MS Treatment by ONX 0914

Ryan Ferrie                   ChemicalBio  441                  Week 9 Blog Spotlight

Basler, M., Mundt, S., Muchamuel, T., Moll, C., Jiang, J., Groettrup, M., and Kirk, C.J. (2014). Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis. EMBO Mol. Med. 6, 226-238.  doi: 10.1002/emmm.201303543

~~~*Foreword: Please click on posted photos to enlarge at a higher resolution.*~~~

Multiple sclerosis (MS) is result of chronic immune mediated inflammation in the brain and spinal cord that degrades myelin sheaths. The loss of insulin disrupts the progression of action potentials through neurons and has observable phenotypic consequences. MS has been characterized by damage to important areas of white matter. Other researchers have created a model to study MS by inducing experimental autoimmune encephalomyelitis (EAE). EAE can be described as triggered brain inflammation by increased permeability of the BBB and administration of antigens into the brain, ultimately resulting in myelin deterioration. Overactive T-cells lose their affinity to bind antigens and instead induce inflammation in the brain by binding with self.

Proteins are typically destroyed following ubiquitination by large protease complexes called proteasomes. The 20S proteasome is a barrel structure with two outter rings composed of alpha subunits and two inner rings composed of beta subunits (7 subunits each ring). The catalytic site of proteolysis is found in the interior beta subunits at serine and threonine residues. Immunoproteasome is the name of the product when the catalytic subunits on regular proteasomes are replaced. Immunoproteasomes have been implicated in T cell overactivation and autoimmune diseases. In previous studies, a protein called ONX 0914 was found to reduce cytokine production from overactive T-cells, which decreased the progression of a variety of diseases in mice. ONX 0914 was able to hinder overactive T-cells by acting as an LMP7-selective epoxyketone inhibitor, which is to say that ONX 0914 lowers the activity of immunoproteasomes by blocking one of their substituted active site subunits (LMP7).

The purpose of this experiment was to take the mouse model of EAE and treat it with ONX to confirm or deny previous hypotheses about ONX. The researchers are in search of a treatment or management for multiple sclerosis in humans.

The researchers performed a number of tests on their mice to validate their findings. The authors looked into whether any of their mice were susceptible to EAE, and their controls were found not to be. In order to determine that ONX effectively acted as an LMP7 selective inhibitor, the authors immunized their mice, and then treated them with ONX three times a week. Inflammation from spinal tissue was observed microscopically. To see how ONX altered cytokine expression after EAE induction, mRNA levels for a variety of interleukines were recorded after 19 days with real time RT-PCR. This allowed them to show that ONX treated mice showed significantly lower levels of cytokines. The authors used similar methodology to show that their results are reproducible in PLP139-151 and MOG35-55 cell types.

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Figure 3 (below) illustrates that inhibition of LMP7 by ONX can cause a decreased passage of T-cells through the brain, and activated lymphocyte and myeloid cells, which would contribute to the building of recently damaged areas. Furthermore, cross-sections of spinal cord were taken and stained to observe that ONX treated mice showed less severe levels of inflammation.

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The best summarization of the ability of ONX to lessen the severity of EAE symptoms is Figure 6A&B (below). In this example, the authors induced EAE, but did not administer ONX until the organisms showed symptoms. Once this happened, they were treated with varying concentrations of ONX, and the figure shows that a greater dose, but not necessarily more frequent dosing, signficantly lessened the effects of the disease from that of the control.

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Figure 7 (below) also gives a nice tissue stain, qualitatively showing how well ONX can reduce inflamation. Figures 6 & 7 indicate the same results in two different cell types.

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The researchers found that ONX reduced symptoms of EAE and prevented brain and spinal cord inflammation. ONX is believed to be selectively targeting chymotrypsin-like activity in cells originating from the hematopoietic system. This system is the one that is mostly responsible for immunoproteasomes and the induction of EAE. Since this enzyme is so selective, there are minimal side effects associated with its treatment. The effective dose used in this study, 10 mg/kg, is below the ‘maximal tolerated’ dose of 30 mg/kg, lending to safe use of this enzyme. ONX was found not only to prevent progression of the disease, but also to inhibit its onset.

