Mechanistic insights into dopaminergic and serotonergic neurotransmission – concerted interactions with helices 5 and 6 drive the functional outcome

Brain functions rely on neurotransmitters that mediate communication between billions of neurons. Disruption of this communication can result in a plethora of psychiatric and neurological disorders. In this work, we combine molecular dynamics simulations, live-cell biosensor and electrophysiological assays to investigate the action of the neurotransmitter dopamine at the dopaminergic D₂ receptor (D₂R). The study of dopamine and closely related chemical probes reveals how neurotransmitter binding translates into the activation of distinct subsets of D₂R effectors (i.e.: G_i2, G_oB, G_z and β-arrestin 2). Ligand interactions with key residues in TM5 (S5.42) and TM6 (H6.55) in the D₂R binding pocket yield a dopamine-like coupling signature, whereas exclusive TM5 interaction is typically linked to preferential G protein coupling (in particular G_oB) over β-arrestin. Further experiments for serotonin receptors indicate that the reported molecular mechanism is shared by other monoaminergic neurotransmitter receptors. Ultimately, our study highlights how sequence variation in position 6.55 is used by nature to fine-tune β-arrestin recruitment and in turn receptor signaling and internalization of neurotransmitter receptors.

Neurotransmitter contacts within the receptor binding site differentially contribute to the overall functional response: transmembrane helix (TM) 5 contacts promote G protein coupling whereas concerted TM5–TM6 contacts enhance β-arrestin recruitment.     

Cooperativity as quantification and optimization paradigm for nuclear receptor modulators

Nuclear Receptors (NRs) are highly relevant drug targets, for which small molecule modulation goes beyond a simple ligand/receptor interaction. NR–ligands modulate Protein–Protein Interactions (PPIs) with coregulator proteins. Here we bring forward a cooperativity mechanism for small molecule modulation of NR PPIs, using the Peroxisome Proliferator Activated Receptor γ (PPARγ), which describes NR–ligands as allosteric molecular glues. The cooperativity framework uses a thermodynamic model based on three-body binding events, to dissect and quantify reciprocal effects of NR–coregulator binding (K^I_D) and NR–ligand binding (K^II_D), jointly recapitulated in the cooperativity factor (α) for each specific ternary ligand·NR·coregulator complex formation. These fundamental thermodynamic parameters allow for a conceptually new way of thinking about structure–activity-relationships for NR–ligands and can steer NR modulator discovery and optimization via a completely novel approach.

A cooperativity framework describes the formation of nuclear receptor ternary complexes and deconvolutes ligand and cofactor binding into intrinsic affinities and a cooperativity factor, providing a conceptually new understanding of NR modulation.     

PET Diagnostic Molecules Utilizing Multimeric Cyclic RGD Peptide Analogs for Imaging Integrin αvβ3 Receptors

Multimeric ligands consisting of multiple pharmacophores connected to a single backbone have been widely investigated for diagnostic and therapeutic applications. In this review, we summarize recent developments regarding multimeric radioligands targeting integrin α_vβ₃ receptors on cancer cells for molecular imaging and diagnostic applications using positron emission tomography (PET). Integrin α_vβ₃ receptors are glycoproteins expressed on the cell surface, which have a significant role in tumor angiogenesis. They act as receptors for several extracellular matrix proteins exposing the tripeptide sequence arginine-glycine-aspartic (RGD). Cyclic RDG peptidic ligands c(RGD) have been developed for integrin α_vβ₃ tumor-targeting positron emission tomography (PET) diagnosis. Several c(RGD) pharmacophores, connected with the linker and conjugated to a chelator or precursor for radiolabeling with different PET radionuclides (¹⁸F, ⁶⁴Cu, and ⁶⁸Ga), have resulted in multimeric ligands superior to c(RGD) monomers. The binding avidity, pharmacodynamic, and PET imaging properties of these multimeric c(RGD) radioligands, in relation to their structural characteristics are analyzed and discussed. Furthermore, specific examples from preclinical studies and clinical investigations are included.     

Enantioenriched Positive Allosteric Modulators Display Distinct Pharmacology at the Dopamine D1 Receptor

(1) Background: Two first-in-class racemic dopamine D₁ receptor (D₁R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular, due to its selectivity for the D₁R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states; (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (d- and l-pantolactone, respectively); (3) Results: We confirm both the racemate and enantiomers of 2 are active and selective for the D₁R, but that the respective stereoisomers show a significant difference in their affinity and magnitude of positive allosteric cooperativity with dopamine; (4) Conclusions: These data warrant further investigation of asymmetric syntheses of optically pure analogues of 2 for the development of D₁R PAMs with superior allosteric properties.     

