Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs.

The fundamental importance of membrane protein (MP) targets in central biological and cellular events has driven a marked increase in the use of membrane mimetics for exploring these proteins as therapeutic targets. The main challenge associated with biophysical analysis of membrane protein is the need for detergent extraction from the bilayer environment, which in many cases causes the proteins to become insoluble, unstable or display altered structure or activity. Recent technological advances have tried to limit the exposure of purified membrane protein to detergents. One such method involves the amphipathic co-polymer of styrene and maleic acid (SMA), which can release lipids and integral membrane proteins into water soluble native particles (or vesicles) termed SMALPs (Styrene Maleic Acid Lipid Particles). In this study, assay conditions that leverage SMA for membrane protein stabilization were developed to perform kinetic analysis of antibody binding to integral membrane protein and complexes in SMALPs in both purified and complex mixture settings using multiple biosensor platforms. To develop a robust and flexible platform using SMALPs technology, we optimized various SPR assay formats to analyze SMALPs produced with cell membrane pellets as well as whole cell lysates from the cell lines overexpressing membrane protein of interest. Here we emphasize the extraction of model membrane proteins of diverse architecture and function from native environments to encapsulate with SMALPs. Given the importance of selected membrane targets in central biological events and therapeutic relevance, MP-specific or tag-specific antibodies were used as a proof-of-principal to validate the SMALPs platform for ligand binding studies to support drug discovery or tool generation processes. MP-SMALPs that retain specific binding capability in multiple assay formats and biosensors, such as waveguide interferometry and surface plasmon resonance, would be a versatile platform for a wide range of downstream applications.

Applications of parallel synthetic lead hopping and pharmacophore-based virtual screening in the discovery of efficient glycine receptor potentiators.

Glycine receptors (GlyRs) are pentameric glycine-gated chloride ion channels that are enriched in the brainstem and spinal cord where they have been demonstrated to play a role in central nervous system (CNS) inhibition. Herein we describe two novel classes of glycine receptor potentiators that have been developed using similarity- and property-guided scaffold hopping enabled by parallel synthesis and pharmacophore-based virtual screening strategies. This effort resulted in the identification of novel, efficient and modular leads having favorable in vitro ADME profiles and high CNS multi-parameter optimization (MPO) scores, exemplified by azetidine sulfonamide 19 and aminothiazole sulfone (ent2)-20.

Crystal structures of human glycine receptor α3 bound to a novel class of analgesic potentiators.

Current therapies to treat persistent pain and neuropathic pain are limited by poor efficacy, side effects and risk of addiction. Here, we present a novel class of potent selective, central nervous system (CNS)-penetrant potentiators of glycine receptors (GlyRs), ligand-gated ion channels expressed in the CNS. AM-1488 increased the response to exogenous glycine in mouse spinal cord and significantly reversed mechanical allodynia induced by nerve injury in a mouse model of neuropathic pain. We obtained an X-ray crystal structure of human homopentameric GlyRα3 in complex with AM-3607, a potentiator of the same class with increased potency, and the agonist glycine, at 2.6-Å resolution. AM-3607 binds a novel allosteric site between subunits, which is adjacent to the orthosteric site where glycine binds. Our results provide new insights into the potentiation of cysteine-loop receptors by positive allosteric modulators and hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.

The Discovery and Hit-to-Lead Optimization of Tricyclic Sulfonamides as Potent and Efficacious Potentiators of Glycine Receptors.

Current pain therapeutics suffer from undesirable psychotropic and sedative side effects, as well as abuse potential. Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels expressed in nerves of the spinal dorsal horn, where their activation is believed to reduce transmission of painful stimuli. Herein, we describe the identification and hit-to-lead optimization of a novel class of tricyclic sulfonamides as allosteric GlyR potentiators. Initial optimization of high-throughput screening (HTS) hit 1 led to the identification of 3, which demonstrated ex vivo potentiation of glycine-activated current in mouse dorsal horn neurons from spinal cord slices. Further improvement of potency and pharmacokinetics produced in vivo proof-of-concept tool molecule 20 (AM-1488), which reversed tactile allodynia in a mouse spared-nerve injury (SNI) model. Additional structural optimization provided highly potent potentiator 32 (AM-3607), which was cocrystallized with human GlyRα3cryst to afford the first described potentiator-bound X-ray cocrystal structure within this class of ligand-gated ion channels (LGICs).

