Molecular-Based Therapies and Technologies Transcript PDF

Transcript Molecular-based Therapies and Technologies Slide 1: Introduction............................................................................................................ 3 Slide 2: Overview of Drugs and Drug Targets.............................................................

Transcript Molecular-based Therapies and Technologies Slide 1: Introduction............................................................................................................ 3 Slide 2: Overview of Drugs and Drug Targets.................................................................... 3 Slide 3: A Brief History of Biologics..................................................................................... 4 Slide 4: Top 20 Drugs in 2021........................................................................................... 5 Slide 5: Section 1: Small Interfering RNA (siRNA)............................................................. 7 Slide 6: Revisiting Molecular Biology................................................................................. 7 Slide 7: Our Antiviral Defense............................................................................................. 8 Slide 8: How siRNA Works................................................................................................... 9 Slide 9: siRNA Market Story.............................................................................................. 10 Tab 1: Target Treatment Areas....................................................................................... 10 Tab 2: Market Activity...................................................................................................... 11 Tab 3: Benefits and Challenges..................................................................................... 11 Slide 10: Section 2: Aptamers............................................................................................ 12 Slide 11: Introducing Aptamers.......................................................................................... 13 Slide 12: Making Aptamers................................................................................................. 13 Tab 1: Step 1................................................................................................................... 14 Tab 2: Step 2................................................................................................................... 14 Tab 3: Step 3................................................................................................................... 15 Tab 4: Step 4................................................................................................................... 16 Tab 5: Step 5................................................................................................................... 16 Tab 6: Step 6................................................................................................................... 17 Tab 7: Step 7................................................................................................................... 17 Slide 13: Benefits and Challenges...................................................................................... 18 Slide 14: Uses of Aptamers................................................................................................. 19 Tab 1: Target Discovery................................................................................................... 19 Tab 2: Target Delivery..................................................................................................... 20 Tab 3: Toxin Removal...................................................................................................... 21 Tab 4: Drug Labelling...................................................................................................... 22 Slide 15: Aptamer Market Story.......................................................................................... 22 Slide 16: Section 3: Blocking Peptides............................................................................... 23 Slide 17: Receptors as Drug Targets.................................................................................. 24 Slide 18: Novel Methods to Alter Receptor Function........................................................ 24 1  Transcript Slide 19: Transport Proteins Control Surface Expression................................................. 25 Slide 20: Multiple Domain Protein Scaffolds..................................................................... 