RESUMO
Recent clinical trials have demonstrated the potential of adeno-associated virus (AAV)-based vectors for treating rare diseases. However, significant barriers remain for the translation of these vectors into widely available therapies. In particular, exposure to the AAV capsid can generate an immune response of neutralizing antibodies. One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization. To accomplish this, we isolated a monoclonal antibody against AAV9 following immunization of BALB/c mice and hybridoma screening. This antibody, PAV9.1, is specific for intact AAV9 capsids and has a high neutralizing titer of >1:160,000. We used cryo-electron microscopy to reconstruct PAV9.1 in complex with AAV9. We then mapped its epitope to the 3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588-QAQAQT-592. Capsid mutagenesis demonstrated that even a single amino acid substitution within this epitope markedly reduced binding and neutralization by PAV9.1. In addition, in vivo studies showed that mutations in the PAV9.1 epitope conferred a "liver-detargeting" phenotype to the mutant vectors, unlike AAV9, indicating that the residues involved in PAV9.1 interactions are also responsible for AAV9 tropism. However, we observed minimal changes in binding and neutralizing titer when we tested these mutant vectors for evasion of polyclonal sera from mice, macaques, or humans previously exposed to AAV. Taken together, these studies demonstrate the complexity of incorporating mapped neutralizing epitopes and previously identified functional motifs into the design of novel capsids able to evade immune response.IMPORTANCE Gene therapy utilizing viral vectors has experienced recent success, culminating in U.S. Food and Drug Administration approval of the first adeno-associated virus vector gene therapy product in the United States: Luxturna for inherited retinal dystrophy. However, application of this approach to other tissues faces significant barriers. One challenge is the immune response to viral infection or vector administration, precluding patients from receiving an initial or readministered dose of vector, respectively. Here, we mapped the epitope of a novel neutralizing antibody generated in response to this viral vector to design a next-generation capsid to evade immune responses. Epitope-based mutations in the capsid interfered with the binding and neutralizing ability of the antibody but not when tested against polyclonal samples from various sources. Our results suggest that targeted mutation of a greater breadth of neutralizing epitopes will be required to evade the repertoire of neutralizing antibodies responsible for blocking viral vector transduction.
Assuntos
Anticorpos Neutralizantes/imunologia , Capsídeo/imunologia , Dependovirus/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Microscopia Crioeletrônica , Análise Mutacional de DNA , Dependovirus/genética , Dependovirus/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estados Unidos , Tropismo ViralRESUMO
Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.
Assuntos
Aminoácidos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Substituição de Aminoácidos , Animais , Asparagina/química , Asparagina/metabolismo , Capsídeo/química , Proteínas do Capsídeo/química , Dependovirus/classificação , Engenharia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional , Sorogrupo , Relação Estrutura-Atividade , Transdução Genética , Tropismo ViralRESUMO
Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.
Assuntos
Proteínas do Capsídeo/genética , DNA Viral/análise , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Fígado/virologia , Sequência de Aminoácidos , Aminoácidos , Animais , Biblioteca Gênica , Terapia Genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Análise de Sequência de DNA , Transdução GenéticaRESUMO
The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between ßH and ßI of the core ß-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/ultraestrutura , Dependovirus/química , Dependovirus/ultraestrutura , Cristalografia por Raios X , Eletroquímica , Modelos Moleculares , Conformação ProteicaRESUMO
The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel ß-barrel with large loop regions between the strands. The ß-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for â¼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors.
Assuntos
Capsídeo/química , Parvovirus H-1/química , Vírion/química , Sequência de Aminoácidos , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Parvovirus H-1/genética , Parvovirus H-1/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Vírion/genética , Vírion/metabolismoRESUMO
Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Distrofia Muscular de Duchenne/terapia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Linhagem Celular , Criança , Pré-Escolar , Dependovirus/fisiologia , Distrofina/genética , Distrofina/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Conformação Proteica , Alinhamento de Sequência , Linfócitos T/imunologia , Transdução Genética , Tropismo ViralRESUMO
We report the first identification, genetic characterization and disease association studies of several novel species of canine bocaviruses (CBoV). Evolutionary analysis confirmed that CBoV are genetically distinct from the only other known canine bocavirus, minute virus of canines, with which they share less than 63, 62 and 64â% protein identity in NS, NP and VP genes, respectively. Comparative genetic analysis of 37 VP gene variants found in diseased and healthy animals showed that these novel viruses are genetically highly diverse and are common in canine respiratory infections that have remained undetected until now. Interestingly, we observed that a CBoV genotype with a unique deletion in the VP2 gene was significantly more prevalent in animals with respiratory diseases compared with healthy animals.
