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Recent advances in mass spectrometry coupled with chemical crosslinking (CX-MS) can be applied for the structural interrogation of macromolecular complexes to identify statedependent distance constraints and provides a powerful complementary technique to other structural methods. In this study, we develop a CX-MS approach to identify the sites of crosslinking from a single targeted location within the human glycine receptor (GlyR) in a single apo/resting state. The GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neuronal transmission. A single cysteine residue was re-introduced intoCys nullGlyR construct at position 41 within the extracellular domain of an overexpressed human a1 homomeric GlyR. After purification and reconstitution into vesicles, a methanethiosulfonate benzophenone heterobifunctional crosslinker was attached via formation of a disulfide bond, and the resting receptor is subsequently photocrosslinked. Monomeric and oligomeric bands from SDS-PAGE gels were then trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intra- and inter-subunit sites of crosslinking were differentiated and identified from single gel bands (pmols of purified GlyR), showing the utility of this approach to identify a diverse array of distance constraints of GlyR in its resting state. These studies highlight the potential of CX-MS as an experimental approach to identify state-dependent crosslinks of full length integral membrane protein assemblies in a native-like lipid environment.

The metabotropic glutamate receptor 2 (mGlu2) is a transmembrane-spanning class C G protein-coupled receptor that is an attractive therapeutic target for multiple psychiatric and neurological disorders. A key challenge has been deciphering the contribution of mGlu2 relative to other closely related mGlu receptors in mediating different physiological responses, which could be achieved through the utilization of subtype selective pharmacological tools. In this respect, allosteric modulators that recognize ligand-binding sites distinct from the endogenous neurotransmitter glutamate offer the promise of higher receptor-subtype selectivity. We hypothesized that mGlu2-selective positive allosteric modulators could be derivatized to generate bifunctional pharmacological tools. Here we developed clickable photoaffinity probes for mGlu2 based on two different positive allosteric modulator scaffolds that retained similar pharmacological activity to parent compounds. We demonstrate successful probe-dependent incorporation of a commercially available clickable fluorophore using bioorthogonal conjugation. Importantly, we also show the limitations of using these probes to assess in situ fluorescence of mGlu2 in intact cells where significant nonspecific membrane binding is evident.

Allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) represent an attractive therapeutic strategy for multiple CNS disorders. Chemically distinct mGlu5 positive allosteric modulators (PAMs) that interact with a common binding site can demonstrate biased allosteric agonism relative to the orthosteric agonist, DHPG, when comparing activity in signaling assays such as IP1 accumulation, ERK1/2 phosphorylation (pERK1/2) and iCa2+ mobilization. However, the structural basis for such biased agonism is not well understood. Therefore, we evaluated biased allosteric agonism mediated by four mGlu5 PAM-agonists from diverse chemical scaffolds (i.e., DPFE, VU0409551, VU29, and VU0424465) for three measures of mGlu5 activation (i.e., iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation) at eight single-point mutations within the common allosteric binding pocket of mGlu5. In particular, mGlu5 allosteric site mutations had differential effects on the intrinsic efficacy of mGlu5 PAMs for multiple signaling pathways. Specifically, a loss of agonism for iCa2+ mobilization was evident for DPFE and VU0409551 for most mutants, whereas IP1 accumulation and ERK phosphorylation were retained, albeit with reduced maximal responses. Additionally, bias profiles between iCa2+ mobilization and IP1/ERK pathways remained similar to wild type for most mutants. However, W784A and A809G mutants lost bias between IP1 accumulation and ERK phosphorylation for VU0424465, whereas a loss of bias between iCa2+ mobilization and ERK1/2 phosphorylation was evident for F787A, S808A and A809G mutants. These data provide further insight into the structural requirements for allosteric agonism across multiple mGlu5-mediated signaling pathways. An understanding of mGlu5 biased agonism at a structural level may provide the foundation for rational structure-based design of biased allosteric ligands for the treatment of neurological disorders.