The results are significant because these authors were able to demonstrate the effectiveness of previously proposed, but never executed interactions between ONX 0914 and immunoproteasomes. While these results are significant, and were produce in vivo, experimentation was nonetheless performed on mice. Results in mice do not always translate well into the same results found in humans. The next step in this study is obviously to experiment in higher order organisms to ultimately develop an effective treatment for humans.

18 Replies to “MS Treatment by ONX 0914”

  1. Nice job, Ryan! I enjoyed reading this paper and your summary because of its direct clinical relevance to our studies of amino acids. It also made me recall that an antigen is merely just a peptide that is embedded in the membrane and recognized by another cell. You mentioned the role of the immunoproteasome in antigen presentation, but do you know how exactly this leads to the manifestation of MS? Thanks!

    1. Hey Zach,

      Thank you for taking the time to comment on my post. Previous studies have have found a catalytic site of immunoproteasomes that is responsible for MHC1 antigen presentation. (Diaz-Hernandez et. al. 2003) The problem with immunoproteasomes is that they deviate from the functions of regular proteasomes and cause cytotoxic T-cell overactivation and do not properly degrade/recycle other proteins. Increased t-cell activation sensitizes the immune system and increases the likelihood of developing an autoimmune response. Furthermore, the persistence of unwanted proteins can accumulate to toxic levels and also contribute to the destruction of the BBB. Lastly, since the activity and antigen presentation capabilities of immunoproteasomes is different from that of regular proteasomes, immunoproteasomes may be presenting improper targets for degradation. I would hypothesize that if immunoproteasomes were to present somatic molecules as antigens, they could condition the overactive cytotoxic T-cells to attack the self. All of these effects are in relation to breaking down the BBB so that inflammatory molecules can enter the brain and induce MS. I hope that helped!

  2. Good job with your summary! In the discussion section of the paper, the authors state that “LMP7 inhibition blocks cytokine production in multiple immune effector cells.” While this is clearly important for stopping the development and production of MS and related diseases, cytokines also play an extremely important role in the healthy body, such as by controlling cell cycle regulation and modulating the immune response. Since cytokines play such an important role in numerous processes, do you think that blocking LMP7, and the subsequent production of cytokines, could cause unwanted side effects in human patients? Clearly there are great benefits to this type of treatment, but this must be balanced against potential detrimental effects, including the risk of decreased immunity and even cancer, which could be deadly. Is the risk worth it? Do you think there might be a way to supplement certain cytokines via a pill or blood infusion in a way that might minimize side effects while also still controlling MS?

    1. Hey Mike,

      I appreciate your well thought out comment. The sentence you quoted ends with ‘production by autoreactive T cells.’ The T cells that are mentioned are those which are overproduced by the immunoproteasomes that contribute to the autoimmune response associated with inflammation in the brain. That is, the cytokine production in this case is harmful, but is solely the result of the different morphology and activity of immunoproteasomes. While inhibition of the immunoproteasomes does block its subsequent production of cytokines, this is far from a universal mechanism and is not implying that the production of beneficial cytokines will be inhibited. To this extent, I do not think that the decrease of excess harmful cytokines will have any unwanted side effects, nor should there be a need to supplement with other types of cytokines because they should be relatively unaffected by inhibition of LMP7.

  3. Hi Ryan. What does “reduction of the BBB” mean? Reducing its size, its permeability, its overall presence….?

    1. Hey Dr. Colabroy,

      Thank you for responding to my post. This article itself does not delve much into the specifics of the interaction with the BBB. However, Constantinescu et. al. (2011) describe a ‘disruption’ or ‘breakdown’ of the BBB through small molecule interactions that increase the permeability of the barrier and allow antigens, cytotoxic T-cells, and cytokines into the CNS. The article did not go further than this, but if the permeability of the BBB is increasing, I would imagine that these voids would likewise be decreasing its density and/or thickness.

  4. Hi Ryan! Nice job with the summary! I see that you highlighted the role of immunoproteosomes in MS and described the basic structure. You also stated that immunoproteosomes have been implicated in several autoimmune diseases. Are there several types of immunoproteosomes that are each specific a certain autoimmune disease, or are all autoimmune diseases regulated by the same kind immunoproteosome? If they are similar, is it possible that inhibition of LMP7 with ONX0914 could be an effective treatment for other autoimmune diseases, too? Thanks!

