An optimized activity-based probe for the study of caspase-6 activation

Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.     

A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors

Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS.     

An activity-based fluorogenic probe for sensitive and selective monoamine oxidase-B detection

We report the design and synthesis of a new class of fluorogenic probes based on monoamine oxidase-triggered oxidative C-O bond cleavage. The selectivity of probe P1 towards MAO-B was 22-fold higher than that towards MAO-A.     

Potentials of mean force for acetylcholine unbinding from the alpha7 nicotinic acetylcholine receptor ligand-binding domain

The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here, we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.     

Using physical chemistry to differentiate nicotinic from cholinergic agonists at the nicotinic acetylcholine receptor

The binding of three distinct agonists-acetylcholine (ACh), nicotine, and epibatidine-to the nicotinic acetylcholine receptor has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp alpha149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond. A simple structural model that considers only possible interactions with Trp alpha149 suggests that a novel aromatic C-H...O=C hydrogen bond further augments the binding of epibatidine. These studies illustrate the subtleties and complexities of the interactions between drugs and membrane receptors and establish a paradigm for obtaining detailed structural information.     

In silico models for the human alpha4beta2 nicotinic acetylcholine receptor

The neuronal alpha4beta2 nicotinic acetylcholine receptor (nAChR) is one of the most widely expressed nAChR subtypes in the brain. Its subunits have high sequence identity (54 and 46% for alpha4 and beta2, respectively) with alpha and beta subunits in Torpedo nAChR. Using the known structure of the Torpedo nAChR as a template, the closed-channel structure of the alpha4beta2 nAChR was constructed through homology modeling. Normal-mode analysis was performed on this closed structure and the resulting lowest frequency mode was applied to it for a "twist-to-open" motion, which increased the minimum pore radius from 2.7 to 3.4 A and generated an open-channel model. Nicotine could bind to the predicted agonist binding sites in the open-channel model but not in the closed one. Both models were subsequently equilibrated in a ternary lipid mixture via extensive molecular dynamics (MD) simulations. Over the course of 11 ns MD simulations, the open channel remained open with filled water, but the closed channel showed a much lower water density at its hydrophobic gate comprised of residues alpha4-V259 and alpha4-L263 and their homologous residues in the beta2 subunits. Brownian dynamics simulations of Na+ permeation through the open channel demonstrated a current-voltage relationship that was consistent with experimental data on the conducting state of alpha4beta2 nAChR. Besides establishment of the well-equilibrated closed- and open-channel alpha4beta2 structural models, the MD simulations on these models provided valuable insights into critical factors that potentially modulate channel gating. Rotation and tilting of TM2 helices led to changes in orientations of pore-lining residue side chains. Without concerted movement, the reorientation of one or two hydrophobic side chains could be enough for channel opening. The closed- and open-channel structures exhibited distinct patterns of electrostatic interactions at the interface of extracellular and transmembrane domains that might regulate the signal propagation of agonist binding to channel opening. A potential prominent role of the beta2 subunit in channel gating was also elucidated in the study.     

Molecular dynamics of nicotinic acetylcholine receptor correlating biological functions

The nicotinic acetylcholine receptor (nAChR) that mediates fast intercellular communication in response to neurotransmitters is a paradigm of ligand-gated ion channels. Molecular dynamics (MD) simulations are valuable in understanding membrane protein function at atomic level, providing useful clues for further experimental/theoretical studies. In this brief review, recent progress in MD simulations of the nAChR has been illustrated, mainly focusing on the latest simulation of the whole transmembrane domain of the receptor. On the basis of MD simulations, asymmetrical and asynchronous motions of five subunits were observed both in the ligand binding and transmembrane domains; a closed-to-open conformational shift of the gate was captured in different simulation systems; the contributions from the lipid molecules and other transmembrane segments rather than M2 to the gate switch as well as the conformational change of the whole channel were assessed; the dynamic behavior and related physical/chemical properties of the water molecules and cations within the ion channel were examined; and an experimentally comparable single-channel conductance and ion selectivity were obtained.     

An affinity-based probe for the proteomic profiling of aspartic proteases

We have developed an affinity-based probe for the proteomic profiling of aspartic proteases. Our probe was shown to be selective towards aspartic proteases over other proteins. It was also shown that the strategy may be used to label selectively aspartic proteases in the presence of a large excess of other proteins, thus making it useful for future proteome profiling experiments.     

Investigation on native vesicles containing the nicotinic acetylcholine receptor using FTIR-spectroscopy

The ligand gated ion channel nicotinic acetylcholine receptor is responsible for the electrochemical signal transduction in nerve cells and at the motor endplates. In the recent years the structure of the channel has emerged to a resolution of 4.6 Å [J. Mol. Biol. 288 (1999) 765]. We have used ATR–FTIR and SEIRA spectroscopy to investigate the extramembraneous structure of the receptor. The adsorption of nicotinic acetylcholine receptor rich vesicles on the surface of Ag-cluster leads to the detection of high content of helical structure in the extra membranous parts of the receptor. Spectra indicate a β-sheet structure perpendicular to the crystal plane.     