This paper highlights a bifunctional isocyanide that contains a photoreactive aliphatic diazirine for protein target capture and a terminal alkyne for click chemistry-based proteomics. Specifically, this isocyanide was employed in five different multicomponent reactions to produce 10 different fully functionalized small-molecule probes (FFSMPs) containing eight different chemical scaffolds. We anticipate this bifunctional isocyanide can be used to create FFSMP libraries of much greater chemical diversity toward identifying compounds with novel mechanisms of action via integrated phenotypic screening and target identification.

G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively "tuning" GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand-GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu5 NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu5 via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu5-bound probe via tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu5 and negative cooperativity with glutamate, interacted with the "common allosteric-binding site," displayed slow binding kinetics, and could irreversibly label mGlu5 following UV exposure. We depleted the number of functional mGlu5 receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes via click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu5 allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily.

The pharmacological properties of (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13') and serine (position 6') rings. However, an additional domain, between the outer (position 20') and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.

Photoaffinity labeling has a longstanding history as a powerful biochemical technique. However, photoaffinity labeling has significantly evolved over the past decade principally due to its coupling with bioorthogonal/click chemistry reactions. This review aims to highlight tandem photoaffinity labeling-bioorthogonal conjugation as a chemical approach in medicinal chemistry and chemical biology. In particular, recent examples of using this strategy for affinity-based protein profiling (AfBPP), drug target identification, binding ensemble profiling, studying endogenous biological molecules, and imaging applications will be presented. Additionally, recent advances in the development of 'all-in-one' compact moieties possessing a photoreactive group and clickable handle will be discussed.

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.

Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4'-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4β2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4β2 neuronal nAChRs. Co-incubation of (±)-[(125)I]-3 with non-radioactive (±)-bupropion or (-)-cocaine blocked labeling of these proteins. Compound (±)-[(125)I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels.

Studies regarding glycine receptor (GlyR) allostery can help provide insight into receptor function and allow us to develop novel therapeutics to modulate its activity. To conduct these studies, single active thiols are introduced into a Cys null background (C41S/C290A/C345S) or in the same background with a double mutation (F207G/A288G) that causes GlyR to be activated in a non-desensitizing manner to ivermectin (IVM). The crosslinker, MTS-Benzophenone, is attached via disulfide linkage to a single active Cys and the receptor is enriched in an allosteric state, resting (no ligand), desensitized (excess glycine) or open (F207G/A288G + ivermectin), before photoactivation and generation of non-specific crosslinks that examine the local topography near the sites of attachment. The crosslinked peptides, generated by trypsin digestion of monomeric and oligomeric bands, are analyzed using crosslinking mass spectrometry (CX-MS) and tandem mass spectrometry (MSMS) to sensitively identify sites of intra- and intermolecular crosslinking, respectively. In this study, we examine the network of interactions generated by three mutations, introduced singly at A41C, H419C or M287C in the resting, open and desensitized states. Recent studies using M287C, a position near the top of TM3, potentially provides information on how this helix moves and how the membrane regions are involved in the opening and closing of the channel.

Ligand virtual screening (VS) using the vestibular binding pocket of a 3-D monoamine transporter (MAT) computational model followed by in vitro pharmacology led to the identification of a human serotonin transporter (hSERT) inhibitor with modest affinity (hSERT K(i) = 284 nM). Structural comparison of this VS-elucidated compound, denoted MI-17, to known SERT ligands led to the rational design and synthesis of DJLDU-3-79, a molecular hybrid of MI-17 and dual SERT/5-HT(1A) receptor antagonist SSA-426. Relative to MI-17, DJLDU-3-79 displayed 7-fold improvement in hSERT binding affinity and a 3-fold increase in [(3)H]-serotonin uptake inhibition potency at hSERT/HEK cells. This hybrid compound displayed a hSERT:hDAT selectivity ratio of 50:1, and a hSERT:hNET (human norepinephrine transporter) ratio of200:1. In mice, DJLDU-3-79 decreased immobility in the tail suspension test comparable to the SSRI fluvoxamine, suggesting that DJLDU-3-79 may possess antidepressant properties. This proof of concept study highlights MAT virtual screening as a powerful tool for identifying novel inhibitor chemotypes and chemical fragments for rational inhibitor design.

