assignment
Rationale of project Multiple Sclerosis is a neurodegenerative disease that abruptly onsets during one of the most eventful and productive stages of life. The disease is the most common neurological disability of young adults and onsets between the ages of 20-40 when people usually begin their careers and start their families (1). Therefore, the emotional burden of this disease is great. Progression of the disease is slow and can begin with fatigue, cognitive decline, neuropathic or musculoskeletal pain, mobility problems and visual impairment (2). This can dramatically impair one’s quality of life and may prevent one from performing their normal daily functions (3). 85% of cases present with relapsing remitting MS (RRMS) which causes episodes of inflammation leading to neurological damage. Of those people who present with (RRMS), 50% of people with RRMS will progress to secondary progressive MS which causes a continual accumulation of neurological damage (4). Although current therapies aim to slow disease progression or manage symptoms, there is no cure and no treatments to reverse the damages to cognition in MS. Concerningly, the prevalence of the disease is increasing. Data shows a 30% increase in MS cases from 2013 to 2021 (5). Current treatments aim to either focus on repairing MS relapses through steroid treatment or treat specific MS symptoms or modify the disease through delaying the progression of MS. Corticosteroids like methyl prednisone act to quiet the immune response however they have no long-term effect and cause secondary problems such as a heightened risk of infection, osteoporosis, and diabetes (6,7). Treating specific symptoms may help relieve the patient of some uncomfortable symptoms but cannot address the underlying problem. Treatments such as donepezil used to treat cognitive impairment cannot repair the damage caused to the myelin sheath and will be untreatable as the disease progresses (6). Lastly, disease modifying therapies only focus on targeting the immune system to delay the severity and number of relapses. Unfortunately, drugs like Cladribine can have secondary consequences to use such as the development of cancer. Interferon beta is a commonly used MS treatment however up to 50% are non-responsive due to the development of neutralising antibodies to IFb. Patients also suffer unpleasant symptoms such as flu-like symptoms which negatively impact the patient’s quality of life (8). Therefore there is a need for treatments that can be more tolerable and more effective in reversing the damage caused in the progression of MS. As previously stated, there are no current drugs on the market that target the underlying causes of MS to lead to disease reversal. Demyelination of neurons is the cause of the neurological symptoms associated with MS and can be triggered by a range of different mechanisms within the patient (https://dx.doi.org/10.1016%2FB978-0-444-52001-2.00004-2). By disrupting one of these mechanisms with a new drug therapy, remyelination could occur. The trigger for demyelination is still not fully understood, but it is known that remyelination is inhibited by a disease-altered microenvironment surrounding the neurons (https://doi.org/10.1038/s41467-021-22263-4). This microenvironment contains various extracellular components that cause different inhibitory pathways of remyelination. One of the most common pathways is through the interaction of these extracellular components with oligodendrocyte progenitor cells (OPCs)—cells which differentiate into oligodendrocyte cells—to inhibit their differentiation into myelinating cells, thereby preventing remyelination (https://doi.org/10.1093/brain/awab106). A pathway of interest is LINGO-1. LINGO-1 forms a protein complex expressed on neurons and OPCs that comprises of the Nogo-66 receptor (NgR1) and either a p75 NT receptor or a TNF receptor orphan Y (TROY) receptor (doi: 10.1517/14712598.8.10.1561). This protein complex is bound by myelin derived proteins, leading to an intracellular activation of RhoA GTPase, triggering inhibition of axonal outgrowth and oligodendrocyte differentiation. Mechanisms triggered include blocking of axon growth cone to guide extension of axons (10.4103/1673-5374.213538) and inhibition of the PI3K/AKT/m-TOR signalling pathway to stop myelin membrane wrapping of CNS axons. (10.1038/srep14235) Hypothesis The proposed treatment of interest involves the development of a cyclic peptide through RaPID mRNA display. This peptide needs to sufficiently bind to LINGO-1, thereby preventing binding of activator molecules and allowing for remyelination to occur. This is a significant area of research since no current drugs on the market target this niche as a treatment, nor do they reverse the symptoms of MS. If this binding is successful, then patients could heal from the demyelination inflicted upon their neurons, and potentially revert to a non-disease state. This drug would be taken in conjugation with another current MS drug in order to manage the neurological symptoms of the disease. LINGO-1 is a good drug target because Aims of the project The overall aim is to develop a drug that stimulates remyelination in the CNS for MS patients. This drug will be developed as a cyclic peptide/bicycle inhibitor for LINGO-1 using RaPID. The drug of interest will be selected by using chemical binding assays to discover a binding peptide which sufficiently disrupts the Nogo-66 receptor. Once the drug has been synthesised, it will be tested in vitro to see whether it’s inhibition of Nogo-66 is sufficient to allow OPC differentiation into oligodendrocytes, and this can be done through OPC assays which allows the visualisation of this cell’s growth (https://dx.doi.org/10.1186%2Fs13104-016-2220-2). Following this, the drug needs to be shown to be able to cross the blood-brain barrier (BBB) before it can undergo in vivo studies. The most promising method for this is through conjugation with dNP2, a cell permeable peptide, which has previously been shown to carry cargo up to 120 kDa across the BBB (https://dx.doi.org/10.1602%2Fneurorx.2.1.1). Once