    1. Hey Kelly,

      Great question.. I did a little digging and found that immunoproteasomes are involved in a variety of different diseases as a result of alterations at the subunits of the complex. If you look at table 1. of Basler et. al. (2013), link below, you can see that even the same subunit change can give a slew of different results! With that being said, ONX was only messing around with LMP7 and would not be effective at immunoproteasome alterations of LMP2 or MECL-1, for example. Also, a polymorphism in the PSMB8 catalytic subunit of immunoproteasomes has been associated with other diseases such as Nakajo-Nishimura syndrome, CANDLE syndrome, and intestinal M. tuberculosis infection. (Gomes, 2013) In conclusion to your question, this treament of LMP7 cannot be used to treat ALL autoimmune diseases regulated by immunoproteasomes because the immunoproteasomes differ in the alterations of their catalytic subunit, but this treatment would be effective in other diseases that have the LMP7 subunit mutation.

      Basler et. al. (2013) link: http://ac.els-cdn.com/S0952791512001859/1-s2.0-S0952791512001859-main.pdf?_tid=afe32c8e-d42e-11e4-9de7-00000aab0f6c&acdnat=1427425951_b642337b7d0502b0d38f168c5434f9b4

  5. Hi Ryan- nice job. Something that intrigues me in the paper is this small part of the discussion:

    “Interestingly, we found that mice genetically deficient in any of the immunoproteasome active site subunits developed EAE following MOG35–55 immunization that was indistinguishable from wild type C57BL/6 mice
    (Fig 1). Similar results using LMP2 (-/-) mice have been reported by Frausto et al (2007) while Seifert et al have reported that MOG 35–55 immunization of LMP7 (-/-)
    mice results in more severe inflammation
    compared to wild type mice (Seifert et al, 2010), which could not be confirmed by others (Nathan et al, 2013).”

    The main claim of the paper seems to include the idea that the immunoproteasome plays an integral part in the inflammation associated with EAE, and that inhibiting it helps to reduce the symptoms of EAE (specifically, inhibiting LMP7). It is curious, then, that a mouse in which LMP7 is knocked out would experience greater inflammation. To me, this seems to provide some complications for the main claim of the paper. Do you think so? If so, do you think the authors could have reconciled this disparate data better?

    Thanks!

    1. Hey Gabby,

      Thanks for taking the time to comment. I do not think that these findings very significantly impact the claims of the paper. The authors find it odd that the degree of induced EAE can change with genetic alterations in the immunoproteasomes’ subunits, but nonetheless, EAE was induced. These do appear to be interesting anomalies, but they are not taking away from the main impact of the paper. If anything, these obscurities will contribute to future work and indicate that there are other pathways through which EAE is induced that also need to be looked into. This work still has thorough methodology and results and should not be discredited from the few sentences you quoted. I hope you enjoyed the feedback!

  6. Ryan – Well done. I was wondering who would be the first non-JBC poster, and you did not disappoint! I am wondering about the strategy used by the authors in using both MOG35-55- and PLP139-151- to induce EAE. It seems odd that they highlight the induction of EAE based on two models. Are there more MS models that they aren’t telling us about? Are these two generally widespread and valid? My question is ultimately about the confidence we should have in the results based on their methodology. All things considered, how far should we ride these results?

    1. Hey Zach,

      Petro (2011), link below, gives a good description of how these two methods work in his introduction. MOG stands for Myelin Oligodendrocyte Glycoprotein and PLP stands for encephalitogenic ProteoLipoProtein peptide. Typically, these two proteins function to form the myelin sheaths around axons to transmit electircal signals. From what I could gather, when these proteins are injected into the brain, they function as targets rather than therapeutic agents, and will trigger an autoimmune response to the protein to attack those composing the myelin sheaths. The destruction of neurons associated with EAE from these protein injections is comparable to the symptoms of MS. EAE induced by MOG is characterized by a progress worsening of symptoms followed by stabilization and maintenance of symptoms similar to progress MS. On the otherhand, PLP induced EAE is more similar to ‘relapsing-remitting MS,’ whereby symptoms increase then dissipate cyclically. To answer your specific question, yes, we can be confident in these results because these methods are well validated and are primary tools in creating EAE to model and treat the symptoms of MS.

