ABSTRACT
Calcium signaling plays a major role in the function of cells. Measurement of intracellular calcium mobilization is a robust assay that can be performed in a high-throughput manner to study the effect of compounds on potential drug targets. Pharmaceutical companies frequently use calcium signaling assays to screen compound libraries on G-protein-coupled receptors (GPCRs). In this chapter we describe the application of FLIPR technology to the evaluation of GPCR-induced calcium mobilization. We also include the implications of GPCR hetero-oligomerization and the identification of heteromeric receptors as novel drug targets on high-throughput calcium screening.
Subject(s)
Benzylidene Compounds/pharmacology , Drug Evaluation, Preclinical/methods , Enkephalin, D-Penicillamine (2,5)-/pharmacology , High-Throughput Screening Assays/methods , Naltrexone/analogs & derivatives , Receptors, Opioid, delta/agonists , Calcium Signaling , Cell Culture Techniques , HEK293 Cells , Humans , Naltrexone/pharmacology , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/biosynthesis , Receptors, Opioid, delta/antagonists & inhibitors , Receptors, Opioid, delta/biosynthesis , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Spectrometry, Fluorescence , TransfectionABSTRACT
Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/µ-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.
Subject(s)
Receptors, G-Protein-Coupled/physiology , Signal Transduction/drug effects , Amino Acid Sequence , Benzamides/pharmacology , Calcium/metabolism , Calcium Signaling , Cloning, Molecular , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enkephalin, Leucine/pharmacology , Enzyme-Linked Immunosorbent Assay , GTP-Binding Protein alpha Subunits, Gi-Go/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/drug effects , GTP-Binding Protein alpha Subunits, Gs/metabolism , HEK293 Cells , Humans , Microscopy, Fluorescence , Molecular Sequence Data , Mutant Chimeric Proteins/chemistry , Mutant Chimeric Proteins/genetics , Piperazines/pharmacology , Polymerase Chain Reaction , Receptors, G-Protein-Coupled/genetics , Receptors, Opioid, delta/drug effects , Receptors, Opioid, delta/genetics , Recombinant Fusion ProteinsABSTRACT
The bioconjugation of organometallic complexes with peptides has proven to be a novel approach for drug discovery. We report the facile and chemoselective reaction of tyrosine-containing G-protein-coupled receptor (GPCR) peptides with [Cp*Rh(H(2)O)(3)](OTf)(2), in water, at room temperature, and at pH 5-6. We have focused on three important GPCR peptides; namely, [Tyr(1)]-leu-enkephalin, [Tyr(4)]-neurotensin(8-13), and [Tyr(3)]-octreotide, each of which has a different position for the tyrosine residue, together with competing functionalities. Importantly, all other functional groups present, i.e., amino, carboxyl, disulfide, phenyl, and indole, were not prominent sites of reactivity by the Cp*Rh tris aqua complex. Furthermore, the influence of the Cp*Rh moiety on the structure of [Tyr(3)]-octreotide was characterized by 2D NMR, resulting in the first representative structure of an organometallic-peptide complex. The biological consequences of these Cp*Rh-peptide complexes, with respect to GPCR binding and growth inhibition of MCF7 and HT29 cancer cells, will be presented for [(η(6)-Cp*Rh-Tyr(1))-leu-enkephalin](OTf)(2) and [(η(6)-Cp*Rh-Tyr(3))-octreotide](OTf)(2).
Subject(s)
Models, Molecular , Organometallic Compounds/chemistry , Peptides/chemistry , Receptors, G-Protein-Coupled/chemistry , Rhodium/chemistry , Tyrosine/chemistry , Binding, Competitive , Breast Neoplasms/drug therapy , Female , HT29 Cells , Humans , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Organometallic Compounds/pharmacology , Protein Binding/drug effects , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/metabolismABSTRACT
Opioid receptors, including the mu and delta opioid receptors (MOR and DOR) are important targets for the treatment of pain. Although there is mounting evidence that these receptors form heteromers, the functional role of the MOR/DOR heteromer remains unresolved. We have designed and synthesized bivalent ligands as tools to elucidate the functional role of the MOR/DOR heteromer. Our ligands (L2 and L4) are comprised of a compound with low affinity at the DOR tethered to a compound with high affinity at the MOR, with the goal of producing ligands with "tuned affinity" at MOR/DOR heteromers compared to DOR homomers. Here we show that both L2 and L4 demonstrate enhanced affinity at MOR/DOR heteromers compared to DOR homomers, thereby providing unique pharmacological tools to dissect the role of the MOR/DOR heteromer in pain.
ABSTRACT
We have observed reversible light-induced mechanical switching for individual organic molecules bound to a metal surface. Scanning tunneling microscopy (STM) was used to image the features of individual azobenzene molecules on Au(111) before and after reversibly cycling their mechanical structure between trans and cis states using light. Azobenzene molecules were engineered to increase their surface photomechanical activity by attaching varying numbers of tert-butyl (TB) ligands ("legs") to the azobenzene phenyl rings. STM images show that increasing the number of TB legs "lifts" the azobenzene molecules from the substrate, thereby increasing molecular photomechanical activity by decreasing molecule-surface coupling.
ABSTRACT
The synthesis of a new fluorescein carboxaldehyde asymmetrically substituted on the xanthene (top) ring is reported. This molecule is a key precursor for two of three monofunctionally derivatized fluorescein-based Zn(II) sensors presented in this work. Detailed preparative routes to, and photophysical characterization of, these sensors are described. The sensors are based on the previously reported ZP4 motif (Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1778-1787) and incorporate a di(2-picolyl)amine-containing aniline-derivatized ligand framework. By varying the nature of the substituent (X) para to the aniline nitrogen atom, which is responsible for PET quenching of the unbound ZP dye, we investigated the extent to which such electronic tuning might improve the fluorescent properties of asymmetrical ZP sensors. Although a comparison of probes with X = H, F, Cl, OMe reveals that the photophysical behavior of these dyes is not readily predictable, our methodology illustrates the ease with which aniline-based ligands may be linked to fluorescein dyes.