Assuntos
Bocavirus/classificação , Bocavirus/isolamento & purificação , Doenças do Cão/virologia , Infecções por Parvoviridae/veterinária , Animais , Bocavirus/genética , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Cães , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Infecções por Parvoviridae/virologia , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
Pyruvate dehydrogenase (PDH) of Escherichia coli is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in lpd led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a pflB, ldhA double mutant of E. coli utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic growth. With the exception of E354M, all changes also restored PDH-dependent anaerobic growth of and fermentation by an ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K(i) for NADH compared with the native complex. The apparent K(m) for pyruvate or NAD(+) for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.
Assuntos
Di-Hidrolipoamida Desidrogenase/genética , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , NAD/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Substituição de Aminoácidos , Di-Hidrolipoamida Desidrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Etanol/metabolismo , Fermentação , Ácido Glutâmico/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Complexo Piruvato Desidrogenase/genética , Ácido Pirúvico/metabolismoRESUMO
Crystals of H-1 Parvovirus (H-1PV), an antitumor gene-delivery vector, were obtained for DNA-containing capsids and diffracted X-rays to 2.7â Å resolution using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=255.4, b=350.4, c=271.6â Å, ß=90.34°. The unit cell contained two capsids, with one capsid per crystallographic asymmetric unit. The H-1PV structure has been determined by molecular replacement and is currently being refined.
Assuntos
Parvovirus H-1/química , Proteínas do Capsídeo/química , Cristalização , Cristalografia por Raios X , Parvovirus H-1/isolamento & purificação , Difração de Raios XRESUMO
We report the generation of a new class of adeno-associated virus serotype 9 (AAV9)-derived vectors displaying selective loss of liver tropism and demonstrating potential for cardiac and musculoskeletal gene transfer applications. Random mutagenesis of residues within a surface-exposed region of the major AAV9 capsid protein yielded a capsid library with mutations clustered at the icosahedral threefold symmetry axis. Using a combination of sequence analysis, structural models, and in vivo screening, we identified several functionally diverse AAV9 variants. The latter were classified into three functional subgroups, with respect to parental AAV9 displaying: (i) decreased transduction efficiency across multiple tissues; (ii) a selective decrease in liver transduction, or (iii) a similar transduction profile. Notably, variants 9.45 and 9.61 (subgroup II) displayed 10- to 25-fold lower gene transfer efficiency in liver, while transducing cardiac and skeletal muscle as efficiently as AAV9. These results were further corroborated by quantitation of vector genome copies and histological analysis of reporter (tdTomato) gene expression. The study highlights the feasibility of generating AAV vectors with selectively ablated tissue tropism, which when combined with other targeting strategies could allow sharply segregated gene expression. Liver-detargeted AAV9 variants described herein are excellent candidates for preclinical evaluation in animal models of cardiac and musculoskeletal disease.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Técnicas de Transferência de Genes , Células HEK293 , Humanos , CamundongosRESUMO
Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Retina/metabolismo , Tirosina/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Mutação , Mutação Puntual/genética , Retina/patologiaRESUMO
Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Policitemia Vera/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Estilbenos/farmacologia , Substituição de Aminoácidos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Mutação de Sentido Incorreto , Policitemia Vera/enzimologia , Policitemia Vera/genética , Inibidores de Proteínas Quinases/química , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Estilbenos/química , Relação Estrutura-AtividadeRESUMO
The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (ßB to ßI) ß-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.
Assuntos
Proteínas do Capsídeo/química , Dependovirus/química , Dependovirus/metabolismo , Polissacarídeos/metabolismo , Vírion/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Dependovirus/classificação , Humanos , Modelos Moleculares , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.
Assuntos
Dependovirus/genética , Vetores Genéticos , Mutação Puntual , Transdução Genética , Tirosina/genética , Animais , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Terapia Genética , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , UbiquitinaçãoRESUMO
Recently, a convincing body of evidence has accumulated suggesting that the overexpression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently, there is no available X-ray crystal structure of CA IX, and this lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using (18)O-exchange and structurally using X-ray crystallography, alone and in complex with five CA sulfonamide-based inhibitors (acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide), and compared to CA II. This structural information has been evaluated by both inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX, inducing an active-site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme-specific CA IX inhibitors, which may lead to development of new therapeutic treatments of some cancers.