      Petro 2011: http://ac.els-cdn.com/S1567576910002389/1-s2.0-S1567576910002389-main.pdf?_tid=9970a0aa-d436-11e4-949c-00000aacb362&acdnat=1427429349_4a3a4ef071b06c02e2163c2c877dad3f

  7. Hey Ryan,

    As I was reading the conclusion, I noticed that a couple of the inflammatory agents (Like IFN-gamma) that ONX 0914 helps to prevent are also present in Celiac disease, which suggests to me that they might be common players in many autoimmune diseases, and therefore ONX0914 might effectively combat symptoms in multiple autoimmune diseases, not just MS. The authors seemed to recognize this in their conclusion, stating “but [proteasome inhibitor’s] therapeutic potential may also extend to autoimmune diseases due to their
    effects on cytokine production and proliferating lymphocytes”, however I didn’t feel like they expanded upon the idea enough to draw major attention to the fact that ONX0914 might have more diverse applications. Do you think the authors addressed this point sufficiently and organized their paper well enough to highlight this point? If not, do you think addressing the potential range of autoimmune diseases ONX 0914 could impact could have increased the impact factor of their paper?

    1. Hey Tommy,

      Thanks for highlighting some of the strategic maneuvers of the authors.

      To summarize, I copied and pasted the titles of the results sections in order:
      1. EAE induction in immunoproteasome-deficient mice
      2. An LMP7-selective inhibitor prevents symptoms of EAE
      3. LMP7 inhibition prevents brain and spinal cord inflammation
      4.Therapeutic treatment with ONX0914 inhibits progression of MOG35–55-induced EAE
      5.ONX0914 blocks differentiation to autoreactive T cells
      6.ONX0914 reduces clinical symptoms in the PLP139–151-induced relapsing-remitting EAE model in SJL/J Mice

      The authors organized the results in order to build their argument and highlight their results. The first couple of steps was to validate their method and show correlations. The final step was to highlight the specific interactions with the enzyme and small molecules. The authors made a point to show that this enzyme is effective in both types of MS models.

      Please not that an immunoproteasome is a proteasome with alterations at the catalytic subunits. While this enzyme is effective in both models, it is not effective against multiple immunoproteasome alterations. Furthermore, even with the same subunit alteration, multiple behavioral differences may be observed. Table 1. of Basler et. al. (2013) demonstrates this concept.

      I think that the paper can not conclusively emphasize the application of ONX to other diseases without experimenting with other disease models. The immunoproteasomes are not universal, and neither is the applicability of ONX to other subunits. If the authors could connect this work to other immunoinflammatory diseases, then that would significantly increase the impact of this study in the future!

      I talked about this a little bit in Kelly’s comment too if you want to check that out. Thanks again for the comment.

      Basler et. al. (2013) link: http://ac.els-cdn.com/S0952791512001859/1-s2.0-S0952791512001859-main.pdf?_tid=afe32c8e-d42e-11e4-9de7-00000aab0f6c&acdnat=1427425951_b642337b7d0502b0d38f168c5434f9b4

  8. Hey Ryan, great choice of article and great job highlighting the main points in a manageable way. I have a pretty big-picture question about MS and other auto-immune diseases that I haven’t considered before. Auto-immunity is basically immunity gone bad and targeting one’s own cells, but can auto-immune disorders (MS included) be categorized as “innate” or “acquired”, or is it a mix of the two? I see at least 1 issue if MS were to be classified as either – if MS is “innate immunity gone bad”, and innate immunity is more or less the same for everyone, do healthy individuals get milder MS symptoms over time naturally? If MS is “acquired immunity gone bad”, how come these “overactive” T-cells, which should have high specificity, are attacking the myelin sheath rather than binding antigens? Any input would be great, thanks!

  9. Hey Besher,

    Very good questions and unfortunately I don’t think I will be able to give you the clear cut answer you want.

    We could categorize MS as a mix of auto-innate and auto-acquired immunities. Cytokines are typically considered a first line of defense, with a rapid response time to a variety of foreign bodies. On the other hand, cytotoxic T cells are specific and require an antigen presented to them for destruction. As for your question about the change of target, I think we should look again at Basler et. al. (2013), but this time turn to figure 1.

    The figure shows the different cleavage patterns between a proteasome and an immunoproteasome. The peptide generated from the immunoproteasome can then act as an epitope and can flag cytotoxic T cells to targets that they wouldn’t otherwise attack.

    Does this help at all? Are you reading the figure the same way I outlined it?

    Thanks for comment!

    link: http://www.sciencedirect.com/science/article/pii/S0952791512001859

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