An ATPase Assay Using Scintillation Proximity Beads for High-Throughput Screening or Kinetic Analysis

Jeffery, Jamie A.; Sharom, Jeffrey R.; Fazekas, Monika; Rudd, Penny A.; Welchner, Ewald; Thauvette, Louise; White, Peter W.

doi:10.1006/abio.2002.5632

Cited by 16 publications

(8 citation statements)

References 26 publications

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“…ATPase Assays-ATPase activity of purified recombinant E1 (40) was measured using a scintillation proximity assay as described previously (40,41). The concentration of HPV11 E1 was 3 nM and ATP and magnesium acetate were used at 20 and 500 M, respectively.…”

Section: E1-e2-ori Complex Formationmentioning

confidence: 99%

Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor

Wang

Coulombe²,

Cameron

et al. 2004

Journal of Biological Chemistry

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Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5-and 2.4-Å resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.Human papillomaviruses (HPVs) 1 are the etiological agents of malignant and benign lesions of the differentiating squamous or mucosal epithelium, notably of cervical cancer. Approximately 25 HPV types replicate in mucosal tissues of the anogenital tract. HPV16, -18, and -31 are the most prevalent "high-risk" types found in pre-cancerous or malignant lesions of the cervix. HPV6 and -11 are the most common "low-risk" types, which cause benign genital warts (condyloma acuminata), a less serious condition but one of the most common sexually transmitted diseases (1). Currently, no specific antivirals are available for the treatment of HPV infections.The small circular double-stranded DNA genome of papillomavirus is actively maintained as a multicopy episome in the nucleus of infected epithelial cells. This process is dependent on replication of the viral genome by the viral E1 and E2 proteins, in conjunction with the host DNA replication machinery. E2 is a sequence-specific DNA-binding protein that has a number of functions in the viral lifecycle. In addition to its role in the initiation of viral DNA replication, E2 is involved in regulating the transcription of viral genes (2-7), and in the segregation of the viral genome during cell division (8, 9). As a replication initiation factor, E2 binds with high affinity to specific sites located within the viral origin (ori) to help recruit it to the E1 helicase (10 -13). Formation of a ternary complex between E1, E2, and the origin relies not only on the interaction of E1 and E2 with specific DNA sequences at the origin but is also critically dependent on a direct interaction between these two proteins (14 -18).The 40-kDa E2 prote...

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Section: E1-e2-ori Complex Formationmentioning

confidence: 99%

Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor

Wang

Coulombe²,

Cameron

et al. 2004

Journal of Biological Chemistry

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“…In addition this assay can be modified for other ATPases and it also offers the ability to identify ATPase stimulators. Although there are other ATPase assays that have been optimised for high throughput screening, these rely largely upon undesirable methods to detect ATPase activity [33][34][35]. Examples include use of radioactive scintillation proximity assay [33] or colorimetric assays [35], as well as coupled-enzyme systems [34] that detect phosphate production linked stoichiometrically to ATP hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

“…Although there are other ATPase assays that have been optimised for high throughput screening, these rely largely upon undesirable methods to detect ATPase activity [33][34][35]. Examples include use of radioactive scintillation proximity assay [33] or colorimetric assays [35], as well as coupled-enzyme systems [34] that detect phosphate production linked stoichiometrically to ATP hydrolysis. These assays depend on the hydrolysis of ATP to ADP and inorganic phosphate under the action of PMCA ATPase activity with the resulting inorganic phosphate forming a complex with the malachite green dye to produce a green colour which can be detected [36].…”

Section: Discussionmentioning

confidence: 99%

Optimisation and Validation of a High Throughput Screening Compatible Assay to Identify Inhibitors of the Plasma Membrane Calcium ATPase Pump - a Novel Therapeutic Target for Contraception and Malaria

et al. 2013

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-Purpose. ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF ® Transcreener ® ADP (TR-FRET) assay to screen a drug library. Methods and Results. PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/μl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1μM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. Conclusions. We have optimised the HTRF ® Transcreener ® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.