We report here the synthesis of an aziridine tethered to a substituted allylsilane via a substituted tether. These tether-substituted aziridine–allylsilanes cyclize differently upon treatment with BF 3 ·OEt 2 than tether-unsubstituted aziridine–allylsilanes and provide a 6- endo -type product. We show that steric interactions between the N -substituent of the aziridine and substitution on the tether can control the product distribution.

Photoaffinity labeling is a well-known biochemical technique that has grown significantly since the turn of the century, principally due to its combination with bioorthogonal/click chemistry reactions. This review highlights new developments and applications of clickable photoprobes in medicinal chemistry and chemical biology. In particular, recent examples of clickable photoprobes for target identification, activity- or affinity-based protein profiling (ABPP or AfBPP), characterization of sterol– or lipid–protein interactions and characterization of ligand-binding sites are presented.

We have shown that the Suzuki cross-coupling reaction of olefinic aziridines is an effective route for the synthesis of substituted aziridines. This is the first example of a palladium coupling reaction applied to an aziridine-containing molecule. This method is complementary to other methods of aziridine synthesis utilizing organocuprate reagents.

We have prepared ring E analogs of the diterpenoid alkaloid methyllycaconitine. These compounds have been assayed for nicotinic activity and were found to act as functional antagonists on adrenal nicotinic receptors.

Adrenal secretion and binding studies were performed using ring E analogues of methyllycaconitine to assess structural determinants affecting activity on bovine adrenal alpha3beta4* nicotinic receptors. The most potent analogues are as potent as many inhibitors of adrenal secretion. Our data support the potential use of methyllycaconitine analogues to generate nicotinic receptor subtype-specific compounds.

In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive D-(+)-methylphenidate or (−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters.

We have shown that the Suzuki cross-coupling reaction of olefinic aziridines is an effective route for the synthesis of substituted aziridines. This is the first example of a palladium coupling reaction applied to an aziridine-containing molecule. This method is complementary to other methods of aziridine synthesis utilizing organocuprate reagents.

Non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored in contrast to tropane-based compounds such as cocaine. In order to fill this knowledge gap, a ligand was synthesized in which the aromatic ring of pyrovalerone was substituted with a photoreactive azido group. The analog 1-(4-azido-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-1-one demonstrated appreciable binding affinity for the DAT (Ki = 78 ± 18 nM), suggesting the potential utility of a radioiodinated version in structure-function studies of this protein.

Publisher Summary Three-membered ring systems, epoxides and aziridines in particular, are excellent synthetic intermediates. This is largely because of their ability to be converted into other functional groups, such as diols, diamines, and amino alcohols to name a few. While the synthesis of aziridines and epoxides can be quite challenging, the rewards for a selective and high-yielding synthesis can be substantial. Epoxides are possibly the most studied of the three-membered heterocycles. While host methods for the synthesis of epoxides have been developed, work continues, especially in the development of more chemo-, regio-, and stereoselective methods. The development of new metal-based epoxidation catalysts continues to garner significant levels of activity. The use of a Mn-based catalyst with a water-soluble ligand provides excellent yields of the corresponding epoxides. In earlier studies, the use of a hydrazinium salt as a nitrogen transfer agent in combination with base proved to be a synthetically useful method for the synthesis of aziridine. Hydrazinium salt can be readily prepared as either the iodide or nitrate salt, although the iodide synthesis is more convenient.

We have shown that connection of C -2 of an allylsilane to a tethered aziridine ring yields exocyclic γ-amino olefins and desilylated azabicyclo[ x .2.1]-systems upon cyclization with BF 3 ·OEt 2 . Furthermore, manipulation of a specific exocyclic γ-amino olefin provided access to an azabicyclo[3.3.1]nonane. This methodology should be useful for the preparation of natural products and pharmacologically active agents containing these bicyclic heterocyclic systems.

