Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by ∼10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target. Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by ∼10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target. Papillomaviruses are a family of small double-stranded DNA viruses that induce benign and malignant hyperproliferative lesions of the differentiating epithelium (reviewed in Refs. 1Chow L.T. Broker T.R. Nathanson N. Viral Pathogenesis. Lippincott-Raven Publishers, Philadelphia1997: 267-301Google Scholar, 2Howley P.M. Fields B.N. Knipe D.M. Fields Virology. 3rd Ed. Raven Press, Ltd., New York1996: 2045-2076Google Scholar, 3Shah K.V. Howley P.M. Fields B.N. Knipe D.M. Fields Virology. 3rd Ed. Raven Press, Ltd., New York1996: 2077-2109Google Scholar, 4Zur Hausen H. de Villiers E.M. Annu. Rev. Microbiol. 1994; 48: 427-447Crossref PubMed Scopus (477) Google Scholar). Approximately 25 types of human papillomavirus (HPV) 1The abbreviations used are: HPV, human papillomavirus; TAD, transactivation domain; CRPV, cottontail rabbit papillomavirus; HSV, herpes simplex virus; SV40, simian virus 40; GST, glutathione S-transferase; SPA, scintillation proximity assay; SEAP, secreted alkaline phosphatase; ELISA, enzyme-linked immunosorbent assay; DBD, DNA-binding domain; TCEP, tris(2-carboxyethyl)phosphine; BrdUrd, 5-bromo-2′-deoxyuridine; PBS, phosphate-buffered saline; ori, origin. infect the anogenital region. These HPV types have been classified as low risk or high risk types depending on whether they cause benign warts or lesions that can progress to invasive cancer, respectively. Current therapies to remove HPV-induced lesions include a variety of ablative or cytodestructive procedures and the use of immunomodulatory molecules such as imiquimod (5Beutner K.R. Am. J. Med. 1997; 102: 28-37Abstract Full Text Full Text PDF PubMed Google Scholar, 6Beutner K.R. Tyring S.K. Trofatter Jr., K.F. Douglas Jr., J.M. Spruance S. Owens M.L. Fox T.L. Hougham A.J. Schmitt K.A. Antimicrob. Agents Chemother. 1998; 42: 789-794Crossref PubMed Google Scholar) to stimulate a host immune response. Small molecule antivirals for the treatment of HPV infections do not currently exist. The life cycle of HPV is coupled to the cellular differentiation program that occurs in the epithelium (7Stubenrauch F. Laimins L.A. Semin. Cancer Biol. 1999; 9: 379-386Crossref PubMed Scopus (185) Google Scholar). Maintenance of the viral genome in infected cells of the basal layer is essential for the viral life cycle and the ensuing pathology. Maintenance of the HPV episome in primary keratinocyte cultures depends on the function of E1 and E2, a 3′-5′ helicase (8Sverdrup F. Myers G. Myers G. Baker C. Munger K. Sverdrup F. McBride A. Bernard H.-U. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos, NM1997: 37-53Google Scholar) and a sequence-specific DNA-binding protein (9McBride A. Myers G. Myers G. Baker C. Munger K. Sverdrup F. McBride A. Bernard H.-U. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos, NM1997: 54-73Google Scholar), respectively, which are required for replication of the genome (10Chow L.T. Broker T.R. Intervirology. 1994; 37: 150-158Crossref PubMed Scopus (119) Google Scholar). HPV DNA replication is initiated by the co-operative binding of E1 and E2 to specific DNA sequences within the viral origin (11Yang L. Li R. Mohr I. Clark R. Botchan M.R. Nature. 1991; 353: 628-633Crossref PubMed Scopus (240) Google Scholar, 12Seo Y.-S. Müller F. Lusky M. Gibbs E. Kim H.-Y. 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McBride A.A. Virology. 1995; 211: 385-396Crossref PubMed Scopus (60) Google Scholar, 20Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar, 21Yasugi T. Benson J.D. Sakai H. Vidal M. Howley P.M. J. Virol. 1997; 71: 891-899Crossref PubMed Google Scholar, 22Zou N. Liu J.-S. Kuo S.-R. Broker T.R. Chow L.T. J. Virol. 1998; 72: 3436-3441Crossref PubMed Google Scholar). Assembly of this initial E1-E2-ori complex serves as a starting point for the recruitment of additional E1 molecules (23Lusky M. Hurwitz J. Seo Y.-S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8895-8899Crossref PubMed Scopus (79) Google Scholar, 24Sanders C.M. Stenlund A. EMBO J. 1998; 17: 7044-7055Crossref PubMed Scopus (93) Google Scholar) and their assembly into hexamers and double hexamers that have ATPase and helicase activity (25Fouts E.T. Egelman E.H. Botchan M.R. J. Biol. Chem. 1999; 274: 4447-4458Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 26Sedman J. Stenlund A. J. Virol. 