Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

Discovery of a Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase with Oral Anti-Inflammatory Activity.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

Development of phenotypic assays for identifying novel blockers of L-type calcium channels in neurons.

L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCC-blocking drugs have a long and successful record in the clinic for treating cardiovascular disorders. In contrast, establishment of their efficacy for indications of the central nervous system remains challenging given the tendency of existing LTCC drugs being functionally and mechanistically more selective for peripheral tissues. LTCCs in vivo are large macromolecular complexes consisting of a pore-forming subunit and other modulatory proteins, some of which may be neuro-specific and potentially harbor mechanisms for neuronal selectivity. To exploit the possibility of identifying mechanistically novel and/or neuro-selective blockers, we developed two phenotypic assays-a calcium flux-based primary screening assay and a patch clamp secondary assay, using rat primary cortical cultures. We screened a library comprised of 1278 known bioactive agents and successfully identified a majority of the potent LTCC-blocking drugs in the library. Significantly, we identified a previously unrecognized LTCC blocker with a novel mechanism, which was corroborated by patch clamp and binding studies. As such, these phenotypic assays are robust and represent an important step towards identifying mechanistically novel and neuro-selective LTCC blockers.

α7 nicotinic acetylcholine receptor upregulation by anti-apoptotic Bcl-2 proteins.

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.

A Genome-Wide Arrayed cDNA Screen to Identify Functional Modulators of α7 Nicotinic Acetylcholine Receptors.

Cellular signaling is in part regulated by the composition and subcellular localization of a series of protein interactions that collectively form a signaling complex. Using the α7 nicotinic acetylcholine receptor (α7nAChR) as a proof-of-concept target, we developed a platform to identify functional modulators (or auxiliary proteins) of α7nAChR signaling. The Broad cDNA library was transiently cotransfected with α7nAChR cDNA in HEK293T cells in a high-throughput fashion. Using this approach in combination with a functional assay, we identified positive modulators of α7nAChR activity. We identified known positive modulators/auxiliary proteins present in the cDNA library that regulate α7nAChR signaling, in addition to identifying novel modulators of α7nAChR signaling. These included NACHO, SPDYE11, TCF4, and ZC3H12A, all of which increased PNU-120596-mediated nicotine-dependent calcium flux. Importantly, these auxiliary proteins did not modulate GluR1(o)-mediated Ca flux. To elucidate a possible mechanism of action, we employed an α7nAChR-HA surface staining assay. NACHO enhanced α7nAChR surface expression; however, the mechanism responsible for the SPDYE11-, TCF4-, and ZC3H12A-dependent modulation of α7nAChR has yet to be defined. This report describes the development and validation of a high-throughput, genome-wide cDNA screening platform coupled to FLIPR functional assays in order to identify functional modulators of α7nAChR signaling.

Pharmacological Modulation of GluK1 and GluK2 by NETO1, NETO2, and PSD95.

The association between the kainate receptors (KARs) GluK1 and GluK2 and the modifying proteins neuropilin- and tolloid-like 1 (NETO1), neuropilin- and tolloid-like 2 (NETO2), and postsynaptic density protein 95 (PSD95) is likely to produce distinct GluK1 and GluK2 pharmacology in postsynaptic neurons. However, little is known about their corresponding modulatory effects on GluK1 and GluK2 activity in high-throughput assays for cell-based drug discovery. Using heterologous cells that potentially mimic the response in native cells in a fluorescence imaging plate reader (FLIPR) assay, we have investigated assays that incorporate (1) coexpression of GluK1 or GluK2 with their modulatory proteins (NETO1, NETO2, PSD95) and/or (2) enablement of assays with physiological concentration of native GluK1 and GluK2 agonist (glutamate) in the absence of an artificial potentiator (e.g., concanavalin A [Con A]). We found that in the absence of Con A, both NETO1 and NETO2 accessory proteins are able to potentiate kainate- and glutamate-evoked GluK1-mediated Ca(2+) influx. We also noted the striking ability of PSD95 to enhance glutamate-stimulated potentiation effects of NETO2 on GluK1 without the need for Con A and with a robust signal that could be utilized for high-throughput FLIPR assays. These experiments demonstrate the utility of heterologous cells coexpressing PSD95/NETO2 with GluK1 or GluK2 in native cell-mimicking heterologous cell systems for high-throughput assays and represent new avenues into the discovery of KAR modulating therapies.

