An Autoreactive Myelin Oligodendrocyte Glycoprotein Protein Inhibitor for Multiple Sclerosis

MCRO 433 Fall 2014

Alicia Romero, Kristina Koster and Steven Liu

A. Project Summary

Multiple sclerosis (MS) is an inflammatory disease characterized by demyelination in the central nervous system. The underlying pathogenesis of MS remains unclear, but one proposed immunological mechanism of MS involves a demyelinating antibody response against the myelin oligodendrocyte glycoprotein (MOG). MOG is a myelin component expressed at the outer surface of the myelin sheath and oligodendrocyte plasma membrane [1]. The glycoprotein contains an immunoglobulin (Ig)-like domain which is exposed at the membrane surface and targeted by T- and B-cells leading to eventual degradation of the myelin sheath [1]. No current cure exists for MS and disease symptoms are primarily treated by physiotherapy, disease-modifying treatments and drug therapy for treatment of MS symptoms.

Our project focuses on designing a novel protein inhibitor that selectively binds autoreactive MOG antibodies. Therefore, anti-MOG antibodies will be unable to bind the Ig-like domain because the protein inhibitor will occupy the binding site of the paratope. The protein inhibitor will be engineered based on a set of rules that relate secondary structure patterns with protein tertiary motifs leading into the target folded state. The protein inhibitor will covalently modify the autoreactive MOG antibodies such that they are unable to bind the epitope of the extracellular Ig-like domain of MOG.

Prediction of protein folding is an integral component of protein design. While the principles of protein design and prediction of protein folding are not fully understood, it is a promising field of research because it will allow us to design new protein molecules that will fold into the desired structure with a novel function. Given our current knowledge of protein design principles, many examples of successfully designed proteins with therapeutic properties already exist. For example, dorzolamide (Truspot®) is a carbonic anhydrase inhibitor which decreases aqueous humor secretion by reducing formation of bicarbonate ions. The drug was approved in 1995 by the FDA as an anti-glaucomal agent. Dorzolamide was formulated by structure-based drug design which relies on understanding the three-dimensional structure of the target molecule [2]. Drugs can then be designed that will be predicted to bind with high affinity to the target molecule. Another successful example of a rational drug design is imatinib, a tyrosine kinase inhibitor used in the treatment of multiple cancers [3]. Imantinib prevents the initiation of the signaling cascade necessary for cancer development.

Given these previous successful examples of protein design, we are confident that we can design a protein inhibitor for autoreactive MOG antibodies. A beneficial treatment for MS may be designed by targeting the T- and B-cells ability to bind to MOG, thereby preventing the gradual degradation of myelin.

B. Introduction

Mutliple sclerosis is an autoimmune disease characterized by demyelination, formation of lesions and inflammation of the myelin sheath. Demyelination occurs when the myelin sheaths of the CNS are damaged. The myelin sheath is composed of multiple concentrically wrapped layers of glial membrane that insulate the axon of the CNS [4]. It plays a critical role in increasing the velocity of nerve-impulse propagations. The loss of the myelin sheath results in impairment or loss of conduction of impulses along the nerve and eventual breakdown of the axon. When the myelin sheath is lost a repair process, remyelination, is initiated in which oligodendrocytes rebuild the myelin sheath. However, repeated degradation of the myelin sheath leads to successively less effective remyelinations because the process creates a thinner myelin sheath [5][6]. Plaque accumulates around the damaged axons with each remyelination attempt until their presence completely prevents remyelination leading to the formation of lesions [5].

Inflammation is another hallmark of MS which occurs during lymphocytic infiltration of the blood brain barrier (BBB) which typically restricts immune cell invasion because it may trigger an autoimmune response. The autoreactive lymphocytes fail to apoptose due to overexpression of β-arrestin 1, a crucial promoter of CD4+ T-cell survival [7][8]. Inflammation is further driven by secretion of interleukin 17 and 22 (cytokines), which facilitate the disruption and breakdown of the BBB. Interleukins are a type of cell signaling molecules expressed by lymphocytes that promote the development and differentiation of T and B lymphocytes [9]. Once the BBB breakdowns, the migration of autoreactive lymphocytes across the barrier attacks the myelin sheaths [10][11]. The attack of myelin triggers the release of proinflammatory cytokines and antibodies which amplifies the immune response by recruiting destructive proteins.

