A CD25-biased interleukin-2 for autoimmune therapy engineered via a semi-synthetic organism.

BACKGROUND: Natural cytokines are poorly suited as therapeutics for systemic administration due to suboptimal pharmacological and pharmacokinetic (PK) properties. Recombinant human interleukin-2 (rhIL-2) has shown promise for treatment of autoimmune (AI) disorders yet exhibits short systemic half-life and opposing immune responses that negate an appropriate therapeutic index. METHODS: A semi-synthetic microbial technology platform was used to engineer a site-specifically pegylated form of rhIL-2 with enhanced PK, specificity for induction of immune-suppressive regulatory CD4 + T cells (Tregs), and reduced stimulation of off-target effector T and NK cells. A library of rhIL-2 molecules was constructed with single site-specific, biorthogonal chemistry-compatible non-canonical amino acids installed near the interface where IL-2 engages its cognate receptor ßγ (IL-2Rßγ) signaling complex. Biorthogonal site-specific pegylation and functional screening identified variants that retained engagement of the IL-2Rα chain with attenuated potency at the IL-2Rßγ complex. RESULTS: Phenotypic screening in mouse identifies SAR444336 (SAR'336; formerly known as THOR-809), rhIL-2 pegylated at H16, as a potential development candidate that specifically expands peripheral CD4+ Tregs with upregulation of markers that correlate with their suppressive function including FoxP3, ICOS and Helios, yet minimally expands CD8 + T or NK cells. In non-human primate, administration of SAR'336 also induces dose-dependent expansion of Tregs and upregulated suppressive markers without significant expansion of CD8 + T or NK cells. SAR'336 administration reduces inflammation in a delayed-type hypersensitivity mouse model, potently suppressing CD4+ and CD8 + T cell proliferation. CONCLUSION: SAR'336 is a specific Treg activator, supporting its further development for the treatment of AI diseases.

Interleukin-2 (IL-2) is a protein that functions as a master regulator of immune responses. A key function of IL-2 is the stimulation of immune-regulatory cells that suppress autoimmune disease, which occurs when the body's immune system mistakenly attacks healthy tissues. However, therapeutic use of IL-2 is limited by its short duration of action and incomplete selectivity for immune-suppressive cells over off-target immune-stimulatory cells. We employ a platform that we have previously developed, which is a bacterial organism with an expanded DNA code, to identify a new version of IL-2, SAR444336 (SAR'336), with an extended duration of activity and increased selectivity for immune-suppressive cells. In mice and monkeys, SAR'336 was a specific activator of immune suppression, with minimal effect on immune cells that stimulate autoimmunity. Our results support further development of SAR'336 for treatment of autoimmune disorders.

Distinct Predictive Immunogenomic Profiles of Response to Immune Checkpoint Inhibitors and IL2: A Real-world Evidence Study of Patients with Advanced Renal Cancer.

Recombinant human high-dose IL2 (HD-IL2; aldesleukin) was one of the first approved immune-oncology agents based upon clinical activity in renal cell carcinoma (RCC) and metastatic melanoma but use was limited due to severe toxicity. Next-generation IL2 agents designed to improve tolerability are in development, increasing the need for future identification of genomic markers of clinical benefit and/or clinical response. In this retrospective study, we report clinical and tumor molecular profiling from patients with metastatic RCC (mRCC) treated with HD-IL2 and compare findings with patients with RCC treated with anti-PD-1 therapy. Genomic characteristics common and unique to IL2 and/or anti-PD-1 therapy response are presented, with insight into rational combination strategies for these agents. Residual pretreatment formalin-fixed paraffin embedded tumor samples from n = 36 patients with HD-IL2 mRCC underwent RNA-sequencing and corresponding clinical data were collected. A de novo 40-gene nearest centroid IL2 treatment response classifier and individual gene and/or immune marker signature differences were correlated to clinical response and placed into context with a separate dataset of n = 35 patients with anti-PD-1 mRCC. Immune signatures and genes, comprising suppressor and effector cells, were increased in patients with HD-IL2 clinical benefit. The 40-gene response classifier was also highly enriched for immune genes. While several effector immune signatures and genes were common between IL2 and anti-PD-1 treated patients, multiple inflammatory and/or immunosuppressive genes, previously reported to predict poor response to anti-PD-L1 immunotherapy, were only increased in IL2-responsive tumors. These findings suggest that common and distinct immune-related response markers for IL2 and anti-PD-1 therapy may help guide their use, either alone or in combination. Significance: Next-generation IL2 agents, designed for improved tolerability over traditional HD-IL2 (aldesleukin), are in clinical development. Retrospective molecular tumor profiling of patients treated with HD-IL2 or anti-PD-1 therapy provides insights into genomic characteristics of therapy response. This study revealed common and distinct immune-related predictive response markers for IL2 and anti-PD-1 therapy which may play a role in therapy guidance, and rational combination strategies for these agents.

