Synthesis, 3D‐QSAR,and Structural Modeling of Benzolactam Derivatives with Binding Affinity for the D2 and D3 Receptors

A series of 37 benzolactam derivatives were synthesized, and their respective affinities for the dopamine D₂ and D₃ receptors evaluated. The relationships between structures and binding affinities were investigated using both ligand‐based (3D‐QSAR) and receptor‐based methods. The results revealed the importance of diverse structural features in explaining the differences in the observed affinities, such as the location of the benzolactam carbonyl oxygen, or the overall length of the compounds. The optimal values for such ligand properties are slightly different for the D₂ and D₃ receptors, even though the binding sites present a very high degree of homology. We explain these differences by the presence of a hydrogen bond network in the D₂ receptor which is absent in the D₃ receptor and limits the dimensions of the binding pocket, causing residues in helix 7 to become less accessible. The implications of these results for the design of more potent and selective benzolactam derivatives are presented and discussed.     

MS Binding Assays for D1 and D5 Dopamine Receptors

MS Binding Assays are a label‐free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D₁ and D₅ dopamine receptors. A highly sensitive, rapid and robust LC–ESI‐MS/MS quantification method for the selective D₁ dopamine receptor antagonist SCH23390 ((5R)‐8‐chloro‐3‐methyl‐5‐phenyl‐1,2,4,5‐tetrahydro‐3‐benzazepin‐7‐ol) was established and validated, using its 8‐bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D₁ and D₅ receptors. Following the concept of MS Binding Assays, saturation experiments for D₁ and D₅ receptors were performed, as well as competition experiments for D₁ receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D₁ and D₅ receptors are well‐suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.     

Selective Pharmacophore Models of Dopamine D1 and D2 Full Agonists Based on Extended Pharmacophore Features

This study is focused on the identification of structural features that determine the selectivity of dopamine receptor agonists toward D₁ and D₂ receptors. Selective pharmacophore models were developed for both receptors. The models were built by using projected pharmacophoric features that represent the main agonist interaction sites in the receptor (the Ser residues in TM5 and the Asp in TM3), a directional aromatic feature in the ligand, a feature with large positional tolerance representing the positively charged nitrogen in the ligand, and sets of excluded volumes reflecting the shapes of the receptors. The sets of D₁ and D₂ ligands used for modeling were carefully selected from published sources and consist of structurally diverse, conformationally rigid full agonists as active ligands together with structurally related inactives. The robustness of the models in discriminating actives from inactives was tested against four ensembles of conformations generated by using different established methods and different force fields. The reasons for the selectivity can be attributed to both geometrical differences in the arrangement of the features, e.g., different tilt angels of the π system, as well as shape differences covered by the different sets of excluded volumes. This work provides useful information for the design of new D₁ and D₂ agonists and also for comparative homology modeling of D₁ and D₂ receptors. The approach is general and could therefore be applied to other ligand–protein interactions for which no experimental protein structure is available.     

Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

Herein we report the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective agonists of the dopamine‐3 (D₃) receptor. A number of these new compounds bind to the D₃ receptor with sub‐nanomolar affinity and show excellent selectivity (>10 000) for the D₃ receptor over the D₁ and D₂ receptors. For example, compound 23 (N‐(cis‐3‐(2‐(((S)‐2‐amino‐4,5,6,7‐tetrahydrobenzo[d]thiazol‐6‐yl)(propyl)amino)ethyl)‐3‐hydroxycyclobutyl)‐3‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)benzamide) binds to the D₃ receptor with a K_i value of 0.53 nM and shows a selectivity of >20 000 over the D₂ and D₁ receptors in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes. Moreover, in vitro functional assays showed it to be a full agonist for the human D₃ receptor.     

