Interferons

By: Katy Nassif

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

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

• 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.

General Action of Interferons

• Three types of interferons: alpha, beta and gamma.

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

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.

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.

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.

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 ) chains interact with IFN- 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-R1.

This leads to the recruitment and phosphorylation of STAT1, which forms homodimers and translocates to the nucleus to activate a range of IFN--responsive genes.

After this, the ligand-binding chains are internalised and dissociate.

The chains are then recycled to the cell surface.

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

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.

Pegylated Interferon-2a (Pegasys)

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.

Pegasys is recombinant interferon alpha-2a that is covalently conjugated with bis-monomethoxy polyethylene glycol (PEG)

Structure:

CH3—(OCH2CH2)n--OH

mPEG—O—O2C—C—NH

mPEG—O—O2C—NH—(CH2)4

O

•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

• 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

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

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

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

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.