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A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials

View Article: PubMed Central - PubMed

ABSTRACT

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds’ activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.

No MeSH data available.


Activity of chemical analogs of edaravone and methoxyisoflavone and identification of edaravone chemical features.(a) The four edaravone analogs 1a–d selected from commercial chemical libraries were used to confirm the structural class of edaravone structure and to identify its functional groups. Among these, only analogs 1b and 1d showed activity by MTT assay (see results). The activity of both analogs was confirmed in dose-response experiments and demyelinated cerebellar slices (Supplementary Figs S3 and S4). (b) The analogs antypyrine, aminopyrine and ramifenazone which were present within the Spectrum Collection library did not overcome the MTT test in both primary and re-testing experiments. (c) Basing on these findings four functional groups essential for the activity of edaravone were identified: in red 2 H bond acceptors (in position 1 and 3) plus in green hydrophobic group (the methyl in position 5) and in orange an aromatic ring (in position 2). (d) Nine isoflavones and (e) four flavones selected as analogs of 5-methyl-7-methoxyisoflavone were tested by MTT assay to confirm the compound activity. The analogs 2a and 3a–d were selected from commercial libraries, all the other compounds were present within the Spectrum Collection library. None of methoxyisoflavone analogs showed significant activity in both primary and re-testing experiments.
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f6: Activity of chemical analogs of edaravone and methoxyisoflavone and identification of edaravone chemical features.(a) The four edaravone analogs 1a–d selected from commercial chemical libraries were used to confirm the structural class of edaravone structure and to identify its functional groups. Among these, only analogs 1b and 1d showed activity by MTT assay (see results). The activity of both analogs was confirmed in dose-response experiments and demyelinated cerebellar slices (Supplementary Figs S3 and S4). (b) The analogs antypyrine, aminopyrine and ramifenazone which were present within the Spectrum Collection library did not overcome the MTT test in both primary and re-testing experiments. (c) Basing on these findings four functional groups essential for the activity of edaravone were identified: in red 2 H bond acceptors (in position 1 and 3) plus in green hydrophobic group (the methyl in position 5) and in orange an aromatic ring (in position 2). (d) Nine isoflavones and (e) four flavones selected as analogs of 5-methyl-7-methoxyisoflavone were tested by MTT assay to confirm the compound activity. The analogs 2a and 3a–d were selected from commercial libraries, all the other compounds were present within the Spectrum Collection library. None of methoxyisoflavone analogs showed significant activity in both primary and re-testing experiments.

Mentions: To identify a relationship between the structure and activity of two hits, we selected and tested structurally related chemical analogs. We selected new chemical analogs from commercial chemical libraries by means of a public database (Zinc, PubChem and ChemSpider), finding four available analogs for edaravone (Figs 1a–d and 6a,b) and five for 5-methyl-7-methoxyisoflavone, of which only one was an isoflavone and four were flavones (Figs 2a, 3a–d and 6d,e,). The activity of the selected analogs (10 μM, 48 h) was evaluated in OPC purified cultures by MTT test, identifying two active analogs for edaravone (1b ER = 1.36 ± 0.029; and 1d ER = 1.44 ± 0.06) and none for 5-methyl-7-methoxyisoflavone. The biological activity of edaravone analogs was then confirmed in mixed glial cell cultures with dose-response curves at six different concentrations (0.001–30 μM; Supplementary Fig. S3) and in demyelinated cerebellar slices (Supplementary Fig. S4). By searching for chemical analogs in the Spectrum Collection library we found three analogs of edaravone (the antipyretic and analgesic drugs antipyrine, aminopyrine and ramifenazone; Fig. 6b) and 7 isoflavones (Fig. 6d) that did not overcome the MTT test in both primary and re-testing experiments. Since in the Spectrum Collection library many flavones and isoflavones are present, we selected only molecules with the same scaffold of the reference compound (Fig. 6d).