Design,Sustainable Synthesis and Biological Evaluation of a Novel Dual α2A/5-HT7 Receptor Antagonist with Antidepressant-Like Properties

The complex pathophysiology of depression, together with the limits of currently available antidepressants, has resulted in the continuous quest for alternative therapeutic strategies. Numerous findings suggest that pharmacological blockade of α₂-adrenoceptor might be beneficial for the treatment of depressive symptoms by increasing both norepinephrine and serotonin levels in certain brain areas. Moreover, the antidepressant properties of 5-HT₇ receptor antagonists have been widely demonstrated in a large set of animal models. Considering the potential therapeutic advantages in targeting both α₂-adrenoceptors and 5-HT₇ receptors, we designed a small series of arylsulfonamide derivatives of (dihydrobenzofuranoxy)ethyl piperidines as dually active ligands. Following green chemistry principles, the designed compounds were synthesized entirely using a sustainable mechanochemical approach. The identified compound 8 behaved as a potent α_2A/5-HT₇ receptor antagonist and displayed moderate-to-high selectivity over α₁-adrenoceptor subtypes and selected serotonin and dopaminergic receptors. Finally, compound 8 improved performance of mice in the forced swim test, displaying similar potency to the reference drug mirtazapine.     

Pharmacological Characterization of µ-Opioid Receptor Agonists with Biased G Protein or β-Arrestin Signaling,and Computational Study of Conformational Changes during Receptor Activation

In recent years, G protein vs. β-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF₃-Phe-Asp]NH₂ (F-81), and the β-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH₂ (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [³⁵S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure β-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand–receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or β-arrestin.     

Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-β-ACPC) Scaffolds

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified α_vβ₃ and α₅β₁ receptors using biotinylated vitronectin (α_vβ₃) and fibronectin (α₅β₁) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for α_vβ₃ over α₅β₁. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin α_vβ₃). The two RGD ligands showed IC₅₀ values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC₅₀ value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the α_Vβ₃ integrin active site, provided a rationale for the behavior of these ligands toward the receptor.     

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.     

Allosteric Modulation of the CB1 Cannabinoid Receptor by Cannabidiol—A Molecular Modeling Study of the N-Terminal Domain and the Allosteric-Orthosteric Coupling

The CB₁ cannabinoid receptor (CB₁R) contains one of the longest N termini among class A G protein-coupled receptors. Mutagenesis studies suggest that the allosteric binding site of cannabidiol (CBD) involves residues from the N terminal domain. In order to study the allosteric binding of CBD to CB₁R we modeled the whole N-terminus of this receptor using the replica exchange molecular dynamics with solute tempering (REST2) approach. Then, the obtained structures of CB₁R with the N terminus were used for ligand docking. A natural cannabinoid receptor agonist, Δ⁹-THC, was docked to the orthosteric site and a negative allosteric modulator, CBD, to the allosteric site positioned between extracellular ends of helices TM1 and TM2. The molecular dynamics simulations were then performed for CB₁R with ligands: (i) CBD together with THC, and (ii) THC-only. Analyses of the differences in the residue-residue interaction patterns between those two cases allowed us to elucidate the allosteric network responsible for the modulation of the CB₁R by CBD. In addition, we identified the changes in the orthosteric binding mode of Δ⁹-THC, as well as the changes in its binding energy, caused by the CBD allosteric binding. We have also found that the presence of a complete N-terminal domain is essential for a stable binding of CBD in the allosteric site of CB₁R as well as for the allosteric-orthosteric coupling mechanism.     

