Atpase assay

Details Atpase assay Atpase assay

2000-06-16

2002-12-10

Venkat, Jyothsna (Department: 1627)

Chemistry: analytical and immunological testing

Phosphorus containing

C436S534000, C436S800000, C436S804000, C435S004000

Reexamination Certificate

active

06492181

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a new ATPase assay. This invention particularly relates to a new method for the detection and measurement of the amount of orthophosphate released by hydrolysis of ATP or any other phosphate containing molecule. More particularly, this invention relates to a new method for the measurement of the ATPase activity of the E1 helicase enzyme from the human papilloma virus (HPV).
BACKGROUND OF THE INVENTION
HPV-associated disease
The human papillomaviruses (HPVS) are small DNA viruses that infect cells of the cutaneous and mucosal epidermis. Over 80 different HPV genotypes have been characterized. Some types, such as HPV-1, -2, -3, -4 and -10, cause cutaneous lesions known as warts or papillomas. These growths are benign and self-limiting, and are found on the hands and feet of 7-10% of the general population. Of greater medical concern are those HPV types that infect the anogenital tract. These genotypes are designated as either “low-risk” or “high-risk” based on their correlation with malignant progression.
So-called low-risk HPVs are associated with genital warts, or condyloma acuminata. For instance, HPV types 6 and 11 are found in more than 90% of benign genital lesions, and very rarely associated with malignant transformation. However, they nonetheless represent a serious public health problem. Approximately 1% of sexually active adults in the U.S.A. have visible genital warts, but in many more cases the infection is sub-clinical. In fact, an estimated further 15% of people aged 15-49 display molecular evidence of HPV infection, in the form of viral DNA detectable by polymerase chain reaction (PCR) assay. Indeed, HPV is ranked as the most common sexually-transmitted viral agent in the U.S.A. and U.K., and its incidence is increasing steadily.
Infection with high-risk HPV types such as 16,18, 31 and 33, has been strongly linked to the development of anogenital malignancies, most notably cervical cancer. In fact, HPV types 16 and 18, while rarely found in benign genital lesions, are detectable in about 70% of all invasive carcinomas of the cervix. The link between HPV and anogenital cancer is well documented—recent studies have found that almost 90% of cervical carcinomas contain HPV DNA.
Current Therapies and the Need for a Virus-Specific Treatment
In spite of the pervasiveness of HPV infection and its possibly life-threatening consequences, no virus-specific inhibitor has yet been described. Antiviral drug discovery for HPV has proven quite difficult thus far as a result of difficulties encountered in propagating the virus in the laboratory.
All current therapies for HPV infection rely on the non-specific destruction or removal of infected tissue. Accepted surgical procedures include the use of dry ice, liquid nitrogen, CO
2
laser therapy, electrocautery or local excision. Various cytotoxic agents are also used to destroy tissue, such as salicylic acid, tricholoroacetic acid, podophyllin, colchicine, bleomycin and cantharidine.
While the risk of cancer makes these procedures the most prudent for the treatment of high-risk HPVs, less invasive treatments are being sought to manage the low-risk genotypes. Compounds that stimulate the immune system have been investigated with the goal of reproducing the spontaneous regression often seen with benign lesions. Imiquimod, such an immune response modifier, has recently passed clinical trials and been approved for treatment of HPV-associated genital warts.
Patients with genital warts often experience high recurrence rates—usually 30-90%—following non-specific treatments such as surgery. Such poor efficiency is a result of the incomplete elimination of HPV DNA, or the presence of virus in normal-appearing tissue adjacent to the papilloma. Obviously, there is a substantial need for an effective, virus-specific therapy for HPV infection, which has thus far gone unmet.
Viral DNA Replication and E1
Semi-conservative DNA replication is an intricate process mediated by many enzymes and accessory proteins. Helicases are enzymes that function during DNA replication, catalyzing the unwinding of duplex DNA ahead of the replication fork. They are very common in prokaryotic and eukaryotic cells, as well as most viruses. The exact mechanism by which helicases convert the binding and hydrolysis of ATP into mechanical energy to power the unwinding of DNA and their own simultaneous motion along the nucleic acid stand is still not completely understood.
The 72 kDa HPV E1 protein has been classified as a member of helicase superfamily III along with the T antigen of Simian Virus 40 (SV40 TAg), with which it is structurally and functionally homologous. E1 and Tag belong to a noteworthy subgroup of viral DNA helicases which have the ability to recognize and bind specific DNA sequences at the viral origin of replication (ori). Also, while most DNA helicases require a region of single-stranded DNA for entry, these proteins can initiate unwinding from completely double-stranded DNA, provided it contains an ori.
Molecular Events at the HPV Origin of Replication
Human papillomaviruses contain approximately 8 kb of double-stranded circular DNA. In the basal cells of the epidermis, the genome is replicated and maintained extra-chromosomally at a steady-state level of about 20-100 copies per cell. High-level amplification of the genome only occurs once the cell has terminally differentiated and migrated to the upper layers of the epithelium.
In a cell-free DNA replication system, the E1 protein can direct origin-specific DNA replication by itself at sufficient concentrations, when provided with the full complement of host replication proteins including the DNA polymerase a primase enzyme. However, replication is greatly stimulated by the viral E2 protein, and at limiting concentrations of E1 the in vitro replication becomes completely E2-dependent. This is a consequence of E1 having a relatively low affinity for its DNA binding site. E2 helps to localize E1 to the origin by acting as an accessory protein . The E1 and E2 binding sites at the viral ori are in close proximity, falling within about 100 bp of each other. The carboxy terminus of E2 binds its palindromic site on DNA, while the amino terminus binds E1, thus bringing E1 to its binding site.
E1 as a Target for Antiviral Therapy
Recently, pharmaceutical companies have been able to substantially expand and accelerate their antiviral compound screening programs as a consequence of advances in molecular biology. Viruses are now routinely examined at the molecular level to find specific inhibitors of virus-encoded gene products.
For several viruses, enzymes such as polymerases, kinases and proteases have been targets for inhibition. In contrast, of the approximately 8 distinct proteins encoded by the HPV genome, the E1 helicase is the only one with enzymatic activity (Fields et a., 1996, Fields Virology, 3
rd
Ed. Lippincott-Raven, Philadelphia, Chap. 65 and refs. therein). E1 displays or-specific DNA-binding activity, E2-binding activity, ATPase activity, and DNA helicase activity—all of which can be assayed independently for potential inhibitors. In addition, it is the most highly conserved of all papillomavirus proteins, so an inhibitor of E1 would likely be effective against multiple HPV types.
High throughput screens are known that allow the discovery of inhibitors of the helicase activity of E1 (WO 99/157283, Nov. 11, 1999). Even though ATP is needed to drive E1 helicase activity and is included in the reaction, this helicase assay cannot be used to identify competitive inhibitors of E1 ATPase function. This is a direct result of very low K
m
of the ATPase, for example approximately 10 &mgr;M for HPV-11 E1, and the fact that the helicase assay is routinely run with 300 &mgr;m-1 mM ATP). A more sensitive assay must be developed if the ATP binding site of E1 is to be targeted for inhibition.
Existing ATPase Assays
Helicase activity is virtually always associated with nucleoside triphosphatase activity (Matson et al., Ann. Rev. Biochem., 1990, 59, 289)

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