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MECHANISMS OF ACTION OF INTERFERON AND NUCLEOSIDE ANALOGUES

Howard Thomas, Graham Foster, Dimitris Platis, Department of Medicine A, Imperial College and Liver and Anti-viral Centre, St Mary’s Hospital, London, UK

The alpha interferons (alpha 2a and b, consensus and lymphoblastoid), lamivudine and adefovir are licensed for therapy of chronic hepatitis B in various parts of the world. I will focus on these and mention briefly those additional compounds that are just entering phase1, 2 or 3 studies in man.

INTERFERON ALPHA.

Interferons are a family of pleiotropic cytokines with antiviral, anti-proliferative and immuno-modulatory properties. The interferon family is subdivided into two subfamilies: Type I and Type II. Type I interferons are a family of monomeric cytokines with an aminoacid similarity of 30-80%, very similar three-dimensional structure (5-alpha helix-bundle), that use the same receptor (IFNAR) to initiate a signalling response.

The 12 interferon alpha protein subtypes exhibit very high amino-acid similarity (over 75%). However, the functional role for the existence of so many distinct proteins is unknown. Their relative antiviral and antiproliferative activities are different depending on the target cell involved.

The alpha and B interferons bind to the same membrane receptor (IFNAR).

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The Interferon a/b Receptor (IFNAR)

The interferon a/B receptor has two components (IFNARI and IFNAR2). The first receptor component (IFNAR1) was cloned in 1990. It has an apparent molecular weight of 110-130kD and encodes for a transmembrane protein with a large extracellular portion with a duplication of a structural domain composed of repeated fibronectin III motif (class II receptor family) and a very short intracellular region (100 aminoacids) with no well-defined motifs. IFNAR1 has a very low affinity for interferon on its own and it is presumed to complement and enhance binding only when accompanied by its companion receptor chain (IFNAR2).

A splice variant of IFNAR1 has been discovered with a deletion in the extracellular region, with a preferential sensitivity to interferon alpha-2 relative to alpha-8. The tissue specific expression of this variant form and its physiological role are still unknown. Phosphatases (SHP-1 and SHP-2) also associate with IFNAR1, supposedly playing a negative regulatory role in the activation of the JAK-STAT pathway.

The second component of the Type I interferon receptor (IFNAR2) was isolated in 1994 and again shown to be composed of both an extracellular and an intracellular region with a molecular weight of 55 –95 kD. It was discovered that IFNAR2 exists in three splice variant forms depending on the length of the intracellular region: IFNAR2a (short), IFNAR2b (soluble) and IFNAR2c (long). The IFNAR2c variant is necessary and sufficient for JAK-STAT activation and an antiviral response, although both IFNAR1 and IFNAR2c are required for interferon signalling. The role of the short and soluble variant forms are not yet known, but the presence of the short form in some human and murine cells might suggested a modulatory role.

The interferon ligand is presumed to bind to IFNAR2 first and then to IFNAR1, which stabilizes the complex. There is no direct evidence for this model, however, it is a logical hypothesis based on the calculated binding constants for each receptor component. While IFNAR2 binds interferon with moderate affinity (KD Ê2-10nM for interferon alpha-2), the intrinsic affinity of IFNAR1 is low (KD>100nM), stabilizing the complex by approximately 10-20 fold. Taken from http://bioinfo.weizmann.ac.il/

The interferons regulate a variety of important biological functions, through interaction with the specific receptors on the surface of cells. These receptors are responsible for carrying the signal through the cell membrane and re-directing it to various cytoplasmic and nuclear compartments. Most of the nuclear signals lead to the induction of specific genes. Upon stimulation with interferon, protein complexes are formed that are translocated to the nucleus, bind to DNA regulatory elements and activate specific genes. These cytoplasmic factors are called STATs (Signal Transducers and Activators of Transcription).

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The Signal Transduction System (Jak-Stat):

Binding of interferon a/ß? ?initiates the signalling cascade by causing dimerization of the receptor subunits, IFNAR1 and IFNAR2. It is almost certain that this initial step triggers a conformation change that is propagated through the cell membrane and is responsible for the initiation of the phosphorylation cascade.

The first intracellular component of the signalling pathway that receives the “transduction pulse” is the JAK kinase, Tyk2, which is preassociated with IFNAR1. Upon interferon stimulation, Tyk2 is immediately phosphorylated by JAK1, another JAK kinase, which is bound to IFNAR2. Activated Tyk2 then in turn phosphorylates JAK1.