One of the leading proposed mechanism of MS pathogenicity is antibody-mediated demyelination [12]. An ideal antigen candidate which marks the myelin sheath for attack by the immune system is myelin oligodendrocyte glycoproteins (MOG). MOG is a transmembrane protein expressed on the outermost surface of myelin sheaths. Its function is to maintain the integrity of the myelin sheath. But MOG has a single Ig-domain that is exposed to the extracellular space, increasing its susceptibility to attacks due to the ease of access for autoantibodies [1][13].

Currently, no known cure exists for MS which affects 2-2.5 million people worldwide. Most therapies are aimed at management of multiple sclerosis by disease-modifying drugs which reduce the number of inflammatory cells that cross the BBB [14], compete for or disrupt antigen presentation to T-cells by antigen presenting cells [15] and suppressing the immune system [16].

Our research group aims to develop a unique therapy based on designing a novel protein inhibitor that will inhibit T- and B-cells ability to bind to MOG thereby preventing initiation of an immune response leading to destruction of the myelin sheath.

C. Rationale And Significance

As of 2010, there are between 2 and 2.5 million people globally affected by MS resulting 18,000 deaths [17][18]. The rates of MS also appear to be increasing though it is unclear whether this is due to more accurate detection or MS if it is affecting more people [17]. Regardless of these differences, the fact that MS rates are increasing represents a growing concern. The need to develop an effective therapy becomes even more urgent in light of these statistics. As previously mentioned, most treatment methods revolve around treating and alleviating the symptoms of MS. Our research aims to tackle this neurodegenerative disorder by developing a protein inhibitor that will alter the autoreactive MOG antibodies ability to bind to MOG and initiate an immune response. By targeting the causation of the disease, we are able to prevent MS symptoms from manifesting. This unique approach shifts the focus from treating MS symptoms to treatment of the cause of MS. In addition to potentially eliminating MS disease symptoms, our research will also be beneficial in advancing the field of protein engineering and further exploring the antibody-mediated demyelination mechanism of MOG.

Protein engineering is a valuable tool because it allows for the development of unique proteins with novel behavior and function. The therapeutic uses for designing proteins are manifold and may potentially offer new avenues of treating diseases. For example, scientists approached malaria with a structure-based drug design and have developed “novel selective and irreversible mosquito acetylcholinesterase inhibitors for controlling malaria and other mosquito-borne diseases” [19]. Our research will also further elucidate the antibody-mediated demyelination mechanism and the role of MOG as a primary contributor in the pathogenicity of MS. MOG has not been conclusively shown to be the direct cause of MS because studies have shown that the increasing risk of developing multiple sclerosis may be due to cross-reactivity between MOG and Epstein-Barr nuclear antigen [20]. Overall, our research will advance current knowledge of protein engineering, conclusively identify the role of MOG and develop a more targeted treatment for MS.

D. Research Objectives And Hypotheses

Primary Objectives:

Obtain three dimensional structure of MOG and autoreactive MOG antibodies in order to identify critical active MOG and antibody interaction sites for inhibitor modeling and design

Design several functional competitive inhibitor protein for autoreactive MOG antibodies by incrementally assembling small molecular pieces within the constraint of the binding pocket. Use comparative modeling to create several target proteins from a homologous (related) protein

Use hybrid scoring functions, which combine the strength of intermolecular forces between all atoms and the assumption that certain groups or atoms are more likely to bind because they are energetically favorable in the complex, to determine the docking affinity of our protein inhibitors and the autoreactive MOG antibodies

Select the protein with the highest affinity for the autoreactive MOG antibodies

Hypothesis:

Our novel competitive inhibitor protein will selectively bind to autoreactive MOG antibodies thereby inhibiting their ability to target the extracellular Ig-domain of MOG and inducing a signal cascade leading to the inflammation and degradation of the myelin sheath of the axon

