Interferons

1. Interferons By: Katy Nassif

2. Discovery of Interferons • 1957 • Isaacs and Lindenmann • Did an experiment using chicken cell cultures • Found a substance that interfered with viral replication and was therefore named interferon • Nagano and Kojima also independently discovered this soluble antiviral protein

3. What are Interferons? • Naturally occurring proteins and glycoproteins • Secreted by eukaryotic cells in response to viral infections, tumors, and other biological inducers • Produce clinical benefits for disease states such as hepatitis, various cancers, multiple sclerosis, and many other diseases • Strucurally, they are part of the helical cytokine family which are characterized by an amino acid chain that is 145-166 amino acids long

4. General Action of Interferons are small proteins released by macrophages, lymphocytes, and tissue cells infected with a virus. When a tissue cell is infected by a virus, it releases interferon. Interferon will diffuse to the surrounding cells. When it binds to receptors on the surface of those adjacent cells, they begin the production of a protein that prevents the synthesis of viral proteins. This prevents the spread of the virus throughout the body. Interferons • Three types of interferons: alpha, beta and gamma.

5. Type I Interferons • Type I: alpha and beta • Alpha interferons are produced by leukocytes • Beta interferons are produced by fibroblasts • Both bind to interferon cell receptors type 1 and both encoded on chromosome 9 • They have different binding affinities but similar biological effects • Viral infection is the stimulus for alpha and beta expression • Used to mobilize our 1st line of defense against invading organisms • Largest group and are secreted by almost all cell types

6. The exact mechanism of type I interferons are not fully understood, but this is an idea of what happens: • Alpha and beta bind to heterodimeric receptor on cell surface. • Alpha receptor is made up of at least 2 polypeptide chains: IFNa-R1 and IFNa-R2 • IFNa-R1 is involved in signal transduction • IFNa-R2 is the ligand-binding chain that also plays a role in signal transduction • Ligation induces oligomerisation and initiation of the signal transduction pathway • This results in phosphorylation of signal transductors and activators of transcription proteins, which translocate to the nucleous as a trimeric complex, ISGF-3. • ISGF-3 activates transcription of interferon stimulated genes, with many biological effects.

7. Type II Interferon (gamma) • Bind to type 2 receptors and its genes are encoded on chromosome 12 • Initially believed that T helper cell type 1 lymphocytes, cytotoxic lymphocytes and natural killer cells only produced IFNg, now evidence that B cells, natural killer T cells and professional antigen-presenting • cells secrete IFNg also. • Gamma production follows activation with immune and inflammatory stimuli rather than viral infection. • This production is controlled by cytokines secreted by interleukin 12 and 18.

8. Interferon Gamma Receptor • Composed of two ligand binding IFNg-R1 chains associated with two signal transducing IFNg-R2 chains • The IFNg-R2 chain is generally the limiting factor in IFNg responsiveness, as the IFNg-R1 chain is usually in excess. • The IFNg-R1 intracellular domain contains binding spots for the Jak 1, latent cytosolic factor, signal transducer and activator of transcription (Stat1) • IFNg only associates with IFNg-R2 when the IFNg-R1 chain is present.

9. Interferon Gamma Receptor and Signalling Pathway Receptors are encoded by separate genes (IFNGR1 and IFNGR2, respectively) that are located on different chromosomes. As the ligand-binding (or a) chains interact with IFN-g they dimerise and become associated with two signal-transducing chains. Receptor assembly leads to activation of the Janus kinases JAK1 and JAK2 and phosphorylation of a tyrosine residue on the intracellular domain of IFN-gR1. This leads to the recruitment and phosphorylation of STAT1, which forms homodimers and translocates to the nucleus to activate a range of IFN-g-responsive genes. After this, the ligand-binding chains are internalised and dissociate. The chains are then recycled to the cell surface.

10. Different Interferon Drugs • Interferons are broken down into recombinant versions of a specific interferon subtype and purified blends of natural human interferon. • Many of these are in clinical use and are given intramuscularly or subcutaneously • Recombinant forms of alpha interferon include: • Alpha-2a drug name Roferon • Alpha-2b drug name Intron A • Alpha-n1 drug name Wellferon • Alpha-n3 drug name AlferonN • Alpha-con1 drug name Infergen • Recombinant forms of beta interferon include: • Beta-1a drug name Avonex • Beta-1b drug name Betaseron • Recombinant forms of gamma interferon include: • Gamma-1b drug name Acimmune

11. Alpha Interferon-2a (Roferon A) • Protein chain that is 165 amino acids long • Produced using recombinant DNA technology • Non-glycosylated protein • Short half life, short terminal elimination of half life, a large volume of distribution, and a larger reduction in renal clearance. • These problems were resolved by pegylating alpha-2a resulting in peginterferon alpha-2a that is named Pegasys.