26 Slide 21: Receptor Trafficking and Anchoring Proteins..................................................... 27 Slide 22: Postsynaptic Density: Full of Proteins................................................................. 27 Slide 23: Creating Blocking Peptides to Inhibit Interactions............................................. 28 Slide 24: Blocking Peptides as Receptor Inhibitors........................................................... 29 Slide 25: Blocking Peptides as Receptor Activators.......................................................... 30 Slide 26: Benefits and Challanges...................................................................................... 30 Slide 27: Blocking Peptides Market Story.......................................................................... 31 Slide 28: Conclusion............................................................................................................ 32 Slide 29: Summary............................................................................................................... 32 2  Transcript Slide 1: Introduction Welcome to this presentation on molecular-based therapies and technologies. My name is Professor Dev and I will lead this presentation where we will explore the mechanisms of action of technologies such as siRNA, aptamers and blocking peptides. We will look at how these technologies work as well as their current status on the market. Slide 2: Overview of Drugs and Drug Targets Let’s start by reviewing the major drugs and drug targets. The major drug targets are considered to be receptors and enzymes. However, molecular drug targets such as DNA and mRNA as well as proteins can also be exploited as drug targets. There has been a focus on small molecules such as drugs, including receptor agonists, receptor antagonists and enzyme inhibitors being the major types of drugs on the 3  Transcript market. More recently, the therapeutic sector has seen an explosion in the research and development of biologics including antibodies, proteins, peptides and molecular drugs. The field has also seen a substantial increase in the commercialisation of cell therapy and medical devices. Perhaps take a moment here to consider which drug target and drug type you would invest in and how can you create novelty? When you are ready, click Next to continue. Slide 3: A Brief History of Biologics As a brief history, the first biologic drug was humanised insulin, which became available in 1982. These days, biologics include large peptides, recombinant proteins and antibodies, DNA/RNA-based molecules, synthetic vaccines and others. There has been a rapid rise in the number of approved biologicals. In 2010, only six biologics were approved, with 15 biologics being approved in 2022, getting closer to the number of small molecules being approved. Reference(s): 1. US Food & Drug Administration. (2023). New Drugs at FDA: CDER’s New Molecular Entities and New Therapeutic Biological Products. https://www.fda.gov/drugs/development-approval-process-drugs/new-drugs-fda-cders- new-molecular-entities-and-new-therapeutic-biological-products 4  Transcript Slide 4: Top 20 Drugs in 2021 Biologic drugs are not only being approved more now than ever, they are also dominating the commercial market space. Market research of the top 20 selling products in 2021 shows that 12 of the top 20 drugs were biologics, namely antibodies, vaccines or protein- based. Use the scrollbar to look through this table. When you are ready, click Next to continue. Note: Table contents Company Product Sales Disease Target/Mechanism Pfizer, Comirnaty $36.8 COVID-19 Vaccine BioNtech billion AbbVie Humira $20.7 Arthritis, Antibody TNFa mAB (Adalimumab) billion Crohn’s, psoriasis Moderna Spikevax $17.7 COVID-19 Vaccine - billion Merck & Co Keytruda $17.2 Cancers Antibody PD1 mAB (Penbrolizumab) billion BMS, Pfizer Eliquis $16.7 Deep vein Drug Anticoagulant (Apixaban) billion thrombosis BMS Revlimid $12.8 Blood Drug CD 28 T cell (Lenalidomide) billion cancers activator AbbVie, J&J Impruvica $9.8 Blood Drug BTK inhibitor (B (Ibruntinib) billion cancers cells) J&J Stelara $9.1 Crohn’s, Antibody IL23/IL12 mAB (Usekinumab) billion psoriasis, colitis Regeneron, Eylea $ 8.9 Macular Protein VEGF inhib Bayer (Afibercept) billion degeneration Gilead Biktarvy $8.6 HIV Drug HIV-1 integrase 5  Transcript billion strand transfer inhibitor BMS, Ono Opdivo $8.5 Cancers Antibody PD1 mAB (Nivolumab) billion Bayer, J&J Xarelto $7.5 Deep vein Drug Anticoagulant (Rivaroxaban) billion thrombosis Regeneron, REGEN-COV, $7.5 COVID-19 Vaccine - Roche Ronapreve billion Eli Lily Trulicity $6.5 Type 2 Protein Analogue of (Dulaglutide) billion diabetes human