The activated JAK kinases, Tyk2 and JAK1, are responsible for the subsequent phosphorylation of IFNAR1 and IFNAR2 at specific tyrosine residues. STAT2 then binds to specific phosphorylated residues on IFNAR1. Upon docking, STAT2 is phosphorylated at a conserved tyrosine residue (Y701) by the JAK kinases thereby creating an additional docking port for STAT1, which is also subsequently phosphorylated at Y690. The phosphorylated STATs then dissociate from the receptor heterodimer and bind to p48 (IRF9), a member of the interferon regulatory factor (IRF) family, forming the major interferon transcription factor, known as ISGF-3.

ISGF-3 translocates to the nucleus and binds to specific regulatory DNA sequences (ISRE-Interferon stimulated response elements) and initiates transcription of several interferon-inducible genes. STAT1:2 heterodimers as well as STAT1:1 homodimers also form and are capable of driving the expression of a minority of interferon stimulated genes (ISGs), independently of p48. In this case, they bind to different DNA regulatory sequences than ISRE called GAS (gamma activated sequence elements), which are usually found in the promoters of interferon gamma stimulated genes.

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Functions of Interferons

Interferons are pleiotropic proteins, able to initiate and regulate a variety of responses, either directly or by stimulating the induction/activation of additional proteins. Different interferon subtypes intrinsically have the ability to stimulate different but overlapping sets of genes. However, the overall phenotypic responses appear to be similar.

Antiviral properties

The ability of interferon to establish an “antiviral state” is the distinctive fundamental property of interferons, essential for the survival of higher vertebrates against viral infection (Figure 3).

There are multiple redundant antiviral pathways that allow interferons to combat multiple different viruses in every vulnerable step in their replication cycle, starting from entry/uncoating (SV40, retroviruses), transcription (influenza, VSV), RNA stability (picornaviruses), translation initiation (reoviruses, adenovirus, vaccinia), maturation and assembly/release (retroviruses, VSV).

The vital antiviral role of interferons has been demonstrated in interferon alpha/beta-deficient mice that are extremely susceptible to viral infections. It should be noted that interferons have no antiviral activity of their own but rather induce the expression of potent antiviral genes.

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i The Protein Kinase R (PKR) system:

This is perhaps the most well characterized interferon-induced antiviral pathway. PKR is a 551 aminoacid serine-threonine kinase mainly found in the cytoplasm of most cells and partly in the nucleus, where it normally lies inactive. The expression of PKR is rapidly stimulated by interferons and reaches levels approximately 5-10 fold higher that in resting cells. This action of interferon is mediated through an ISRE (interferon-stimulated regulatory element) and GAS (gamma interferon-activated site) in the promoter region of the PKR gene.

PKR contains two conserved dsRNA-binding motifs at the amino-terminus, RI (aminoacids 55-75) and RII (aminoacids 145-166). Both regions possess a similar core sequence, but RI alone appears to be necessary and sufficient to mediate dsRNA binding. It is believed that a combination of both domains creates a single binding site where the different domains bind to different regions of the dsRNA molecule. No RNA sequence specificity has been identified.

PKR is activated by double stranded RNA (viral RNA or secondary RNA structures), which results in a conformational change that uncovers the carboxy-terminal catalytic domain of PKR. This activates the kinase activity of PKR, which then undergoes autophosphorylation/activation in several serine and threonine residues. The activated kinase phosphorylates in turn the translation initiation factor eIF2- aa t Ser51. eIF2--Ga TP is required for initiation of translation. It is recycled from eIF2-a-GDP, which is produced after each round of initiation, by eIF-2B, which mediates the guanine nucleotide exchange step. However, eIF-2B binds preferentially to the phosphorylated form of eIF-2a. Therefore, the cellular stock of eIF-2B is sequestered in the inactive complex eIF2a-GDP-eIF-2B resulting in rapid inhibition of translation.

PKR has also been involved in regulating cell proliferation, playing a role as tumour suppressor and in signal transduction by regulating the serine phosphorylation of STAT1 and by phosphorylating IkB, which in turn results in activation of NF-kB-dependent genes.

ii The 2-5A oligosynthetase/RNAse L system

This multienzyme system is composed of three separate components: 2-5A synthetase, endoribonuclease RNAse L and 2-5A phosphodiesterase. The different 2-5A synthetases (40, 46, 69 and 100kD) are encoded by multiple genes and reside in different subcellular compartments. The components of the 2-5A system are present at low levels in resting cells but are strongly induced by interferons.