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.

Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

We reported previously that ethanol treatment regulates D(1) receptor phosphorylation and signaling in a protein kinase C (PKC) delta- and PKCgamma-dependent fashion by a mechanism that may involve PKC isozyme-specific interacting proteins. Using a PKC isozyme-specific coimmunoprecipitation approach coupled to mass spectrometry, we report the identification of RanBP9 and RanBP10 as novel interacting proteins for both PKCgamma and PKCdelta. Both RanBP9 and RanBP10 were found to specifically coimmunoprecipitate with both PKCgamma and PKCdelta; however, this association did not seem to mediate the ethanol regulation of the PKCs. It is noteworthy that the D(1) receptor was also found to specifically coimmunoprecipitate with RanBP9/10 from human embryonic kidney (HEK) 293T cells and with endogenous RanBP9 from rat kidney. RanBP9 and RanBP10 were also found to colocalize at the cellular level with the D(1) receptor in both kidney and brain tissue. Although overexpression of RanBP9 or RanBP10 in HEK293T cells did not seem to alter the kinase activities of either PKCdelta or PKCgamma, both RanBP proteins regulated D(1) receptor phosphorylation, signaling, and, in the case of RanBP9, expression. Specifically, overexpression of either RanBP9 or RanBP10 enhanced basal D(1) receptor phosphorylation, which was associated with attenuation of D(1) receptor-stimulated cAMP accumulation. Moreover, treatment of cells with select PKC inhibitors blocked the RanBP9/10-dependent increase in basal receptor phosphorylation, suggesting that phosphorylation of the receptor by PKC is regulated by RanBP9/10. These data support the idea that RanBP9 and RanBP10 may function as signaling integrators and dictate the efficient regulation of D(1) receptor signaling by PKCdelta and PKCgamma.

Ethanol regulation of D(1) dopamine receptor signaling is mediated by protein kinase C in an isozyme-specific manner.

Ethanol consumption potentiates dopaminergic signaling that is partially mediated by the D(1) dopamine receptor; however, the mechanism(s) underlying ethanol-dependent modulation of D(1) signaling is unclear. We now show that ethanol treatment of D(1) receptor-expressing cells decreases D(1) receptor phosphorylation and concurrently potentiates dopamine-stimulated cAMP accumulation. Protein kinase C (PKC) inhibitors mimic the effects of ethanol on D(1) receptor phosphorylation and dopamine-stimulated cAMP levels in a manner that is non-additive with ethanol treatment. Ethanol was also found to modulate specific PKC activities as demonstrated using in vitro kinase assays where ethanol treatment attenuated the activities of lipid-stimulated PKCgamma and PKCdelta in membrane fractions, but did not affect the activities of PKCalpha, PKCbeta(1), or PKCvarepsilon. Importantly, ethanol treatment potentiated D(1) receptor-mediated DARPP-32 phosphorylation in rat striatal slices, supporting the notion that ethanol enhances D(1) receptor signaling in vivo. These findings suggest that ethanol inhibits the activities of specific PKC isozymes, resulting in decreased D(1) receptor phosphorylation and enhanced dopaminergic signaling.

Are Caenorhabditis elegans receptors useful targets for drug discovery: pharmacological comparison of tyramine receptors with high identity from C. elegans (TYRA-2) and Brugia malayi (Bm4).

The biogenic amine, tyramine (TA), modulates a number of key processes in nematodes and a number of TA-specific receptors have been identified. In the present study, we have identified a putative TA receptor (Bm4) in the recently completed Brugia malayi genome and compared its pharmacology to its putative Caenorhabditis elegans orthologue, TYRA-2, under identical expression and assay conditions. TYRA-2 and Bm4 are the most closely related C. elegans and B. malayi BA receptors and differ by only 14aa in the TM regions directly involved in ligand binding. Membranes from HEK-293 cells stably expressing Bm4 exhibited specific, saturable, high affinity, [(3)H]LSD and [(3)H]TA binding with K(d)s of 18.1+/-0.93 and 15.1+/-0.2 nM, respectively. More importantly, both TYRA-2 and Bm4 TA exhibited similar rank orders of potencies for a number of potential tyraminergic ligands. However, some significant differences were noted. For example, chloropromazine exhibited an order of magnitude higher affinity for Bm4 than TYRA-2 (pK(i)s of 7.6+/-0.2 and 6.49+/-0.1, respectively). In contrast, TYRA-2 had significantly higher affinity for phentolamine than Bm4. These results highlight the utility of the nearly completed B. malayi genome and the importance of using receptors from individual parasitic nematodes for drug discovery.