this mechanism has been determined, in vivo testing can commence on mice, using an EAE mice model, the most common experimental model for human MS. Drug Delivery The delivery of drugs to the central nervous system (CNS) and the brain has been a considerable and long-standing pharmacological challenge for the treatment of neurological diseases due to the blood-brain-barrier (BBB). Traditional approaches for traversing the BBB have focused on tailoring the physico-chemical properties of drug molecules to conform with Lipsinki’s CNS Modified “rule of five” emphasising lipophilicity and low molecular weight with approximately 70% of all FDA approved CNS drugs conforming to the modified Lipsinki criteria.1 Recent research has explored the use of peptides, particularly cell-permeable peptides (CPPs) such as cationic CPPs as brain drug delivery vectors due to their versatility, low toxicity and immunogenicity, their facile production, and their ability to cross cell membranes without causing significant membrane damage.1, 2 The human-derived cationic CPP dNP2 first reported by Lim and coworkers3 has been demonstrated to effectively cross the BBB with conjugated cargos of up to 120 kDa in size in in vivo multi-photon microscopy studies of C57BL/6 mice. Staining of the mice brains showed the dNP2-dTomato conjugate had been localised to brain tissue, including co-localisation with neurons, astrocytes and microglial cells, demonstrating dNP2’s effectiveness in delivering cargo to the CNS past the BBB.3 Indeed, this peptide has successfully delivered cargo across the BBB and ameliorated the effects of experimental autoimmune encephalomyelitis (a mouse model for MS) in vivo in mice;3, 4 highlighting its applicability and suitability for delivering our proposed drug to the CNS for the inhibition of LINGO-1. Furthermore, a 14 day repeat dose toxicity study conducted by Lim and colleagues in C57BL/6 mice intraperitoneally administered 5 mg/kg of dNP2-dTomato once every second day reported no adverse effects or signs of toxicity, yielding a preliminary toxicological characterisation of our proposed drug delivery peptide.3 Despite reported experimental success of dNP2 the mechanism by which it—and CPPs—transport large molecular cargos across the BBB remains unclear, however, there is a consensus that the mechanism depends on endocytosis and a direct penetration mechanism.1, 2 One disadvantage of the CPP class of molecular transporter is their broad distribution to other organs as well as the CNS.1 Experimental design Aims for convenience: 1. Develop a cyclic peptide/bicycle inhibitor for LINGO-1 2. Test its binding activity to determine its binding site and whether that would inhibit Nogo receptor/p73?? Interactions 3. Test in vitro to see whether it stimulates OPC differentiation (assays) etc. Immunofluorescence 4. Develop delivery mechanism to get things through the BBB 5. test/analyse multiple drug delivery mechanisms past BBB and optimise 6. Test in vivo - mice, toxicology Chemistry tests: For aim 1: RaPID methods to discover binding ligand · Make synthetic DNA libraries · Ensure the start codon ATG codes for an acyl chloride (use engineered flexizyme) · Transcribe the DNA, ligate puromycin terminator to the 3’ end of the DNA (end codon has puromycin) · Translate the RNA, making the cyclic peptides (Cysteine has to be in it somewhere to cyclise it) · Pan the peptide-RNA conjugates against the immobilised protein · wash/blow off the peptides that don’t bind · Remove the peptides that do bind · Sequence the RNA and determine what the protein sequences were · Mutagenise if necessary, bias DNA library towards more similar sequences, repeat. For aim 2: Alanine scans + SPR: · Mutate specific residues on the cyclic peptide in turn, and test binding to immobilised protein using SPR. · See change in binding upon changing a residue to alanine - the more it decreases, the more important that amino acid was for binding. · Also do SPR on its own to test binding of the RaPID hits first. Aim 2: finding out binding location - X-ray crystallography · Co-crystallise LINGO1 and RaPID ligands, see where they bind after sending them to Melbourne’s synchrotron, data collection (likely at ungodly hours of the morning), structure solving (very tedious) etc. · Maybe even cryoEM? Just protein + ligand tests: X-ray crystallography to determine PPIs, binding location, NMR spectroscopy, fluorescence etc. In vitro – cell culture: Based on testing of previous LINGO inhibiting agents, in vitro testing will be performed to see whether our anti-LINGO agent will be able to stimulate OPC differentiation. Previous analysis of LINGO-1 expression in purified CNS cells via reverse transcription and polymerase chain reaction, showed there is a high expression in neurons, some expression in oligodendrocytes and very little expression in astrocytes. Such research negates the need to investigate LINGO-1 expression further. Certain methods must however be taken to analyse if our anti-LINGO agent promotes oligodendrocyte differentiation, axonal myelination as predicted. For culturing, embryonic dorsal root ganglion neurons dissected from embryonic Long Evans rats and populations of oligodendrocytes from female Long Evans P2 rats will be grown in vitro. Oligodendrocyte precursors (A2B5+) will be collected and allowed to differentiate into O4+ and MBP+ mature oligodendrocytes. A2B5+ cells will be cultured in the presence or absence of our anti-LINGO agent to analyse promotion of axonal myelination while O4+ oligodendrocytes were stained with antibodies in the presence or absence to analyse whether oligodendrocyte differentiation increases due to the anti-LINGO agent. Immunohistochemistry will be used to analyse monoclonal antibodies against factors such as O4, MBP and LINGO-1 to confirm the predicted increase in oligodendrocyte differentiation and myelination in the presence of the anti-LINGO agent. Concluding paragraph - emphasising the importance of above results. – 200 words Of the experiment design^ Limitation of the project + future directions 300 words What