A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials
Activity of chemical analogs of edaravone and methoxyisoflavone and identification of edaravone chemical features.(a) The four edaravone analogs 1a–d selected from commercial chemical libraries were used to confirm the structural class of edaravone structure and to identify its functional groups. Among these, only analogs 1b and 1d showed activity by MTT assay (see results). The activity of both analogs was confirmed in dose-response experiments and demyelinated cerebellar slices (Supplementary Figs S3 and S4). (b) The analogs antypyrine, aminopyrine and ramifenazone which were present within the Spectrum Collection library did not overcome the MTT test in both primary and re-testing experiments. (c) Basing on these findings four functional groups essential for the activity of edaravone were identified: in red 2 H bond acceptors (in position 1 and 3) plus in green hydrophobic group (the methyl in position 5) and in orange an aromatic ring (in position 2). (d) Nine isoflavones and (e) four flavones selected as analogs of 5-methyl-7-methoxyisoflavone were tested by MTT assay to confirm the compound activity. The analogs 2a and 3a–d were selected from commercial libraries, all the other compounds were present within the Spectrum Collection library. None of methoxyisoflavone analogs showed significant activity in both primary and re-testing experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384285&req=5

f6: Activity of chemical analogs of edaravone and methoxyisoflavone and identification of edaravone chemical features.(a) The four edaravone analogs 1a–d selected from commercial chemical libraries were used to confirm the structural class of edaravone structure and to identify its functional groups. Among these, only analogs 1b and 1d showed activity by MTT assay (see results). The activity of both analogs was confirmed in dose-response experiments and demyelinated cerebellar slices (Supplementary Figs S3 and S4). (b) The analogs antypyrine, aminopyrine and ramifenazone which were present within the Spectrum Collection library did not overcome the MTT test in both primary and re-testing experiments. (c) Basing on these findings four functional groups essential for the activity of edaravone were identified: in red 2 H bond acceptors (in position 1 and 3) plus in green hydrophobic group (the methyl in position 5) and in orange an aromatic ring (in position 2). (d) Nine isoflavones and (e) four flavones selected as analogs of 5-methyl-7-methoxyisoflavone were tested by MTT assay to confirm the compound activity. The analogs 2a and 3a–d were selected from commercial libraries, all the other compounds were present within the Spectrum Collection library. None of methoxyisoflavone analogs showed significant activity in both primary and re-testing experiments.
Mentions: To identify a relationship between the structure and activity of two hits, we selected and tested structurally related chemical analogs. We selected new chemical analogs from commercial chemical libraries by means of a public database (Zinc, PubChem and ChemSpider), finding four available analogs for edaravone (Figs 1a–d and 6a,b) and five for 5-methyl-7-methoxyisoflavone, of which only one was an isoflavone and four were flavones (Figs 2a, 3a–d and 6d,e,). The activity of the selected analogs (10 μM, 48 h) was evaluated in OPC purified cultures by MTT test, identifying two active analogs for edaravone (1b ER = 1.36 ± 0.029; and 1d ER = 1.44 ± 0.06) and none for 5-methyl-7-methoxyisoflavone. The biological activity of edaravone analogs was then confirmed in mixed glial cell cultures with dose-response curves at six different concentrations (0.001–30 μM; Supplementary Fig. S3) and in demyelinated cerebellar slices (Supplementary Fig. S4). By searching for chemical analogs in the Spectrum Collection library we found three analogs of edaravone (the antipyretic and analgesic drugs antipyrine, aminopyrine and ramifenazone; Fig. 6b) and 7 isoflavones (Fig. 6d) that did not overcome the MTT test in both primary and re-testing experiments. Since in the Spectrum Collection library many flavones and isoflavones are present, we selected only molecules with the same scaffold of the reference compound (Fig. 6d).

View Article: PubMed Central - PubMed

ABSTRACT

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds’ activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.

No MeSH data available.