12. Pegylated Interferon-2a (Pegasys) Pegasys is recombinant interferon alpha-2a that is covalently conjugated with bis-monomethoxy polyethylene glycol (PEG) Background: • First developed by Davis, Abuchowski and colleagues in the 1970s • In early 1990s PEG attached to alpha-2a, but it lacked the required profile of improving pharmacokinetics • Pegylation of interferon alpha-2b was achieved with the addition of a linear PEG, designed to degrade to allow the full potency of the interferon, while achieving a longer half-life.

13. Structure: • PEG moieties are inert, longchain amphiphilic molecules that are produced by linking repeating units of ethylene oxide. • Can be linear or branched in their structure • Increasing the size with PEG, the absorption and ½ life are prolongued and the clearance of the IFN is decreased. • Goal of pegylation is to decrease clearence, retention of biological activity, get a stable linkage and enhance water solubility CH3—(OCH2CH2)n--OH • Pegylation is achieved by the covalent attachment of PEG derivatives that utilize amino groups of lysines and the N-terminus of polypeptide molecules as the modification site mPEG—O—O2C—C—NH O mPEG—O—O2C—NH—(CH2)4

14. Interferon Beta-2a (Avonex) • FDA approval on May 17 1996 for Relapsing Remitting MS • Clinical trials showed that it slowed MS progression and had an extra benefit of slowing or preventing the development of MS-related brain atropy. • The exact mechanism of IFN beta activity in treating MS is unknown, but studies have shown that interlukin 10 levels in the cerebrospinal fluid were increased in patients • Structurally IFNb-2a is a 166 amino acid glycoprotein. • Produced by recombinant DNA technology using genetically engineered mammalian cells which the human beta gene has been introduced into • Amino acid sequence is the same as human beta interferon. They are both glycosylated at the asparagines residue at position 80 • Some side effects include: • Flu-like symptoms • Muscle aches • Chills

15. Combination Therapy with Ribavirin • Many times interferons and peginterferons are used in combination with Ribavirin • It is a purine nucleoside analogue with a modified base and a D-ribose sugar moiety • 1st made in 1970 by Drs. Joseph Witkowski and Roland Robins • It inhibits the replication of a variety of RNA and DNA viruses and is serves as an immunomodulator to enhance type 1 cytokine production. This increases the end of treatment response and reduces post-treatment relapse. • Mechanism is not well known, but there are 4 proposed mechanisms

16. Conclusion • Interferons have overlapping but different biological activities • Their mechanisms of action are not fully understood, therefore there is a lot of room for future growth within this field • Interferon based strategies can possibly be further tailored to each individual patient according to early response dynamics • Other immunomodulatiors that are being tested include: Zadaxin and Ceplene

17. References • 1. Al-Hasso, Shahla. “Interferons: An Overview.” US Pharmacist 26:06 • 2. Alm, Gunner V. “Role of Natural Interferon-alpha Producing cells (Plasmacytoid Dendritic cells) in Autoimmunity.” Autoimmunity 36 (2003): 463-472. • 3. Decatris, Marios. “Potential of Interferon-alfa in Solid Tumours.” Biodrugs 16 (2002): 261-268. • 4. Goodsell, David S. “The Molecular Perspective: Interferons” The Oncologist 6 (2001): 374-375. • 5. Hertzog, Paul J. “Interferon-gamma: an overview of signals, mechanisms and functions.” Journal of Leukocyte Biology 75 (2004): 163-179. • 6. Lau, Johnson Y.N. “Mechanism of Action of Ribavirin in the Combination Treatment of Chronic HVC Infection.” Perspectives in Clinical Hepatology 35 (2002): 1002-1007. • 7. Matthews, James S. “Peginterferon Alfa-2a: A Review of Approval and Investigational Uses.” Clinical Therapeutics 26 (2004): 991-998. • 8. Pedder, Simon C.J. “Pegylation of Interferon Alfa: Structural and Pharmacokinetic Properties.” Seminars in Liver Disease 23 (2003): 19-21. • 9. Schreiber, Gregory H. “Interferon gamma.” The Cytokine Handbook 4 (2003):567-569. • 10. Vrolijk, J.M. “The treatment of hepatitis C: history, presence and future.” Journal of Medicine 62 (2004): 76-82.