The Structural Basis of the Activity Cliff in Modafinil-Based Dopamine Transporter Inhibitors.

Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead sulfoxide-substituted analogs showing characteristics of atypical inhibition (e.g., JJC8-091). Interestingly, the only distinction between sulfoxide and sulfide substitution is the presence of one additional oxygen atom. To elucidate why such a subtle difference in ligand structure can result in different typical or atypical profiles, we investigated two pairs of analogs. Our quantum mechanical calculations revealed a more negatively charged distribution of the electrostatic potential surface of the sulfoxide substitution. Using molecular dynamics simulations, we demonstrated that sulfoxide-substituted modafinil analogs have a propensity to attract more water into the binding pocket. They also exhibited a tendency to dissociate from Asp79 and form a new interaction with Asp421, consequently promoting an inward-facing conformation of hDAT. In contrast, sulfide-substituted analogs did not display these effects. These findings elucidate the structural basis of the activity cliff observed with modafinil analogs and also enhance our understanding of the functionally relevant conformational spectrum of hDAT.

Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes.

The dopamine transporter (DAT) is one of the key regulators of dopamine (DA) signaling in the central nervous system (CNS) and in the periphery. Recent reports in a model of Parkinson's disease (PD) have shown that dopamine neuronal loss in the CNS impacts the expression of DAT in peripheral immune cells. The mechanism underlying this connection is still unclear but could be illuminated with sensitive and high-throughput detection of DAT-expressing immune cells in the circulation. Herein, we have developed fluorescently labeled ligands (FLL) that bind to surface-expressing DAT with high affinity and selectivity. The diSulfoCy5-FLL (GC04-38) was utilized to label DAT in human and mouse peripheral blood mononuclear cells (PBMCs) that were analyzed via flow cytometry. Selective labeling was validated using DAT KO mouse PBMCs. Our studies provide an efficient and highly sensitive method using this novel DAT-selective FLL to advance our fundamental understanding of DAT expression and activity in PBMCs in health and disease and as a potential peripheral biomarker.

Modifications to 1-(4-(2-Bis(4-fluorophenyl)methyl)sulfinyl)alkyl Alicyclic Amines That Improve Metabolic Stability and Retain an Atypical DAT Inhibitor Profile.

Atypical dopamine transporter (DAT) inhibitors have shown therapeutic potential in the preclinical models of psychostimulant use disorders (PSUD). In rats, 1-(4-(2-((bis(4-fluorophenyl)methyl)sulfinyl)ethyl)-piperazin-1-yl)-propan-2-ol (JJC8-091, 3b) was effective in reducing the reinforcing effects of both cocaine and methamphetamine but did not exhibit psychostimulant behaviors itself. Improvements in DAT affinity and metabolic stability were desirable for discovering pipeline drug candidates. Thus, a series of 1-(4-(2-bis(4-fluorophenyl)methyl)sulfinyl)alkyl alicyclic amines were synthesized and evaluated for binding affinities at DAT and the serotonin transporter (SERT). Replacement of the piperazine with either a homopiperazine or a piperidine ring system was well tolerated at DAT (Ki range = 3-382 nM). However, only the piperidine analogues (20a-d) showed improved metabolic stability in rat liver microsomes as compared to the previously reported analogues. Compounds 12b and 20a appeared to retain an atypical DAT inhibitor profile, based on negligible locomotor activity in mice and molecular modeling that predicts binding to an inward-facing conformation of DAT.

Illuminating the monoamine transporters: Fluorescently labelled ligands to study dopamine, serotonin and norepinephrine transporters.

