Multimodal small-molecule screening for human prion protein binders.

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

Stepwise Aggregation of Cholate and Deoxycholate Dictates the Formation and Loss of Surface-Available Chirally Selective Binding Sites.

Bile salts are facially amphiphilic, naturally occurring chemicals that aggregate to perform numerous biochemical processes. Because of their unique intermolecular properties, bile salts have also been employed as functional materials in medicine and separation science (e.g., drug delivery, chiral solubilization, purification of single-walled carbon nanotubes). Bile micelle formation is structurally complex, and it remains a topic of considerable study. Here, the exposed functionalities on the surface of cholate and deoxycholate micelles are shown to vary from one another and with the micelle aggregation state. Collectively, data from NMR and capillary electrophoresis reveal preliminary, primary, and secondary stepwise aggregation of the salts of cholic (CA) and deoxycholic (DC) acid in basic conditions (pH 12, 298 K), and address how the surface availability of chirally selective binding sites is dependent on these sequential stages of aggregation. Prior work has demonstrated sequential CA aggregation (pH 12, 298 K) including a preliminary CMC at ca. 7 mM (no chiral selection), followed by a primary CMC at ca. 14 mM that allows chiral selection of binaphthyl enantiomers. In this work, DC is also shown to form stepwise preliminary and primary aggregates (ca. 3 mM DC and 9 mM DC, respectively, pH 12, 298 K) but the preliminary 3 mM DC aggregate is capable of chirally selective solubilization of the binaphthyl enantiomers. Higher-order, secondary bile aggregates of each of CA and DC show significantly degraded chiral selectivity. Diffusion NMR reveals that secondary micelles of CA exclude the BNDHP guests, while secondary micelles of DC accommodate guests, but with a loss of chiral selectivity. These data lead to the hypothesis that secondary aggregates of DC have an exposed binding site, possibly the 7α-edge of a bile dimeric unit, while secondary CA micelles do not present binding edges to the solution, potentially instead exposing the three alcohol groups on the hydrophilic α-face to the solution.

Chemical Shifts of the Carbohydrate Binding Domain of Galectin-3 from Magic Angle Spinning NMR and Hybrid Quantum Mechanics/Molecular Mechanics Calculations.

Magic angle spinning NMR spectroscopy is uniquely suited to probe the structure and dynamics of insoluble proteins and protein assemblies at atomic resolution, with NMR chemical shifts containing rich information about biomolecular structure. Access to this information, however, is problematic, since accurate quantum mechanical calculation of chemical shifts in proteins remains challenging, particularly for 15NH. Here we report on isotropic chemical shift predictions for the carbohydrate recognition domain of microcrystalline galectin-3, obtained from using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, implemented using an automated fragmentation approach, and using very high resolution (0.86 Å lactose-bound and 1.25 Å apo form) X-ray crystal structures. The resolution of the X-ray crystal structure used as an input into the AF-NMR program did not affect the accuracy of the chemical shift calculations to any significant extent. Excellent agreement between experimental and computed shifts is obtained for 13Cα, while larger scatter is observed for 15NH chemical shifts, which are influenced to a greater extent by electrostatic interactions, hydrogen bonding, and solvation.

Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy.

Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin's involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin.

An Edge Selection Mechanism for Chirally Selective Solubilization of Binaphthyl Atropisomeric Guests by Cholate and Deoxycholate Micelles.