An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism.

The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug accumulation in the tumor tissue, stimulates tumor-infiltrating CD8+ T and NK cells, and leads to a dose-dependent reduction of tumor growth. These results support further characterization of the immune modulatory, anti-tumor properties of THOR-707 and represent a fundamental advance in the application of synthetic biology to medicine, leveraging engineered semi-synthetic organisms as cellular factories to facilitate discovery and production of differentiated classes of chemically modified biologics.

Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist.

RATIONALE: Gq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors. OBJECTIVE: Test whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist. METHODS AND RESULTS: In recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the α1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor. CONCLUSIONS: Gq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.

Structural Basis of Small-Molecule Aggregate Induced Inhibition of a Protein-Protein Interaction.

A prevalent observation in high-throughput screening and drug discovery programs is the inhibition of protein function by small-molecule compound aggregation. Here, we present the X-ray structural description of aggregation-based inhibition of a protein-protein interaction involving tumor necrosis factor α (TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor (JNJ525) induces a quaternary structure switch of TNFα that inhibits the protein-protein interaction between TNFα and TNFα receptors. SPD-304 may employ a similar mechanism of inhibition.

Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo.

Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNFα Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNFα, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNFα secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFα in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.

A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors.

5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4 Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds.

Polypharmacology of Small-Molecule Modulators of the 5-Lipoxygenase Activating Protein (FLAP) Observed via a High-throughput Lipidomics Platform.

Leukotrienes (LTs) and related species are proinflammatory lipid mediators derived from arachidonic acid (AA) that have pathological roles in autoimmune and inflammatory conditions, cardiovascular diseases, and cancer. 5-Lipoxygenase activating protein (FLAP) plays a critical accessory role in the conversion of AA to LTA4, and its subsequent conversion to LTC4 by LTC4 synthase. Pharmacological inhibition of FLAP results in a loss of LT production by preventing the biosynthesis of both LTB4 and LTC4, making it an attractive target for the treatment of inflammatory diseases in which LTs likely play a role. Small-molecule (SM) drugs often exhibit polypharmacology through various pathways, which may explain the differential therapeutic efficacies of compounds sharing structural similarity. We have profiled a series of SM FLAP modulators for their selectivity across enzymes of AA cascade in human whole blood (HWB), using a recently developed LC/MS (liquid chromatography-mass spectrometry)-based high-throughput lipidomics platform that monitors 122 eicosanoids in multiplex. Highly efficient data acquisition coupled with fast and accurate data analysis allowed facile compound profiling from ex vivo study samples. This platform allowed us to quantitatively map the effects of those SMs on the entire AA cascade, demonstrating its potential to discriminate structurally related compounds.

Phenotypic Approaches to Identify Inhibitors of B Cell Activation.

An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin's lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton's tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt's lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation.

Phenotyping drug polypharmacology via eicosanoid profiling of blood.

It is widely accepted that small-molecule drugs, despite their selectivity at primary targets, exert pharmacological effects (and safety liabilities) through a multiplicity of pathways. As such, it has proved extremely difficult to experimentally assess polypharmacology in an agnostic fashion. Profiling of metabolites produced as part of physiological responses to pharmacological stimuli provides a unique opportunity to explore drug pharmacology. A total of 122 eicosanoid lipids in human whole blood were monitored from 10 different donors upon stimulation with several inducers of immunological responses and treatment with modulators of prostaglandin (PG) and leukotriene biosynthesis, including clinical and investigational molecules. Such analysis revealed differentiation between drugs nominally targeting different eicosanoid biosynthetic enzymes, or even those designed to target the same enzyme. Profiled agents, some of them marketed products, affect eicosanoid biosynthesis in ways that cannot be predicted from information on their intended targets. As an example, we used this platform to discriminate drugs based on their ability to silence PG biosynthesis in response to bacterial lipopolysaccharide, resulting in differential pharmacological activity in an in vivo model of endotoxemia. Some of the observed effects are subject to variability among individuals, indicating a potential application of this methodology to the patient stratification, based on their responses to benchmark drugs and experimental compounds read on the eicosanome via a simple blood test.

Isoproterenol acts as a biased agonist of the alpha-1A-adrenoceptor that selectively activates the MAPK/ERK pathway.

