Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments.;Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation.;This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover, applications of immobilized P450 enzyme constructs will also be used for monitoring protein-protein interaction and metabolite production with the use of immobilized-P450 bioreactor constructs. This work provides insight into the effect on catalytic activity caused by both P450 aggregation as well as isoform-specific protein-protein interactions and provides insight in the production of biosynthetically produced drug metabolites

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016

Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism.

Cytochrome P450 (P450) protein-protein interactions have been shown to alter their catalytic activity. Furthermore, these interactions are isoform specific and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show that these effects are also dependent on the protein partner cytochrome P450 reductase (CPR) and the order of protein addition to purified reconstituted enzyme systems. In this study, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homomeric/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real time utilizing surface plasmon resonance. Finally, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems. Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4, in rank order.KDvalues obtained via surface plasmon resonance show strong binding, in the nanomolar range, for the above pairs, with CYP2C9-CYP2D6 yielding the lowestKD, followed by CYP2C9-CYP2C9, CYP2C9-CPR, and CYP2C9-CYP3A4. Metabolic incubations show that immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 at saturating CPR concentrations in solution but was inhibited when CYP2C9 was immobilized. The order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced the magnitude of inhibition for CYP3A4 and CYP2D6. These results indicate isoform-specific P450 interactions and effects on P450-mediated metabolism

A phase Ib/II study of the BRAF inhibitor encorafenib plus the MEK inhibitor binimetinib in patients with BRAFV600E/K-mutant solid tumors

Purpose: This open-label, dose-finding phase Ib/II study reports the safety and activity of the first combination use with BRAF inhibitor (BRAFi) encorafenib plus MEK inhibitor (MEKi) binimetinib in patients with BRAF V600E-mutant solid tumors. Patients and methods: In phase I, the recommended phase 2 doses (RP2D) were established (primary objective). In phase II, the clinical activity of the combination at the RP2D was assessed (primary objective) in patients with BRAF-mutant metastatic colorectal cancer (mCRC), BRAFi-treated BRAF-mutant melanoma, and BRAFi-naïve BRAF-mutant melanoma. Results: A total of 126 patients with BRAF-mutant solid tumors were enrolled (phase I: 47 patients; phase II: 79 patients). The RP2D was encorafenib 450 mg once daily plus binimetinib 45 mg twice daily and pharmacokinetic data suggest that drug exposures of each agent were similar in combination compared with single-agent studies. In the phase II cohorts, confirmed responses were seen in two of 11 (18%) evaluable patients with mCRC, 11 of 26 (42%) evaluable patients with BRAFi-pretreated melanoma, and 28 of 42 (67%) BRAFi-naïve patients with melanoma. The most common grade 3/4 adverse event in phase II was increased alanine aminotransferase. Conclusions: The combination of encorafenib (450 mg) plus binimetinib (45 mg) showed acceptable tolerability and encouraging activity in patients with BRAF V600-mutant tumors, which led to the dose selection for the melanoma COLUMBUS study. The safety profile of the combination was consistent with other approved BRAFi plus MEKi regimens, with several differences, including lower rates of dose-limiting pyrexia, arthralgia, and photosensitivity

Measurement of Electron Transfer through Cytochrome P450 Protein on Nanopillars and the Effect of Bound Substrates

Electron transfer in cytochrome P450 enzymes is a fundamental process for activity. It is difficult to measure electron transfer in these enzymes because under the conditions typically used they exist in a variety of states. Using nanotechnology-based techniques, gold conducting nanopillars were constructed in an indexed array. The P450 enzyme CYP2C9 was attached to each of these nanopillars, and conductivity measurements made using conducting probe atomic force microscopy under constant force conditions. The conductivity measurements were made on CYP2C9 alone and with bound substrates, a bound substrate–effector pair, and a bound inhibitor. Fitting of the data with the Poole–Frenkel model indicates a correlation between the barrier height for electron transfer and the ease of CYP2C9-mediated metabolism of the bound substrates, though the spin state of iron is not well correlated. The approach described here should have broad application to the measurement of electron transfer in P450 enzymes and other metalloenzymes

Global effects of land use on local terrestrial biodiversity

Human activities, especially conversion and degradation of habitats, are causing global biodiversity declines. How local ecological assemblages are responding is less clear--a concern given their importance for many ecosystem functions and services. We analysed a terrestrial assemblage database of unprecedented geographic and taxonomic coverage to quantify local biodiversity responses to land use and related changes. Here we show that in the worst-affected habitats, these pressures reduce within-sample species richness by an average of 76.5%, total abundance by 39.5% and rarefaction-based richness by 40.3%. We estimate that, globally, these pressures have already slightly reduced average within-sample richness (by 13.6%), total abundance (10.7%) and rarefaction-based richness (8.1%), with changes showing marked spatial variation. Rapid further losses are predicted under a business-as-usual land-use scenario; within-sample richness is projected to fall by a further 3.4% globally by 2100, with losses concentrated in biodiverse but economically poor countries. Strong mitigation can deliver much more positive biodiversity changes (up to a 1.9% average increase) that are less strongly related to countries' socioeconomic status