glucagon-like peptide-1 J&J Darzalex $6.0 Multiple Antibody CD38 mAB (Daratumumab) billion myeloma Vertex Trikafta/Kartrio $5.7 Cystic Drug Cystic fibrosis billion fibrosis transmembrane conductance regulator (CFTR) Merck Gardasil 9 $5.7 Human Vaccine - billion papilloma virus Sanofi, Dupixent $5.6 Dermatitis, Antibody IL4/IL13 mAB Regeneron (Dupilumab) billion asthma Gilead Veklury $5.6 COVID-19 Drug SARS-CoV-2- (Remdesivir) billion RNA-dependent RNA polymerase inhibitor Pfizer Ibrance $5.4 Breast Drug CDK 4/6 (Palbociclib) billion cancer inhibitor 6  Transcript Slide 5: Section 1: Small Interfering RNA (siRNA) Let’s turn to the first molecular-based biological of this session, namely siRNA, or small interfering RNA. Slide 6: Revisiting Molecular Biology In order to understand what siRNA is and how it works, we need to consider or remember our basics in molecular biology. This slide provides a summary of how proteins are made in the cell. The diagram shows the process of transcription, namely the encoding of mRNA from DNA, thereafter the generation of proteins using the mRNA code in a process called translation, and the further modification of proteins via post-translational modification events. 7  Transcript In a case where too much of a protein is generated or if a mutated protein function needs to be inhibited, we need a way to stop this over production or misfunction. siRNA technology is a powerful method to prevent protein production. siRNA molecules are small bits of RNA that target and assist in the destruction of mRNA, such that protein translation is limited or blocked. In other words, small interfering RNA (siRNA) target mRNA to stop protein production. We will look at this more closely in a moment. Image(s): 1. Dev, K. (2023). How Proteins are Made. [Graphic]. School of Medicine, Trinity College Dublin Slide 7: Our Antiviral Defense Let’s first turn to our normal antiviral defense system, which is shown in this diagram. Imagine in Step 1 that a mammalian cell is infected by a virus. This virus contains double-stranded RNA. Upon entry into the cell, in Step 2, an enzyme called DICER cuts the viral RNA into smaller parts generating fragments of RNA, which essentially, we can consider as small bits of RNA, or small interfering RNA (siRNA). In Step 3, these small pieces of RNA are recognised by the RISC complex. RISC also known as RNA-induced silencing complex, is a multi-protein complex, that binds to one strand of viral siRNA. In Step 4, the siRNA/RISC complex binds a specific compatible mRNA viral sequence within the mammalian mRNA. In Step 5, the final step, the siRNA/RISC complex cuts the mRNA. This cut mRNA can no longer create the protein thus inducing gene silencing. The discovery of siRNA was so important in the field of biology that the two scientists who discovered this process in 1998, Andrew Fire and Craig Mello, were awarded the Nobel Prize in 2006. 8  Transcript Image(s): 1. Dev, K. (2023). Antiviral Defense System. [Graphic]. School of Medicine, Trinity College Dublin Slide 8: How siRNA Works With the process of gene silencing in mind, lets focus on how siRNA specifically works. In Step 1, small interfering RNA (siRNA) sequence must be created that matches the mRNA encoding the protein that we would like to target. Typically, siRNA is double- stranded RNA of about 20 base pairs and can be synthesised in a machine. There are multiple commercial companies that provide this service. Once created, siRNA is introduced into cells by artificial or viral-based approaches. In Step 2, and similar to our antiviral defense system, the RISC (RNA-induced silencing complex) multi-protein complex, includes the Ago2 protein that binds to one strand of siRNA. In Step 3 the siRNA/RISC complex binds a specific compatible mRNA that we want to target. In Step 4, the siRNA/RISC complex cuts the mRNA. The cut mRNA can no longer create the protein, resulting in gene silencing. Image(s): 1. Dev, K. (2023). How siRNA Works. [Graphic]. School of Medicine, Trinity College Dublin 9  Transcript Slide 9: siRNA Market Story Let’s take a look at how successful this technology has been, siRNA market value and the challenges that lie ahead for this technology. Click the tabs to learn more. When you are ready, click next to continue. Tab 1: Target Treatment Areas The target tissues for siRNA include eyes, lungs, liver, skin, kidney and tumours. These are tissues where siRNA can be delivered by injection. Therapeutic areas that have been tried include age-related macular degeneration (AMD), asthma, metabolic diseases, dermatology (skin-related diseases), renal failure and cancers. You may wish to conduct research on the successes and failures of use of siRNA in these areas. 