Fluorescence microscopy has revolutionized the visualization of physiological processes in live-cell systems. With the recent innovations in super resolution microscopy, these events can be examined with high precision and accuracy. The development of fluorescently labelled small molecules has provided a significant advance in understanding the physiological relevance of targeted proteins that can now be visualized at the cellular level. One set of physiologically important target proteins are the monoamine transporters (MATs) that play an instrumental role in maintaining monoamine signalling homeostasis. Understanding the mechanisms underlying their regulation and dysregulation is fundamental to treating several neuropsychiatric conditions such as attention deficit hyperactivity disorder (ADHD), anxiety, depression and substance use disorders. Herein, we describe the rationale behind the small molecule design of fluorescently labelled ligands (FLL) either as MAT substrates or inhibitors as well as their applications to advance our understanding of this class of transporters in health and disease.

Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux.

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

Differential Activation and Desensitization States Promoted by Noncanonical α7 Nicotinic Acetylcholine Receptor Agonists.

A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential α7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists. Using oocytes expressing human α7 receptors, we found that the DPPs were efficacious activators of the receptor, with currents showing rapid desensitization characteristic of α7 receptors. However, we note that the rate of recovery from this desensitization depends strongly on structural features within the DPP family. Although the activation of receptors by DPP was blocked by the competitive antagonist methyllycaconitine (MLA), MLA had no effect on the DPP-induced desensitization, suggesting multiple modes of DPP binding. As expected, the desensitized conformational states could be reactivated by PAMs. Mutants made insensitive to acetylcholine by the C190A mutation in the agonist binding site were weakly activated by DPPs. The observation that activation of C190A mutants by the DPP compounds was resistant to the allosteric antagonist (-)cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide supports the hypothesis that the activity of these noncanonical agonists in the orthosteric binding sites was not entirely dependent on the classic epitopes controlling activation by typical agonists and that perhaps they may access alternative modes for promoting the conformational changes associated with activation and desensitization. SIGNIFICANCE STATEMENT: This study reports a family of nicotinic acetylcholine receptor agonists that break the rules about what the structure of a nicotinic acetylcholine receptor agonist should be. It shows that the activity of these noncanonical agonists in the orthosteric binding sites is not dependent on the classical epitopes controlling activation by typical agonists and that through different binding poses, they promote unique conformational changes associated with receptor activation and desensitization.

Methylphenidate Analogues as a New Class of Potential Disease-Modifying Agents for Parkinson's Disease: Evidence from Cell Models and Alpha-Synuclein Transgenic Mice.

Parkinson's disease (PD) is characterized by dopaminergic nigrostriatal neurons degeneration and Lewy body pathology, mainly composed of α-synuclein (αSyn) fibrillary aggregates. We recently described that the neuronal phosphoprotein Synapsin III (Syn III) participates in αSyn pathology in PD brains and is a permissive factor for αSyn aggregation. Moreover, we reported that the gene silencing of Syn III in a human αSyn transgenic (tg) mouse model of PD at a pathological stage, manifesting marked insoluble αSyn deposits and dopaminergic striatal synaptic dysfunction, could reduce αSyn aggregates, restore synaptic functions and motor activities and exert neuroprotective effects. Interestingly, we also described that the monoamine reuptake inhibitor methylphenidate (MPH) can recover the motor activity of human αSyn tg mice through a dopamine (DA) transporter-independent mechanism, which relies on the re-establishment of the functional interaction between Syn III and α-helical αSyn. These findings support that the pathological αSyn/Syn III interaction may constitute a therapeutic target for PD. Here, we studied MPH and some of its analogues as modulators of the pathological αSyn/Syn III interaction. We identified 4-methyl derivative I-threo as a lead candidate modulating αSyn/Syn III interaction and having the ability to reduce αSyn aggregation in vitro and to restore the motility of αSyn tg mice in vivo more efficiently than MPH. Our results support that MPH derivatives may represent a novel class of αSyn clearing agents for PD therapy.

Preferential Gs protein coupling of the galanin Gal1 receptor in the µ-opioid-Gal1 receptor heterotetramer.

Recent studies have proposed that heteromers of µ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Illuminating the norepinephrine transporter: fluorescent probes based on nisoxetine and talopram.