Combining micellar electrokinetic capillary chromatography (MEKC) and nuclear magnetic resonance (NMR) experimentation, we shed light on the structural basis for the chirally selective solubilization of atropisomeric binaphthyl compounds by bile salt micelles comprised of cholate (NaC) or deoxycholate (NaDC). The model binaphthyl analyte R,S-BNDHP exhibits chirally selective interactions with primary micellar aggregates of cholate and deoxycholate, as does the closely related analyte binaphthol (R,S-BN). Chiral selectivity was localized, by NMR chemical shift analysis, to the proton at the C12 position of these bile acids. Correspondingly, MEKC results show that the 12α-OH group of either NaC or NaDC is necessary for chirally selective resolution of these model binaphthyl analytes by bile micelles, and the S isomer is more highly retained by the micelles. With NMR, the chemical shift of 12ß-H was perturbed more strongly in the presence of S-BNDHP than R-BNDHP. Intermolecular NOEs demonstrate that R,S-BNDHP and R,S-BN interact with a similar hydrophobic planar pocket lined with the methyl groups of the bile salts, and are best explained by the existence of an antiparallel dimeric unit of bile salts. Finally, chemical shift data and intermolecular NOEs support different interactions of the enantiomers with the edges of dimeric bile units, indicating that R,S-BNDHP enantiomers sample the same binding site preferentially from opposite edges of the dimeric bile unit. Chirality 28:525-533, 2016. © 2016 Wiley Periodicals, Inc.

NMR crystallography of an oxovanadium(V) complex by an approach combining multinuclear magic angle spinning NMR, DFT, and spin dynamics simulations.

Bioinorganic vanadium(V) solids are often challenging for structural analysis. Here, we explore an NMR crystallography approach involving multinuclear (13) C/(51) V solid-state NMR spectroscopy, density functional theory (DFT), and spin dynamics numerical simulations, for the spectral assignment and the 3D structural analysis of an isotopically unmodified oxovanadium(V) complex, containing 17 crystallographically inequivalent (13) C sites. In particular, we report the first NMR determination of C-V distances. So far, the NMR observation of (13) C-(51) V proximities has been precluded by the specification of commercial NMR probes, which cannot be tuned simultaneously to the close Larmor frequencies of these isotopes (100.6 and 105.2 MHz for (13) C and (51) V, respectively, at 9.4 T). By combining DFT calculations and (13) C-(51) V NMR experiments, we propose a complete assignment of the (13) C spectrum of this oxovanadium(V) complex. Furthermore, we show how (13) C-(51) V distances can be quantitatively estimated.

NMR crystallography for structural characterization of oxovanadium(V) complexes: deriving coordination geometry and detecting weakly coordinated ligands at atomic resolution in the solid state.

NMR crystallography is an emerging method for atomic-resolution structural analysis of ubiquitous vanadium(V) sites in inorganic and bioinorganic complexes as well as vanadium-containing proteins. NMR crystallography allows for characterization of vanadium(V) containing solids, based on the simultaneous measurement of (51)V-(15)N internuclear distances and anisotropic spin interactions, described by (13)C, (15)N, and (51)V chemical shift anisotropy and (51)V electric field gradient tensors. We show that the experimental (51)V, (13)C, and (15)N NMR parameters are essential for inferring correct coordination numbers and deriving correct geometries in density functional theory (DFT) calculations, particularly in the absence of single-crystal X-ray structures. We first validate this approach on a structurally known vanadium(V) complex, ((15)N-salicylideneglycinate)-(benzhydroxamate)oxovanadium(V), VO(15)NGlySalbz. We then apply this approach to derive the three-dimensional structure of (methoxo)((15)N-salicylidene-glycinato)oxovanadium(V) with solvated methanol, [VO((15)NGlySal)(OCH3)]·(CH3OH). This is a representative complex with potentially variable coordination geometry depending on the solvation level of the solid. The solid material containing molecules of CH3OH, formally expressed as [VO((15)NGlySal)(OCH3)]·(CH3OH), is found to have one molecule of CH3OH weakly coordinated to the vanadium. The material is therefore best described as [VO((15)NGlySal)(OCH3)(CH3OH)] as deduced by the combination of multinuclear solid-state NMR experiments and DFT calculations. The approach reported here can be used for structural analysis of systems that are not amenable to single-crystal X-ray diffraction characterization and which can contain weakly associated solvents.

Phase-modulated LA-REDOR: a robust, accurate and efficient solid-state NMR technique for distance measurements between a spin-1/2 and a quadrupole spin.

