Fumaric acid esters (FAE) are a group of compounds beneficial in systemic treatment of psoriasis1-6 and recently FAEs have also been suggested as a new therapeutic option to treat relapsing remitting multiple sclerosis7-9.
Various combinations of FAEs for oral treatment of psoriasis have been used for more than 50 years10,11. Although a controlled study demonstrated the efficacy of dimethylfumarate (DMF) in psoriasis in 198912, it was an empirically composed mixture of dimethylfumarate (DMF) with calcium, magnesium and zinc salts of ethylhydrogen fumarate that was registered as Fumaderm® in Germany in 1994. Fumaderm® has since then become the leading drug for systemic therapy of psoriasis in Germany11. One limitation in the use of FAE's is the reported side effects with flushing and gastrointestinal symptoms such as diarrhoea, nausea and cramps. Thus, overall FAE's have been shown to have a favourable long-term safety and clinical-efficacy profile2 and in particular no long-term toxicity nor a higher risk for infections or malignancies have been observed in more than 100,000 patient years3.
A second generation fumaric acid derivative (BG-12) was developed as an enteric-coated microtablet to improve gastrointestinal tolerability13. BG-12 has shown very promising result in patients with relapsing-remitting multiple sclerosis in a multi-centre, randomized, double-blind, placebo-controlled phase IIb study9,14 and most recently two phase III clinical trials in patients with relapsing-remitting multiple sclerosis including more than 2600 patients have been reported to confirm these results although the complete data set has not yet been published15.
Fumaric acid esters have been shown to be effective in several dermatological diseases including: Necrobiosis16-19, granuloma annulare18,20-22, alopecia areata23, cheilitis granulomatosa18,24, recurrent oral aphthae25, pityriasis rubra pilaris18, and annular elastolytic giant cell granuloma18, as well as a range of non-dermatological diseases: Sarcoldosis18,26 and non-infectious chronic uveitis27. Fumaric acid esters have also shown potential for the use in treatment of cancer28-30, Huntington's disease31, malaria32, human immunodeficiency virus33, bronchial asthma34, myocardial infarction35 and for use as an immunosuppressor in organ transplantation36.
Despite a clear clinical effect of FAE's in psoriasis and relapsing-remitting multiple sclerosis and numerous in vitro and in vivo studies with FAE's, the precise mechanism of action had not been fully understood. FAEs have been shown to inhibit the expression of TNF-α induced adhesion molecules37 as well as various cytokines including psoriasis associated cytokines like IL-IP, IL-6, IL-8, IL-20 and TNF-α38-40. DMF also suppresses the expression of VEGFR2 in human endothelial cells indicating a possible anti-angiogenic action41. Other possible mechanisms causing an anti-psoriatic effect are FAE induced apoptosis of purified human T-cells42 and a FAE induced shift in the immunological balance from a Th1- towards a Th2-like43. Because there is a lack of detectable DMF plasma concentrations after oral intake it has been suggested that there is a reaction of DMF with glutathione (GSH) in the portal vein blood44. This hypothesis has recently been supported by novel findings of Ghoreschi and co-workers45.
Both multiple sclerosis (MS) and psoriasis are considered autoimmune CD4+ T-cell driven disorders with predominance of a Th1 and Th17 phenotype of pathogenic T-cells46. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) bridging innate and acquired immunity, recognizing infections, secreting proinflammatory cytokines and orchestrating the maturation of naïve T-cells and to create the cytokine microenvironment regulating T-cell differentiation47. FAE have previously been shown to induce a shift in the immunological balance from a Th1- toward a Th2-like response43 and to inhibit the differentiation of dendritic cells48. More recently Ghoreschi at al. 201145 suggested that DMF depletes glutathione (GSH) followed by increased hemoxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. HO-1 interact with AP-1 and NF-κB binding sites in the IL-23p19 promoter inhibiting its expression and IL-23 is a key driver of Th17 maturation. STAT1 inhibition prevented IL-12p35 expression leading to decreased expression of the Th1 driver, IL-12. It was therefore suggested that DMF improved MS and psoriasis through inhibition of Th1 and Th17 responses. In this model, the DMF induced GSH depletion is essential.