The system is activated by dsRNAs, probably of viral origin. Initially, 2-5A synthetase is stimulated by dsRNA and produces a series of short 2’-5’-oligoadenylates that bind to inactive, monomeric RNAse L converting it into a dimeric, active enzyme. RNAse L degrades all single-stranded RNA inhibiting, in theory, all viral replication that uses an RNA intermediate step. 2-5A phosphodiesterase regulates the entire process by catalysing the degradation of the 2’-5’ oligoadenylates thereby “switching off” the system.

The efficiency of the 2-5A system has been shown for EMC virus as well as vaccinia and HIV-1, but not for VSV or HSV. Since the 2-5A system operates mostly in the cytoplasm it is possible that viruses that replicate in the nucleus, such as VSV and HSV, might escape the antiviral actions is the system.

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iii The Mx system

Mx proteins are interferon-inducible, high-abudance 70-80kD GTPases of the dynamin superfamily. Human MxA proteins assemble into oligomeric complexes in cell-free systems. They interfere with replication at the transcriptional and other levels in influenza and other negative-stranded viruses replication.

Initially the Mx system was studied in mice, in which Mx1 was found to be responsible for resistance to orthomyxovirus infection. Two homologues of Mx1 were found in humans, MxA and MxB, which appear to be functionally different from Mx1 and are also localized in the cytoplasm as opposed to Mx1, which is mainly a nuclear protein. The exact mode of action of Mx protein is not completely known, but they are believed to interfere with trafficking or activity of viral polymerases.

iv Other anti-viral systems

Several proteins are been known to interfere with virus replication. Guanylate Binding Protein (GBP) is an interferon-stimulated protein with demonstrates antiviral properties, although the mechanism of action is unknown. Interferon inducible protein 9-27 inhibits VSV replication and nitric oxide synthase (iNOS) protects macrophages from infection by several viruses. A protein with very high homology to 9-27 has been identified to bind to the rev-response element of the human immunodeficiency virus HIV-1 and inhibits rev-mediated transcription. Additionally, experiments in yeast have shown proteins to bind to viral mRNAs and prevent their translation.

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Antiproliferative properties

Interferons have the ability to arrest cell growth, which is why they are used as treatment for cancer. It seems that both direct and indirect (via the immune system) inhibition of the tumour cells might be involved, however, the exact mechanism of action is unclear. No specific genes have been linked to the antiproliferative activity of interferons, however, STAT1 is believed to be involved since it is often deficient in tumours. The mechanism of cellular arrest in not known, but it is likely to target components of the cell-cycle control apparatus, including induction of CDK inhibitors such as p15/16 and p21WAF1/Cip or the decrease in levels of cyclin D and cdc25A. PKR is also suspected to play a role in the regulation of cell growth.

Control of apoptosis

Regulation of apoptosis and control of cell growth is very important for host responses against viruses. Interferons in conjunction with dsRNA, TNF or LPS are a potent inducer of cell death. Overexpression of PKR induces apoptosis through a mechanism dependent on bcl-2 and ICE. Additionally, PKR-deficient mice resist apoptotic death through a mechanism linked to a defect in activating the DNA-binding activity of IRF-1. Novel interferon regulated genes have been identified that encode for death associated proteins (DAPs) and are necessary for interferon-induced apoptosis in HeLa cells.

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Immunomodulatory properties

Interferon gamma is the predominant immunomodulatory interferon. Type I interferons are mainly antiviral cytokines but also utilize the immune system to eradicate viral infection.

NK cells are a heterogeneous subpopulation of lymphocytes capable of lysing target cells without prior sensitisation. They act very rapidly upon viral infection and together with interferons are considered the first line of defence against viral infections and tumours. Alpha interferons augment NK activity in vitro, except for subtype alpha-7, which appears to be relatively inactive despite the fact it has normal antiviral and antiproliferative properties.

It has been suggested that interferon alpha enhances humoral immunity by increasing proliferation of B cells, which secrete antibodies against invading antigens. Interferon alpha also enhances the CD8+ cytotoxic T cell (CTL) response by upregulating the expression of MHC class I molecules on the surface of the cells. MHC I molecules are responsible for presenting viral antigens to CTLs, thereby triggering the destruction of the infected cell. Additionally, Th1 cells are responsible for amplifying CD8+ cytotoxic T cells in reponse to IL-12 and induce proliferation. Type I interferons appear to upregulate IL-12Rb, a component of IL-12 receptor on Th cells, thereby upregulating the production of CTLs and enhancing the Th1 responses.

MHC class II molecules located on the surface of antigen-presenting cells (APCs), such as dendritic cells and macrophages, present viral antigens to helper T cells. Certain interferon alpha subtypes may increase the expression of MHC class II molecules by APCs.