10  Transcript Tab 2: Market Activity The siRNA technology held great promise in early days. In 2006, Merck acquired Sirna Therapeutics for $1.1 billion and only to sell it in 2014 to Alnylam for a mere $175 million. Soon alongside, Novartis sold its decades-long RNAi business in a fire sale to Arrowhead for $35 million. In 2018, FDA approved the first siRNA by Alnylam, for a rare disease (mutations “transthyretin” which leads to neuropathic pain). Tab 3: Benefits and Challenges Since we know the genetic code of every protein that exists in the human cell, the key benefit of siRNA technology is our ability to design specific siRNA for each of these 11  Transcript proteins. In principle, therefore, with siRNA we can downregulate the expression of any drug target that we wish. However, significant challenges still exist, including, for example, how to deliver siRNA molecules to the right place in the body, how to get siRNA inside the cells of interest and the stability of siRNA? Novel methods are being developed for siRNA which are addressing some of these hurdles and may bring this technology for better use as therapeutics. Slide 10: Section 2: Aptamers Let’s turn to the second molecular-based biologic of this presentation, namely Aptamers. 12  Transcript Slide 11: Introducing Aptamers Aptamers are single-stranded DNA or RNA that selectively bind to a target, for example a specific protein, drug, toxin, or cell type. Aptamers are normally around 20-100 base pairs in length. Aptamers create a 3D structure, which is an extremely important property of these molecules which is perfectly shaped to the drug target. Slide 12: Making Aptamers The process of finding an Aptamer for your drug target of interest is shown in the diagram. The process is called SELEX and in simple terms, it involves seven key steps. Click the tabs to learn more. When you are ready, click Next to continue. 13  Transcript Image(s): 1. BasePair Biotechnologies Incorporated. (2020). SELEX Aptamer Selection [Graphic]. https://www.basepairbio.com/what-is-an-aptamer/ Tab 1: Step 1 In step one of the process, a single stranded DNA or RNA library of aptamers is made that contains around a million billion different aptamers. This library of aptamers is a little like a library of drugs that is used to screen for the best aptamer that binds the target choice. Tab 2: Step 2 14  Transcript In step 2, the mixture of around a million billion different aptamers is poured down a column which is coated with the target of interest. Tab 3: Step 3 In step 3, some aptamers will bind the target of interest because they fit perfectly in a pocket, domain or groove of the drug target. Remember it is the 3D structure created by each 20-100 base pair aptamer that is important for binding to the drug target. On the other hand, aptamers that don’t bind, known as non-binders, will flow through the column and be discarded. 15  Transcript Tab 4: Step 4 The bound aptamers are then eluted or washed off the column with an elution buffer - a solution designed to release the bound aptamer from the drug target. Tab 5: Step 5 The aptamer sequences are now eluted from the column. Because only very small amounts of aptamers are used from the screening library, the eluted aptamers need to be amplified. Amplification of the bound eluted aptamers can be done by polymerase chain reaction (PCR), which makes multiple copies of each aptamer that bound to the drug target and had been eluted from the column. 16  Transcript Tab 6: Step 6 The steps 2-5 are repeated with increasing and more stringent or harsh wash steps. This is done by increasing, for example, the pH or salt content of the elution buffer. Increasing the stringency of wash steps is done to isolate the aptamers that bind most strongly. Upon increasing the stringency of wash steps weak binding aptamers are washed away eventually leaving only the strongest binding aptamers. Tab 7: Step 7 Finally, the highest affinity aptamer (the one that binds the most strongly to the drug target) is selected and is deemed to be the most efficient for targeting the molecule. 