ABSTRACT
Pharmacological activation of the E3 ligase Parkin represents a rational therapeutic intervention for the treatment of Parkinson's disease. Here we identify several compounds that enhance the activity of wildtype Parkin in the presence of phospho-ubiquitin and act as positive allosteric modulators (PAMs). While these compounds activate Parkin in a series of biochemical assays, they do not act by thermally destabilizing Parkin and fail to enhance the Parkin translocation rate to mitochondria or to enact mitophagy in cell-based assays. We conclude that in the context of the cellular milieu the therapeutic window to pharmacologically activate Parkin is very narrow.
ABSTRACT
Time-dependent inhibition (TDI) of cytochrome P450 enzymes is an important cause of drug-drug interactions. The standard approach to characterize the kinetics of TDI is to determine the rate of enzyme loss, kobs, at various inhibitor concentrations, [I], and replot the kobs versus [I] to obtain the key kinetic parameters, KI and kinact. In our companion manuscript (Part 1; Nagar et al., 2014) in this issue of Drug Metabolism and Disposition, we used simulated datasets to develop and test a new numerical method to analyze in vitro TDI data. Here, we have applied this numerical method to five TDI datasets. Experimental datasets include the inactivation of CYP2B6, CYP2C8, and CYP3A4. None of the datasets exhibited Michaelis-Menten-only kinetics, and the numerical method allowed use of more complex models to fit each dataset. Quasi-irreversible as well as partial inhibition kinetics were observed and parameterized. Three datasets required the use of a multiple-inhibitor binding model. The mechanistic and clinical implications provided by these analyses are discussed. Together with the results in Part 1, we have developed and applied a new numerical method for analysis of in vitro TDI data. This method appears to be generally applicable to model in vitro TDI data with atypical and complex kinetic schemes.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors/metabolism , Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/metabolism , Drug Interactions/physiology , Humans , In Vitro Techniques , Kinetics , Statistics as TopicABSTRACT
A series of 2-[(2,6)-dimethylphenyl]benzimidazole analogs displayed strong potential for mutagenicity following metabolic activation in either TA98 or TA100 Salmonella typhimurium strains. The number of revertants was significantly reduced by replacing the 2,6-dimethylphenyl group with a 2,6-dichlorophenyl moiety. Time-dependent CYP3A4 inhibition was also observed with a compound containing a 2-[(2,6)-dimethylphenyl] benzimidazole ring, implying risk for this scaffold to generate reactive metabolites.
Subject(s)
Anthelmintics/pharmacology , Benzimidazoles/pharmacology , Cytochrome P-450 CYP3A Inhibitors , Mutagens/pharmacology , Salmonella typhimurium/drug effects , Albendazole/pharmacology , Cytochrome P-450 CYP3A , Mutagenicity Tests , Salmonella typhimurium/genetics , Time FactorsSubject(s)
Cytochrome P-450 Enzyme Inhibitors , Drug Discovery/methods , Enzyme Inhibitors/chemistry , High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Automation, Laboratory/methods , Chromatography, Liquid/methods , Cytochrome P-450 Enzyme System/chemistry , Enzyme Inhibitors/pharmacology , Linear Models , Midazolam/analogs & derivatives , Midazolam/chemistryABSTRACT
The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.