The development of new agents that selectively interact with subtypes of neuronal nicotinic receptors (nAChRs) is of primary importance for the study of physiological processes and pathophysiological conditions involving these receptors. Our laboratory has evidence that simple ring E analogues of methyllycaconitine (MLA) act as antagonists to bovine adrenal alpha3beta4* nAChRs. The following studies were designed to characterize the concentration-response effects of several ring E analogues of MLA in order to assess structural requirements involved with their inhibitory activity on bovine adrenal alpha3beta4* nAChRs. Ring E analogues with various substitutions on the ring E nitrogen were tested for their ability to inhibit nicotinic stimulated adrenal catecholamine release and [3H]epibatidine binding to a bovine adrenal membrane preparation. Several N-alkyl derivatives inhibited secretion with IC50 values in the low micromolar range. The N-phenpropyl analogue was the most potent of the analogues tested (IC50, 11 microM) on adrenal secretion. Competition binding studies suggest a noncompetitive interaction of the analogues with bovine adrenal nAChRs. These studies identify several structural features of ring E analogues of MLA which significantly affect their inhibitory activity on bovine adrenal alpha3beta4* nAChRs.

A wealth of high-resolution structural information regarding pentameric ligand-gated ion channels is now available, but less is known of the molecular details underlying complex allosteric mechanisms involved in channel gating and desensitization. Receptor allostery can be studied by identifying state-dependent distance constraints that may be used in molecular modeling of these receptors. Systematically generated single Cys mutations of the human α1 glycine receptor (GlyR) expressed in insect cells were labeled with a clickable methanethiosulfonate-benzophenone crosslinker. After covalent ligation to Cys, crosslinks may then be introduced in the resting, open, or desensitized states by photoactivation. Including an alkyne tag on the crosslinker permits click chemistry addition of biotin, which allows for enrichment by avidin chromatography. Mass spectrometry (MS) fingerprinting of monomeric and higher-order GlyR bands on SDS-PAGE using ESI-QTOF MS/MS then allows for determination of the site of crosslinking. Our initial proof-of-principle studies conducted on purified GlyR have provided state-dependent information on this receptor. This approach may be broadly applicable to studies of any allosteric complex.

This chapter covers the chemistry of three-membered heterocycles, epoxides, aziridines, azirines, and oxaziridines for the calendar year 2012. For each of the different three-membered heterocycles, a listing of new methods of synthesis and new examples of reactions are presented. This review is not comprehensive for 2012 but covers methods and reactions that should be synthetically useful for the practicing chemist and those interested in the study of three-membered heterocyclic ring systems.

Bupropion is clinically prescribed for the treatment of depression (Wellbutrin) and for smoking cessation (Zyban). While there is consensus that the primary mechanism of action involves increased synaptic concentrations of dopamine and norepinephrine via inhibition of the respective reuptake transporters, DAT and NET, there is growing evidence that some of the therapeutic benefits of bupropion may result from non-competitive antagonism of neuronal nicotinic acetylcholine receptors (nAChRs), in particular α4β2 and α3β4 subtypes. To aid in elucidating the mechanism of bupropion action and the development of new therapeutic agents for smoking cessation and depression, our goal is to determine the specific molecular interactions between bupropion and several nAChR subtypes (Torpedo, α4β2, α3β4). We first established that bupropion dose-dependently and reversibly inhibits ACh-induced currents in Xenopus laevis oocytes injected with affinity-purified and lipid-reconstituted Torpedo nAChRs (IC50 0.34 μM). Using a photoreactive analog of bupropion ([125I]-SADU-3-72), we next established that bupropion inhibits the binding of [125I]-SADU-3-72 to the Torpedo nAChR (IC50 closed state, 3.6 μM; desensitized state, 1.2 μM) and that binding to the closed nAChR is fully inhibited by tetracaine (IC50 0.42 μM) consistent with a bupropion/[125I]-SADU-3-72 binding site within the nAChR channel. [125I]-SADU-3-72 photolabeled Torpedo nAChR subunits in an agonist-sensitive and bupropion-inhibitable (specific) manner. Finally for the closed nAChR, we identified sites of specific [125I]-SADU-3-72 labeling within δM2 (δLeu265/δM2-9) and βM2 (βLeu257/βM2-9). In the desensitized state, TCP-inhibitable (specific) labeling was identified within δM2 (δLeu265, δM2-9 with minor labeling of δSer258, δM2-2). Our results establish that bupropion binds in the middle (M2-9) of the Torpedo nAChR channel, with a slightly broader binding locus in the desensitized channel. Currently, studies are underway to identify the site(s) of [125I]-SADU-3-72 labeling in affinity-purified α4β2 and α3β4 nAChRs.