17  Transcript Slide 13: Benefits and Challenges Similar to siRNA there are a number of benefits and limitations of aptamers. The benefits include that aptamers are: Small size, about a tenth of the size of antibodies and have a good distribution and may cross the blood brain barrier (BBB) They have low or no immunogenicity They are stable at heat and pH allowing survival throughout the various organs of the body, for example the stomach, and They are easy to manufacture with good batch-to-batch variation The limitations of aptamers include: They are not as well-characterised or as widely used as antibody and so there is limited availability of reagents and expertise Their specificity and affinity for target molecules can vary, and They can be expensive to produce 18  Transcript Slide 14: Uses of Aptamers There are many suggested uses of aptamers. Let’s now look at four major uses of this technology. Click the tabs to learn more. When you are ready, click Next to continue. Tab 1: Target Discovery Aptamers can be used for target discovery. Imagine we have a healthy cell and a diseased cell. We can create a column of cells that are healthy and a column of cells that are diseased, for example cancer cells. We can then take our million billion different aptamer libraries and expose them to these two cell types. The hope is that we identify specific aptamers that bind diseased cells versus healthy cells. 19  Transcript In other words, that we identify aptamers that are specific for diseased versus healthy cells. We can then isolate these specific aptamers that bind diseased cells and not healthy cells. Using purification and isolation methods, we can use these specific aptamers to isolate ‘drug targets’ that are specific to aptamers which were recognised by diseased cells versus healthy cells. In this way, we can create drug targets or biomarkers linked to diseased cells. Tab 2: Target Delivery Aptamers can also be used for target delivery. In this example we show, firstly attach (or conjugate) the aptamer to the drug. This aptamer has already been identified to bind a specific cell, tissue or even cross the blood brain barrier. Once the aptamer is attached to the drug, it can direct the drug to specific drug targets, cells or tissue. Such targeted delivery can reduce the amount of drug required and thus help in reducing drug toxicity. 20  Transcript Tab 3: Toxin Removal Aptamers can also be used to remove toxins. Imagine you had a bad reaction to a specific drug, or have been exposed to some toxins, or perhaps a venomous snake bite. In this case, we use the example of opioid overdose. About 60% of drug overdoses are due to opioid abuse equating to about 100 deaths/day in US, which has over US$75bn per year economic impact. Also, 1-2% of newborns experience opioid damage. We can create aptamers that bind specific drugs or toxins and are coupled to removal tags. In this case, the aptamer binds the toxin and the removal tag assists in the elimination of the toxin once bound to the aptamer. Removal tags can, for example, target the toxin-aptamer complex for elimination via the kidneys, and/or result in faster liver enzyme metabolism. Together the aptamer and removal tag can thus promote removal and can neutralise or assist in elimination. This approach can be used for many toxins, drug-induced toxicity or an overdose. 21  Transcript Tab 4: Drug Labelling The last use of aptamers we will look at is to label drugs. We can attach a fluorescent tag onto an aptamer that binds our drug of interest. We can therefore use the aptamer to see where the drug is distributed in the body. We can therefore use the aptamer to see how the drug is metabolised, for example, for PK/metabolism analysis of drugs. Slide 15: Aptamer Market Story Similar to siRNA, aptamers hold great promise, although this has not materialised just yet in the marketplace. Some of the key companies involved in aptamer research and development are shown on this slide. 22  Transcript You make wish to visit the websites of these companies and see how they are doing. Are they successful or are these companies struggling? There are links to these companies’ websites in the Extend section. Note: Table Contents Company Aptamer name Drug target Indication Status OSI Pegaptanib/Macugen VEGF AMD (Macular FDA Pharma/Astellas Degeneration) approved Ophthotech Zimura C5 AMD Phase 2 Corp complement Noxxon NOX-12 SDF-1 Cancer Phase 1-2 Avacta Life AVA-004 PDL1 Cancer Pre-clinical Sciences Ribomic RBM004 NGF Pain Pre-clinical Slide 16: Section 3: Blocking Peptides Another biologic technology we will look at in this presentation are blocking peptides. We will specifically cover the use of blocking peptides for use in regulating receptor function. 23  Transcript Slide 17: Receptors as Drug Targets Typical methods to regulate receptor function are listed here and include agonists, partial agonists, antagonists, inverse agonists and modulators. Can you think of other ways to regulate receptor function? When you are ready, click Next to continue. Slide 18: Novel Methods to Alter Receptor Function So how else can we alter receptor function? Have you considered what would happen if we regulate receptor trafficking to the cell surface? What would happen if we kept receptors inside cells to stop function? Also, have you considered why specific placement of a receptor is important? 