The utilization of fluorescent ligands to study the monoamine transporters (MATs) has increased our knowledge of their function and distribution in live cell systems. In this study, we extend SAR for nisoxetine and talopram as parent compounds, to identify high affinity rhodamine-labeled fluorescent probes for the norepinephrine transporter (NET). Nisoxetine-based fluorescent probe 6 demonstrated high binding affinity (K i = 43 nM) for NET and an overall selectivity compared to the other transporters for dopamine (DAT; K i = 1540 nM) and serotonin (SERT; K i = 785 nM) in competitive radioligand binding assays. Using confocal microscopy, compound 6 was shown to stain both NET and SERT, but not DAT, at low nanomolar concentrations, in transporter-expressing cells.

Ligand design for human acetylcholinesterase and nicotinic acetylcholine receptors, extending beyond the conventional and canonical.

We detail here distinctive departures from lead classical cholinesterase re-activators, the pyridinium aldoximes, to achieve rapid CNS penetration and reactivation of AChE in the CNS (brain and spinal cord). Such reactivation is consistent with these non-canonical re-activators enhancing survival parameters in both mice and macaques following exposure to organophosphates. Thus, the ideal cholinesterase re-activator should show minimal toxicity, limited inhibitory activity in the absence of an organophosphate, and rapid CNS penetration, in addition to its nucleophilic potential at the target, the conjugated AChE active center. These are structural properties directed to reactivity profiles at the conjugated AChE active center, reinforced by the pharmacokinetic and tissue disposition properties of the re-activator leads. In the case of nicotinic acetylcholine receptor (nAChR) agonists and antagonists, with the many existing receptor subtypes in mammals, we prioritize subtype selectivity in their design. In contrast to nicotine and its analogues that react with panoply of AChR subtypes, the substituted di-2-picolyl amine pyrimidines possess distinctive ionization characteristics reflecting in selectivity for the orthosteric site at the α7 subtypes of receptor. Here, entry to the CNS should be prioritized for the therapeutic objectives of the nicotinic agent influencing aberrant CNS activity in development or in the sequence of CNS ageing (longevity) in mammals, along with general peripheral activities controlling inflammation.

Lessons from nature: Structural studies and drug design driven by a homologous surrogate from invertebrates, AChBP.

It has been almost 20 years since the discovery and crystallization of a structural surrogate, the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP), comprising the extracellular domain of the nicotinic acetylcholine receptors (nAChRs). Structural characterization of this soluble protein has increased our understanding of the requirements for agonist and antagonist interactions at the ligand recognition site of the nAChRs. Application can be extended to orthologs in the pentameric ligand-gated ion channel superfamily, encompassing receptors that depolarize or hyperpolarize upon neurotransmitter association. Despite the lack of transmembrane and intracellular motifs, the highly conserved binding or recognition loci have made these soluble binding proteins, and mutants derived from them, prototypic tools for molecular recognition and structural studies of pentameric ligand-gated ion channels. Targeting nAChRs has been a major goal as this family is associated with neurodegenerative diseases and disorders. Accordingly, the ligand binding site has played a key role to the development of selective ligands for modulation of this transmembrane proteins. In this review article, we cover both the potential and limitations of soluble surrogates, termed the AChBP family, in drug development. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

Synthesis, Pharmacological Characterization, and Structure-Activity Relationships of Noncanonical Selective Agonists for α7 nAChRs.

A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC50's between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay. After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine analogues, thereby distinguishing this subclass of noncanonical agonists from previously defined types of agonists (agonists, partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed pKa values for ionization of lead candidates, demonstrating distinctive modes of interaction for this landscape of ligands.

Assessment of ionizable, zwitterionic oximes as reactivating antidotal agents for organophosphate exposure.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

Substituted 2-Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins.

Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4ß2-nAChR, and a serotonin receptor (5-HT3AR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 µM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC50 values of 70 nM and Kd values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4ß2-nAChR or 5-HT3AR at concentrations up to 10 µM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.