Resonance Raman Spectroscopy Reveals That Substrate Structure Selectively Impacts the Heme-Bound Diatomic Ligands of CYP17

An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the corticosteroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17α-hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17α-hydroxyprogesterone (17-OH PROG). The hydroxylated products then either are used for production of corticoids or undergo a second CYP17 catalyzed transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well-known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein, resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions

Kinetic Solvent Isotope Effect in Human P450 CYP17A1-Mediated Androgen Formation: Evidence for a Reactive Peroxoanion Intermediate

Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydro­epi­andro­sterone, preg­nenol­one is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17–C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C–C “lyase” activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1. Our experiments revealed the expected small positive (∼1.3) isotope effect for the hydroxylase chemistry. However, a surprising result was the large inverse isotope effect (∼0.39) observed for the C–C bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion

Drug–Drug Interactions between Atorvastatin and Dronedarone Mediated by Monomeric CYP3A4

Heterotropic interactions between atorvastatin (ARVS) and dronedarone (DND) have been deciphered using global analysis of the results of binding and turnover experiments for pure drugs and their mixtures. The <i>in vivo</i> presence of atorvastatin lactone (ARVL) was explicitly taken into account by using pure ARVL in analogous experiments. Both ARVL and ARVS inhibit DND binding and metabolism, while a significantly higher affinity of CYP3A4 for ARVL makes the latter the main modulator of activity (effector) in this system. Molecular dynamics simulations reveal significantly different modes of interactions of DND and ARVL with the substrate binding pocket and with a peripheral allosteric site. Interactions of both substrates with residues F213 and F219 at the allosteric site play a critical role in the communication of conformational changes induced by effector binding to productive binding of the substrate at the catalytic site

Native Mass Spectrometry Characterization of Intact Nanodisc Lipoprotein Complexes

We describe here the analysis of nanodisc complexes by using native mass spectrometry (MS) to characterize their molecular weight (MW) and polydispersity. Nanodiscs are nanoscale lipid bilayers that offer a platform for solubilizing membrane proteins. Unlike detergent micelles, nanodiscs are native-like lipid bilayers that are well-defined and potentially monodisperse. Their mass spectra allow peak assignment based on differences in the mass of a single lipid per complex. Resultant masses agree closely with predicted values and demonstrate conclusively the narrow dispersity of lipid molecules in the nanodisc. Fragmentation with collisionally activated dissociation (CAD) or electron-capture dissociation (ECD) shows loss of a small number of lipids and eventual collapse of the nanodisc with release of the scaffold protein. These results provide a foundation for future studies utilizing nanodiscs as a platform for launching membrane proteins into the gas phase

Nanodiscs as a Modular Platform for Multimodal MR-Optical Imaging

Nanodiscs are monodisperse, self-assembled discoidal particles that consist of a lipid bilayer encircled by membrane scaffold proteins (MSP). Nanodiscs have been used to solubilize membrane proteins for structural and functional studies and deliver therapeutic phospholipids. Herein, we report on tetramethylrhodamine (TMR) tagged nanodiscs that solubilize lipophilic MR contrast agents for generation of multimodal nanoparticles for cellular imaging. We incorporate both multimeric and monomeric Gd­(III)-based contrast agents into nanodiscs and show that particles containing the monomeric agent (<b>ND2</b>) label cells with high efficiency and generate significant image contrast at 7 T compared to nanodiscs containing the multimeric agent (<b>ND1</b>) and Prohance, a clinically approved contrast agent

Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

<div><p>Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery for membrane protein targets.</p></div

Screening strategy effectively eliminates false positives.