Predictions:

The competitive inhibitor protein will be assembled and folded into a structure with high affinity for a binding pocket of the autoreactive MOG antibodies. Even if the protein does not bind the autoreactive MOG antibodies, it will provide more data and information about the principles of protein folding and design. This may further the discipline of protein engineering and offer new perspectives regarding design of novel therapeutic proteins

The competitive inhibitor protein will inhibit the ability of autoreactive MOG antibodies to bind to MOG. MS disease symptoms should not be observed because the autoreactive MOG antibodies will not be able to initiate an immune attack on the myelin sheath of the axon. This will also further elucidate the role of MOG in mediating the demyelination of the axon. It will allow us to investigate the role of MOG as a primary contributor to the pathogenicity of MS.

E. Methods

Overview of Methods

This research will be broken down into three overlapping phases of research. In Phase I, biochemists will use The Human Genome Project database and other forms of literature to identify the MOG gene sequence and the nine gene variations of the isotopes. These sequences will be used to design amino acid sequences of the epitope of the extracellular Ig-domain where antibodies will bind. Because MOG is a transmembrane protein, our biochemists will focus on “removing” the bulk of hydrophobic transmembrane regions of the protein without compromising the structure of the extracellular epitope region. These modifications will be designed using molecular modeling programs, mathematical programs, and computational chemistry programs. Based on these analyses, a new protein will be designed with a similar amino acid sequence such that it folds to resemble the epitope where the paratope of the antibody binds.

Phase II begins when a gene sequence is be deduced from the newly engineered inhibitor protein’s amino acid sequence and this gene segment is inserted into the pUC19 plasmid through restriction enzyme cloning procedures as the coding region. This plasmid will be used to transform the Origami E. coli strain and transformation will be confirmed by bacterial growth on media containing the plasmid’s resistance marker’s antibiotic. Colonies will continue to be cloned and subjected to “pressured” expression of the plasmid’s genes. The protein will be harvested and purified using Ni-NTA magnetic beads. The proteins will then be tested for accuracy through Western Blot analysis using anti-MOG antibodies.

Phase III begins if the proteins run through the Western Blot and successfully bind to the anti-MOG antibodies consistently after repeated trials. Phase III will evaluate the effectiveness of the novel protein inhibitor by analysis in a living mouse model. The newly engineered protein will be introduced to an EAE Induced mouse model to analyze the efficiency of the inhibitor in a living system by observation of regressing EAE symptoms in mice over time. Ability of the protein inhibitor to sustain subdued symptoms over time will also be monitored and used to test the efficiency of the novel protein inhibitor.

Phase II:

Restriction Enzyme Cloning

The designed primers will be used to add EcoR1 to the end of the PCR fragment. Agarose gel electrophoresis will be performed to verify the desired product. The cloning plasmid and DNA fragment to be cloned will then be digested with EcoR1 to generate a linearized plasmid and DNA with overhanging ends. The linearized plasmid and the

digested fragment to be cloned will then be analyzed by agarose gel electrophoresis. Uncut plasmid and DNA fragment will be included as a control in order to identify the digested versions. The sequence of interest will then be ligated into the linear plasmid. The transformation kit will be used to transform 1-5 µl of ligation reaction into competent cells. These will be plated onto petri plates containing ampicillin and then we will perform a blue/white screening to verify the presence of the DNA. Several white colonies will be selected for PCR and then plasmids will be isolated using a miniprep kit and the insert will be sequenced to confirm the correct insert.

Transformation of Origami E.coli

Origami E. coli will be prepared by growing a 4 mL broth overnight at 37C from a single colony isolate. 1:50 Dilutions will be prepared using the overnight culture and SUPER-COMP media. These will be incubated to reach an OD of 0.5-0.6 and then kept on ice for 15 minutes. The cultures will then be spun for 15 minutes at 3000x g and the pellet will be resuspended with 0.10 mL of Transform-It for each reaction that will be run. 0.10 mL of the resuspension will be aliquotted into Eppendorf tubes and kept on ice. Our recombinant pUC19 plasmids will then be added and will be kept on ice for 10 minutes, moved to room temperature for 10 minutes, and put on ice once more for 10 minutes. The solution will then be added to 0.90 mL of SUPER-COMP media and incubated for 60 minutes at 37C in a shaker. 0.10 mL will then be added to an LB-Ampicillin plate and spread using Roll& Grow Plating Beads. The plates will then be incubated overnight at 37C and growth will indicate a successful transformation.

