Nitro-benzylideneoxymorphone, a bifunctional mu and delta opioid receptor ligand with high mu opioid receptor efficacy.

Introduction: There is a major societal need for analgesics with less tolerance, dependence, and abuse liability. Preclinical rodent studies suggest that bifunctional ligands with both mu (MOPr) and delta (DOPr) opioid peptide receptor activity may produce analgesia with reduced tolerance and other side effects. This study explores the structure-activity relationships (SAR) of our previously reported MOPr/DOPr lead, benzylideneoxymorphone (BOM) with C7-methylene-substituted analogs. Methods: Analogs were synthesized and tested in vitro for opioid receptor binding and efficacy. One compound, nitro-BOM (NBOM, 12) was evaluated for antinociceptive effects in the warm water tail withdrawal assay in C57BL/6 mice. Acute and chronic antinociception was determined, as was toxicologic effects on chronic administration. Molecular modeling experiments were performed using the Site Identification by Ligand Competitive Saturation (SILCS) method. Results: NBOM was found to be a potent MOPr agonist/DOPr partial agonist that produces high-efficacy antinociception. Antinociceptive tolerance was observed, as was weight loss; this toxicity was only observed with NBOM and not with BOM. Modeling supports the hypothesis that the increased MOPr efficacy of NBOM is due to the substituted benzylidene ring occupying a nonpolar region within the MOPr agonist state. Discussion: Though antinociceptive tolerance and non-specific toxicity was observed on repeated administration, NBOM provides an important new tool for understanding MOPr/DOPr pharmacology.

Delta Opioid Receptor-Mediated Antidepressant-Like Effects of Diprenorphine in Mice.

Major depressive disorder is a highly common disorder, with a lifetime prevalence in the United States of approximately 21%. Traditional antidepressant treatments are limited by a delayed onset of action and minimal efficacy in some patients. Ketamine is effective and fast-acting, but there are concerns over its abuse liability. Thus, there is a need for safe, fast-acting antidepressant drugs. The opioid buprenorphine shows promise but also has abuse liability due to its mu-agonist component. Preclinical evidence indicates that the delta-opioid system contributes to mood disorders, and delta-opioid agonists are effective in preclinical models of depression- and anxiety-like states. In this study, we test the hypothesis that the mu-opioid antagonist diprenorphine by virtue of its partial delta opioid agonist activity may offer a beneficial profile for an antidepressant medication without abuse liability. Diprenorphine was confirmed to bind with high affinity to all three opioid receptors, and functional experiments for G protein activation verified diprenorphine to be a partial agonist at delta- and kappa-opioid receptors and a mu-antagonist. Studies in C57BL/6 mice demonstrated that an acute dose of diprenorphine produced antidepressant-like effects in the tail suspension test and the novelty-induced hypophagia test that were inhibited in the presence of the delta-selective antagonist, naltrindole. Diprenorphine did not produce convulsions, a side effect of many delta agonists but rather inhibited convulsions caused by the full delta agonist SNC80; however, diprenorphine did potentiate pentylenetetrazole-induced convulsions. Diprenorphine, and compounds with a similar pharmacological profile, may provide efficient and safe rapidly acting antidepressants. SIGNIFICANCE STATEMENT: The management of major depressive disorder, particularly treatment-resistant depression, is a significant unmet medical need. Here we show that the opioid diprenorphine, a compound with mu-opioid receptor antagonist activity and delta- and kappa-opioid receptor partial agonist activities, has rapid onset antidepressant-like activity in animal models. Diprenorphine and compounds with a similar pharmacological profile to diprenorphine should be explored as novel antidepressant drugs.

Opioid Antagonists from the Orvinol Series as Potential Reversal Agents for Opioid Overdose.

The opioid crisis continues to claim many lives, with a particular issue being the ready availability and use (whether intentional or accidental) of fentanyl and fentanyl analogues. Fentanyl is both potent and longer-acting than naloxone, the standard of care for overdose reversal, making it especially deadly. Consequently, there is interest in opioid reversal agents that are better able to counter its effects. The orvinol series of ligands are known for their high-affinity binding to opioid receptors and often extended duration of action; generally, compounds on this scaffold show agonist activity at the kappa and the mu-opioid receptor. Diprenorphine is an unusual member of this series being an antagonist at mu and only a partial agonist at kappa-opioid receptors. In this study, an orvinol antagonist, 14, was designed and synthesized that shows no agonist activity in vitro and is at least as good as naloxone at reversing the effects of mu-opioid receptor agonists in vivo.

Finding the Perfect Fit: Conformational Biosensors to Determine the Efficacy of GPCR Ligands.