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e., not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and ß2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as ß-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Characterization of RO5126946, a Novel α7 nicotinic acetylcholine receptor-positive allosteric modulator.

Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor activation (α7nAChR) improves cognitive function, the decline of which is associated with conditions such as Alzheimer's disease and schizophrenia. Moreover, allosteric modulation of α7nAChR is an emerging therapeutic strategy in an attempt to avoid the rapid desensitization properties associated with the α7nAChR after orthosteric activation. We used a calcium assay to screen for positive allosteric modulators (PAMs) of α7nAChR and report on the pharmacologic characterization of the novel compound RO5126946 (5-chloro-N-[(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl]-2-methoxy-benzamide), which allosterically modulates α7nAChR activity. RO5126946 increased acetylcholine-evoked peak current and delayed current decay but did not affect the recovery of α7nAChRs from desensitization. In addition, RO5126946's effects were absent when nicotine-evoked currents were completely blocked by coapplication of the α7nAChR-selective antagonist methyl-lycaconitine. RO5126946 enhanced α7nAChR synaptic transmission and positively modulated GABAergic responses. The absence of RO5126946 effects at human α4ß2nAChR and 5-hydroxytryptamine 3 receptors, among others, indicated selectivity for α7nAChRs. In vivo, RO5126946 is orally bioavailable and brain-penetrant and improves associative learning in a scopolamine-induced deficit model of fear conditioning in rats. In addition, procognitive effects of RO5126946 were investigated in the presence of nicotine to address potential pharmacologic interactions on behavior. RO5126946 potentiated nicotine's effects on fear memory when both compounds were administered at subthreshold doses and did not interfere with procognitive effects observed when both compounds were administered at effective doses. Overall, RO5126946 is a novel α7nAChR PAM with cognitive-enhancing properties.

A highly efficient, high-throughput lipidomics platform for the quantitative detection of eicosanoids in human whole blood.

We have developed an ultra-performance liquid chromatography-multiple reaction monitoring/mass spectrometry (UPLC-MRM/MS)-based, high-content, high-throughput platform that enables simultaneous profiling of multiple lipids produced ex vivo in human whole blood (HWB) on treatment with calcium ionophore and its modulation with pharmacological agents. HWB samples were processed in a 96-well plate format compatible with high-throughput sample processing instrumentation. We employed a scheduled MRM (sMRM) method, with a triple-quadrupole mass spectrometer coupled to a UPLC system, to measure absolute amounts of 122 distinct eicosanoids using deuterated internal standards. In a 6.5-min run, we resolved and detected with high sensitivity (lower limit of quantification in the range of 0.4-460 pg) all targeted analytes from a very small HWB sample (2.5 µl). Approximately 90% of the analytes exhibited a dynamic range exceeding 1000. We also developed a tailored software package that dramatically sped up the overall data quantification and analysis process with superior consistency and accuracy. Matrix effects from HWB and precision of the calibration curve were evaluated using this newly developed automation tool. This platform was successfully applied to the global quantification of changes on all 122 eicosanoids in HWB samples from healthy donors in response to calcium ionophore stimulation.

Molecular analysis of model gut microbiotas by imaging mass spectrometry and nanodesorption electrospray ionization reveals dietary metabolite transformations.

The communities constituting our microbiotas are emerging as mediators of the health-disease continuum. However, deciphering the functional impact of microbial communities on host pathophysiology represents a formidable challenge, due to the heterogeneous distribution of chemical and microbial species within the gastrointestinal (GI) tract. Herein, we apply imaging mass spectrometry (IMS) to localize metabolites from the interaction between the host and colonizing microbiota. This approach complements other molecular imaging methodologies in that analytes need not be known a priori, offering the possibility of untargeted analysis. Localized molecules within the GI tract were then identified in situ by surface sampling with nanodesorption electrospray ionization Fourier transform ion cyclotron resonance-mass spectrometry (nanoDESI FTICR-MS). Products from diverse structural classes were identified including cholesterol-derived lipids, glycans, and polar metabolites. Specific chemical transformations performed by the microbiota were validated with bacteria in culture. This study illustrates how untargeted spatial characterization of metabolites can be applied to the molecular dissection of complex biology in situ.

AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist.

BACKGROUND AND PURPOSE: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. EXPERIMENTAL APPROACH: The antagonistic potencies (pIC(50)) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. KEY RESULTS: The pIC(50) estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t(1/2) = 1.63 h) and plasma-free fraction (98.2% protein bound). CONCLUSIONS AND IMPLICATIONS: The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.