Ring-Opening Polymerization of ε-Caprolactone and Styrene Oxide–CO2 Coupling Reactions Catalyzed by Chelated Dehydroacetic Acid–Imine Aluminum Complexes

A series of chelated dehydroacetic acid–imine-based ligands L¹H~L⁴H was synthesized by reacting dehydroacetic acid with 2-t-butylaniline, (S)-1-phenyl-ethylamine, 4-methoxylbenzylamine, and 2-(aminoethyl)pyridine, respectively, in moderate yields. Ligands L¹H~L⁴H reacted with AlMe₃ in toluene to afford corresponding compounds AlMe₂L¹ (1), AlMe₂L² (2), AlMe₂L³ (3), and AlMe₂L⁴ (4). All the ligands and aluminum compounds were characterized by IR spectra, ¹H and ¹³C NMR spectroscopy. Additionally, the ligands L¹H~L⁴H and corresponding aluminum derivatives 1, 3, and 4 were characterized by single-crystal X-ray diffractometry. The catalytic activities using these aluminum compounds as catalysts for the ε-caprolactone ring-opening polymerization (ROP) and styrene oxide-CO₂ coupling reactions were studied. The results show that increases in the reaction temperature and selective solvent intensify the conversions of ε-caprolactone to polycaprolactone. Regarding the coupling reactions of styrene oxide and CO₂, the conversion rate is over 90% for a period of 12 h at 90 °C. This strategy dispenses the origination of cyclic styrene carbonates, which is an appealing concern because of the transformation of CO₂ into an inexpensive, renewable and easy excess carbon feedstock.     

Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor

Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β₁ adrenergic receptor model in complex with (S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D₂ receptor were produced with the selective and rigid agonist (R)-N-propylapomorphine ((R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D₂ receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.     

Design and Synthesis of Arylpiperazine Serotonergic/Dopaminergic Ligands with Neuroprotective Properties

Long-chain arylpiperazine scaffold is a versatile template to design central nervous system (CNS) drugs that target serotonin and dopamine receptors. Here we describe the synthesis and biological evaluation of ten new arylpiperazine derivatives designed to obtain an affinity profile at serotonin 5-HT_1A, 5-HT_2A, 5-HT₇ receptor, and dopamine D₂ receptor of prospective drugs to treat the core symptoms of autism spectrum disorder (ASD) or psychosis. Besides the structural features required for affinity at the target receptors, the new compounds incorporated structural fragments with antioxidant properties to counteract oxidative stress connected with ASD and psychosis. All the new compounds showed CNS MultiParameter Optimization score predictive of desirable ADMET properties and cross the blood–brain barrier. We identified compound 12a that combines an affinity profile compatible with antipsychotic activity (5-HT_1A K_i = 41.5 nM, 5-HT_2A K_i = 315 nM, 5-HT₇ K_i = 42.5 nM, D₂ K_i = 300 nM), and compound 9b that has an affinity profile consistent with studies in the context of ASD (5-HT_1A K_i = 23.9 nM, 5-HT_2A K_i = 39.4 nM, 5-HT₇ K_i = 45.0 nM). Both compounds also had antioxidant properties. All compounds showed low in vitro metabolic stability, the only exception being compound 9b, which might be suitable for studies in vivo.     

Theasaponin E1 Inhibits Platinum-Resistant Ovarian Cancer Cells through Activating Apoptosis and Suppressing Angiogenesis

Novel therapeutic strategies for ovarian cancer treatment are in critical need due to the chemoresistance and adverse side effects of platinum-based chemotherapy. Theasaponin E₁ (TSE1) is an oleanane-type saponin from Camellia sinensis seeds. Its apoptosis-inducing, cell cycle arresting and antiangiogenesis activities against platinum-resistant ovarian cancer cells were elucidated in vitro and using the chicken chorioallantoic membrane (CAM) assay. The results showed that TSE1 had more potent cell growth inhibitory effects on ovarian cancer OVCAR-3 and A2780/CP70 cells than cisplatin and was lower in cytotoxicity to normal ovarian IOSE-364 cells. TSE1 significantly induced OVCAR-3 cell apoptosis via the intrinsic and extrinsic apoptotic pathways, slightly arresting cell cycle at the G2/M phase, and obviously inhibited OVCAR-3 cell migration and angiogenesis with reducing the protein secretion and expression of vascular endothelial growth factor (VEGF). Western bolt assay showed that Serine/threonine Kinase (Akt) signaling related proteins including Ataxia telangiectasia mutated kinase (ATM), Phosphatase and tensin homolog (PTEN), Akt, Mammalian target of rapamycin (mTOR), Ribosome S6 protein kinase (p70S6K) and e IF4E-binding protein 1(4E-BP1) were regulated, and Hypoxia inducible factor-1α (HIF-1α) protein expression was decreased by TSE1 in OVCAR-3 cells. Moreover, TSE1 treatment potently downregulated protein expression of the Notch ligands including Delta-like protein 4 (Dll4) and Jagged1, and reduced the protein level of the intracellular domain (NICD) of Notch1. Combination treatment of TSE1 with the Notch1 signaling inhibitor tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate (DAPT), or the Akt signaling inhibitor wortmannin, showed a stronger inhibition toward HIF-1α activation compared with single compound treatment. Taken together, TSE1 might be a potential candidate compound for improving platinum-resistant ovarian cancer treatment via Dll4/Jagged1-Notch1-Akt-HIF-1α axis.     

TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca²⁺ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca²⁺-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca²⁺ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca²⁺-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without α1_SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in α1_SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or α1_SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca²⁺ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.     

Synthesis and Antibacterial Properties of Oligomeric Dehydrogenation Polymer from Lignin Precursors

The lignin precursors of coniferin and syringin were synthesised, and guaiacyl-type and guaiacyl-syringyl-type oligomeric lignin dehydrogenation polymers (DHP and DHP-GS) were prepared with the bulk method. The carbon-13 nuclear magnetic resonance spectroscopy showed that both DHP-G and DHP-GS contained β-O-4, β-5, β-β, β-1, and 5-5 substructures. Extraction with petroleum ether, ether, ethanol, and acetone resulted in four fractions for each of DHP-G (C₁₁–C₁₄) and DHP-GS (C₂₁–C₂₄). The antibacterial experiments showed that the fractions with lower molecular weight had relatively strong antibacterial activity. The ether-soluble fractions (C₁₂ of DHP-G and C₂₂ of DHP-GS) had strong antibacterial activities against E. coli and S. aureus. The C₁₂ and C₂₂ fractions were further separated by preparative chromatography, and 10 bioactive compounds (G₁–G₅ and GS₁–GS₅) were obtained. The overall antibacterial activities of these 10 compounds was stronger against E. coli than S. aureus. Compounds G₁, G₂, G₃, and GS₁, which had the most significant antibacterial activities, contained β-5 substructures. Of these, G₁ had the best antibacterial activity. Its inhibition zone diameter was 19.81 ± 0.82 mm, and the minimum inhibition concentration was 56.3 ± 6.20 μg/mL. Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) showed that the antibacterial activity of G₁ was attributable to a phenylcoumarin dimer, while the introduction of syringyl units reduced antibacterial activity.     

σ2 Receptor and Its Role in Cancer with Focus on a MultiTarget Directed Ligand (MTDL) Approach

Sigma-2 (σ₂) is an endoplasmic receptor identified as the Endoplasmic Reticulum (ER) transmembrane protein TMEM97. Despite its controversial identity, which was only recently solved, this protein has gained scientific interest because of its role in the proliferative status of cells; many tumor cells from different organs overexpress the σ₂ receptor, and many σ₂ ligands display cytotoxic actions in (resistant) cancer cells. These properties have shed light on the σ₂ receptor as a potential druggable target to be bound/activated for the diagnosis or therapy of tumors. Additionally, diverse groups have shown how the σ₂ receptor can be exploited for the targeted delivery of the anticancer drugs to tumors. As the cancer disease is a multifactorial pathology with multiple cell populations, a polypharmacological approach is very often needed. Instead of the simultaneous administration of different classes of drugs, the use of one molecule that interacts with diverse pharmacological targets, namely MultiTarget Directed Ligand (MTDL), is a promising and currently pursued strategy, that may overcome the pharmacokinetic problems associated with the administration of multiple molecules. This review aims to point out the progress regarding the σ₂ ligands in the oncology field, with a focus on MTDLs directed towards σ₂ receptors as promising weapons against (resistant) cancer diseases.     

