RESUMEN
Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC.
Asunto(s)
Antígenos de Superficie/metabolismo , Aptámeros de Nucleótidos/administración & dosificación , Glutamato Carboxipeptidasa II/metabolismo , Terapia Molecular Dirigida/métodos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Animales , Aptámeros de Nucleótidos/farmacocinética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. A significant obstacle to the clinical adoption of RNA aptamers is the high cost of manufacturing long RNA sequences through chemical synthesis. Therapeutic aptamers are often truncated postselection by using a trial-and-error process, which is time consuming and inefficient. Here, we used a "rational truncation" approach guided by RNA structural prediction and protein/RNA docking algorithms that enabled us to substantially truncateA9, an RNA aptamer to prostate-specific membrane antigen (PSMA),with great potential for targeted therapeutics. This truncated PSMA aptamer (A9L; 41mer) retains binding activity, functionality, and is amenable to large-scale chemical synthesis for future clinical applications. In addition, the modeled RNA tertiary structure and protein/RNA docking predictions revealed key nucleotides within the aptamer critical for binding to PSMA and inhibiting its enzymatic activity. Finally, this work highlights the utility of existing RNA structural prediction and protein docking techniques that may be generally applicable to developing RNA aptamers optimized for therapeutic use.
Asunto(s)
Aptámeros de Nucleótidos/química , Simulación por Computador , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Modelos Moleculares , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/farmacología , Línea Celular Tumoral , Diseño de Fármacos , Pruebas de Enzimas , Glutamato Carboxipeptidasa II/química , Glutamato Carboxipeptidasa II/metabolismo , Humanos , Masculino , Conformación de Ácido Nucleico , Neoplasias de la Próstata , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20) with an abbreviated variable region. The Sel2N20 yields RNAs 51 nt in length, which unlike longer RNAs, are more amenable to large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine, resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in control, "nontargeted" selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or more of the amplification and propagation steps of the SELEX process.
Asunto(s)
Aptámeros de Nucleótidos/química , Pirimidinas/química , Técnica SELEX de Producción de Aptámeros , Animales , Composición de Base , Secuencia de Bases , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Receptor EphA2/química , Proteínas Recombinantes/química , Análisis de Secuencia de ARN , Termodinámica , Transcripción GenéticaRESUMEN
DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Prueba de Complementación Genética , Cinética , Modelos Moleculares , Polifosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
The yeast Rev1 protein (Rev1p) is a member of the Y family of DNA polymerases that specifically catalyzes the incorporation of C opposite template G and several types of DNA damage. The X-ray crystal structure of the Rev1p-DNA-dCTP ternary complex showed that Rev1p utilizes an unusual mechanism of nucleotide incorporation whereby the template residue is displaced from the DNA double helix and the side chain of Arg-324 forms hydrogen bonds with the incoming dCTP. To better understand the impact of this protein-template-directed mechanism on the thermodynamics and kinetics of nucleotide incorporation, we have carried out pre-steady-state kinetic studies with Rev1p. Interestingly, we found that Rev1p's specificity for incorporating C is achieved solely at the initial nucleotide-binding step, not at the subsequent nucleotide-incorporation step. In this respect, Rev1p differs from all previously investigated DNA polymerases. We also found that the base occupying the template position in the DNA impacts nucleotide incorporation more at the nucleotide-binding step than at the nucleotide-incorporation step. These studies provide the first detailed, quantitative information regarding the mechanistic impact of protein-template-directed nucleotide incorporation by Rev1p. Moreover, on the basis of these findings and on structures of the unrelated Escherichia coli MutM DNA glycosylase, we suggest the possible structures for the ternary complexes of Rev1p with the other incoming dNTPs.