G protein-coupled receptors (GPCRs) are highly druggable targets that adopt numerous conformations. A ligand's ability to stabilize specific conformation(s) of its cognate receptor determines its efficacy or ability to produce a biological response. Identifying ligands that produce different receptor conformations and potentially discrete pharmacological effects (e.g., biased agonists, partial agonists, antagonists, allosteric modulators) is a major goal in drug discovery and necessary to develop drugs with better effectiveness and fewer side effects. Fortunately, direct measurements of ligand efficacy, via receptor conformational changes are possible with the recent development of conformational biosensors. In this review, we discuss classical efficacy models, including the two-state model, the ternary-complex model, and multistate models. We describe how nanobody-, transducer-, and receptor-based conformational biosensors detect and/or stabilize specific GPCR conformations to identify ligands with different levels of efficacy. In particular, conformational biosensors provide the potential to identify and/or characterize therapeutically desirable but often difficult to measure conformations of receptors faster and better than current methods. For drug discovery/development, several recent proof-of-principle studies have optimized conformational biosensors for high-throughput screening (HTS) platforms. However, their widespread use is limited by the fact that few sensors are reliably capable of detecting low-frequency conformations and technically demanding assay conditions. Nonetheless, conformational biosensors do help identify desirable ligands such as allosteric modulators, biased ligands, or partial agonists in a single assay, representing a distinct advantage over classical methods.

OREX-1038: a potential new treatment for pain with low abuse liability and limited adverse effects.

Drugs targeting mu opioid receptors are the mainstay of clinical practice for treating moderate-to-severe pain. While they can offer excellent analgesia, their use can be limited by adverse effects, including constipation, respiratory depression, tolerance, and abuse liability. Multifunctional ligands acting at mu opioid and nociceptin/orphanin FQ peptide receptors might provide antinociception with substantially improved adverse-effect profiles. This study explored one of these ligands, OREX-1038 (BU10038), in several assays in rodents and nonhuman primates. Binding and functional studies confirmed OREX-1038 to be a low-efficacy agonist at mu opioid and nociceptin/orphanin FQ peptide receptors and an antagonist at delta and kappa opioid receptors with selectivity for opioid receptors over other proteins. OREX-1038 had long-acting antinociceptive effects in postsurgical and complete Freund's adjuvant (CFA)-induced thermal hyperalgesia assays in rats and a warm water tail-withdrawal assay in monkeys. OREX-1038 was active for at least 24 h in each antinociception assay, and its effects in monkeys did not diminish over 22 days of daily administration. This activity was coupled with limited effects on physiological signs (arterial pressure, heart rate, and body temperature) and no evidence of withdrawal after administration of naltrexone or discontinuation of treatment in monkeys receiving OREX-1038 daily. Over a range of doses, OREX-1038 was only transiently self-administered, which diminished rapidly to nonsignificant levels; overall, both OREX-1038 and buprenorphine maintained less responding than remifentanil. These results support the concept of dual mu and nociceptin/orphanin FQ peptide receptor partial agonists having improved pharmacological profiles compared with opioids currently used to treat pain.

In silico analysis of SARS-CoV-2 proteins as targets for clinically available drugs.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires treatments with rapid clinical translatability. Here we develop a multi-target and multi-ligand virtual screening method to identify FDA-approved drugs with potential activity against SARS-CoV-2 at traditional and understudied viral targets. 1,268 FDA-approved small molecule drugs were docked to 47 putative binding sites across 23 SARS-CoV-2 proteins. We compared drugs between binding sites and filtered out compounds that had no reported activity in an in vitro screen against SARS-CoV-2 infection of human liver (Huh-7) cells. This identified 17 "high-confidence", and 97 "medium-confidence" drug-site pairs. The "high-confidence" group was subjected to molecular dynamics simulations to yield six compounds with stable binding poses at their optimal target proteins. Three drugs-amprenavir, levomefolic acid, and calcipotriol-were predicted to bind to 3 different sites on the spike protein, domperidone to the Mac1 domain of the non-structural protein (Nsp) 3, avanafil to Nsp15, and nintedanib to the nucleocapsid protein involved in packaging the viral RNA. Our "two-way" virtual docking screen also provides a framework to prioritize drugs for testing in future emergencies requiring rapidly available clinical drugs and/or treating diseases where a moderate number of targets are known.

Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling.

ABSTRACT: The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.

Allosteric Modulator Leads Hiding in Plain Site: Developing Peptide and Peptidomimetics as GPCR Allosteric Modulators.