Bacterial Cell Culture Techniques

Untransformed cultures of bacteria will be grown on plates of LB Agar and single colony isolates will be obtained. Single colony isolates will be used to grow turbid liquid cultures in Luria Broth. Frozen stocks will be prepared by adding ~15 ml of liquid culture to ~20 ml of glycerol and storing in falcon tubes at -80C.

Verified transformed colonies from growth on ampicillin-LB Agar, will be grown on LB Agar to obtain single colony isolates. These transformants will also be stored at -80C by the same procedure as mentioned above except 1g of ampicillin will be added to each mL of Luria Broth in broth cultures used for frozen stock prep. All LB Agar plates will be grown overnight at 35C. All broths will be grown overnight at 35C in shakers.

His-tag protein purification using Ni-NTA magnetic beads

30 µl of magnetic suspension will be placed in a magnetic rack (Dynal MPC-S) and the magnet will be applied to remove the supernatant. The magnetic beads (Qiagen Ni-NTA) will be washed with 300 µl of magnetic PBS, and then resuspended in 50 µl of extraction buffer. 1.3 ml of the bacterial culture will be spun for 5 min at 13,000 rpm and the pellet will be stored at -20° C. 300 µl of extraction buffer will then be added to resuspend the

cells and the suspension will incubate at room temperature for 30 min. The cell suspension will then be homogenized by sonication in the ultrasonic water bath (Branson 200) for 3 min followed by centrifugation for 10 min at 13,000 rpm. The supernatant will be removed and transferred to the prepared magnetic beads that were allowed to incubate in the rocking device (Dynal) for 30 min at room temperature. Again, the magnet will be applied to remove the supernatant and then the beads will wash with 300 µl of washing buffer for 30 min at room temperature using the rocking device (Dynal). The magnetic beads will be washed again with 300 µl of PBS for 10 min and then 12 µl of SDS sample buffer will be added to the beads and allowed to boil for 3 min to elute our protein of interest from the magnetic beads. The protein will be stored at -20°C and an equal volume of pure glycerol will be added to the protein to avoid freezing.

Western Blot Analyses of Protein

Purified Protein samples and standards will be added to a12% SDS-PAGE gel and run for one hour at 100 volts. The gel will then be transferred to a PVDF membrane with anti-MOG antibodies and developed in order to show protein purification. Protein “functionality” will be determined qualitatively by analysis of the developed PVDF membrane. Multiple purified protein samples can be run in the same SDS-PAGE gel.

Phase III:

EAE Induction by Active Immunization in C57BL/6 Mice

To induce the inflammatory demyelinating disease of the CNS, EAE will be induced in C57BL/6 mice using the Hooke Kit TM MOG1-125/CFA Emulsion PTX. We chose the MOG1-125

antigen kit because the MOG (1-125) peptide is especially relevant for testing therapeutics that specifically target B-cells. The MOG protein is also highly conserved between mice/ rats and humans, which allows us to utilize our novel anti-MOG antibody protein inhibitor in the mouse model. Our protein inhibitor seeks to bind the paratope of the antibodies secreted by B-cells. Female C57BL/6 mice of 10 weeks or older were used in all models and allowed to acclimate for at least 7 days prior to immunization. The antigen emulsion was administered subcutaneously at two sites: on the upper back with 0.1 ml of emulsion and then on the lower back with another 0.1 ml of emulsion. After two hours, a 0.1 ml/dose of pertussis toxin solution (PTX) was administered intraperitoneally. Twenty-four hours later, PTX administration was repeated for all mice. On day 7, we began to monitor the mice for signs of EAE. When symptoms of EAE began to manifest in the mice, 0.1 ml of our protein inhibitor was administered intravenously. If our protein inhibitor is successful then we would expect the symptoms of EAE to regress.