24  Transcript Receptor localisation governs function. But how does a receptor know where to be placed at the cell surface? What happens if this goes wrong in disease? What happens if a receptor that should be placed in the centre of the synapse is now placed at the extrasynaptic site? Our understanding of receptor trafficking and placement as well as developing methods to regulate receptor trafficking is therefore important and can lead to new methods to regulate receptor function. Blocking peptides can be used to regulate receptor trafficking. Slide 19: Transport Proteins Control Surface Expression Surface expression is controlled by three events: Endocytosis, which is the removal of receptors from cell surface Exocytosis, which is the insertion of receptors into the cell surface Localisation, which is the exact location of the receptor at the cell surface for example synaptic/extrasynaptic Protein interactions are important for all these events as they regulate receptor surface expression. In a broad sense, there are three types of proteins that regulate receptor trafficking and placement into the cell surface: Proteins that remove receptors from the membrane Proteins that insert receptors into the membrane Proteins that stabilise receptors at the membrane, also called scaffolding proteins 25  Transcript Slide 20: Multiple Domain Protein Scaffolds There are many different types of trafficking and scaffold proteins that are involved in receptor insertion, removal and stabilisation at the cell surface. These proteins often have multiple domains that allow them to bind receptors as well as bind themselves or other proteins or other parts of the cell. In this way, trafficking and scaffold proteins can bind to and regulate receptor trafficking and placement. The slide here shows some examples of trafficking and scaffold proteins, as well as their domains. Multiple domains allow for scaffolds that can also bring a number of different receptors together in close proximity. Take some time to review this slide. When you are ready, click Next to continue. Image(s): 1. Dev, K. (2023). Protein Domains. [Graphic]. School of Medicine, Trinity College Dublin 26  Transcript Slide 21: Receptor Trafficking and Anchoring Proteins This slide shows more examples of receptor trafficking and scaffolding proteins. We can see here three examples of receptors and how each receptor can be bound to many differing types of receptor trafficking and scaffold proteins, which are all working in a co- ordinated manner to regulate receptor surface expression and localisation. Take some time to review this slide. When you are ready, click Next to continue. Image(s): 1. Dev, K. (2023). Receptor trafficking and anchoring of proteins. [Graphic]. School of Medicine, Trinity College Dublin Slide 22: Postsynaptic Density: Full of Proteins 27  Transcript The cell surface is decorated with multiple receptors and other surface-expressed proteins. Just underneath the membrane also reside hundreds and perhaps thousands of proteins that are working to hold these surface expressed proteins in place. In fact, the proteins under the membrane can be so dense they can be visualised by Electron Microscopy. This diagram shows the high density of proteins that are expressed at the post-synaptic density, which is the central site of the postsynaptic neuron. This electron micrograph shows the postsynaptic density which appears as a dense structure due to the very high content of transport and scaffolding proteins as well as receptors and surface proteins. Interestingly, you can also see synaptic vesicles in the Electron Micrograph. You are more than welcome to research some of these transport and scaffolding proteins shown in this diagram and in the diagrams on the previous slides. However, it is not a must for you to learn every single trafficking or scaffolding protein. We now have enough information to return to discuss how we can use blocking peptides to regulate receptor surface expression. Image(s): 1. Dev, K. (2023). Proteins Expressed at Postsynaptic Density. [Graphic]. School of Medicine, Trinity College Dublin 2. Heupel, K. (2007). Electron Micrograph. [Photograph]. Neural Development 3:25 doi:10.1186/1749-8104-3-25et al. Sourced from: https://commons.wikimedia.org/wiki/File:Ultra- structural_analysis_of_synapses_in_the_brainstem_of_wild- type_%28WT%29_and_transforming_growth_factor_%28TGF%29-%CE%B22_knock- out_%28KO%29_mice_at_embryonic_day_18.5.jpg