1998; 72: 6893-6897Crossref PubMed Google Scholar). Any of the protein-protein and protein-DNA interactions occurring at the origin could in principle be targeted for the development of small molecule antivirals to treat HPV infections. In reality, however, not all targets are readily amenable to inhibition by small molecules. Protein interactions for example have been considered difficult to inhibit because they often involve large surfaces devoid of small molecule binding pockets (27Way J.C. Curr. Opin. Chem. Biol. 2000; 4: 40-46Crossref PubMed Scopus (43) Google Scholar, 28Gadek T.R. Nicholas J.B. Biochem. Pharmacol. 2003; 65: 1-8Crossref PubMed Scopus (107) Google Scholar). In the absence of structural information, one often has to rely on screening large compound collections to determine whether a protein can be antagonized by small molecules and for the identification of lead inhibitors. Here we present a class of small molecules that bind reversibly to the E2 transactivation domain and inhibit the E1-E2 protein interaction in vitro and in vivo. The chemical features and synthetic procedures for these molecules will be described elsewhere. 2C. Yoakim, N. J. B. J. A. M. G. J. and P. for our these are the small molecule inhibitors of HPV DNA replication with cellular such they highlight the potential of the E1-E2 interaction as an antiviral target. of inhibitors and will be described elsewhere. 2C. Yoakim, N. J. B. J. A. M. G. J. and P. for was by procedures the and was used for of E2 at to a acid was by a the and the in The to E2 by in vitro was by a to of the genome in The the origin of replication was by a to of into and respectively, the origin and of secreted alkaline by a of the genome or the into The E1 to the transactivation domain of was by a E1 into E2 by as described A. Pelletier A. J. 1994; Scopus Google Scholar) and into of E2 was with the the described of the and the of the used for or will be was and by and and in a of 25 and at was 11 of and was as by or of Virology. PubMed Scopus Google Scholar), E1 and P.W. Pelletier A. Brault K. S. E. L. M. Cordingley M.G. Archambault J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), E2 P.W. Pelletier A. Brault K. S. E. L. M. Cordingley M.G. Archambault J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar) have been described The coupled was used for in vitro of E1 and E2 as described S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar). Formation and complex was as described P.W. Pelletier A. Brault K. S. E. L. M. Cordingley M.G. Archambault J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). all E1-E2-ori complex with and in the assay and used at a E1 or E2 as In with in vitro E2, of was used binding In this E2 but E1 used as because the present the assay to binding of E2 to the origin in the absence of E1 was in a to and the E2. assay to binding of large to origin was of large and an of the This was by which the ori, and and was the J. J. Virol. PubMed Google Scholar) and protein in vitro E2 proteins or E2 the DNA binding a for the origin was was used for the in 6 to for the obtained in with proteins the DNA binding the binding the was to to which an been the for with protein-DNA by which the that and also because was was and the for and a scintillation is by a of the inhibition the program or E1-E2 was used as the for all with as at the in at with of at on a in a of in with and the and with 25 of E2 or of in vitro E2. E2 with or E1 with in vitro and inhibitors and of for this E1 was by with the used in the complex assay and with with and activity was to the an with a in and and to of proteins at at and the was the to the and for the is the a Isothermal calorimetry was the at a of 25 was into an of and TCEP, was a to a at in the and the protein was a and the was in a for at to remove The of the protein was to be by at on a of initial was by a of at a of the to a of the of by the at of of inhibition was a of the E1-E2-ori complex assay described E2 was with in assay at a to or the The was and with E1 and such that the of was in and used as the for of The of E2 was in to for the in on of E2. HPV DNA HPV DNA replication was as described S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar, S. Pelletier A. Brault K. Wardrop E. White P.W. Cordingley M.G. Archambault J. J. Virol. 2000; PubMed Scopus Google Scholar, S. Brault J. White P.W. Archambault J. J. Virol. 2003; PubMed Scopus Google Scholar). of HPV DNA was by on a and to the E1 inhibitors to the in a of compound was in DNA replication was the E1-E2 cells transfected with respectively, E2 and a the of the origin of DNA replication of cells with and in inhibitors to the in a of The of in the was by the to the by the was in which by a for inhibitors also on cells transfected with respectively, an of and the of of the DNA-binding domain to the domain of of a of Small the of E1 and E2 to the binding of E1 and E2 to the viral origin is an essential in the of HPV DNA inhibitors of this a high screening assay was that the binding of E1 and E2 to origin of E1 to the origin was by of an coupled to scintillation proximity assay S. Brault J. White P.W. Archambault J. J. Virol. 