Recognition Properties and Competitive Assays of a Dual Dopamine/Serotonin Selective Molecularly Imprinted Polymer

A molecularly imprinted polymer (MIP) with dual dopamine/serotonin-like binding sites (DS-MIP) was synthesized for use as a receptor model of study the drug-interaction of biological mixed receptors at a molecular level. The polymer material was produced using methacrylic acid (MAA) and acrylamide (ACM) as functional monomers, N,N′-methylene bisacrylamide (MBAA) as cross-linker, methanol/water mixture (4:1, v/v) as porogen and a mixture of dopamine (D) and serotonin (S) as templates. The prepared DS-MIP exhibited the greatest rebinding of the template(s) in aqueous methanol solution with decreased recognition in acetonitrile, water and methanol solvent. The binding affinity and binding capacity of DS-MIP with S were found to be higher than those of DS-MIP with D. The selectivity profiles of DS-MIP suggest that the D binding site of DS-MIP has sufficient integrity to discriminate between species of non-optimal functional group orientation, whilst the S binding site of DS-MIP is less selective toward species having structural features and functional group orientations different from S. The ligand binding activities of a series of ergot derivatives (ergocryptine, ergocornine, ergocristine, ergonovine, agroclavine, pergolide and terguride) have been studied with the DS-MIP using a competitive ligand binding assay protocol. The binding affinities of DS-MIP were demonstrated in the micro- or submicro-molar range for a series of ergot derivatives, whereas the binding affinities were considerably greater to natural receptors derived from the rat hypothalamus. The DS-MIP afforded the same pattern of differentiation as the natural receptors, i.e. affinity for the clavines > lysergic acid derivatives > ergopeptines. The results suggest that the discrimination for the ergot derivatives by the dopamine and serotonin sites of DS-MIP is due to the structural features and functional orientation of the phenylethylamine and indolylethylamine entities at the binding sites, and the fidelity of the dopamine and serotonin imprinted cavities.     

Indoloquinolizidine–Peptide Hybrids as Multiple Agonists for D1 and D2 Dopamine Receptors

Multiple‐specificity ligands are considered promising pharmacological tools that may show higher efficacy in the treatment of diseases for which the modulation of a single target is therapeutically inadequate. We prepared a set of novel ligands for D₁ and D₂ dopamine receptors by combining two indolo[2,3‐a]quinolizidine scaffolds with various tripeptide moieties. The binding and functional properties of these molecules were determined by radioligand binding studies in brain striatum membranes and by intracellular cAMP production assays in cells expressing different dopamine receptor subtypes. Some indoloquinolizidine–peptide hybrids, mainly with the trans configuration, showed dual agonist activity at both D₁ and D₂ dopamine receptors and may therefore be useful for testing the therapeutic potential of multivalent drugs on these targets.     

Chiral Aryloxyalkylamines: Selective 5‐HT1B/1D Activation and Analgesic Activity

A series of chiral 2,3‐dichlorophenoxy and 1‐naphthyloxy alkylamines were synthesized, and their binding affinities towards 5‐HT_1D and h5‐HT_1B receptors were evaluated. In the naphthyloxy series, the (R)‐prolinol derivative was the most selective 5‐HT_1D ligand, while (S)‐N‐methyl‐2‐(1‐naphthyloxy)propan‐1‐amine showed the highest selectivity for h5‐HT_1B. Both compounds performed as 5‐HT_1D agonists in the isolated guinea pig assay and showed higher analgesic activity than both sumatriptan and the achiral analogue 20 b in the mouse hot‐plate test. Neither ligand displayed any affinity for nicotinic ACh receptors present in mouse brain membranes, thus indicating that their analgesic activity does not arise through interaction with these receptors.     

Efficient cell surface display of organophosphorous hydrolase using N-terminal domain of ice nucleation protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant Escherichia coli systems expressing organophosphorous hydrolase (OPH) have been used for detoxifying toxic organophosphate compounds. However, a whole cell biocatalyst system has an intrinsic problem due to substrate diffusion limitation by its cell membrane. As a strategy for reducing this diffusion barrier limitation to enhance whole cell biocatalytic activity, we engineered E. coli cells to target OPH on cell surface using ice nucleation protein (InaK) as a surface targeting motif, especially N-terminal domain of InaK (InaK-N). The whole cell OPH activities of the cells expressing InaK/OPH fusion constructs were higher (∼2.5-fold for InaK-N and ∼5.7-fold for combined N-and C-terminal domain of InaK (InaK-NC)) than that of the cells expressing cytosolic OPH. Interestingly, the membrane targeting efficiency of the cells expressing InaK-N/OPH fusion proteins was ∼2.2-fold higher compared to the cells expressing InaK-NC/OPH even though both whole cell and total cell lysate OPH activities were lower. Therefore, we found that the small size N-terminal domain of InaK is more efficient for targeting OPH on the cell surface, and the surface display of OPH using N-terminal InaK domain can reduce the mass-transfer problem in whole cell bioconversion system. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007     