<p>(a) Screening assays used to evaluate the effect of Spectrum Collection molecules on AβO binding. The arrows indicate the reduction in compounds resulting at each step. (b) Compiled data from the primary AlphaScreen assay are shown in a single graph normalized to POPC and SMPL in-plate controls. (c) Schematics of counterscreening assays designed to identify false positive compounds acting on off-target elements of the primary screening assay (dashed red lines). Assays use AlphaScreen donor and acceptor beads linked together by either biotinylated hexahistidine (top) or Nanodiscs containing biotinylated synaptic proteins (bottom). (d) Data from the biotinylated hexahistidine counterscreen. Black symbols denote compounds classified as likely false positives. Blue symbols denote compounds that were retested in dose-response format (Examples shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125263#pone.0125263.g007" target="_blank">Fig 7</a>), and the compounds showing significant signal reduction at 1 μM are shown as open red circles. (e) Secondary, orthogonal assays to verify compound efficacy in preventing AβO binding include a dot immunoblot test for AβO binding to rat cortical synaptosomes (Top; shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125263#pone.0125263.g008" target="_blank">Fig 8</a>) and an immunocytochemical analysis of AβO binding to cultures of rat hippocampal neurons (Bottom; shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125263#pone.0125263.g009" target="_blank">Fig 9</a> for ATA). Red squares in panels b and d identify the data points associated with ATA.</p

AβO binding is receptor-mediated but PrP^C-independent in Nanodiscs and mature neurons.

<p>Immobilized SMPL Nanodiscs were titrated with AβOs and bound AβOs were detected with NU2 oligomer-specific antibody coupled to an HRP-based colorimetric assay. Nonspecific binding was measured in the presence of excess ATA, which blocks AβO/receptor binding, and used to calculate the specific binding component (blue). n = 3; mean +/- SD. (a). To analyze Nanodisc proteins co-immunoprecipitating with AβOs, Nanodiscs containing biotinylated synaptic plasma membranes were affinity precipitated and visualized by SDS-PAGE immunoblot with biotin detection (b). To test the prediction that PrP^C mediates AβO binding, immobilized Nanodiscs were split into four equivalent reactions and pre-treated with 0, 0.05, 0.1, or 0.2 units of PIPLC to remove PrP^C before exposing to AβOs and probing with NU2 as in (a). PrP^C removal was verified by Western blotting after the colorimetric assay was complete. NU2 bound to each immobilized Nanodisc/AβO complex is detected in the blot by anti-mouse secondary antibodies used to probe for the mouse antibody against PrP^C (c). The effect of PrP^C removal on AβO binding was tested using mature hippocampal cultures treated with PIPLC (d). PrP^C was detected using an antibody (red) and fluorescence-conjugated AβOs were visualized directly (green).</p

SMPL Nanodiscs provide the basis for a high-throughput assay for AβO binding antagonists.

<p>A schematic of the AlphaScreen assay adapted to measure AβO binding to synaptic Nanodiscs (a). Biotinylated AβOs and His-tagged MSP molecules link Nanodiscs to AlphaScreen donor and acceptor beads. The proof-of-concept assay produces high dynamic range (b). NU2 oligomer-specific antibodies were used as a drug stand-in to test the assay’s response to an applied treatment (c).</p

Nanodiscs preserve synaptic protein composition and structure.

<p>(a) Schematic of SMPL Nanodisc formation using synaptic plasma membranes. (b) Nanodiscs containing biotinylated synaptic membranes (solid curve) or POPC (dashed curve) were separated by size exclusion chromatography. Fractions eluting from the synaptic Nanodisc run were collected and analyzed by dot blot to locate biotinylated synaptic proteins (red curve; Mean +/-SD; n = 2). The population of biotinylated membrane proteins inserted into Nanodiscs was analyzed by SDS-PAGE, probing for biotin (c) or using antibodies against specific proteins related to AβO binding (d). ³H glutamate binding to SMPL Nanodiscs was assessed in the absence and presence of a 100-fold excess of cold glutamate (Mean +/- SD; n = 3; * p<0.05) (e). Enzymatic activity was assessed in synaptic plasma membranes (Syn PM) and SMPL Nanodiscs (SMPL) by probing for tyrosine phosphorylation in the absence and presence of ATP (f). Insulin receptor activity was probed using an antibody recognizing IR_βpTyr^1162/1163 (g).</p