-target effects of molecules identified in repurposing screens. A. EBP enzymatic activity in a biochemical assay at 10 μM. n = 2 replicates per condition, representative of two independent experiments. B. Quantification of the area of electrospun microfibers wrapped by MBP+ oligodendrocytes. n = 2 wells per condition. C. Total DAPI+ cell number for the experiment in B. D. Representative confocal images of data shown in B. Scale bar, 100 μM. E. Inhibition of muscarinic receptor M1 by TASIN-1 (100 nM) and indicated small molecules at 1 μM, n = 2 replicated per condition. F, G. Effects of tamoxifen (F), TASIN-1 (100 nM), or EBP inhibitors at 1 μM on the estrogen dependent growth of T47D cells, n = 8 replicates per condition. H. Activation of κ-opioid receptor (OPRD1) by U50488 (2 μM), TASIN-1 (100 nM) and indicated small molecules at 1 μM, n = 2 replicates per condition. I. Structures and canonical targets of molecules that can inhibit EBP. Data in panels C, D and H are represented as mean ± s.d.