2003; PubMed Scopus Google Scholar) in the of an of DNA to that binding of E1 to the origin was dependent on with E2. that a was E1 and E2 to the This was dependent on the of E1 with E2 because was to by the amino acid in E2 that E1 binding Virology. 1998; PubMed Scopus Google Scholar, Botchan M.R. J. Virol. PubMed Google Scholar, H. T. Benson J.D. Howley P.M. J. Virol. PubMed Google Scholar). In the substitution, which the transactivation function of E2 and not to bind to E1 Botchan M.R. J. Virol. PubMed Google Scholar, H. T. Benson J.D. Howley P.M. J. Virol. PubMed Google Scholar), Screening of our corporate compound collection with this assay in of this to such as and These compounds or in a that binding of large to origin that they do not with the scintillation proximity or bind to DNA or also E1 and E2 the low risk anogenital at a the The E1-E2 Protein the of binding of E1 and E2 to DNA the interaction of both proteins with and with The inhibitors found to be or in that the binding of E2 to DNA was also at the helicase activity of E1 this class of compounds not the binding of E1 or E2 to These results that the E1-E2 protein interaction be the target of This was an The E2 protein used in this assay was cells or obtained by in vitro both of E1 to E2 was an by a coupled to in the was dependent on the interaction of E1 with E2 because was by the in E2. the can be in and all compounds binding of E1 to E2 with with that in the E1-E2-ori complex The compounds the E1-E2 interaction of whether the assay was with E1 cells or with E1 by in vitro not the target of these inhibitors is the E1-E2 protein to the of found that and antagonize the E1-E2 protein we to determine to which protein these compounds we whether of compound on the of E1 or E2. amino such as are often found in or to binding and the binding of to such has been to protein fluorescence in M.R. 1997; PubMed Scopus Google Scholar). was into of E1 or E2 and a was on the fluorescence of a of was for E2, with this compound binding to E2. The on E1 and the of for E2 at high compound could be by binding of compound to protein of the low of activity in the DNA binding assay E2 can be into an N-terminal transactivation domain amino and a C-terminal DNA domain the amino by a acid with (9McBride A. Myers G. Myers G. Baker C. Munger K. Sverdrup F. McBride A. Bernard H.-U. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos, NM1997: 54-73Google Scholar). for is that the TAD, but not the DBD, interacts with E1 M. Stenlund A. J. Virol. 1997; 71: 3853-3863Crossref PubMed Google Scholar, C.M. Stenlund A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we the titration with a protein the E2 TAD, as as with as a and The results binding of to the but not to GST, that the compound to the results also obtained with a of the E2 not we the binding of to a titration calorimetry of was obtained by in with the obtained in the E1-E2-ori complex The is with the of a because compound is and has been for compounds in this that one of the is S. K. L. A. E. L. L. C. Yoakim, M. G. and J. the results evidence that this class of the E1-E2 protein interaction by binding to the E2 Functional the E2 the of the of this we found that in vitro E2 can bind with E1 to the or E2, however, was found to be resistant to inhibition by compound not the low of amino acid between the of and that of we found that the inhibitors are the E2 protein of the high risk HPV types and not which at the amino acid are to E2. use of the that E2 types are resistant to inhibition to the that the is the target of the inhibitors. we E2 proteins the with the and DNA-binding domain CRPV, or the These in vitro and in the E1-E2-ori complex assay E1. could bind with E1 on the but the one the was sensitive to inhibition by compound This evidence that the is the target of is to be difficult to inhibitors of protein interactions that by binding by with the target protein or by (27Way J.C. Curr. Opin. Chem. Biol. 2000; 4: 40-46Crossref PubMed Scopus (43) Google Scholar). determine whether inhibition of E1-E2-ori complex was we the in In this E2, the target was with a high of and into the E1-E2-ori complex assay to a compound the that the is by a the activity of E2 be of the the of inhibition is inhibition be a we used the with this compound in inhibition to a the In of E2 with compound not