Slide 23: Creating Blocking Peptides to Inhibit Interactions So how do we create blocking peptides to prevent trafficking or scaffold proteins from binding their receptors? There are three key parts to this: 28  Transcript First, we need to identify trafficking or scaffolding proteins that interact with our receptors of choice. There are many ways to do this, for example using yeast two-hybrid experiments. You may look into this type of experiment if you wish to in your own time. Second, identify and define discrete binding sites between the receptor and the trafficking or scaffolding proteins. Interactions that occur using 3-10 amino acids are suitable for blockade by blocking peptides. Interactions that occur over larger surfaces with multiple interaction sites are not suitable for blockade by blocking peptides. These type of interactions are more suitable for blockade by antibodies. Third, once trafficking or scaffolding proteins have been found and discrete binding sites have been defined, blocking peptides can be made. These blocking peptides are essentially the same 3-10 amino acids of the receptor sequence that binds the trafficking or scaffolding protein. Thus, blocking peptides are easy to design. These blocking peptides compete with the receptor, they bind the trafficking or scaffolding protein and thus prevent the receptor from binding the trafficking or scaffolding protein. By preventing the binding of certain trafficking or scaffolding proteins with the receptor we can regulate the surface expression. Slide 24: Blocking Peptides as Receptor Inhibitors The diagram shows how blocking peptides can be used to inhibit receptor function. In this case, a blocking peptide is used to prevent a trafficking protein from inserting its receptor into the membrane. No receptor reaches the membrane and resulting in an inhibitory effect. Image(s): 1. Dev, K. (2023). Blocking Peptides as Receptor Inhibitors. [Graphic]. School of Medicine, Trinity College Dublin 29  Transcript Slide 25: Blocking Peptides as Receptor Activators In contrast to our last slide, in this diagram you can see how blocking peptides can be used to promote receptor function. In this case, a blocking peptide is used to prevent a trafficking protein from removing its receptor from the membrane. There is a build-up of receptors at the membrane, resulting in an activation effect. Image(s): 1. Dev, K. (2023). Blocking Peptides as Receptor Activators. [Graphic]. School of Medicine, Trinity College Dublin Slide 26: Benefits and Challanges The key advantage of receptor blocking peptides is their ability to quickly design them 30  Transcript without needing to conduct large screening studies. Once we identify receptor trafficking proteins, we can quickly identify binding sites between the receptor and the trafficking protein. If the binding sites are located over a short number of amino acids, for example 5-10 amino acids, the blocking peptide can be created that matches this 5-10 amino acid sequence. At high concentrations, the blocking peptide will bind to the trafficking protein and competitively block the receptor from binding its trafficking protein. There are unfortunately, multiple disadvantages of receptor blocking peptides. These include: Poor stability of peptides, fast metabolism and poor distribution The possibility of producing an immune response, where the immune system will see these peptides as foreign (i.e. high immunogenicity) Difficulty in delivering these peptides to the right place in the body and inside the cells of interest Specificity of these peptides for one receptor-protein interaction over another receptor- protein interaction, in this case of where a trafficking protein binds multiple receptors. Slide 27: Blocking Peptides Market Story At present, this receptor blocking peptide technology remains at the level of the academic research lab. This technology has not reached market commercialisation. 31  Transcript Slide 28: Conclusion So, to conclude the presentation, I have provided an overview of siRNA technology, aptamer technology and the potential use of blocking peptides to target receptor trafficking. All three of these represent a novel approach to classic receptor pharmacology. Slide 29: Summary In summary, having completed the presentation, you should be able to: Describe mechanisms of action of siRNA technology Explain the mechanism of action of aptamer technology 32  Transcript Describe the mechanism of action of receptor blocking peptides List the pros and cons of each of these three technologies State the therapeutic status of these three technologies I hope you enjoyed this presentation. 33