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NUCLEOSIDE ANALOGUES.

The main target is the HBV polymerase gene product which has many enzymic and other activities:

  • priming of reverse transcription (priming)
  • Reverse transcriptase
  • RNAse H
  • DNA polymerase(DNA Pol)
  • chaperone – like function in nucleocapsid assembly.
  • All anti-HBV analogues inhibit DNA and RT Pol and some that are deoxyguanosine analogues inhibit priming. None are known to inhibit RNAse H

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Reverse Transcription Inhibition

Lamivudine was developed as a reverse transcriptase inhibitor for use in HIV infection. It also has activity against HBV at lower concentrations. Lamivudine (2’, 3’ dideoxythiacytidine) is a minus enantomer and it is thought that this may help explain the very low rates of side-effects noted with this agent.

Chain Termination

Deoxyadenosine Monophoshate Analogues

Adefovir dipivoxil (9-(2-phoshonyl-methoxyethyl)-adenine) is an acyclic adenine nucleoside analogue with a broad range of antiviral activity. It has inhibitory effects on herpes viruses, retroviruses and hepadnaviruses. Trials in HBV have shown considerable activity against HBV. Perhaps most importantly adefovir has in vivo activity against lamivudine-resistant strains, with potent antiviral activity in vitro against YMDD mutants, and for this reason adefovir is now the subject of ongoing clinical trials.

However, there have been some concerns regarding toxicity with some reports of deranged liver function tests in some patients, and reports of possible renal tubular damage in patients receiving more prolonged higher dose therapy.

These compounds are incorporated into the growing strand of HBV DNA and prevent further strand growth

Deoxyguanosine Analogues

This group includes ganciclovir, penciclovir and now entecavir. These compounds inhibit priming and also chain extension.

Ganciclovir: has been shown to be active against hepatitis B virus replication

Famciclovir: the prodrug for penciclovir, is mainly used as therapy of herpes virus infections such as herpes simplex virus types 1 and 2 and varicella zoster virus infection. Famciclovir requires the presence of the viral thymidine kinase enzyme for the first phosphorylation towards it active triphosphate form. HBV does not possess thymidine kinase activity and cellular kinases are involved in activation of the drug. Famciclovir is well tolerated and may be of benefit as maintenance therapy. Viral resistance is being recognised, although these mutants appear to remain sensitive to other antiviral agents.

Entecavir: is a guanine nucleoside analogue which is a potent and selective inhibitor of hepatitis B virus. It has been evaluated in Hep G2.2.15 cells, ducks and woodchucks with hepadna virus infection. Against HBV it has a 50% effective concentration of 0.00375 micromols which is circa 30 fold more active than lamivudine. It is phosphorylated by cellular kinases. It has activity against lamivudine resistant strains of HBV in vitro. It is currently in phase 3 studies in man.

Other antiviral agents:

Trials are also underway with several nucleoside analogues, including emtricitabine, which has demonstrated promising activity.

Studies of fialuridine (FIAU) were suspended when several patients developed severe toxicities, including lactic acidosis, pancreatitis liver and renal failure. It is thought that these severe side-effects, not predicted by short-term treatment studies, are related to the toxic effect of FIAU on host mitochondrial DNA. A similar syndrome has been reported as a rare adverse event with other nucleoside analogues such as zidovudine. The morphology and function of mitochondria have been carefully studied in patients receiving lamivudine with no evidence, as yet, of similar phenomena.

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REFERENCES:

Signalling pathways in interferon system

Muller M; Ibelgaufts.H; Kerr IM 1994 Interferon response pathways – a paradigm for cytokine signalling? J Viral Hepatitis 1:87-103

Interferon Receptor

Mogensen, K. E., M. Lewerenz, J. Reboul, G. Lutfalla, and G. Uze. 1999. The type I interferon receptor: structure, function, and evolution of a family business. J Interferon Cytokine Res 19:1069-98.

Domanski, P. and O. R. Colamonici. 1996. The type-I interferon receptor. The long and short of it. Cytokine Growth Factor Rev. 7:143-151.

Antiviral effects of Interferons

Sen, G. C. 2001. Viruses and interferons. Annu.Rev.Microbiol. 55:255-281.

JAKs and STATs

Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, and R. D. Schreiber. 1998. How cells respond to interferons. Annu.Rev.Biochem. 67:227-264.

Leonard, W. J. and J. J. O'Shea. 1998. Jaks and STATs: biological implications. Annu.Rev.Immunol. 16:293-322.

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