Structure‐Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics

Structure‐based virtual screening using a D₂ receptor homology model was performed to identify dopamine D₂ receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D₂ ligands were identified (47.6 % success rate, among them D₂ receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5‐HT_2A receptors. The affinity (K_i values) of the compounds ranged from 58 nm to about 24 μm . Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D₂ receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D₂ pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20‐fold binding selectivity for the D₂ receptor over the D₃ receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction.     

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K_D=42 and 84 μM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K_D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.     

Molecular Recognition of Two 2,4‐syn‐Functionalized (S)‐Glutamate Analogues by the Kainate Receptor GluK3 Ligand Binding Domain

The kainate receptors are the least studied subfamily of ionotropic glutamate receptors. These receptors are thought to have a neuromodulatory role and have been associated with a variety of disorders in the central nervous system. This makes kainate receptors interesting potential drug targets. Today, structures of the ligand binding domain (LBD) of the kainate receptor GluK3 are only known in complex with the endogenous agonist glutamate, the natural product kainate, and two synthetic agonists. Herein we report structures of GluK3 LBD in complex with two 2,4‐syn‐functionalized (S)‐glutamate analogues to investigate their structural potential as chemical scaffolds. Similar binding affinities at GluK3 were determined for the 2‐(methylcarbamoyl)ethyl analogue (K_i=4.0 μM ) and the 2‐(methoxycarbonyl)ethyl analogue (K_i=1.7 μM ), in agreement with the similar positioning of the compounds within the binding pocket. As the binding affinity is similar to that of glutamate, this type of Cγ substituent could be used as a scaffold for introduction of even larger substituents reaching into unexplored binding site regions to achieve subtype selectivity.     

Bivalent Argininamide‐Type Neuropeptide Y Y1 Antagonists Do Not Support the Hypothesis of Receptor Dimerisation

Bivalent ligands are potential tools to investigate the dimerisation of G‐protein‐coupled receptors. Based on the (R)‐argininamide BIBP 3226, a potent and selective neuropeptide Y Y₁ receptor (Y₁R) antagonist, we prepared a series of bivalent Y₁R ligands with a wide range of linker lengths (8–36 atoms). Exploiting the high eudismic ratio (>1000) of the parent compound, we synthesised sets of R,R‐, R,S‐ and S,S‐configured bivalent ligands to gain insight into the “bridging” of two Y₁Rs by simultaneous interaction with both binding sites of a putative receptor dimer. Except for the S,S isomers, the bivalent ligands are high‐affinity Y₁R antagonists, as determined by Ca²⁺ assays on HEL cells and radioligand competition assays on human Y₁R‐expressing SK‐N‐MC and MCF‐7 cells. Whereas the R,R enantiomers are most potent, no marked differences were observed relative to the corresponding meso forms. The difference between R,R and R,S diastereomers was most pronounced (about sixfold) in the case of the Y₁R antagonist containing a spacer of 20 atoms in length. Among the R,R enantiomers, linker length and structural diversity had little effect on Y₁R affinity. Although the bivalent ligands preferentially bind to the Y₁R, the selectivity toward human Y₂, Y₄, and Y₅ receptors was markedly lower than that of the monovalent argininamides. The results of this study neither support the presence of Y₁R dimers nor the simultaneous occupation of both binding pockets by the twin compounds. However, as the interaction with Y₁R dimers cannot be unequivocally ruled out, the preparation of a bivalent radioligand is suggested to determine the ligand–receptor stoichiometry. Aiming at such radiolabelled pharmacological tools, prototype twin compounds were synthesised, containing an N‐propionylated amino‐functionalised branched linker (K_i≥18 nM ), a tritiated form of which can be easily prepared.     