F. Research Timeline

1. The timeline will be broken down into three overlapping Phases as described in the

methods section. Movement into subsequent phases will be dependent on the

productivity of the previous phase, and timelines will be arranged around processing

and analysis of each novel gene sequence generated in Phase I. The majority of the

work done in Phase I will be conducted by Biochemists. The majority of the work

done in Phase II and Phase III will be conducted by Cell/Molecular Biologists.

Researchers involved will work full-time on this project for a total of three years.

2. Phase I: Researchers will engineer anti-MOG antibody protein inhibitors and develop

gene sequences to be inserted into plasmids for protein production during the

first eighteen months or until a protein has reached Phase III. Once a protein has

reached Phase III, researchers will aim to gather more information on the protein’s

structure, function, and isoforms for the remainder of the project timeline.

3. Phase II: No more than 6 gene sequences will be processed through Phase II at any

given time. Researchers will begin Phase II once provided with gene sequences from

Phase I and will continue to process, analyze, and move proteins into Phase III until

four months before the 3 year timeline is complete. Gene sequences will be inserted

into the pUC19 plasmid using restriction enzyme cloning. Plasmids will then be

amplified and used to transform E. coli. Transformed strains will then be maintained

and the proteins will be harvested/purified from the cultures. Purified proteins will

then be analyzed using Western Blot. Ten weeks will be allowed for a protein gene

sequence to be processed through Phase II. (including experimental error; these

sequences will be stored and noted for future consideration) By the tenth week, the

protein must move into Phase III or be deemed “nonfunctional”. The

“nonfunctional” protein sequences will be sent back to Phase I and further

molecular analyses will be conducted.

4. Phase III: Phase III of this research will begin once an efficient anti-MOG antibody

protein inhibitor has been generated from Phase II and analyzed using the Western

Blot analysis. The final phase will include testing for anti-MOG antibody inhibition

through EAE Induction by active immunization in mice. This will take the remainder

of the allotted research timeline. The final phase may not be conducted if the

previous three steps are unsuccessful at yielding our desired novel protein inhibitor.

Analysis of each protein in the mouse model should take no longer than eight

weeks.

G. Budget

Equipment:

New England BioLabs Custom Sequence $5/sequenceNew England BioLabs Taq DNA Polymerase with ThermoPol®Buffer

$250

New England BioLabs pUC19 Vector $260New England BioLabs EcoR1 $440New England Biolabs Taq DNA Ligase $296ThermoScientific TransformAid Bacterial Transformation Kit

$58

Fisher Scientific Ampicillin (25 g) $100Fisher Scientific Petri plates (500) $130Sigma Aldrich Lysogeny broth (1 kg) $123Fisher Scientific Bacto Agar (2 kg) $850EMD Millipore Origami™ Competent Cell Set $223Qiagen Ni-NTA Magnetic Agarose Beads (2 x 1 ml)

$216

LifeTechnologies Dynabeads®MPC®-S (Magnetic Particle Concentrator)

$598

LifeTechnologies Western-Star Immunodetection System with Goat Anti-Mouse IgG + IgM AP Conjugate

$392

EMD Millipore Anti Myelin-Oligodendrocyte Glycoprotein Antibody (MAB5680)

$399

Jackson Laboratories C57BL/6 Mice (30 females)

$900

Hooke Kit™ MOG1-125/CFA Emulsion PTX $1714Mice cage (30) $539.70LabDiet 5001 mice food (50 lb) $30.99Total minimum budget $7,519.69

Labor:

Biochemistry Director $5-7,000/monthBiochemists (10) $2,900/monthLab Technician – part time ($25 hrs/week) undergraduate students (10)

$1,000/month

Cell Biologist/Microbiologist Director $7,500/monthCell Biologist (10) $2,900/monthTotal yearly wage $220,800/yr

Facility:

