Oxidation of HMG-CoA reductase inhibitors by tert-butoxyl and 1, 1-diphenyl-2-picrylhydrazyl radicals: model reactions for predicting oxidatively sensitive compounds during preformulation.

Hydrogen atom abstraction rate constants for the reaction of tert-butoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical with the HMG-CoA reductase inhibitors lovastatin, simvastatin, and statins I-IV were measured. This series of diene-containing drugs is known to be prone to oxidation. The tert-butoxyl radical was generated by the thermolysis of di-tert-butylperoxyoxalate at 40 degrees C. A competitive kinetic method was used to determine the relative rate of hydrogen atom abstraction by tert-butoxyl radical to beta-scission. The absolute rate constants were calculated using the experimentally determined product ratios of t-butanol to acetone and the known rate of beta-scission of tert-butoxyl radical. The rate constants for the reaction with DPPH radical were measured spectrophotometrically by monitoring the loss of DPPH radical as a function of substrate concentration. The rate constants correlate well with the structure of the molecules studied. These kinetic techniques allow for oxidatively sensitive compounds to be identified early in the drug development cycle. The tert-butoxyl radical, a strong hydrogen atom abstractor, is representative of the hydroxyl (. OH) and alkoxyl (. OR) radicals; in contrast the DPPH radical, a much weaker radical, is a good kinetic model for hydroperoxyl (. OOH) and peroxyl (. OOR) radicals. These kinetic methods can be used to quantitatively assess the lability of drug candidates towards reaction with oxygen-centered radicals at an early stage of development and facilitate the design of inhibiting strategies.

The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport.

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150(Glued) subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150(Glued) disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150(Glued) antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.

On the mechanism of amine oxidations by P450.

1. Experimental data previously used to support an electron/proton transfer mechanism for oxidative dealkylation of amines by P450 are critically analysed with the conclusion that the mechanistic evidence is indecisive. 2. A new mechanistic criterion recently proposed to distinguish between electron/proton transfer and hydrogen atom transfer mechanisms is discussed. It is based on isotope effect profiles determined for the deprotonation of a series of para-substituted N-methyl-N-trideuteriomethyl)aniline cation radicals by pyridine and for hydrogen atom abstraction from the corresponding neutral amines by the tert-butoxyl radical. These reactions model the steps proposed in the two P450 mechanisms. 3. Isotope effect profiles measured for the demethylation of substituted NN-bis(dideuteriomethyl)anilines by four different forms of P450 were found to be experimentally indistinguishable from the hydrogen atom transfer profile, and distinctly different from the cation radical deprotonation profile. This provides strong evidence that P450 oxidatively dealkylates the amines by a hydrogen atom transfer mechanism and not by an electron/proton transfer mechanism.