TYRA-2 (F01E11.5): a Caenorhabditis elegans tyramine receptor expressed in the MC and NSM pharyngeal neurons.

Tyramine appears to regulate key processes in nematodes, such as pharyngeal pumping, and more complex behaviors, such as foraging. Recently, a Caenorhabditis elegans tyramine receptor, SER-2, was identified that is involved in the TA-dependent regulation of these processes. In the present study, we have identified a second C. elegans gene, tyra-2 (F01E11.5) that encodes a tyramine receptor. This is the first identification of multiple tyramine receptor genes in any invertebrate. Membranes from COS-7 cells expressing TYRA-2 bind [(3)H]tyramine with high affinity with a K(d) of 20 +/- 5 nM. Other physiologically relevant biogenic amines, such as octopamine and dopamine, inhibit [(3)H]tyramine binding with much lower affinity (K(i)s of 1.55 +/- 0.5 and 1.78 +/- 0.6 microM, respectively), supporting the identification of TYRA-2 as a tyramine receptor. Indeed, tyramine also dramatically increases GTPgammaS binding to membranes from cells expressing TYRA-2 (EC(50) of 50 +/- 13 nM) and the TA-dependent GTPgammaS binding is PTX-sensitive suggesting that TYRA-2 may couple to Galpha(i/o). Based on fluorescence from tyra::gfp fusion constructs, TYRA-2 expression appears to be exclusively neuronal in the MC and NSM pharyngeal neurons, the AS family of amphid neurons and neurons in the nerve ring, body and tail. Taken together, these results suggest that TYRA-2 encodes a second Galpha(i/o)-coupled tyramine receptor and suggests that TA-dependent neuromodulation may be mediated by multiple receptors and more complex than previously appreciated.

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal pumping and foraging behavior in Caenorhabditis elegans.

Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical Ki values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC50 values. Tyramine, but not octopamine, also elevates Ca2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonin-dependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.

Biogenic amine receptors in parasitic nematodes: what can be learned from Caenorhabditis elegans?

The biogenic amines, serotonin, octopamine, tyramine and dopamine regulate many essential processes in parasitic nematodes, such as pharyngeal pumping, muscle contraction, and egg-laying, as well as more complex behaviors, such as mechanosensation and foraging, making biogenic amine receptors excellent targets for drug discovery. This review is designed to summarize our knowledge of nematode biogenic amine signaling and preliminarily identify some of the key receptors involved in the regulation of biogenic amine-dependent behaviors through an analysis of the free-living nematode, Caenorhabditis elegans.

Functional characterization of alternatively spliced 5-HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode, Ascaris suum.

Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.

Characterization of a tyramine receptor from Caenorhabditis elegans.

Octopamine (OA) plays an important role in the regulation of a number of key processes in nematodes, including pharyngeal pumping, locomotion and egg-laying. However, while putative OA receptors can be tentatively identified in the Caenorhabditis elegans database, no OA receptors have been functionally characterized from any nematode. We have isolated two cDNAs, ser-2 and ser-2a, encoding putative C.elegans serotonin/OA receptors (C02D4.2, ser-2). The sequences of these cDNAs differ from that predicted by GeneFinder and lack 42 bp of exon 2. In addition, ser-2a appears to be alternatively spliced and lacks a predicted 23 amino acids in the third intracellular loop. COS-7 cells expressing SER-2 bind [3H]LSD in the low nM range and exhibit Kis for tyramine, octopamine and serotonin of 0.07, 2, and 13.7 micro m, respectively. Significantly, tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells stably expressing SER-2 with an IC50 of about 360 nm, suggesting that SER-2 is a tyramine receptor.