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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.


Compound induced MBP mRNA expression in cerebellar slices.Slices were prepared from P7 mouse cerebellum. Two hours after plating, slices were treated with compounds (20 μM) or DMSO (0.002% vehicle) for 5 DIV. Expression of MBP transcript was evaluated by real time RT-PCR. The results show that four compounds significantly (p* ≤ 0.05; p** ≤ 0.01) stimulated the expression of MBP mRNA compared to untreated control, demonstrating their ability to promote OPC differentiation. Data are expressed as 2−ΔΔCt value relative to untreated control, using GAPDH as reference gene. Progesterone (40 μM) was used as positive control. 5-methyl-7-methoxyisoflavone is abbreviated as methoxyisoflavone. Bars represent the mean ± SEM of 3–5 independent experiments. (Student’s T-Test; *p ≤ 0.05; p** ≤ 0.01).
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f3: Compound induced MBP mRNA expression in cerebellar slices.Slices were prepared from P7 mouse cerebellum. Two hours after plating, slices were treated with compounds (20 μM) or DMSO (0.002% vehicle) for 5 DIV. Expression of MBP transcript was evaluated by real time RT-PCR. The results show that four compounds significantly (p* ≤ 0.05; p** ≤ 0.01) stimulated the expression of MBP mRNA compared to untreated control, demonstrating their ability to promote OPC differentiation. Data are expressed as 2−ΔΔCt value relative to untreated control, using GAPDH as reference gene. Progesterone (40 μM) was used as positive control. 5-methyl-7-methoxyisoflavone is abbreviated as methoxyisoflavone. Bars represent the mean ± SEM of 3–5 independent experiments. (Student’s T-Test; *p ≤ 0.05; p** ≤ 0.01).

Mentions: We next tested whether the five hit compounds could promote the differentiation of endogenous OPC in central nervous system (CNS) tissue. Cerebellar slices were generated from mice at postnatal day 7, a time that corresponds to the onset of myelination. Two hours after plating, slices were treated with compounds for 5 DIV and the expression MBP mRNA was evaluated by real time RT-PCR in 3–5 experiments. We focused on MBP transcripts since this myelin marker shows a wider range of expression than CGT during the developmental period analyzed. Progesterone (40 μM) was used as a positive control due to its myelin stimulating activity in this ex vivo model26. We found that only 5-methyl-7-methoxyisoflavone, edaravone, losartan and lovastatin significantly (p ≤ 0.05) increased MBP transcript levels relative to control slices, demonstrating their ability to promote OPC differentiation also in this experimental model (Fig. 3).


A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials
Compound induced MBP mRNA expression in cerebellar slices.Slices were prepared from P7 mouse cerebellum. Two hours after plating, slices were treated with compounds (20 μM) or DMSO (0.002% vehicle) for 5 DIV. Expression of MBP transcript was evaluated by real time RT-PCR. The results show that four compounds significantly (p* ≤ 0.05; p** ≤ 0.01) stimulated the expression of MBP mRNA compared to untreated control, demonstrating their ability to promote OPC differentiation. Data are expressed as 2−ΔΔCt value relative to untreated control, using GAPDH as reference gene. Progesterone (40 μM) was used as positive control. 5-methyl-7-methoxyisoflavone is abbreviated as methoxyisoflavone. Bars represent the mean ± SEM of 3–5 independent experiments. (Student’s T-Test; *p ≤ 0.05; p** ≤ 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384285&req=5

f3: Compound induced MBP mRNA expression in cerebellar slices.Slices were prepared from P7 mouse cerebellum. Two hours after plating, slices were treated with compounds (20 μM) or DMSO (0.002% vehicle) for 5 DIV. Expression of MBP transcript was evaluated by real time RT-PCR. The results show that four compounds significantly (p* ≤ 0.05; p** ≤ 0.01) stimulated the expression of MBP mRNA compared to untreated control, demonstrating their ability to promote OPC differentiation. Data are expressed as 2−ΔΔCt value relative to untreated control, using GAPDH as reference gene. Progesterone (40 μM) was used as positive control. 5-methyl-7-methoxyisoflavone is abbreviated as methoxyisoflavone. Bars represent the mean ± SEM of 3–5 independent experiments. (Student’s T-Test; *p ≤ 0.05; p** ≤ 0.01).
Mentions: We next tested whether the five hit compounds could promote the differentiation of endogenous OPC in central nervous system (CNS) tissue. Cerebellar slices were generated from mice at postnatal day 7, a time that corresponds to the onset of myelination. Two hours after plating, slices were treated with compounds for 5 DIV and the expression MBP mRNA was evaluated by real time RT-PCR in 3–5 experiments. We focused on MBP transcripts since this myelin marker shows a wider range of expression than CGT during the developmental period analyzed. Progesterone (40 μM) was used as a positive control due to its myelin stimulating activity in this ex vivo model26. We found that only 5-methyl-7-methoxyisoflavone, edaravone, losartan and lovastatin significantly (p ≤ 0.05) increased MBP transcript levels relative to control slices, demonstrating their ability to promote OPC differentiation also in this experimental model (Fig. 3).

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.