Reconstitution of the Torpedo californica nicotinic acetylcholine receptor into planar lipid bilayers

Detergent-solubilized acetylcholine receptor (nAcChR) proteins can be purified by affinity chromatography and reconstituted into lipid vesicles and afterwards into planar lipid bilayer membranes via, in principle, two methods: fusion or assembly from two vesicle-spread monolayers. In the presence of agonists (carbamoylcholine, suberoyldicholine) different kinds of channel openings are recorded: fast single channels, bursts and long openings with short closures in between. Similar results have been obtained with reconstituted membrane fragments rich in nAcChR. In addition, Torpedo californica nAcChR proteins give rise to fuzzy channels and less defined events of conductivity, which “reemerge” again all the time. Frequently the channel events have conductance levels of about 200 to 300 pS, obviously simultaneous openings of several aggregated receptors. Under these conditions 40 pS conductance events occur also. It appears that the conductance of the channels measured is a multiple of 6.3 pS. Often, with the same sample, no channel openings are seen. Contrary to patch-clamp investigations on whole cells, AcChR-channel openings in reconstituted systems occur only several minutes after agonist application and not immediately. It is not clear whether the “reconstituted channels” reflect rapid activation or whether they result from desensitized receptor states only. Although a clear-cut correlation of channel event and channel protein unit is only possible by reconstitution of the biochemically characterized protein, e.g. monomer, dimer or higher oligomers, the reconstitution technique is still in its infancy.     

A stereochemical test of a proposed structural feature of the nicotinic acetylcholine receptor

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the alphaVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the alphaVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism.     

Synthesis of two fluoro analogues of the nicotinic acetylcholine receptor agonist UB-165

Two racemic fluoropyridine analogues 4 and 5 of the potent nicotinic agonist UB-165 have been synthesized. Halogenated pyridines 7 and 12 provided the organometallic reagents needed for the Negishi and Suzuki coupling reactions used for the preparation of 4 and 5, and the N-vinyloxycarbonyl protecting group of 8 and 15 was cleaved using a novel trifluoroacetic acid-mediated deprotection protocol. Analogue 4 retained high binding affinity at rat brain alpha4beta2 and alpha7 nicotinic receptors.     

Epibatidine and its analogues as nicotinic acetylcholine receptor agonist: an update

Epibatidine (EPB) (1), an alkaloid isolated from the skin of the Ecuadorian poison frog, Epipedobates tricolor has attracted attention because of its exceptionally powerful analgesic properties: several hundred times greater than those of morphine, and the fact that it acts at nicotine rather than opiate receptors. Although the substance is toxic, it does serve as a lead compound in the development of drugs for pain relief as well as treatment of disorders whose pathogenesis involves nicotinic receptors. In this article, isolation, synthetic methods, effect on neuronal and neuromuscular nicotinic receptors, therapeutic potential, toxicity, nicotinic pharmacophore structural modifications related issues of 1 are discussed.     

An improved nicotinic pharmacophore and a stereoselective CoMFA-model for nicotinic agonists acting at the central nicotinic acetylcholine receptors labelled by [3H]-N-methylcarbamylcholine

A study of a series of compounds with agonistic effect at the alpha4beta2 nicotinic acetylcholine receptors resulted in an improved pharmacophore model as well as a CoMFA model. The pharmacophore was composed of three pharmacophoric elements: (1) a site point (a) corresponding to a protonated nitrogen atom, (2) a site point (b) corresponding to an electronegative atom capable of forming a hydrogen bond, and (3) the centre of a heteroaromatic ring or a C=O bond (c). The pharmacophoric elements were related by the following parameters: (a-b) 7.3-8.0 A, (a-c) 6.5-7.4 A, and the angle between the two distance vectors (delta bac) 30.4-35.8 degrees. In addition to this, a stereoselective CoMFA model was developed, which showed good predictability even for compound classes not present in the training set.     

Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes

Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.     

Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.     

The alphaM1 transmembrane segment of the nicotinic acetylcholine receptor interacts strongly with model membranes

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) plays a role in the regulation of the activity of this important ligand-gated ion channel. The lipid composition of the host membrane affects conformational equilibria of the nAChR and several classes of inhibitors, most notably anaesthetics, interact directly or indirectly with the four transmembrane M-segments, M1-M4, of the nAChR subunits. It has proven difficult to gain insight into structure-function relationships of the M-segments in the context of the entire receptor and the biomembrane environment. However, model membrane systems are well suited to obtain detailed information about protein-lipid interactions. In this solid-state NMR study, we characterized interactions between a synthetic alphaM1 segment of the T. californica nAChR and model membranes of different phosphatidylcholine (PC) lipids. The results indicate that alphaM1 interacts strongly with PC bilayers: the peptide orders the lipid acyl chains and induces the formation of small vesicles, possibly through modification of the lateral pressure profile in the bilayer. The multilamellar vesicle morphology was stabilized by the presence of cholesterol, implying that either the rigidity or the bilayer thickness is a relevant parameter for alphaM1-membrane interactions, which also has been suggested for the entire nAChR. Our results suggest that the model systems are to a certain extent sensitive to peptide-bilayer hydrophobic matching requirements, but that the lipid response to hydrophobic mismatch alone is not the explanation. The effect of alphaM1 on different PC bilayers may indicate that the peptide is conformationally flexible, which in turn would support a membrane-mediated modulation of the conformation of transmembrane segments of the nAChR.