Characterization of More Selective Central Nervous System Nrf2-Activating Novel Vinyl Sulfoximine Compounds Compared to Dimethyl Fumarate

Abstract

The Nrf2 transcription factor is a key regulator of redox reactions and considered the main target for the multiple sclerosis (MS) drug dimethyl fumarate (DMF). However, exploration of additional Nrf2-activating compounds is motivated, since DMF displays significant off-target effects and has a relatively poor penetrance to the central nervous system (CNS). We de novo synthesized eight vinyl sulfone and sulfoximine compounds (CH-1-CH-8) and evaluated their capacity to activate the transcription factors Nrf2, NFκB, and HIF1 in comparison with DMF using the pTRAF platform. The novel sulfoximine CH-3 was the most promising candidate and selected for further comparison in vivo and later an experimental model for traumatic brain injury (TBI). CH-3 and DMF displayed comparable capacity to activate Nrf2 and downstream transcripts in vitro, but with less off-target effects on HIF1 from CH-3. This was verified in cultured microglia and oligodendrocytes (OLs) and subsequently in vivo in rats. Following TBI, DMF lowered the number of leukocytes in blood and also decreased axonal degeneration. CH-3 preserved or increased the number of pre-myelinating OL. While both CH-3 and DMF activated Nrf2, CH-3 showed less off-target effects and displayed more selective OL associated effects. Further studies with Nrf2-acting compounds are promising candidates to explore potential myelin protective or regenerative effects in demyelinating disorders.

Keywords: HIF; NFκB; Nrf2; dimethyl fumarate; microglia; multiple sclerosis; pTRAF; redox regulation; traumatic brain injury; sulfoximine.

Fig. 1

Experimental outline and structures of newly synthetized compounds. (A) Initial screening of compound CH-1–8 and DMF using pTRAF and qPCR. (B) Stimulation of primary glial cultures with DMF and CH-3. (C) Evaluation of CH-3 and DMF in an experimental model for traumatic brain injury. (D) Chemical structure of CH-1–8

Fig. 2

Stable transfected HEK(pTRAF^{Nrf2/HIF/NFkB}) reporter cell line enables simultaneous detection of Nrf2, NFκB, and HIF1 activation. (A) Schematic illustration of analysis approach using pTRAF and qPCR. (B–D) Stimulation for 24 h with 10 μM of CH-1, CH-2, and CH-3 or 40 μM DMF, with and without 20 ng/mL TNF (n = 3). (E) Examples of transcription levels following 3 h of stimulations with DMF (40 μM), CH-3 (10 μM) with and without TNF (20 ng/mL). Error-bars show S.D. Two group comparisons with a control group were done with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Transcriptional profile in HEK293 cells and primary cultures of microglia and oligodendrocyte following DMF and CH-3. (A) Heat-map of transcript fold-change (Log₂) following CH-3 and DMF stimulation. (B) PCA reduction plot based on the transcription of genes indicated in the heat-map after 3 h of stimulations with CH-3 (red), DMF (blue), or unstimulated (white). (C) Heat-map of transcription levels following 3 h of stimulations with DMF (15 μM), CH-3 (10 μM) (n = 6). (D) PCA reduction plot based on the transcription of genes indicated in the heat-map after 3 h of stimulations (n = 6)

Fig. 4

Naïve in vivo characterization of DMF and CH-3 and following TBI. (A) Experimental outline for routes of administration. (B) Transcriptional pattern in peripheral blood cells (dash) and brain (solid). (C) Transcription pattern in corpus callosum (CC) 5 h following intra cisterna injections of DMF (4 mM) and CH-3 (4 mM) (n = 5). (D) Experimental outline for TBI in combination with intervention. (E) Flow cytometric valuation of Cd45⁺ (leukocytes) in the spleen compared to vehicle (n = 6). (F, G) Microglia in brain following CH-3 or DMF treatment assessed by transcription of Cd11b (n = 4) (F), and following CH-3 or DMF treatment and TBI (g) assessed by flow cytometry (n = 7 + 5). (H, I) Oligodendrocytes (OL) in brain following CH-3 or DMF treatment assessed by the OL lineage marker Sox10 (n = 4) (H), and following CH-3 or DMF treatment and TBI assessed by flow cytometry (n = 7) (I). (J) Levels of NFL in the CSF in sham and following TBI in combination with treatment, detected with ELISA. Error-bars show S.D. Two group comparisons with a control group were done with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001