RESUMEN
UNLABELLED: The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE: While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 11/fisiología , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Papillomavirus Humano 11/genética , Humanos , Estructura Terciaria de Proteína , Eliminación de Secuencia , Proteínas Virales/genéticaRESUMEN
An anthranilic acid series of allosteric thumb pocket 2 HCV NS5B polymerase inhibitors exhibited hindered rotation along a covalent bond axis, and the existence of atropisomer chirality was confirmed by NMR, HPLC analysis on chiral supports, and computational studies. A thorough understanding of the concerted rotational properties and the influence exerted by substituents involved in this steric phenomenon was attained through biophysical studies on a series of truncated analogues. The racemization half-life of a compound within this series was determined to be 69 min, which was consistent with a class 2 atropisomer (intermediate conformational exchange). It was further found by X-ray crystallography that one enantiomer of a compound bound to the intended HCV NS5B polymerase target whereas the mirror image atropisomer was able to bind to an unrelated HIV matrix target. Analogues were then identified that selectively inhibited the former. These studies highlight that atropisomer chirality can lead to distinct entities with specific properties, and the phenomenon of atropisomerism in drug discovery should be evaluated and appropriately managed.
Asunto(s)
Antivirales/farmacología , VIH/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Antivirales/química , Antivirales/farmacocinética , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Hepacivirus/enzimología , Espectroscopía de Resonancia Magnética , EstereoisomerismoRESUMEN
The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/metabolismo , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/química , Línea Celular , Cristalografía por Rayos X , VIH-1/fisiología , Humanos , Espectroscopía de Resonancia Magnética , Reacción en Cadena de la Polimerasa , Replicación Viral/efectos de los fármacosRESUMEN
Despite truly impressive achievements in the global battle against HIV there remains a need for new drugs directed against novel targets, and the viral capsid protein (CA) may represent one such target. Intense structural characterization of CA over the last two decades has provided unprecedented insight into the structure and assembly of this key viral protein. Furthermore, several inhibitor-binding sites that elicit antiviral activity have been reported on CA, two of which are located on its N-terminal domain (CANTD). In this work, the binding of a novel capsid-assembly inhibitor that targets a unique inhibitory site on CANTD is reported. Moreover, whereas cocrystallization of CANTD in complex with ligands has proven to be challenging in the past, the use of this inhibitor as a tool compound is shown to vastly facilitate ternary cocrystallizations with CANTD. This improvement in crystallization is likely to be achieved through the formation of a compound-mediated homodimer, the intrinsic symmetry of which greatly increases the prospect of generating a crystal lattice. While protein engineering has been used in the literature to support a link between the inherent symmetry of a macromolecule and its propensity to crystallize, to our knowledge this work represents the first use of a synthetic ligand for this purpose.
Asunto(s)
Antivirales/química , Proteínas de la Cápside/química , Cápside/química , VIH-1/química , Antivirales/metabolismo , Sitios de Unión , Cápside/metabolismo , Proteínas de la Cápside/antagonistas & inhibidores , Proteínas de la Cápside/metabolismo , Cristalización , VIH-1/metabolismo , Modelos Moleculares , Difracción de Rayos XRESUMEN
The optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly that possess a labile stereocenter at C3 is described. Quaternization of the C3 position of compound 1 in order to prevent racemization gave compound 2, which was inactive in our capsid disassembly assay. A likely explanation for this finding was revealed by in silico analysis predicting a dramatic increase in energy of the bioactive conformation upon quaternization of the C3 position. Replacement of the C3 of the diazepine ring with a nitrogen atom to give the 1,5-dihydro-benzo[f][1,3,5]triazepine-2,4-dione analog 4 was well tolerated. Introduction of a rigid spirocyclic system at the C3 position gave configurationally stable 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione analog 5, which was able to access the bioactive conformation without a severe energetic penalty and inhibit capsid assembly. Preliminary structure-activity relationships (SAR) and X-ray crystallographic data show that knowledge from the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly can be transferred to these new scaffolds.
Asunto(s)
Fármacos Anti-VIH/química , Benzodiazepinas/química , Proteínas de la Cápside/antagonistas & inhibidores , VIH-1/metabolismo , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Benzodiazepinas/síntesis química , Benzodiazepinas/farmacología , Sitios de Unión , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Terciaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , Ensamble de Virus/efectos de los fármacosRESUMEN
Detailed structure-activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position. The molecular basis of how compound 27 inhibits mature CA assembly is rationalized using high-resolution structural information. Our understanding of how compound 27 may inhibit immature Gag assembly is also discussed.