A Molecular and Chemical Perspective in Defining Melatonin Receptor Subtype Selectivity

Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT₁ and MT₂ transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT₁ and MT₂ receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT₁ and MT₂ that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT₁ and MT₂ receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands.     

Development of a novel strategy for engineering high-affinity proteins by yeast display

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.     

Dopamine D4 Receptor Counteracts Morphine-Induced Changes in μ Opioid Receptor Signaling in the Striosomes of the Rat Caudate Putamen

The mu opioid receptor (MOR) is critical in mediating morphine analgesia. However, prolonged exposure to morphine induces adaptive changes in this receptor leading to the development of tolerance and addiction. In the present work we have studied whether the continuous administration of morphine induces changes in MOR protein levels, its pharmacological profile, and MOR-mediated G-protein activation in the striosomal compartment of the rat CPu, by using immunohistochemistry and receptor and DAMGO-stimulated [³⁵S]GTPγS autoradiography. MOR immunoreactivity, agonist binding density and its coupling to G proteins are up-regulated in the striosomes by continuous morphine treatment in the absence of changes in enkephalin and dynorphin mRNA levels. In addition, co-treatment of morphine with the dopamine D₄ receptor (D₄R) agonist PD168,077 fully counteracts these adaptive changes in MOR, in spite of the fact that continuous PD168,077 treatment increases the [³H]DAMGO B_max values to the same degree as seen after continuous morphine treatment. Thus, in spite of the fact that both receptors can be coupled to G_i/0 protein, the present results give support for the existence of antagonistic functional D₄R-MOR receptor-receptor interactions in the adaptive changes occurring in MOR of striosomes on continuous administration of morphine.     

Shuttling of Peptide-Drug Conjugates by G Protein-Coupled Receptors Is Significantly Improved by Pulsed Application

G protein-coupled receptors (GPCRs) can be used to shuttle peptide-drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y₁ receptor (Y₁R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150–250 % to 300–400 %). Accordingly, in this well-suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general.     

The interaction of 1,25-dihydroxyvitamin D3 with its intestinal mucosa receptor: Kinetic parameters and structural requirements

Vitamin D₃ and its metabolites comprise an endocrine system which plays a critical role in calcium homeostasis. The active form of vitamin D₃ is 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Chromatin localization of 1,25(OH)₂D₃ and sucrose density gradient centrifutation have demonstrated the presence of an intestinal mucosa cytosol receptor which specifically binds 1,25(OH)₂D₃. The kinetic parameters of 1,25(OH)₂D₃ binding to its receptor have been determined by hydroxylapatite and reconstituted chromatin cytosol assays. Utilization of these assays has also permitted a determination of the precise structural requirements of the vitamin D ligand for the intestinal receptor. Furthermore, it has been possible to propose two receptor-ligand models which are capable of accommodating the conformationaly modile A ring of the vitamin D seco-steroids.     

Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling

G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively ¹⁹F-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.     

Functional Cell Adhesion Receptors (Integrins) in Polymeric Architectures

Integrins, as transmembrane heterodimeric receptors, have important functions in cell adhesion, migration, proliferation, survival apoptosis and signal transduction, in many physio‐ as well as pathophysiological settings. Characterisation of integrins and their ligand/antagonist binding is notoriously difficult, due to high integrin redundancy and ubiquity. Bypassing the intrinsic difficulties of cell‐based integrin expression, purification and reconstitution, we present for the first time the synthesis of a heterodimeric integrin receptor and its assembly into a block‐copolymeric membrane mimic. We present comprehensive data to demonstrate the synthesis of functionally active integrin α_vβ₃, generated by in vitro membrane‐assisted protein synthesis (iMAPS). This work represents the first step towards a robust and adaptable polymer‐based platform for characterisation of integrin–ligand interactions.