Assuntos
Antígenos de Neoplasias/metabolismo , Antineoplásicos/análise , Antineoplásicos/farmacologia , Inibidores da Anidrase Carbônica/análise , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Desenho de Fármacos , Mimetismo Molecular/efeitos dos fármacos , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antineoplásicos/química , Western Blotting , Anidrase Carbônica IX , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Adeno-associated virus serotype 2 (AAV2) uses heparan sulfate proteoglycan as a cell surface-attachment receptor. In this study the structures of AAV2 alone and complexed with heparin were determined to approximately 18A resolution using cryo-electron microscopy and three-dimensional image reconstruction. A difference map showed positive density, modeled as heparin, close to the icosahedral twofold axes and between the protrusions that surround the threefold axes of the capsid. Regions of the model near the threefold place the receptor in close proximity to basic residues previously identified as part of the heparin binding site. The region of the model near the twofold axes identifies a second contact site, not previously characterized but which is also possibly configured by heparin binding. The difference map also revealed two significant conformational changes: (I) at the tops of the threefold protrusions, which have become flattened in the complex, and (II) at the fivefold axes where the top of the channel is widened possibly in response to movement of the HI loops in the capsid proteins. Ordered density in the interior of the capsid in the AAV2-heparin complex was interpreted as nucleic acid, consistent with the presence of non-viral DNA in the expressed capsids.
Assuntos
Dependovirus/química , Heparina/química , Capsídeo/química , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nucleocapsídeo/química , Conformação Proteica , Vírion/químicaRESUMO
The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Dependovirus/metabolismo , Sequência de Aminoácidos , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Sequência Conservada , Dependovirus/genética , Dependovirus/ultraestrutura , Deleção de Genes , Glicina/genética , Glicina/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Fenótipo , Estrutura Quaternária de Proteína , Alinhamento de SequênciaRESUMO
A new adeno-associated virus (AAV), referred to as AAV(VR-942), has been isolated as a contaminant of adenovirus strain simian virus 17. The sequence of the rep gene places it in the AAV serotype 2 (AAV2) complementation group, while the capsid is only 88% identical to that of AAV2. High-level AAV(VR-942) transduction activity requires cell surface heparan sulfate proteoglycans, although AAV(VR-942) lacks residues equivalent to the AAV2 R585 and R588 amino acid residues essential for mediating the interaction of AAV2 with the heparan sulfate proteoglycan receptor. Instead, AAV(VR-942) uses a distinct transduction region. This finding shows that distinct domains on different AAV isolates can be responsible for the same activities.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Dependovirus/isolamento & purificação , Proteoglicanas de Heparan Sulfato/metabolismo , Transdução Genética , Sequência de Aminoácidos , Aminoácidos Básicos , Animais , Células COS , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Dependovirus/classificação , Dependovirus/genética , Evolução Molecular , Proteínas de Fluorescência Verde/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
The crystal structure of the Mycoplasma hyorhinis protein Mh-p37 has been solved and refined to 1.9 A resolution. This is the first de novo structure to be determined using the recently described heavy-atom reagent [Beck et al. (2008), Acta Cryst. D64, 1179-1182] 5-amino-2,4,6-triiodoisophthalic acid (I3C), which contains three I atoms arranged in an equilateral triangle, by SIRAS methods. Data collection was performed in-house at room temperature. SHELXD and SHELXE were used to determine the I-atom positions and phase the native protein and PHENIX AutoBuild software was used to automatically fit the amino-acid sequence to the electron-density map. The structure was refined using SHELX97 to an R(cryst) of 18.6% and an R(free) of 24.0%. Mh-p37 is an alpha/beta protein with two well defined domains which are separated by a deep cleft. An unanticipated ligand bound in the center of the molecule at the base of the cleft has been modeled as thiamine pyrophosphate or vitamin B(1). Retrospective attempts to solve the crystal structure by Patterson search methods using either isomorphous or anomalous differences failed. Additionally, attempts to use proteins with the highest structural homology in the Protein Data Bank to phase the data by molecular replacement were unsuccessful, most likely in hindsight because of their poor structural agreement. Therefore, the I3C reagent offers an alternative, quick and inexpensive method for in-house phasing of de novo structures where other methods may not be successful.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Mycoplasma hyorhinis , Neoplasias/metabolismo , Proteínas Recombinantes/química , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X/métodos , Humanos , Neoplasias/patologia , Ligação Proteica/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia Estrutural de Proteína , Tiamina Pirofosfato/metabolismo , Transferrina/metabolismo , Ácidos Tri-Iodobenzoicos/metabolismoRESUMO
In our quest for HIV-1 protease inhibitors that are not affected by the V82A resistance mutation, we have synthesized and tested a second generation set of C2-symmetric HIV-1 protease inhibitors that contain a cyclohexane group at P1 and/or P1'. The binding affinity results indicate that these compounds have an improved response to the appearance of the V82A mutation than the parent compound. The X-ray structure of one of these compounds with the V82A HIV-1 PR variant provides the structural rationale for the better resistance profile of these compounds. Moreover, scrutiny of the X-ray structure suggests that the ring of the Cha side chain might be in a boat rather than in the chair conformation, a result supported by molecular dynamics simulations.