NEW YORK (GenomeWeb) – Researchers from the Scripps Research Institute and the La Jolla Institute for Allergy and Immunology have completed a chemical proteomic analysis investigating the behavior of the autoimmune drug dimethyl fumarate (DMF).
Detailed in a paper published this week in Science Signaling, the work provides new insights into the drug's mode of action and identifies potential new drug targets for diseases like multiple sclerosis, Megan Blewett, a graduate student in the lab of Scripps researcher Benjamin Cravatt and first author on the study, told GenomeWeb.
Originally identified as a treatment for psoriasis in the late 1950s, DMF has more recently become used to treat multiple sclerosis and is currently sold by Biogen under the brand name Tecfidera. Traditionally, DMF has been thought to work by promoting an antioxidant response in the brain and creating a neuroprotective effect, Blewett said. But, in the last several years research has suggested the drug actually modulates the immune system.
"The prevailing hypothesis was that DMF covalently modified cysteine residues on the Keap1 (Kelch-like erythroid cell-derived protein) complex, which is part of the Nrf2-Keap1 antioxidant pathway," she said. "That promotes an antioxidant response in the brain, and somehow reducing reactive oxygen species in the brain leads to efficacy or clinical benefit for multiple sclerosis patients."
This neuroprotection theory is undermined somewhat by the fact that DMF is also used to treat psoriasis, which is not a neurological disorder. Also, Blewett noted, in 2014, a patient being treated with the drug died of progressive multifocal leukoencephalopathy, a rare viral infection that typically affects people with compromised immune systems.
"At this point I think there is pretty convincing data that DMF is immunomodulatory," she said.
In the Science Signaling paper, Blewett and her colleagues set out to further characterize the mechanisms by which DMF works using a chemical proteomics platform they previously developed to measure the interaction of particular electrophilic molecules like DMF.
Named isoTOP-ABPP (for isotopic tandem orthogonal proteolysis-activity-based protein profiling), the method takes a cell culture or tissue of interest and exposes it to the molecule of interest, in this case DMF. A control sample is exposed to an unreactive analog. Then both samples are exposed to an iodoacetamide probe that reacts with protein cysteine residues, and the amount of these probes that bind to cysteines in the samples is then quantified using mass spec.
Because cysteines that bind to DMF will not be available to bind to the iodoacetamide, measuring the amount of bound iodoacetamide allows the researchers to quantify how strongly DMF binds to particular cysteines.
"The greater the loss of [iodoacetamide] labeling for any given cysteine residue, the more sensitive that residue is to [DMF] treatment," Blewett said. She added that the technique was especially useful in that it allows researchers to "do this in a proteome-wide fashion and… identify the specific cysteine residues in the proteome that are modified."
The approach also uses isobaric labeling to multiplex the cases and controls, which improves quantitation, particularly in the case of low-abundance residues, Blewett said. "It gives us increased confidence with the lower-abundance species, because in cases where you have very few spectral counts you are going to see a lot more percentage variability from run to run. But if we run DMF and [the control] at the same time, then we can compare those very low spectral counts."
In the Science Signaling study, Blewett and her colleagues profiled 2,500 cysteine residues in primary human T cells, identifying 50 residues that were sensitive to DMF treatment. A large proportion of these residues had no prior functional annotation, which, Blewett noted, makes functional validation a challenge but also suggests new lines of inquiry.
The study also turned up some known associations, she said. For instance, several of the affected cysteines were on proteins involved in NF-kβ signaling, which is key to T cell activation. "So inhibition of that pathway might explain some of DMF's immunomodulatory effects," she said.
The researchers did functional validation of one of those proteins, PKCϴ, which Blewett said a number of drug companies are pursuing as a target for diseases including multiple sclerosis.
These efforts have struggled in part due to selectivity issues, which could make the PKCϴ cysteine residue identified in the study an attractive target, she noted.
The nice thing about the cysteine residues on PKCϴ labeled by DMF is that they are unique to PKCϴ, they are not in any other PKC isoforms, of which there are about 10 total," Blewett said. "So we are very excited to follow some of these [hits] up and see if we can use some of these residues to generate next-generation probes and ultimately immunosuppressants that are very selective."
PKCϴ is an important drug development target due to research showing that mice without the protein are not susceptible to animal models of multiple sclerosis. "So even prior to this [study] there was a lot of data [indicating] that targeting PKCϴ could be beneficial for MS patients," Blewett said.
"It's a little ironic," she added. "These [pharma companies] are trying to develop PKCϴ inhibitors, and there is already one on the market."
Even so, DMF isn't an ideal agent for PKCϴ inhibition, she noted. The drug is rapidly hyrdrolyzed in the body, meaning patients must take very high doses. Additionally, it's possible that the hydrolysis product could cause some of the drug's side effects, which include things like headaches and gastrointestinal problems.
A better designed, more selective PKCϴ inhibitor could be more effective with fewer side effects and at a lower dose, Blewett said.
Beyond drug targets, the research also offers basic insights into the biology affected by DMF treatment, Scripps researcher and study co-author John Teijaro said.
"The cysteines of PKCϴ have never been described as having a role in immune modulation," he said. "And as we see other cysteines being modified in these non-active site places, it starts to give us hints as to how these proteins interact with other proteins and how they function in vivo to modulate immune response."