Sphingosine kinase activity is not required for tumor cell viability.

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Structure guided design of a series of sphingosine kinase (SphK) inhibitors.

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other's loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.

Development and implementation of a high-throughput AlphaLISA assay for identifying inhibitors of EZH2 methyltransferase.

The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z'>0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.

Screening for lysine-specific demethylase-1 inhibitors using a label-free high-throughput mass spectrometry assay.

Posttranslational modifications on the N terminus of histone H3 act in a combinatorial fashion to control epigenetic responses to extracellular stimuli. Lysine-specific demethylase-1 (LSD1) represents an emerging epigenetic target class for the discovery of novel antitumor therapies. In this study, a high-throughput mass spectrometry (HTMS) assay was developed to measure LSD1-catalyzed demethylation of lysine-4 on several H3 substrates. The assay leverages RapidFire chromatography in line with a triple stage quadrupole detection method to measure multiple LSD1 substrate and product reactions from an assay well. This approach minimizes artifacts from fluorescence interference and eliminates the need for antibody specificity to methylated lysines. The assay was robust in a high-throughput screen of a focused library consisting of more than 56,000 unique chemical scaffolds with a median Z' of 0.76. Validated hits from the primary screen were followed up by successive rounds of virtual and HTMS screening to mine for related structures in a parent library consisting of millions of compounds. The screen resulted in the rapid discovery of multiple chemical classes amenable to medicinal chemistry optimization. This assay was further developed into a generic platform capable of rapidly screening epigenetic targets that use the N-terminal tail of histone H3 as a substrate.

Discovery of pyridazinopyridinones as potent and selective p38 mitogen-activated protein kinase inhibitors.

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure-activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats.

Discovery and evaluation of 7-alkyl-1,5-bis-aryl-pyrazolopyridinones as highly potent, selective, and orally efficacious inhibitors of p38alpha mitogen-activated protein kinase.

The p38alpha mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38alpha inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED(50) < or = 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.

Part 2: Structure-activity relationship (SAR) investigations of fused pyrazoles as potent, selective and orally available inhibitors of p38alpha mitogen-activated protein kinase.

A novel class of pyrazolopyridazine p38alpha mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC(50) values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38alpha inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38alpha/beta over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of ca. 0.08mg/kg.

Part 1: Structure-Activity Relationship (SAR) investigations of fused pyrazoles as potent, selective and orally available inhibitors of p38alpha mitogen-activated protein kinase.

A novel class of fused pyrazole-derived inhibitors of p38alpha mitogen-activated protein kinase (MAPK) is disclosed. These inhibitors were evaluated for their ability to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in 50% human whole blood. This series was optimized through a SAR investigation to provide inhibitors with IC(50) values in the low single-digit nanomolar range in whole blood. Further investigation of their pharmacokinetic profiles led to the identification of two potent and orally bioavailable p38 inhibitors 10 m and 10 q. Inhibitor 10 m was found to be efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of 0.1mg/kg while 10 q was found to have an ED(50) of 0.05-0.07 mg/kg.

3-amino-7-phthalazinylbenzoisoxazoles as a novel class of potent, selective, and orally available inhibitors of p38alpha mitogen-activated protein kinase.

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.

Discovery of highly selective and potent p38 inhibitors based on a phthalazine scaffold.

Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFalpha production.

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors.

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases.

A potent and selective c-Kit inhibitor 20 was identified through a structure-activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC(50) of 26 nM.

Discovery of aryl aminoquinazoline pyridones as potent, selective, and orally efficacious inhibitors of receptor tyrosine kinase c-Kit.

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

New 'chemical probes' to examine the role of the hFPRL1 (or ALXR) receptor in inflammation.

We report the development of the novel N-substituted benzimidazole 11 as a potent and selective human formyl peptide receptor-like 1 (hFPRL1) agonist. This compound and its less active enantiomer 12 were identified as useful tools for studying receptor function in vitro.

High capacity homogeneous non-radioactive cortisol detection assays for human 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.