Appendage and Scaffold Diverse Fully Functionalized Small-Molecule Probes via a Minimalist Terminal Alkyne-Aliphatic Diazirine Isocyanide.

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.

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram.

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., 3H or 125I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT Ki = 0.16 nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT Ki = 1.77 nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).

Bupropion binds to two sites in the Torpedo nicotinic acetylcholine receptor transmembrane domain: a photoaffinity labeling study with the bupropion analogue [(125)I]-SADU-3-72.

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, ßV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within ßV8-22/23K, 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), ßLeu(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.

(±)-2-(N-tert-Butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban).

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 α4ß2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4ß2 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.

Tandem photoaffinity labeling-bioorthogonal conjugation in medicinal chemistry.

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.

Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling.

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-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane (ß-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.

Development of Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 2 Based on Two Positive Allosteric Modulator Chemotypes.

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.

Differential contribution of metabotropic glutamate receptor 5 common allosteric binding site residues to biased allosteric agonism.

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.

A novel photoaffinity ligand for the dopamine transporter based on pyrovalerone.

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 (K(i)=78+/-18 nM), suggesting the potential utility of a radioiodinated version in structure-function studies of this protein.

Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators.

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.

Recent developments and applications of clickable photoprobes in medicinal chemistry and chemical biology.

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.

Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram.

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 µM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.

Effects of methyllycaconitine and related analogues on bovine adrenal alpha3beta4* nicotinic acetylcholine 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.

Structure-activity studies with ring E analogues of methyllycaconitine on bovine adrenal alpha3beta4* nicotinic receptors.

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.

Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-threo-Methylphenidate.

The development of photoaffinity ligands for determining covalent points of attachment to the dopamine transporter (DAT) has predominantly focused on tropane-based compounds bearing variable-length linkers between the photoreactive group and inhibitor pharmacophore. In order to expand the array of photoprobes useful for mapping inhibitor-binding pockets within the DAT, a compact non-tropane ligand was synthesized featuring a photoreactive azide and iodine tag directly attached to the aromatic ring of (±)-threo-methylphenidate. (±)-threo-4-Azido-3-iodomethylphenidate ((±)-6); K(i) = 4.0 ± 0.8 nM) displayed high affinity for hDAT. Moreover, a radioiodinated analog of (±)-6 demonstrated covalent ligation to the DAT in cultured cells and rat striatal membranes, thus suggesting the potential utility of this photoprobe in DAT structure-function studies.

Structural and functional interaction of (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone, a photoreactive bupropion derivative, with nicotinic acetylcholine receptors.

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. Ca²âº influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1ß1γδ) and adult (hα1ß1εδ) 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.

Identification of a novel selective serotonin reuptake inhibitor by coupling monoamine transporter-based virtual screening and rational molecular hybridization.

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 of >200:1. 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.