Asunto(s)
Fármacos Anti-VIH/química , Benzodiazepinas/química , Benzodiazepinonas/química , Proteínas de la Cápside/antagonistas & inhibidores , VIH-1/metabolismo , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Benzodiazepinas/síntesis química , Benzodiazepinas/farmacología , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacología , Sitios de Unión , Células CACO-2 , Proteínas de la Cápside/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Estructura Terciaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , Ensamble de Virus/efectos de los fármacosRESUMEN
The HIV-1 capsid (CA) protein, a domain of Gag, which participates in formation of both the mature and immature capsid, represents a potential target for anti-viral drug development. Characterization of hits obtained via high-throughput screening of an in vitro capsid assembly assay led to multiple compounds having this potential. We previously presented the characterization of two inhibitor series that bind the N-terminal domain of the capsid (CA(NTD)), at a site located at the bottom of its helical bundle, often referred to as the CAP-1 binding site. In this work we characterize a novel series of benzimidazole hits. Initial optimization of this series led to compounds with improved in vitro assembly and anti-viral activity. Using NMR spectroscopy we found that this series binds to a unique site on CA(NTD), located at the apex of the helical bundle, well removed from previously characterized binding sites for CA inhibitors. 2D (1)H-(15)N HSQC and (19)F NMR showed that binding of the benzimidazoles to this distinct site does not affect the binding of either cyclophilin A (CypA) to the CypA-binding loop or a benzodiazepine-based CA assembly inhibitor to the CAP-1 site. Unfortunately, while compounds of this series achieved promising in vitro assembly and anti-viral effects, they also were found to be quite sensitive to a number of naturally occurring CA(NTD) polymorphisms observed among clinical isolates. Despite the negative impact of this finding for drug development, the discovery of multiple inhibitor binding sites on CA(NTD) shows that capsid assembly is much more complex than previously realized.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , VIH-1 , Fármacos Anti-VIH/metabolismo , Bencimidazoles/química , Sitios de Unión , Unión Competitiva , Cristalografía por Rayos X , Ciclofilina A/metabolismo , Ciclofilina A/farmacología , VIH-1/genética , VIH-1/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Polimorfismo Genético , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The nucleocapsid (NC) protein is an essential factor with multiple functions within the human immunodeficiency virus type 1 (HIV-1) replication cycle. In this study, we describe the discovery of a novel series of inhibitors that targets HIV-1 NC protein by blocking its interaction with nucleic acids. This series was identified using a previously described capsid (CA) assembly assay, employing a recombinant HIV-1 CA-NC protein and immobilized TG-rich deoxyoligonucleotides. Using visible absorption spectroscopy, we were able to demonstrate that this new inhibitor series binds specifically and reversibly to the NC with a peculiar 2:1 stoichiometry. A fluorescence-polarization-based binding assay was also developed in order to monitor the inhibitory activities of this series of inhibitors. To better characterize the structural aspect of inhibitor binding onto NC, we performed NMR studies using unlabeled and (13)C,(15)N-double-labeled NC(1-55) protein constructs. This allowed the determination of the solution structure of a ternary complex characterized by two inhibitor molecules binding to the two zinc knuckles of the NC protein. To the best of our knowledge, this represents the first report of a high-resolution structure of a small-molecule inhibitor bound to NC, demonstrating sub-micromolar potency and moderate antiviral potency with one analogue of the series. This structure was compared with available NC/oligonucleotide complex structures and further underlined the high flexibility of the NC protein, allowing it to adopt many conformations in order to bind its different oligonucleotide/nucleomimetic targets. In addition, analysis of the interaction details between the inhibitor molecules and NC demonstrated how this novel inhibitor series is mimicking the guanosine nucleobases found in many reported complex structures.
Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/metabolismo , VIH-1/efectos de los fármacos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Fármacos Anti-VIH/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidoresRESUMEN
The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)-1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high-throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X-ray co-crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand-based (19)F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N-terminal domain of the capsid (CA(NTD)) as its molecular target. Protein-based NMR ((1)H and (15)N chemical shift perturbation analysis) identified key residues within the CA(NTD) involved in inhibitor binding, while X-ray co-crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.
Asunto(s)
Benzodiazepinas/metabolismo , Proteínas de la Cápside/metabolismo , VIH-1/metabolismo , Benzodiazepinas/química , Sitios de Unión , Proteínas de la Cápside/química , Cristalografía por Rayos X , Flúor/química , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno/química , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
A uHTS campaign led to the discovery of a 5-(5-furan-2-ylpyrazol-1-yl)-1H-benzimidazole series that inhibits assembly of HIV-1 capsid. Synthetic manipulations at N1, C2 and C16 positions improved the antiviral potency by a . The X-ray structure of 33 complexed with the capsid N-terminal domain allowed identification of major interactions between the inhibitor and the protein.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.
Asunto(s)
Fármacos Anti-VIH/farmacología , Cápside/efectos de los fármacos , Productos del Gen gag/antagonistas & inhibidores , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Bencimidazoles/farmacología , Benzodiazepinas/farmacología , Cápside/metabolismo , Línea Celular , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/química , VIH-1/genética , VIH-1/fisiología , Humanos , Estructura Terciaria de Proteína , Ensamble de Virus/efectos de los fármacosRESUMEN
The discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly is described. Synthesis of analogs of the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione hit established structure-activity relationships. Replacement of the enamine functionality of the hit series with either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse transcriptase. Optimization of the bicyclic benzodiazepine scaffold to include a 3-phenyl substituent led to lead compound 48, a pure capsid assembly inhibitor with improved antiviral activity.
Asunto(s)
Fármacos Anti-VIH/química , Benzodiazepinonas/química , Proteínas de la Cápside/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacología , Proteínas de la Cápside/metabolismo , Evaluación Preclínica de Medicamentos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , Humanos , Imidazoles/química , Pirazoles/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 11/enzimología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Papillomavirus Humano 11/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Organismos Modificados Genéticamente , Conformación Proteica , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas Virales/genéticaRESUMEN
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-A 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.
Asunto(s)
Activación Transcripcional , Proteínas Virales/química , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Aminoácidos/química , Sitios de Unión , Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Ácido Glutámico/química , Histidina/química , Leucina/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Tirosina/química , UltracentrifugaciónRESUMEN
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 approximately 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.