Anti-herpesviral agent

Details Anti-herpesviral agent Anti-herpesviral agent

1999-01-25

2002-01-08

Salimi, Ali R. (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S231100, C424S204100, C435S005000, C536S023720, C530S300000

Reexamination Certificate

active

06337074

ABSTRACT:

The present invention relates to an anti-viral agent effective against herpesviruses and to an assay for screening for other suitable anti-viral agents.
Herpesviruses include Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2), Human Cytomegalovirus (HCMV), Epstein-Barr Virus (EBV) and Equine herpesviruses 1 and 4 (EHV-1 and EHV-4). The term “Herpesvirus” is used herein to refer to any virus of the herpesvirus family, including viruses in the &agr; group (e.g. HSV 1 & 2, EHV 1 & 4), the &bgr; group (e.g. HCMV) and in the &ggr; group (e.g. EBV).
Infections due to HSV have been successfully treated for many years through use of the drug acyclovir, a nucleoside analogue. Acyclovir is relatively non-toxic to the human host since it does not adversely affect the activity of the mammalian homologue of the targeted viral protein. However, similar low toxicity regimes for treating all herpesviruses have not yet been found. Whilst HCMV is treatable via use of the drug gancyclovir (Coen, 1992) the application of this drug is limited by its toxicity, poor bioavailability and the emergence of drug-resistant variants (reviewed by Coen 1992; Haffey & Field 1995; Filley et al 1995). A low-toxicity treatment for HCMV is particularly of interest as infection by this virus can cause congenital abnormalities in the newborn exposed to the virus by maternal transmission, and is also extremely problematic to immunocompromised patients, for example patients suffering from AIDS, or those on immunosuppressive therapy for cancer or following organ transplant.
The genome of herpes simplex virus type 1 (HSV-1) encodes seven proteins essential for origin dependent viral DNA synthesis (Wu et al., 1988). The genes encoding these proteins, and their protein products, are known in the art as UL5, UL8, UL9, UL29, UL30, UL42 and UL52. (McGeoch et al., 1988). Frequently the names of the genes are italicized, eg UL5, to avoid possible ambiguities. The UL30 protein, the catalytic subunit of the heterodimeric HSV-1 DNA polymerase, is also known as POL. Homologues of all seven genes have been identified in other alphaherpesviruses and human herpesviruses 6 and 7 (HHV-6 and HHV-7). Other beta- and gammaherpesviruses encode homologues of all these proteins except UL9. For convenience the terminology of the HSV-1 proteins will be used to refer not only to that particular protein but also its equivalent in other herpesviruses. Thus, as used herein the term “UL8” refers not only to UL8 of HSV-1 itself, but also to the HCMV homolgue UL102 and to equivalent homologues in other herpesviruses. Similarly, as used herein the term “POL” (or “UL30”) refers not only to POL of HSV-1 itself, but also to the HCMV homolgue UL54 and to equivalent homolgues in other herpesviruses.
The functions of these proteins and their interactions may be summarised as follows. The UL9 product is an origin-binding protein (OBP) and the UL29 product (ICP8) a single-stranded DNA binding protein. These two proteins can interact via the C-terminus of UL9 (Boehmer and Lehman, 1993; Boehmer et al., 1994). The UL30 protein (POL) and UL42 proteins comprise the catalytic and accessory components, respectively, of a dimeric DNA polymerase (reviewed by Challberg, 1991; Weller, 1991) and interact via residues at or near the C-terminus of POL (Digard & Coen, 1990; Digard et al., 1993, 1995; Marsden at al., 1994; Stow et al., 1993; Tenney et al., 1993). The UL5, UL8 and UL52 proteins form a trimeric complex that exhibits both DNA helicase and DNA primase activities (Dodson et al., 1989; Crute et al., 1989). The UL5 protein is largely responsible for DNA helicase activity (Gorbalenya et al., 1989; Zhu & Weller, 1992), and the UL52 protein contributes an essential role in DNA priming (Klinedinst & Challberg, 1994; Dracheva et al., 1995) and these two proteins can form a stable subassembly that retains both functions (Calder & Stow, 1990; Dodson & Lehman, 1991; Crute et al., 1991). The UL8 component has auxiliary effects on the DNA primase activity, stimulating primer synthesis and/or utilization on a natural-sequence single-stranded DNA template (Sherman et al., 1992; Tenney et al., 1994), and is also required for efficient nuclear entry of the trimeric complex. (Calder et al., 1992; Marsden et al., 1996). UL8 is capable of binding separately to the UL5 and UL52 proteins and can also interact specifically with UL9 (McLean et al., 1994). The latter interaction with OBP may serve to recruit the helicase-primase into an initiation complex at the viral origins.
Further evidence for the occurrence of multiple interactions between DNA replication proteins has been provided by immunofluorescence experiments. In cells infected with HSV-1 in the presence of inhibitors of viral DNA synthesis UL29 (ICP8) localises to punctate structures within the nucleus termed “pre-replicative sites” (Quinlan et al., 1984). The requirement for each of the DNA replication proteins in the formation of these sites has been studied by the use of viral mutants with defects in individual replication proteins (Liptak et al., 1996; Lukonis et al., 1996). It was observed that proteins UL5, UL8, UL9 and UL52 are all necessary for the localisation of UL29 (ICP8) into pre-replicative sites and that mutants with defects in any of the other six DNA replication genes are affected in the ability of POL to localize to these sites. Although these data suggest that the DNA polymerase holoenzyme is the last component to be recruited (Liptak et al., 1996) they do not identify the specific interactions involved in this event.
It has now been found that the protein UL8 interacts with POL. Further, it has been found that disruption of the POL/UL8 interaction is possible. Examples of molecules, monoclonal antibodies and peptides that specifically disrupt the interaction have been identified.
The present invention provides an anti-viral agent capable of combatting replication of a herpesvirus by interfering with the association of UL8 and POL (as defined above).
Both the UL8 and POL proteins of HSV-1 have been previously described in the literature (e.g. Parry et al., 1993; Gottleib et al., 1990).
Furthermore the amino acid/DNA sequences of UL8 and POL from HSV-1 are available from publically accessible Genbank and EMBL databases under Nos. P10192/M19120 and P04293/M12356 (and several other entries), respectively.
The UL8/POL association is an association between two viral proteins, that are significantly different from any protein in the mammalian host organism (for HSV-1, the host is humans). Although homologues of POL are present in mammalian cells they are considerably diverged. No cellular homologue of UL8 is known. For the virus to overcome disruption of such a viral protein: viral protein interaction a double mutation, i.e. a mutation in each of the viral proteins involved, may be required. Alternatively the range of single mutations that overcome disruption, yet allow the two proteins to interact normally may be severely restricted. The probability of such reversion occurring is thus relatively low rendering this type of interaction attractive as a potential target for therapeutic agents. Additionally, as neither UL8 nor POL have close homologues in mammalian cell metabolism, the toxicity of an agent which specifically interacts with these proteins will be low.
The anti-viral agent may be a peptide or more preferably a non-peptidal compound having peptidomimetic properties. Such a non-peptidal compound will be preferred since it will be resistant to enzymic breakdown by peptidases. Suitable anti-viral compounds may include peptides having an amino acid sequence derived from the C-terminal or C-proximal region of UL8, a functional equivalent of such a peptide, or a peptidomimetic compound therefor.
The computer program “Predict-Protein” (EMBL-Heidelberg) makes a strong prediction of the presence of an alpha-helical region near the C-terminus of HSV-1 UL8 (amino acids 709-728) with the very C-terminus (residues 729-750) predicted to be in looped or extended structures (perhaps as a “tail”). The secondary str

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