ABSTRACT
OBJECTIVE: Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-related pain. DESIGN: Neutralizing antibodies to CGRP were generated de novo. One of these antibodies, LY2951742, was characterized in vitro and tested in pre-clinical in vivo models of OA pain. RESULTS: LY2951742 exhibited high affinity to both human and rat CGRP (KD of 31 and 246 pM, respectively). The antibody neutralized CGRP-mediated induction of cAMP in SK-N-MC cells in vitro and capsaicin-induced dermal blood flow in the rat. Neutralization of CGRP significantly reduced pain behavior as measured by weight bearing differential in the rat monoiodoacetate model of OA pain in a dose-dependent manner. Moreover, pain reduction with neutralization of CGRP occurred independently of prostaglandins, since LY2951742 and NSAIDs worked additively in the NSAID-responsive version of the model and CGRP neutralization remained effective in the NSAID non-responsive version of the model. Neutralization of CGRP also provided dose-dependent and prolonged (>60 days) pain reduction in the rat meniscal tear model of OA after only a single injection of LY2951742. CONCLUSIONS: LY2951742 is a high affinity, neutralizing antibody to CGRP. Neutralization of CGRP is efficacious in several OA pain models and works independently of NSAID mechanisms of action. LY2951742 holds promise for the treatment of pain in OA patients.
Subject(s)
Antibodies, Neutralizing/pharmacology , Calcitonin Gene-Related Peptide/drug effects , Osteoarthritis/drug therapy , Pain/prevention & control , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antimicrobial Cationic Peptides , Cathelicidins/metabolism , Disease Models, Animal , Humans , Male , Rats , Rats, Inbred Lew , Regional Blood Flow , Skin/blood supplyABSTRACT
EcoRI DNA methyltransferase was previously shown to bend its cognate DNA sequence by 52 degrees and stabilize the target adenine in an extrahelical orientation. We describe the characterization of an EcoRI DNA methyltransferase mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant revealed only minor functional consequences: DNA binding affinity (KDDNA) was reduced 10-fold, and kcat was decreased 30%. However, in direct contrast to the wild type enzyme, DNA bending within the mutant enzyme-DNA complexes was not observed by scanning force microscopy. The bending-deficient mutant showed enhanced discrimination against the methylation at nontarget sequence DNA. This enhancement of enzyme discrimination was accompanied by a change in the rate-limiting catalytic step. No presteady-state burst of product formation was observed, indicating that the chemistry step (or prior event) had become rate-limiting for methylation. Direct observation of the base flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contributes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can enhance specificity.
Subject(s)
DNA/metabolism , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Amino Acid Sequence , DNA Methylation , Deoxyribonucleases, Type II Site-Specific/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Spectrometry, Fluorescence , Substrate Specificity , ThermodynamicsABSTRACT
The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1 = kon = 2.25 x 10(7) M-1 s-1. Base-flipping kinetics (monitored using 2-aminopurine fluorescence intensity) were essentially synchronous with the binding kinetics, with less than a 4 ms delay between enzyme binding and target base flipping. The 4 ms delay translates into a base-flipping rate of at least 195 s-1, when the data are analyzed in terms of a sequential DNA binding and base-flipping reaction mechanism. Synchrony of binding and base flipping was only observed during the first 80% of the reaction, and an additional 20% base-flipping signal occurred well after DNA binding was complete. This additional 2AP fluorescence change, with an effective rate of 0.55 s-1, is an intramolecular isomerization reaction which greatly accelerates the dissociation of the enzyme from DNA. The correlation between the dissociation of the enzyme-DNA complex and the restacking of the extrahelical base also revealed a very tight coupling of these two events. Both dissociation and base restacking were found to be biphasic. These data are consistent with the following mechanism. The initial binding rate and base-flipping rates map very closely with previously determined pre-steady-state burst-rate kinetics for methyl transfer. Hence, binding, flipping, and methylation appear to occur in nearly a single concerted step. The bound complex then slowly isomerizes (0.1 s-1) to a distinct configuration that accelerates the product-release phase of the reaction. The product-release enzyme configuration dissociates from DNA approximately 8 times faster than the initial bound complex (0.18 s-1 vs 0.024 s-1 ). When the enzyme dissociates from the DNA along the product-release pathway, the target base remains in an extrahelical conformation and restacks at a rate of only 0.6 s-1. This "multicolor" fluorescence kinetic approach directly measures the absolute temporal correlation between DNA binding and base flipping, with millisecond timing resolution. The data reveal that even when the B-DNA structure is altered in a radical manner (e.g., via base flipping), enzymes can perform this operation in a highly efficient, if not completely concerted manner.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Site-Specific DNA-Methyltransferase (Adenine-Specific)/chemistry , Binding Sites , DNA/metabolism , DNA-Binding Proteins/metabolism , Fluorescence Polarization/methods , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Kinetics , Models, Chemical , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Spectrometry, Fluorescence/methodsABSTRACT
DNA methyltransferases are excellent prototypes for investigating DNA distortion and enzyme specificity because catalysis requires the extrahelical stabilization of the target base within the enzyme active site. The energetics and kinetics of base flipping by the EcoRI DNA methyltransferase were investigated by two methods. First, equilibrium dissociation constants (KDDNA) were determined for the binding of the methyltransferase to DNA containing abasic sites or base analogs incorporated at the target base. Consistent with a base flipping mechanism, tighter binding to oligonucleotides containing destabilized target base pairs was observed. Second, total intensity stopped flow fluorescence measurements of DNA containing 2-aminopurine allowed presteady-state real time observation of the base flipping transition. Following the rapid formation of an enzyme-DNA collision complex, a biphasic increase in total intensity was observed. The fast phase dominated the total intensity increase with a rate nearly identical to k(methylation) determined by rapid chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking of the extrahelical base also revealed biphasic kinetics with the recovered amplitudes from these off-rate experiments matching very closely to those observed during the base unstacking process. These results provide the first direct and continuous observation of base flipping and show that at least two distinct conformational transitions occurred at the flipped base subsequent to complex formation. Furthermore, our results suggest that the commitment to catalysis during the methylation of the target site is not determined at the level of the chemistry step but rather is mediated by prior intramolecular isomerization within the enzyme-DNA complex.
Subject(s)
DNA/metabolism , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Enzyme Stability , Isomerism , Models, Chemical , Nucleic Acid Conformation , Plasmids/metabolism , Protein ConformationABSTRACT
We describe a novel fluorescence-based assay for detecting DNA conformational alterations within enzyme-DNA complexes. The target adenine for EcoRI DNA methyltransferase (GAATTC) was replaced with 2-aminopurine, which fluoresces upon excitation at 310 nm. Addition of the methyltransferase to the duplex binding site results in a 14-fold increase in fluorescence intensity with a 10 nm blue shift. The fluorescence is approximately 50% of that observed with equimolar free nucleoside, consistent with extrahelical stabilization of the target base in the enzyme-DNA complex. The shift in lambda max further implies the base is placed into a low dielectric environment. For adenine-specific DNA methyltransferases, a hydrophobic pocket composed of highly conserved amino acids lies proximal to the cofactor binding site. Substitution of 2-aminopurine adjacent to the target base also results in detectable changes in fluorescence emission following complex formation with the methyltransferase. Thus, other classes of enzymes hypothesized to utilize base flipping can be investigated by this method.