Allosteric modulators (AMs) of G-protein coupled receptors (GPCRs) are desirable drug targets because they can produce fewer on-target side effects, improved selectivity, and better biological specificity (e.g., biased signaling or probe dependence) than orthosteric drugs. An underappreciated source for identifying AM leads are peptides and proteins-many of which were evolutionarily selected as AMs-derived from endogenous protein-protein interactions (e.g., transducer/accessory proteins), intramolecular receptor contacts (e.g., pepducins or extracellular domains), endogenous peptides, and exogenous libraries (e.g., nanobodies or conotoxins). Peptides offer distinct advantages over small molecules, including high affinity, good tolerability, and good bioactivity, and specific disadvantages, including relatively poor metabolic stability and bioavailability. Peptidomimetics are molecules that combine the advantages of both peptides and small molecules by mimicking the peptide's chemical features responsible for bioactivity while improving its druggability. This review 1) discusses sources and strategies to identify peptide/peptidomimetic AMs, 2) overviews strategies to convert a peptide lead into more drug-like "peptidomimetic," and 3) critically analyzes the advantages, disadvantages, and future directions of peptidomimetic AMs. While small molecules will and should play a vital role in AM drug discovery, peptidomimetics can complement and even exceed the advantages of small molecules, depending on the target, site, lead, and associated factors.

The Buprenorphine Analogue BU10119 Attenuates Drug-Primed and Stress-Induced Cocaine Reinstatement in Mice.

There are no Food and Drug Administration-approved medications for cocaine use disorder, including relapse. The µ-opioid receptor (MOPr) partial agonist buprenorphine alone or in combination with naltrexone has been shown to reduce cocaine-positive urine tests and cocaine seeking in rodents. However, there are concerns over the abuse liability of buprenorphine. Buprenorphine's partial agonist and antagonist activity at the nociception receptor (NOPr) and κ-opioid receptor (KOPr), respectively, may contribute to its ability to inhibit cocaine seeking. Thus, we hypothesized that a buprenorphine derivative that exhibits antagonist activity at MOPr and KOPr with enhanced agonist activity at the NOPr could provide a more effective treatment. Here we compare the pharmacology of buprenorphine and two analogs, BU10119 and BU12004, in assays for antinociception and for cocaine- and stress-primed reinstatement in the conditioned place preference paradigm. In vitro and in vivo assays showed that BU10119 acts as an antagonist at MOPr, KOPr, and δ-opioid receptor (DOPr) and a partial agonist at NOPr, whereas BU12004 showed MOPr partial agonist activity and DOPr, KOPr, and NOPr antagonism. BU10119 and buprenorphine but not BU12004 lessened cocaine-primed reinstatement. In contrast, BU10119, BU12004, and buprenorphine blocked stress-primed reinstatement. The selective NOPr agonist SCH221510 but not naloxone decreased cocaine-primed reinstatement. Together, these findings are consistent with the concept that NOPr agonism contributes to the ability of BU10119 and buprenorphine to attenuate reinstatement of cocaine-conditioned place preference in mice. The findings support the development of buprenorphine analogs lacking MOPr agonism with increased NOPr agonism for relapse prevention to cocaine addiction. SIGNIFICANCE STATEMENT: There are no Food and Drug Administration-approved medications for cocaine use disorder. Buprenorphine has shown promise as a treatment for cocaine relapse prevention; however, there are concerns over the abuse liability of buprenorphine. Here we show a buprenorphine analogue, BU10119, which lacks µ-opioid receptor agonism and inhibits cocaine-primed and stress-primed reinstatement in a conditioned place-preference paradigm. The results suggest the development of BU10119 for the management of relapse to cocaine seeking.

Comprehensive molecular pharmacology screening reveals potential new receptor interactions for clinically relevant opioids.

Most clinically used opioids are thought to induce analgesia through activation of the mu opioid receptor (MOR). However, disparities have been observed between the efficacy of opioids in activating the MOR in vitro and in inducing analgesia in vivo. In addition, some clinically used opioids do not produce cross-tolerance with each other, and desensitization produced in vitro does not match tolerance produced in vivo. These disparities suggest that some opioids could be acting through other targets in vivo, but this has not been comprehensively tested. We thus screened 9 clinically relevant opioids (buprenorphine, hydrocodone, hydromorphone, morphine, O-desmethyl-tramadol, oxycodone, oxymorphone, tapentadol, tramadol) against 9 pain-related receptor targets (MOR, delta opioid receptor [DOR], kappa opioid receptor [KOR], nociceptin receptor [NOP], cannabinoid receptor type 1 [CB1], sigma-1 receptor [σ1R], and the monoamine transporters [NET/SERT/DAT]) expressed in cells using radioligand binding and functional activity assays. We found several novel interactions, including monoamine transporter activation by buprenorphine and σ1R binding by hydrocodone and tapentadol. Tail flick anti-nociception experiments with CD-1 mice demonstrated that the monoamine transporter inhibitor duloxetine selectively promoted buprenorphine anti-nociception while producing no effects by itself or in combination with the most MOR-selective drug oxymorphone, providing evidence that these novel interactions could be relevant in vivo. Our findings provide a comprehensive picture of the receptor interaction profiles of clinically relevant opioids, which has not previously been performed. Our findings also suggest novel receptor interactions for future investigation that could explain some of the disparities observed between opioid performance in vitro and in vivo.

Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the Mu-Delta Opioid Receptor Heterodimer that Reduces Morphine Withdrawal in Mice.

A major limitation in the study of the mu-delta opioid receptor heterodimer (MDOR) is that few selective pharmacological tools exist and no heteromer-selective antagonists. We thus designed a series of variable-length (15-41 atoms) bivalent linked peptides with selective but moderate/low-affinity pharmacophores for the mu and delta opioid receptors. We observed a U-shaped MDOR potency/affinity profile in vitro, with the 24-atom spacer length (D24M) producing the highest MDOR potency/affinity (<1 nM) and selectivity (≥89-fold). We further evaluated D24M in mice and observed that D24M dose-dependently antagonized tail flick antinociception produced by the MDOR agonists CYM51010 and Deltorphin-II, without antagonizing the monomer agonists DAMGO and DSLET. We also observed that D24M sharply reduced withdrawal behavior in models of acute and chronic morphine dependence. These findings suggest that D24M is a first-in-class high-potency MDOR-selective antagonist both in vitro and in vivo.

Novel Molecular Strategies and Targets for Opioid Drug Discovery for the Treatment of Chronic Pain.

Opioid drugs like morphine and fentanyl are the gold standard for treating moderate to severe acute and chronic pain. However, opioid drug use can be limited by serious side effects, including constipation, tolerance, respiratory suppression, and addiction. For more than 100 years, we have tried to develop opioids that decrease or eliminate these liabilities, with little success. Recent advances in understanding opioid receptor signal transduction have suggested new possibilities to activate the opioid receptors to cause analgesia, while reducing or eliminating unwanted side effects. These new approaches include designing functionally selective ligands, which activate desired signaling cascades while avoiding signaling cascades that are thought to provoke side effects. It may also be possible to directly modulate downstream signaling through the use of selective activators and inhibitors. Separate from downstream signal transduction, it has also been found that when the opioid system is stimulated, various negative feedback systems are upregulated to compensate, which can drive side effects. This has led to the development of multi-functional molecules that simultaneously activate the opioid receptor while blocking various negative feedback receptor systems including cholecystokinin and neurokinin-1. Other novel approaches include targeting heterodimers of the opioid and other receptor systems which may drive side effects, and making endogenous opioid peptides druggable, which may also reduce opioid mediated side effects. Taken together, these advances in our molecular understanding provide a path forward to break the barrier in producing an opioid with reduced or eliminated side effects, especially addiction, which may provide relief for millions of patients.

Mapping Atmospheric Moisture Climatologies across the Conterminous United States.

Spatial climate datasets of 1981-2010 long-term mean monthly average dew point and minimum and maximum vapor pressure deficit were developed for the conterminous United States at 30-arcsec (~800m) resolution. Interpolation of long-term averages (twelve monthly values per variable) was performed using PRISM (Parameter-elevation Relationships on Independent Slopes Model). Surface stations available for analysis numbered only 4,000 for dew point and 3,500 for vapor pressure deficit, compared to 16,000 for previously-developed grids of 1981-2010 long-term mean monthly minimum and maximum temperature. Therefore, a form of Climatologically-Aided Interpolation (CAI) was used, in which the 1981-2010 temperature grids were used as predictor grids. For each grid cell, PRISM calculated a local regression function between the interpolated climate variable and the predictor grid. Nearby stations entering the regression were assigned weights based on the physiographic similarity of the station to the grid cell that included the effects of distance, elevation, coastal proximity, vertical atmospheric layer, and topographic position. Interpolation uncertainties were estimated using cross-validation exercises. Given that CAI interpolation was used, a new method was developed to allow uncertainties in predictor grids to be accounted for in estimating the total interpolation error. Local land use/land cover properties had noticeable effects on the spatial patterns of atmospheric moisture content and deficit. An example of this was relatively high dew points and low vapor pressure deficits at stations located in or near irrigated fields. The new grids, in combination with existing temperature grids, enable the user to derive a full suite of atmospheric moisture variables, such as minimum and maximum relative humidity, vapor pressure, and dew point depression, with accompanying assumptions. All of these grids are available online at http://prism.oregonstate.edu, and include 800-m and 4-km resolution data, images, metadata, pedigree information, and station inventory files.