lead to inhibition 3rd to and the in compound by a of DNA in we to determine whether the compound be to inhibit HPV DNA replication in transiently transfected cells. In this E1 and E2 transfected into cells with a the origin of DNA cells with of compound to 25 and the of DNA was by a for we in the of the compound on cellular DNA replication as by that compound was to inhibit HPV DNA replication in a with a of In cellular DNA replication was at a of of The inhibition of at the is to of the compound specific inhibition of cellular DNA of the E1-E2 Protein in the of of the inhibitors in we a cellular assay that the interaction of E1 and E2. this assay the of a the of the origin of of respectively, E2 and the E1 protein to the transactivation domain of In this E2 could by of the The of transactivation by E2 is with the that E2 of the low risk HPV types are R. M.G. J. Virol. PubMed Google Scholar). of the was dependent on the of both and E2 and on their because was by the in E2 which E1 binding the in E2 that of the was dependent on the interaction between E1 and E2, we the of compounds and in this assay and in a one on E1 and E2 and by the proteins with of and The of compound in the and HPV DNA replication that activity on viral DNA replication is to of the E1-E2 protein and also found to be the with with of and 6 respectively. the in between and protein was in this cellular assay with in vitro The for the in and proteins in vivo is currently but could be to in the of E2 in vivo with in vitro a for we that the inhibitors on the of an by a protein an additional for we showed that an of that is in vitro in the E1-E2-ori complex was also in the cellular assay not additional evidence that the inhibitors in vivo by the as in we use of a E2 protein that has a for this class of inhibitors. This protein a amino in the TAD, E100A, which sensitive to this class of inhibitors type E2 in the in vitro E1-E2-ori complex The was a of the E2 at identifying in compound binding not this amino acid also the of E2 in the assay This evidence that the of of this class of inhibitors is the in vivo as in of of the E1-E2 this we have the identification of the small molecule inhibitors of the E1-E2 protein interaction of HPV DNA replication in vivo. have by changes in protein fluorescence and titration that compounds bind to the transactivation domain of E2. Functional evidence for binding to the E2 was also obtained by swapping this domain between a sensitive E2 type (HPV11) and a resistant one as as by identifying an amino acid within the TAD, E100A, that increases inhibition by in The of inhibitors of the E1-E2 protein interaction HPV types 6 and 11 the potential of E2 as a target for the treatment of anogenital The genome of and is present in in of warts L. L. H. U. Hausen H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. J. Microbiol. 1993; PubMed Google Scholar), and inhibition of the E1-E2 interaction is to Chem. Chemother. 1998; 9: PubMed Scopus Google Scholar). In this is that E1 and E2 are to be at in infections in transfected a that could lead to the of these inhibitors. the antiviral activity of these inhibitors the development of a and HPV viral replication of inhibitors in this are the E2 protein of the low risk HPV 6 and but are the protein by in This in was both with proteins and with proteins by in vitro we have found evidence that E1 and E2 interacts the proteins not we that the inhibitors bind to E2. The and are at the primary amino acid and of the amino acid or could binding of the we found that the in in cellular is the in is in the HPV DNA replication This between type 6 and 11 was also in the E1-E2 interaction assay that this class of compound inhibit the E1-E2 interaction of both HPV types with in cells. is that a for binding has a for both HPV types in vivo in because the of E2 in vivo is as a of interaction with cellular will be required to this in E2 in to this a acid E2 and was to be of the E1-E2 interaction in vitro H. Howley P.M. Benson J.D. J. Virol. 1998; 72: PubMed Google Scholar). have an essential for in interaction with E1. this to was to the of E2 to bind to E1 Virology. 1998; PubMed Scopus Google Scholar, Botchan M.R. J. Virol. PubMed Google Scholar, H. T. Benson J.D. Howley P.M. J. Virol. 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Protein Sci. 1994; PubMed Scopus Google Scholar). protein interaction inhibitors have been identified to because has been to such as and have in in identifying small molecule inhibitors of the E1-E2 interaction by high This that will also small molecule inhibitors of protein interactions as and for critical of the

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