RESUMEN
Several nucleotide-specific phosphohydrolases can cleave P-F bonds in substrate analogues containing a fluorophosphate moiety to release fluoride ions. In this work, by employing a fluoride-sensitive molecular sensor, we harnessed this cleavage reaction to develop a fluorescence assay to screen for phosphohydrolase inhibitors. The assay is rapid, sensitive, and based on simple and synthetically available reagents. The assay was adapted to the high-throughput screening (HTS) format and its utility was demonstrated by screening an 'in-house' library of small nucleotides against two enzymes: DcpS, a metal-independent mRNA decapping pyrophosphatase of the histidine triad (HIT) family; and PDE-I, a divalent cation-dependent nuclease. Our screening results agreed with the known specificities of DcpS and PDE-I, and led to the selection of several inhibitors featuring low-micromolar IC50 values. For DcpS, we also verified the results by using an alternative method with the natural substrate. Notably, the assay presented here is the first fluorescence-based HTS-adaptable assay for DcpS, an established therapeutic target for spinal muscular atrophy. The assay should be useful for phosphohydrolase specificity profiling and inhibitor discovery, particularly in the context of DcpS and other HIT-family enzymes, which play key roles in maintaining cellular functions and have been linked to disease development.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/antagonistas & inhibidores , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Caperuzas de ARN/metabolismo , Animales , Crotalus , Humanos , Concentración 50 Inhibidora , Espectrometría de FluorescenciaRESUMEN
Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5' mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.
Asunto(s)
Vacunas contra el Cáncer/genética , Células Dendríticas/inmunología , Análogos de Caperuza de ARN/química , Estabilidad del ARN , Presentación de Antígeno , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/metabolismo , Células Dendríticas/metabolismo , Genes Reporteros , Semivida , Humanos , Luciferasas/análisis , Oligonucleótidos Fosforotioatos/química , Biosíntesis de Proteínas , ARN/química , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/metabolismoRESUMEN
DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine K(m) and V(max)values. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m(7)Gp(CH(2))ppG and m(7)Gpp(CH(2))pG) and less potently by ARCA (m(7,3' O)GpppG).
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Guanina/análogos & derivados , Pirofosfatasas/química , Análogos de Caperuza de ARN/química , Animales , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Guanina/química , Hidrólisis/efectos de los fármacos , Cinética , Pirofosfatasas/antagonistas & inhibidores , Análogos de Caperuza de ARN/farmacología , Espectrometría de FluorescenciaRESUMEN
The mechanism by which phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the reversible elimination of ammonia from phenylalanine yielding (E)-cinnamic acid has gained much attention in the recent years. Dehydroalanine is essential for the catalysis. It was assumed that this prostetic group acts as the electrophile, leading to a covalently bonded enzyme-intermediate complex with quarternary nitrogen of phenylalanine. Recently, an alternative mechanism has been suggested in which the enzyme-intermediate complex is formed in a Friedel-Crafts reaction between dehydroalanine and orthocarbon of the aromatic ring. Using semiempirical calculations we have shown that these two alternative mechanisms can be distinguished on the basis of the hydrogen secondary kinetic isotope effect when tritium label is placed in the orthopositions. Our calculations indicated also that the kinetic isotope effect measured using ring-labeled d(5)-phenylalanine could not be used to differentiate these alternative mechanisms. Measured secondary tritium kinetic isotope effect shows strong dependence on the reaction progress, starting at the inverse value of k(H)/k(T) = 0.85 for 5% conversion and reaching the normal value of about 1.15 as the conversion increases to 20%. This dependence has been interpreted in terms of a complex mechanism with initial formation of the Friedel-Crafts type intermediate.