In opposition to this, several studies have shown that DMF induces a transient GSH depletion whereas prolonged exposure raised GSH expression11 and therefore depletion of GSH cannot account for all the effects of DMF seen in MS and psoriasis. However, regulation of T-cell differentiation is important in controlling these two diseases and it is therefore interesting that a recent study59 has demonstrated DMF mediated inhibition of Th1 and Th17 differentiation through suppression of NF-κB and the p38 MAPK-MSK1 and ERK1/2-MSK1 signalling pathways39,46. Both NF-κB and MAPK activation have previously been shown to contribute to LPS mediated DC maturation49,50. Although the ERK1/2 MAPK and the NF-κB pathways are independent, they can interact via MSK1. MSK1 enhances NF-κB transcriptional activity through phosphorylation of serine 276 of the NF-κB subunit, p65. Further MSK1 phosphorylates Histone-3 at serine 10 which also enhances NF-κB transcriptional activity. The present inventors and others have previously demonstrated an inhibitory effect of DMF on different signaling pathways including the p38 MAPK-MSK1, ERK1/2-RSK1 and MSK1-NF-κB pathways36, 39 59.
DMF has previously been shown to block the activation of the Ribosomal S6 Kinase family (composed of RSK1 to RSK4 and the homologous kinases MSK1 and MSK2) by the extracellular signal-regulated kinase (ERK)39,49.
MSK1/2 and RSK1/2 have overlapping effects on transcription factors like the cAMP-responsive element (CREB), ATF1 and Histone 346, while RSK1 to RSK4 separately regulate the phosphorylation of c-Fos, c-Jun and JunB. The complexes of c-Fos, c-Jun and JunB, formed as dimers, bind to the activator 1 (AP-1) site and AP-1 DNA binding activity regulates cell proliferation54. The specific inhibitory effect of DMF on RSK1 and MSK1 activation followed by the induction of p-c-Jun (S63) and p-p53 (S15) led to the inhibition of keratinocyte proliferation, partly explaining the anti-psoriatic effect of DMF46. The specificity of DMF's inhibitory effect on RSK1 and MSK1 activation was proved by transfection with small interfering RSK1 and MSK1 RNA instead of DMF which showed the same effects on induction of p-c-Jun (S63) and p-p53 (S15)46.
MSK1/2 and RSK1-4 are activated by pro-inflammatory cytokines and growth factors and their activity is controlled by multiple phosphorylation sites55,56. The serine and threonine kinase activity of MSK1/2 and RSK1-4 is dependent on full length activation by phosphorylation at multiple sites in MSK1/2 and RSK1-457. The alignment of the amino acid sequences shows 43% homology between MSK1/2 and RSK1-458.
MSKs and RSKs are composed of two kinase domains (a N- and a C-terminal) and are activated by either p38 MAPKs and ERK 1/2 for MSK1/2 or only by ERK1/2 for RSK1-455-57. The activation starts for both MSKs and RSKs in a similar way by phosphorylation of an activation loop in the C-terminal kinase domain. This phosphorylation leads to the activation of the hydrophobic linker loop in the middle part and then the N-terminal kinase domain is phosphorylated. The N-terminal kinase domain binds and phosphorylates substrates. DMF has been shown to fully inhibit activation of specific phosphorylation sites at the C-terminal domain, in the linker loop and in the N-terminal domain and this reduced the kinase activity of the N-terminal domain and thereby downstream substrate activations39,46,51.
The MSK/RSK kinases are composed of two catalytic domains (an N- and a C-terminal) separated by a ˜100 amino acid linker. Each of the catalytic domains is composed of a small N-terminal lobe comprising β-sheets and a larger C-terminal lobe mainly comprising α-helices. ATP and substrate are bound in the interface between the two lobes. The kinases are activated by ERK by phosphorylation of an activation loop in the C-terminal catalytic domain. This phosphorylation leads to the activation of the N-terminal catalytic domain by phosphorylation of the linker region. Phosphorylation of the linker region is abolished by DMF and the activation of the N-terminal kinase domain hereby reduced39,46. An apo-structure of the C-terminal kinase domain of murine RSK2 was previously described47 but does not provide any insights to the binding of ligands (e.g. ATP and substrate). Some RSK2 inhibitors have been identified including Staurosporine like compounds48, kaemperol-glycosides49 and 50s. The majority of the identified RSKs inhibitors are ATP-competitive and bind in the ATP pocket between the two lobes in either one or both of the catalytic domains. An irreversible RSK2 inhibitor of the C-terminal catalytic domain has been developed, covalently binding to a cysteine located in the ATP pocket51,52.
ATP-competitive inhibitors generally display reduced selectivity due to the numerous ATP-binding pockets in the cell and poor cellular activity due to the high intracellular ATP concentration. The development of an allosteric RSK/MSK inhibitor will reduce off-target effects and increase efficacy. DMF can form covalent adducts with intracellular thiol containing molecules such as GSH, whereas a RSK/MSK selective inhibitor will only undergo covalent interaction once bound in the allosteric pocket.