Rodger Allen

The inhibition of antigen-induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4.

1 The novel tri‐aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50s: 4–45 nM). CDP840 is stereo‐selective since its S enantiomer (CT 1731) is 10–50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50s: > 100 μm) against PDE types 1, 2, 3 and 5. 2 Oral administration of CDP840 caused a dose‐dependent reduction of interleukin‐5 (IL‐5)‐induced pleural eosinophilia in rats (ED50 = 0.03 mg kg−1). The eosinophils in pleural exudates from CDP840‐treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation. CDP840 was approximately equi‐active with the steroid dexamethasone in this model and was 10–100 times more potent than the known PDE 4‐selective inhibitors rolipram and RP73401. The activity of CDP840 was not influenced by adrenalectomy, β‐sympathomimetics or β‐sympatholytics. 3 Antigen‐induced pulmonary eosinophilia in sensitized guinea‐pigs was reduced dose‐dependently by CDP840 (0.01‐1 mg kg−1, i.p.) and intracellular EPO levels were significantly higher. CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401. 4 Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan‐induced pleurisy in rats and thus do not exhibit general antiinflammatory activity. 5 In sensitized guinea‐pigs, aerosols of the antigen ovalbumin caused a dose‐dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure. Administration of CDP840 (0.001‐1.0 mg kg−1, i.p.), 1 h before antigen challenge, resulted in dose‐dependent reduction in response to antigen. This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg−1) did not significantly change the bronchoconstrictor response to histamine. Rolipram was approximately 10 times less active than CDP840 in preventing antigen‐induced bronchoconstriction. 6 These results confirm the observations that selective PDE 4 inhibitors reduce antigen‐induced bronchoconstriction and pulmonary eosinophilic inflammation. CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4. Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL‐5 model following oral administration. The novel series of tri‐aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma.

PDE 4 inhibitors: the use of molecular cloning in the design and development of novel drugs

Phosphodiesterase 4 (PDE 4) enzymes are the principal phosphodiesterases responsible for the hydrolysis of cAMP in pro-inflammatory leukocytes. The functional consequences of elevating cAMP in these cells suggests that inhibition of PDE 4 offers a novel approach to asthma therapy. However, clinical development of early inhibitors has been limited by the side-effect of nausea. In this review, we detail how the molecular biology of the PDE 4 gene family has been integrated with biochemical, cellular and pharmacological studies. This approach has led to the discovery and development of CDP840, a prototype inhibitor for which efficacy has been demonstrated in a clinical model of asthma in the absence of side-effects.

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor‐ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1‐coupled pathways by known CCR1 agonists. Chemokines leukotactin‐1, macrophage inflammatory protein (MIP)‐1α, monocyte chemotactic peptide (MCP)‐3, RANTES, and MIP‐1δ all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP‐1δ was unable to signal via G14‐, G16‐ or chimeric 16z44‐coupled pathways. In a stable cell line expressing CCR1 and Gα14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)‐1 and myeloid progenitor inhibitory factor (MPIF)‐1 were able to stimulate Gi/o‐coupled pathways, but MIP‐1δ, HCC‐1 and MPIF‐1 were unable to activate G14‐mediated stimulation of phospholipase Cβ activity. In addition, MIP‐1δ was unable to promote the phosphorylation of extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase. This suggests that different chemokines are able to selectively activate CCR1‐coupled pathways, probably because of differentintrinsic ligand efficacies. CCR1 and Gα14 or Gα16 are co‐expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine‐tune intracellular signaling pathways.

mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging

BackgroundThe mammalian target of rapamycin (mTOR) signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization.ResultsIn this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed) fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin.ConclusionsThe results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time.

Differential involvement of Gα16 in CC chemokine-induced stimulation of phospholipase Cβ, ERK, and chemotaxis

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.

CDP840: a novel inhibitor of PDE-4.

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitors activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

Inhibition of Human T Cell Activation by Novel Src Kinase Inhibitors Is Dependent upon the Complexity of the Signal Delivered to the Cell

The activity of a novel series of protein tyrosine kinase inhibitors that are selective for the Src family has been assessed. The activity of these compounds [named CT-SKI (Celltech Src kinase inhibitors)] was investigated by assessing their potential to modulate T cell receptor activation, an event thought to involve the Src kinases Lck and Fyn. This series of compounds contained low-nanomolar inhibitors of Src kinases with selectivity over Csk, epidermal growth factor receptor kinase, protein kinase C, and ζ-associated 70-kDa protein. These compounds were shown to attenuate anti-CD3-induced T cell proliferation and block interleukin (IL)-2, IL-4, and interferon-γ production, and CD25 expression in anti-CD3-activated T cells. In addition, inhibition of anti-CD3-induced, but not phorbol ester and calcium ionophore-induced IL-2 production, correlated with inhibition of in vitro Lck kinase activity. When more complex stimuli were used to activate T cells, as in the mixed lymphocyte reaction (MLR), these inhibitors proved to be less effective and inhibition of the MLR did not correlate with inhibition of isolated Lck enzyme. Interestingly, inhibition of anti-CD3-induced proliferation could be reversed by the addition of exogenous IL-2, indicating that signaling through the IL-2 receptor may not be critically dependent on any functional Src enzymes.

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

Seletalisib: Characterization of a Novel, Potent, and Selective Inhibitor of PI3Kδ

Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3Kδ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3Kδ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide–stimulated but not phorbol myristate acetate–stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3Kδ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.

Enhanced anti-tumour activity of the combination of the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037

PurposeTumours frequently have defects in multiple oncogenic pathways, e.g. MAPK and PI3K signalling pathways, and combinations of targeted therapies may be required for optimal activity. This study evaluated the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037, as single agents and in combination, in colorectal carcinoma cell lines and tumour xenograft-bearing mice.MethodsIn vitro growth inhibition, survival and signal transduction were measured using the Sulforhodamine B, clonogenic and Western blotting assays, respectively, in HCT116 and HT29 cell lines. In vivo anti-tumour efficacy and pharmacokinetic properties were assessed in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice.ResultsThe combination of WX-554 and WX-037 exhibited marked synergistic growth inhibition in vitro, which was associated with increased cytotoxicity and enhanced inhibition of ERK and S6 phosphorylation, compared to either agent alone. Pharmacokinetic analyses indicated that there was no PK interaction between the two drugs at low doses, but that at higher doses, WX-037 may delay the tumour uptake of WX-554. In vivo efficacy studies revealed that the combination of WX-037 and WX-554 was non-toxic and exhibited marked tumour growth inhibition greater than observed with either agent alone.ConclusionThese studies show for the first time that combination treatment with the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037 can induce synergistic growth inhibition in vitro, which translates into enhanced anti-tumour efficacy in vivo.