Discovery of Novel Multifunctional Ligands with µ/δ Opioid Agonist/Neurokinin-1 (NK1) Antagonist Activities for the Treatment of Pain.

Multifunctional ligands with agonist bioactivities at µ/δ opioid receptors (MOR/DOR) and antagonist bioactivity at the neurokinin-1 receptor (NK1R) have been designed and synthesized. These peptide-based ligands are anticipated to produce better biological profiles (e.g., higher analgesic effect with significantly less adverse side effects) compared to those of existing drugs and to deliver better synergistic effects than coadministration of a mixture of multiple drugs. A systematic structure-activity relationship (SAR) study has been conducted to find multifunctional ligands with desired activities at three receptors. It has been found that introduction of Dmt (2,6-dimethyl-tyrosine) at the first position and NMePhe at the fourth position (ligand 3: H-Dmt-d-Ala-Gly-NMePhe-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) displays binding as well as functional selectivity for MOR over DOR while maintaining efficacy, potency, and antagonist activity at the NK1R. Dmt at the first position with Phe(4-F) at the fourth position (ligand 5: H-Dmt-d-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) exhibits balanced binding affinities at MOR and DOR though it has higher agonist activity at DOR over MOR. This study has led to the discovery of several novel ligands including 3 and 5 with excellent in vitro biological activity profiles. Metabolic stability studies in rat plasma with ligands 3, 5, and 7 (H-Tyr-d-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) showed that their stability depends on modifications at the first and fourth positions (3: T1/2 > 24 h; 5: T1/2 ≈ 6 h; 7: T1/2 > 2 h). Preliminary in vivo studies with these two ligands have shown promising antinociceptive activity.

Rapid, Antibody-Free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation.

Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this chapter, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of posttranslational protein modifications such as glycosylation.

Combination of photocatalysis and HC/SCR for improved activity and durability of DeNOx catalysts.

A photocatalytic HC/SCR system has been developed and its high deNOx performance (54.0-98.6% NOx conversion) at low temperatures (150-250 °C) demonstrated by using a representative diesel fuel hydrocarbon (dodecane) as the reductant over a hybrid SCR system of a photocatalytic reactor (PCR) and a dual-bed HC/SCR reactor. The PCR generates highly active oxidants such as O3 and NO2 from O2 and NO in the feed stream, followed by the subsequent formation of highly efficient reductants such as oxygenated hydrocarbon (OHC), NH3, and organo-nitrogen compounds. These reductants are the key components for enhancing the low temperature deNOx performance of the dual-bed HC/SCR system containing Ag/Al2O3 and CuCoY in the front and rear bed of the reactor, respectively. The OHCs are particularly effective for both NOx reduction and NH3 formation over the Ag/Al2O3 catalyst, while NH3 and organo-nitrogen compounds are effective for NOx reduction over the CuCoY catalyst. The hybrid HC/SCR system assisted by photocatalysis has shown an overall deNOx performance comparable to that of the NH3/SCR, demonstrating its potential as a promising alternative to the current urea/SCR and LNT technologies. Superior durability of HC/SCR catalysts against coking by HCs has also been demonstrated by a PCR-assisted regeneration scheme for deactivating catalysts.

Rapid, antibody-free detection of recombinant proteins on blots using enzyme fragment complementation.

Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this article, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of post-translational protein modifications such as glycosylation.

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS.

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.

A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (approximately 4 kDa), optimized alpha fragment peptide (termed ProLink) derived from beta-galactosidase, and beta-arrestin is fused to an N-terminal deletion mutant of beta-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the beta-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active beta-galactosidase enzyme, and thus GPCR activation can be determined by quantifying beta-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Galphai-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified.

Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery.

Many cell-based assays interrogating cell pathway activation employ protocols that require microscopic imaging techniques. However, such assays are not in general widely adopted for primary screening. Protein complementation, particularly of enzymes, provides an alternative approach for cell pathway analysis, with a principal advantage that is amenable to high throughput screening using microtiter plate protocols. Notably, alpha complementation of the enzyme beta-galactosidase has been exploited as a technology in this regard, using substrates that generates luminescent signals. This review describes the various uses of this flexible technology to cell-based assay development.