Distances between a spin-1/2 and a spin>1/2 can be efficiently measured by a variety of magic-angle spinning solid state NMR methods such as Rotational Echo Adiabatic Passage Double Resonance (REAPDOR), Low-Alpha/Low-Amplitude REDOR (LA-REDOR) and Rotational-Echo Saturation-Pulse Double-Resonance (R/S-RESPDOR). In this manuscript we show that the incorporation of a phase modulation into a long quadrupolar recoupling pulse, lasting 10 rotor periods that are sandwiched between rotor-synchronized pairs of dipolar recoupling π pulses, extends significantly the range of the values of the quadrupole moments that can be accessed by the experiment. We show by a combination of simulations and experiments that the new method, phase-modulated LA-REDOR, is very weakly dependent on the actual value of the radio-frequency field, and is highly robust with respect to off-resonance irradiation. The experimental results can be fitted by numerical simulations or using a universal formula corresponding to an equal-transition-probability model. Phase-modulated LA-REDOR (13)C{(11)B} and (15)N{(51)V} dipolar recoupling experiments confirm the accuracy and applicability of this new method.

Effect of Ancillary Ligand on Electronic Structure as Probed by 51V Solid-State NMR Spectroscopy for Vanadium-o-Dioxolene Complexes.

A series of vanadium(V) complexes with o-dioxolene (catecholato) ligands and an ancillary ligand, (N-(salicylideneaminato)ethylenediamine) (hensal), were investigated using 51V solid-state magic angle spinning NMR spectroscopy (51V MAS NMR) to assess the local environment of the vanadium(V). The solid-state 51V NMR parameters of vanadium(V) complexes with a related potentially tetradentate ancillary ligand (N-salicylidene-N'-(2-hydroxyethyl)ethylenediamine) (h2shed) were previously shown to be associated with the size of the HOMO-LUMO gap in the complex, and as such provide insights on the interaction between metal ion and ligand (P. B. Chatterjee, et al., Inorg. Chem 50 (2011) 9794). Our results show that the modification of the ancillary ligand does not impact the observed trend between complexes ranging from catechols with electron rich to electron poor substituents. However, the ancillary ligand does impact the size of the HOMO-LUMO separation in the parent complex and thus the solid-state vanadium NMR chemical shift of the unsubstituted vanadium complex. For these complexes significant changes observed in the isotropic shifts and more modest changes detected in the CQ reflect the electronic changes in the complex as the catechol is varied. However, no obvious trend was observed in the chemical shift anisotropies (δσ and ησ) with the variation in the catechol. The electronic changes in the coordination environment of the vanadium can be described using solid-state 51V NMR spectroscopy.

Determination of total antioxidant capacity of commercial beverage samples by capillary electrophoresis via inline reaction with 2,6-dichlorophenolindophenol.

This paper demonstrates proof-of-concept for the use of electrophoretically mediated microanalysis (EMMA) as a new approach to the determination of total antioxidant capacity (TAC). EMMA is a low-volume, high-efficiency capillary electrophoretic technique that has to date been underutilized for small molecule reactions. Here, nanoliter volumes of 2,6-dichlorophenolindophenol (DCIP) reagent solution are mixed with an antioxidant-containing sample within the confines of a narrow-bore capillary tube. The mixing is accomplished by exploiting differential migration rates of the reagents when a voltage field is applied across the length of the capillary tube. The ensuing electron transfer reaction between DCIP and the antioxidant(s) is then used as a quantitative measure of the TAC of the sample. Linear calibration using either redox form of DCIP is accomplished with standard solutions of ascorbic acid. Several commercial beverage samples are analyzed, and the TAC values obtained with the reported methodology are compared to results obtained with the widely used ferric reducing antioxidant power (FRAP) spectroscopic method. For the analysis of real samples of unknown ionic strength, the method of standard additions is shown to be superior to the use of external calibration. This easily automated EMMA method may represent a useful new approach to TAC determination.