Identification of a µ-δ opioid receptor heteromer-biased agonist with antinociceptive activity.

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to µ-δ opioid receptor (µOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the µOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting µOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in µOR-δOR cells but not µOR or δOR cells alone. Among them, CYM51010 was found to be a µOR-δOR-biased ligand, because its activity is blocked by the µOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a µOR-δOR-biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.

Small-molecule proteostasis regulators for protein conformational diseases.

Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting this mechanism as a promising therapeutic approach. We describe the results of a screen comprised of ∼900,000 small molecules that identified new classes of small-molecule proteostasis regulators that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. These beneficial effects to proteome stability are mediated by HSF-1, FOXO, Nrf-2 and the chaperone machinery through mechanisms that are distinct from current known small-molecule activators of the heat shock response. We suggest that modulation of the proteostasis network by proteostasis regulators may be a promising therapeutic approach for the treatment of a variety of protein conformational diseases.

Design, synthesis and SAR analysis of novel potent and selective small molecule antagonists of NPBWR1 (GPR7).

Novel small molecule antagonists of NPBWR1 (GPR7) are herein reported. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 5-chloro-4-(4-methoxyphenoxy)-2-(p-tolyl)pyridazin-3(2H)-one as a NPBWR1 hit antagonist with micromolar activity. Design, synthesis and structure-activity relationships study of the HTS-derived hit led to the identification of 5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one lead molecule with submicromolar antagonist activity at the target receptor and high selectivity against a panel of therapeutically relevant off-target proteins. This lead molecule may provide a pharmacological tool to clarify the molecular basis of the in vivo physiological function and therapeutic utility of NPBWR1 in diverse disease areas including inflammatory pain and eating disorders.

Label-free LC-MS based assay to characterize small molecule compound binding to cells.

Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.

Global consequences of activation loop phosphorylation on protein kinase A.

Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr(197) in the activation loop when expressed in Escherichia coli. Although mutation of Arg(194) to Ala prevents autophosphorylation, phosphorylation of Thr(197) can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr(197) decreased the K(m) by approximately 15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the alphaH-alphaI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu(208) and Arg(280).

High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives.

For nearly two decades mass spectrometry has been used as a label-free, direct-detection method for both functional and affinity-based screening of a wide range of therapeutically relevant target classes. Here, we present an overview of several established and emerging mass spectrometry platforms and summarize the unique strengths and performance characteristics of each as they apply to high-throughput screening. Multiple examples from the recent literature are highlighted in order to illustrate the power of each individual technique, with special emphasis given to cases where the use of mass spectrometry was found to be differentiating when compared with other detection formats. Indeed, as many of these examples will demonstrate, the inherent strengths of mass spectrometry-sensitivity, specificity, wide dynamic range, and amenability to complex matrices-can be leveraged to enhance the discriminating power and physiological relevance of assays included in screening cascades. It is our hope that this review will serve as a useful guide to readers of all backgrounds and experience levels on the applicability and benefits of mass spectrometry in the search for hits, leads, and, ultimately, drugs.

Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening.

The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC(50) values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (K(i) values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.

High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate.

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n approximately 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.

Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries.

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused 'click' chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine K(i) and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (K(i)=K(i)(')=1.5+/-0.2microM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, K(i)=0.41+/-0.03microM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, K(i)=1.4+/-0.10microM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.

De novo discovery of serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a member of the GCN5 N-acetyltransferase (GNAT) superfamily and catalyzes the penultimate step in the biosynthesis of melatonin; a large daily rhythm in AANAT activity drives the daily rhythm in circulating melatonin. We have used a structure-based computational approach to identify the first druglike and selective inhibitors of AANAT. Approximately 1.2 million compounds were virtually screened by 3D high-throughput docking into the active site of X-ray structures for AANAT, and in total 241 compounds were tested as inhibitors. One compound class, containing a rhodanine scaffold, exhibited low micromolar competitive inhibition against acetyl-CoA (AcCoA) and proved to be effective in blocking melatonin production in pineal cells. Compounds from this class are predicted to bind as bisubstrate inhibitors through interactions with the AcCoA and serotonin binding sites. Overall, this study demonstrates the feasibility of using virtual screening to identify small molecules that are selective inhibitors of AANAT.

Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates.

The Z mutant of alpha1-antitrypsin (Glu342Lys) causes a domain swap and the formation of intrahepatic polymers that aggregate as inclusions and predispose the homozygote to cirrhosis. We have identified an allosteric cavity that is distinct from the interface involved in polymerization for rational structure-based drug design to block polymer formation. Virtual ligand screening was performed on 1.2 million small molecules and 6 compounds were identified that reduced polymer formation in vitro. Modeling the effects of ligand binding on the cavity and re-screening the library identified an additional 10 compounds that completely blocked polymerization. The best antagonists were effective at ratios of compound to Z alpha1-antitrypsin of 2.5:1 and reduced the intracellular accumulation of Z alpha1-antitrypsin by 70% in a cell model of disease. Identifying small molecules provides a novel therapy for the treatment of liver disease associated with the Z allele of alpha1-antitrypsin.

An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C).

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

Structure of E. coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites.

We report the crystal structure of E. coli ketopantoate hydroxymethyltransferase (KPHMT) at 1.9 A resolution, in complex with its product, ketopantoate. KPHMT catalyzes the first step in the biosynthesis of pantothenate (vitamin B(5)), the precursor of coenzyme A and the acyl carrier protein cofactor. The structure of the decameric enzyme was solved by multiwavelength anomalous dispersion to locate 160 selenomethionine sites and phase 560 kDa of protein, making it the largest structure solved by this approach. KPHMT adopts the (betaalpha)(8) barrel fold and is a member of the phosphoenolpyruvate/pyruvate superfamily. The active site contains a ketopantoate bidentately coordinated to Mg(2+). Similar binding is likely for the substrate, alpha-ketoisovalerate, orienting the C3 for deprotonation.

Development of photo-crosslinking reagents for protein kinase-substrate interactions.

The identification of relevant protein kinase-protein substrate partners remains a serious challenge on a genome-wide scale. The design and synthesis of a photo-activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the gamma-phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non-competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein-protein crosslinking that is dependent on UV irradiation. That significant kinase-substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners.

Implementing fluorescence anisotropy screening and crystallographic analysis to define PKA isoform-selective activation by cAMP analogs.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIß, RIIα, and RIIß. Although these isoforms all respond to cAMP, they are functionally nonredundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIß holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIß bound to HE33. The RIIß structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A, and the side chains are displaced to accommodate the HE33 dipropyl groups. Comparison between cAMP-bound structures reveals that RIIß, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent noncyclic nucleotide activators to specifically target PKA iso-holoenyzmes.

Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease.

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure-activity relationships within S(1'), S(1), and S(2) enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.

Small molecule activators of TRPML3.

We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel, which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na(+)]. This synergism revealed the existence of distinct and cooperative activation mechanisms and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed the absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are nonresponsive to the activators identified in this screen.

NH-1,2,3-Triazole-based Inhibitors of the VIM-2 Metallo-ß-Lactamase: Synthesis and Structure-Activity Studies.

Metallo-ß-lactamases (MBL) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 ß-lactamase is the most commonly encountered MBL in clinical isolates worldwide. Described here are potent and selective small molecule inhibitors of VIM-2 containing the arylsulfonyl-NH-1,2,3-triazole chemotype that potentiate the efficacy of the ß-lactam, imipenem, in E. coli.

Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor.

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.