Facilitatory interplay in alpha 1a and beta 2 adrenoceptor function reveals a non-Gq signaling mode: implications for diversification of intracellular signal transduction.

Agonist occupied alpha(1)-adrenoceptors (alpha(1)-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress alpha(1)- and beta-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in alpha(1A)- and beta(2)-AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca(2+) mobilization to monitor in real time the activation of both G(q) and G(s) pathways. We found that coactivation of alpha(1A)- and beta(2)-AR by the nonselective agonist NE or via a combination of the highly selective alpha(1A)-AR agonist A61603 and the beta-selective agonist isoproterenol led to increases in Ca(2+) influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca(2+) from intracellular stores. This effect became more evident upon examination of an alpha(1A)-AR variant exhibiting a partial defect in coupling to G(q), and we attribute it to potentiation of a non G(q)-pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca(2+) entry channel blocker. We also found that stimulation with A61603 of a second alpha(1A)-AR variant entirely unable to signal induced no Ca(2+) unless beta(2)-AR was concomitantly activated. These results may be accounted for by the presence of alpha(1A)/beta(2)-AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting alpha- and beta-adrenoceptors.

Effects of TNFalpha-converting enzyme inhibition on amyloid beta production and APP processing in vitro and in vivo.

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine that is elevated in Alzheimer's disease (AD) brains. Because TNFalpha is released from cell membranes by the TNFalpha-converting enzyme (TACE), inhibition of TACE has the potential to mitigate TNFalpha effects in AD brain. TACE also cleaves amyloid precursor protein (APP) and generates sAPPalpha, precluding the formation of potentially harmful amyloid beta (Abeta) peptides by beta-site APP cleaving enzymes (BACE). Hence, the anti-inflammatory benefits of TACE inhibition might be offset by an increase in Abeta. We have examined the effects of the highly selective TACE inhibitor, BMS-561392, on APP processing in vitro and in vivo. In Chinese hamster ovary cells expressing APP, BMS-561392 significantly reduced secretion of sAPPalpha without a corresponding increase in Abeta production. Conversely, a BACE inhibitor decreased sAPPbeta and Abeta peptides with no change in the secretion of sAPPalpha. These data indicate an absence of TACE and BACE competition for the APP substrate. Despite this, we observed competition for APP when TACE activity was enhanced via phorbol ester treatment or if APP was modified such that it was retained within the trans-Golgi network (TGN). These results suggest that BACE and TACE share a common TGN localization, but under normal conditions do not compete for APP. To confirm this finding in vivo, BMS-561392 was infused into the brains of Tg2576 and wild-type mice. Although decreased brain sAPPalpha levels were observed, steady-state Abeta levels were not significantly changed. Accordingly, it is possible that TACE inhibitors could reduce TNFalpha levels without increasing Abeta levels within the AD brain.

Antidepressants targeting the serotonin reuptake transporter act via a competitive mechanism.

Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099-3108, 1973). Two different SERT-selective radioligands, [(3)H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [(3)H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [(3)H]DASB and [(3)H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC(50)) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-beta-carboxy-3-beta-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.

Kinetic and thermodynamic assessment of binding of serotonin transporter inhibitors.

Several serotonin reuptake inhibitors are in clinical use for treatment of depression and anxiety disorders. However, to date, reported pharmacological differentiation of these ligands has focused mainly on their equilibrium binding affinities for the serotonin transporter. This study takes a new look at antidepressant binding modes using radioligand binding assays with [(3)H]S-citalopram to determine equilibrium and kinetic rate constants across multiple temperatures. The observed dissociation rate constants at 26 degrees C fall into a narrow range for all molecules. Conversely, association rate constants generally decreased with increasing equilibrium binding affinities. Consistent with this, the measured activation energy for S-citalopram association was relatively large (19.5 kcal . mol(-1)), suggesting conformational change upon ligand binding. For most of the drugs, including citalopram, the enthalpy (DeltaH(O)) and entropy (-TDeltaS(O)) contributions to reaction energetics were determined by van't Hoff analyses to be roughly equivalent (25-75% DeltaG(O)) and to correlate (positively for enthalpy) with the polar surface area of the drug. However, the binding of the drug fluvoxamine was predominantly entropically driven. When these data are considered in the context of the physicochemical properties of these ligands, two distinct binding modes can be proposed. The citalopram-type binding mode probably uses a polar binding pocket that allows charged or polar interactions between ligand and receptor with comparatively small loss in enthalpy due to dehydration. The fluvoxamine-type binding mode is fueled by energy released upon burying hydrophobic ligand moieties into a binding pocket that is flexible enough to suffer minimal loss in entropy from conformational constraint.