Cellular Biology Laboratory Rental $216,666.67/yr

H. References and Literature Cited

1. Berger, Thomas and Markus Reindl. “Multiple sclerosis: Disease biomarkers as indicated by pathophysiology.” Journal of the Neurological Sciences, 259 (2007), pp. 21-26

2. Greer J, Erickson JW, Baldwin JJ and Varney MD. “Application of the three-dimensional structures of protein target molecules in structure-based drug design.” Journal of Medicinal Chemistry, 37 (1994), pp. 1035-54

3. Drukey BJ and Lydon NB. “Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia.” Journal of Clinical Investigation, 105 (2000), pp. 3-7

4. Daniel K. Hartline. “What is myelin?” Neuron Glia Biology, 4 (2008), pp. 1531-635. Chari DM. “Remyelination in multiple sclerosis.” International Review of

Neurobiology, 79 (2007), pp. 589-6206. Irvine K.A. and Blakemore WF. “Remyelination protects axons from

demyelination-associated axon degeneration.” Brain, 131 (208), pp, 1464-14777. Y Shi, Y Feng, J Kang et al. “Critical regulation of CD4+ T cell survival and

autoimmunity by beta-arrestin 1.” Nature Immunology, 8 (2007), pp. 817-248. V Viglietta, C Baecher-Allan, HL Weiner and DA Hafler. “Loss of functional

suppression by CD4+ CD25+ regulatory T cells in patients with multiple sclerosis.” Journal of Experimental Medicine, 199 (2004), pp. 971-79

9. CL Langrish, Y Chen, WM Blumenschein et al. “IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.” Journal of Experimental Medicine, 201 (2005), pp. 233-40

10. H Kebir, K Kreymborg, I Ifergan et al. “Human T(H)17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation.” Nature Medicine, 13 (2007), pp. 1173-75

11. JS Tzartos, MA Friese, MJ Craner et al. “Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis.” American Journal of Pathology, 172 (2008), pp. 146-55

12. Tomassini V, De Giglio L, Reindl M, Russo P, Pestalozza I, Pantano P and Pozzilli C. “Anti-myelin antibodies predict the clinical outcome after a first episode suggestive of MS.” Multiple Sclerosis Journal, 9 (2007), pp. 1086-94

13. Roth MP, Malfroy L, Offer C, Sevin J, Enault G, Borot N, Pontarotti P and Coppin H. “The human myelin oligodendrocyte glycoprotein (MOG) gene: complete nucleotide sequence and structural characterization.” Genomics, 2 (1995), pp. 241-50

14. Kieseier, Bernd C. “The Mechanism of Action of Interferon-β in Relapsing Multiple Sclerosis.” CNS Drugs, 6 (2011), pp. 491-502

15. Arnon R and Aharoni. “Mechanism of action of glatiramer acetate in multiple sclerosis and its potential for the development of new applications.” Proceedings of the National Academy of Scienes of the United States of America, 101 (2004), pp. 14593-98

16. Marriot J.J, Miyasaki JM, Gronseth G and O’Connor PW. “Evidence Report: The efficacy and safety of mitoxantrone (Novatrone) in the treatment of multiple sclerosis: Report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology.” Neurology, 74 (2010), pp. 1463-70

17. Milo R and Kahana E. “Multiple sclerosis: geoepidemiology, genetics and the environment.” Autoimmunity Review, 5 (2010), pp. 389-94

18. Lozano R. “Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systemic analysis for the Global Burden of Disease Study 2010.” Lancet, 380 (2012), pp. 2095-128

19. Dengfeng Dou, Jewn Giew Park, Sandeep Rana, Benjamin J. Madden, Haobao Jiang and Yuan-Ping Pang. “Novel Selective and Irreversible Mosquito Acetylcholinesterase Inhibitors for Controlling Malaria and Other Mosquito-Borne Diseases.” Scientific Reports, 3 (2013)

20. H. Wang, KL Munger, M. Reindl, EJ O’Reilly, LI Levin, T. Berger and A. Ascherio. “Myelin oligodendrocyte glycoprotein antibodies and multiple sclerosis in healthy young adults.” Neurology, 71 (2008), pp. 1142-46