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“…Most solution assays were performed using the previously reported scintillation proximity method (19). Briefly, 45-l reactions were carried out in assay buffer (20 mM HEPES, pH 7.5, 1 mM dithiothreitol, 0.05 mM EDTA, 0.005% IGEPAL-CA630 [vol/vol]; Sigma [equivalent to Nonidet P-40]) supplemented with 2% dimethyl sulfoxide.…”

Section: Methodsmentioning

confidence: 99%

“…Because of the multiple steps involved in this procedure and the relatively high variability in quantification by thin-layer chromatography, estimates of the absolute concentrations of phosphate were found to vary by a factor of 2, although the relative concentrations obtained for different time points or reaction wells were determined much more accurately. This procedure has been described in detail previously (19). Kinetic data were analyzed using the equations for linear or hyperbolic competitive inhibition (29) with the program GraFit 3.0 (Erithacus Software Ltd.).…”

Section: Methodsmentioning

confidence: 99%

Biphenylsulfonacetic Acid Inhibitors of the Human Papillomavirus Type 6 E1 Helicase Inhibit ATP Hydrolysis by an Allosteric Mechanism Involving Tyrosine 486

White¹,

Faucher

Massariol³

et al. 2005

Antimicrob Agents Chemother

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Human papillomaviruses (HPVs) are the causative agents of benign and malignant lesions of the epithelium. Despite their high prevalence, there is currently no antiviral drug for the treatment of HPV-induced lesions. The ATPase and helicase activities of the highly conserved E1 protein of HPV are essential for viral DNA replication and pathogenesis and hence are considered valid antiviral targets. We recently described novel biphenylsulfonacetic acid inhibitors of the ATPase activity of E1 from HPV type 6 (HPV6). Based on kinetics and mutagenesis studies, we now report that these compounds act by an allosteric mechanism. They are hyperbolic competitive inhibitors of the ATPase activity of HPV6 E1 and also inhibit its helicase activity. Compounds in this series can also inhibit the ATPase activity of the closely related enzyme from HPV11; however, the most potent inhibitors of HPV6 E1 are significantly less active against the type 11 protein. We identified a single critical residue in HPV6 E1, Tyr-486, substituted by a cysteine in HPV11, which is primarily responsible for this difference in inhibitor potency. Interestingly, HPV18 E1, which also has a tyrosine at this position, could be inhibited by biphenylsulfonacetic acid derivatives, thereby raising the possibility that this class of inhibitors could be optimized as antiviral agents against multiple HPV types. These studies implicate Tyr-486 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts.Papillomaviruses infect the squamous and mucosal epithelia of many different mammals, including humans, often resulting in the development of benign and sometimes malignant lesions (reviewed in references 16, 31, and 42). There are over 100 types of human papillomavirus (HPV), each exhibiting a particular tropism for specific tissue types (8). For example, HPV1 causes plantar warts, HPV6 and -11 cause anogenital warts (condyloma acuminata), and infection with HPV16 and -18, among others, can lead to cervical cancer (2, 42). Among the HPV types that infect the anogenital region, those that are associated with cancer are collectively referred to as "highrisk" types, whereas those that cause only benign warts are known as "low-risk" types (42). Despite the medical burden associated with treating and screening for HPV infections, an HPV-specific antiviral drug is still lacking, and there are only a few reports of HPV-specific inhibitors which could serve as potential leads for drug discovery. To our knowledge, the E1 ATPase inhibitors described in this report and our previously published series of E2 inhibitors (37, 39) are the only potent and selective small molecules targeting HPV DNA replication proteins ever to be reported.All papillomaviruses have a small circular double-stranded DNA genome which encodes for only eight well-characterized proteins (for a recent review, see reference 21). The most highly conserved protein, and the only one with enzymatic activity, is the E1 helicase (review...

show abstract

An ATPase Assay Using Scintillation Proximity Beads for High-Throughput Screening or Kinetic Analysis

Cited by 16 publications

References 26 publications

Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor

Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor

Optimisation and Validation of a High Throughput Screening Compatible Assay to Identify Inhibitors of the Plasma Membrane Calcium ATPase Pump - a Novel Therapeutic Target for Contraception and Malaria

Biphenylsulfonacetic Acid Inhibitors of the Human Papillomavirus Type 6 E1 Helicase Inhibit ATP Hydrolysis by an Allosteric Mechanism Involving Tyrosine 486

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