A pharmacophore model for dopamine D4 receptor antagonists

A pharmacophore model for dopamine D₄ antagonists has been developed on the basis of a previously reported dopamine D₂ model. By using exhaustive conformational analyses (MM3^* force field and the GB/SA hydration model) and least-squares molecular superimposition studies, a set of eighteen structurally diverse high affinity D₄ antagonists have successfully been accommodated in the D₄ pharmacophore model. Enantioselectivities may be rationalized by conformational energies required for the enantiomers to adopt their proposed bioactive conformations. The pharmacophore models for antagonists at the D₄ and D₂ receptor subtypes have been compared in order to get insight into molecular properties of importance for D₂/D₄ receptor selectivity. It is concluded that the bioactive conformations of antagonists at the two receptor subtypes are essentially identical. Receptor essential volumes previously identified for the D₂ receptor are shown to be present also in the D₄ receptor. In addition, a novel receptor essential volume in the D₄ receptor, not present in the D₂ receptor, has been identified. This feature may be exploited for the design of D₄ selective antagonists. However, it is concluded that the major determinant for D₂/D₄ selectivity is the nature of the interactions between the receptor and aromatic ring systems. The effects of the electronic properties of these ring systems on the affinities for the two receptor subtypes differ substantially.     

The Antagonist pGlu-βGlu-Pro-NH2 Binds to an Allosteric Site of the Thyrotropin-Releasing Hormone Receptor

After we identified pGlu-βGlu-Pro-NH₂ as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH’s binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-βGlu-Pro-NH₂. The allosteric binding of this TRH antagonist is more robust than TRH’s binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.     

Functional proteomics,human genetics and cancer biology of GIPC family members

GIPC1, GIPC2 and GIPC3 consist of GIPC homology 1 (GH1) domain, PDZ domain and GH2 domain. The regions around the GH1 and GH2 domains of GIPC1 are involved in dimerization and interaction with myosin VI (MYO6), respectively. The PDZ domain of GIPC1 is involved in interactions with transmembrane proteins [IGF1R, NTRK1, ADRB1, DRD2, TGFβR3 (transforming growth factorβ receptor type III), SDC4, SEMA4C, LRP1, NRP1, GLUT1, integrin α5 and VANGL2], cytosolic signaling regulators (APPL1 and RGS19) and viral proteins (HBc and HPV-18 E6). GIPC1 is an adaptor protein with dimerizing ability that loads PDZ ligands as cargoes for MYO6-dependent endosomal trafficking. GIPC1 is required for cell-surface expression of IGF1R and TGFβR3. GIPC1 is also required for integrin recycling during cell migration, angiogenesis and cytokinesis. On early endosomes, GIPC1 assembles receptor tyrosine kinases (RTKs) and APPL1 for activation of PI3K–AKT signaling, and G protein-coupled receptors (GPCRs) and RGS19 for attenuation of inhibitory Gα signaling. GIPC1 upregulation in breast, ovarian and pancreatic cancers promotes tumor proliferation and invasion, whereas GIPC1 downregulation in cervical cancer with human papillomavirus type 18 infection leads to resistance to cytostatic transforming growth factorβ signaling. GIPC2 is downregulated in acute lymphocytic leukemia owing to epigenetic silencing, while Gipc2 is upregulated in estrogen-induced mammary tumors. Somatic mutations of GIPC2 occur in malignant melanoma, and colorectal and ovarian cancers. Germ-line mutations of the GIPC3 or MYO6 gene cause nonsyndromic hearing loss. As GIPC proteins are involved in trafficking, signaling and recycling of RTKs, GPCRs, integrins and other transmembrane proteins, dysregulation of GIPCs results in human pathologies, such as cancer and hereditary deafness.     

N-Skatyltryptamines—Dual 5-HT6R/D2R Ligands with Antipsychotic and Procognitive Potential

A series of N-skatyltryptamines was synthesized and their affinities for serotonin and dopamine receptors were determined. Compounds exhibited activity toward 5-HT_1A, 5-HT_2A, 5-HT₆, and D₂ receptors. Substitution patterns resulting in affinity/activity switches were identified and studied using homology modeling. Chosen hits were screened to determine their metabolism, permeability, hepatotoxicity, and CYP inhibition. Several D₂ receptor antagonists with additional 5-HT₆R antagonist and agonist properties were identified. The former combination resembled known antipsychotic agents, while the latter was particularly interesting due to the fact that it has not been studied before. Selective 5-HT₆R antagonists have been shown previously to produce procognitive and promnesic effects in several rodent models. Administration of 5-HT₆R agonists was more ambiguous—in naive animals, it did not alter memory or produce slight amnesic effects, while in rodent models of memory impairment, they ameliorated the condition just like antagonists. Using the identified hit compounds 15 and 18, we tried to sort out the difference between ligands exhibiting the D₂R antagonist function combined with 5-HT₆R agonism, and mixed D₂/5-HT₆R antagonists in murine models of psychosis.