Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren's syndrome.

BACKGROUND: The phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies. Primary Sjögren's syndrome (pSS) is a chronic immune-mediated inflammatory disease characterised by exocrine gland lymphocytic infiltration and B-cell hyperactivation which results in systemic manifestations, autoantibody production and loss of glandular function. Given the central role of B cells in pSS pathogenesis, we investigated PI3Kδ pathway activation in pSS and the functional consequences of blocking PI3Kδ in a murine model of focal sialoadenitis that mimics some features of pSS. METHODS AND RESULTS: Target validation assays showed significant expression of phosphorylated ribosomal protein S6 (pS6), a downstream mediator of the phosphatidylinositol 3-kinase delta (PI3Kδ) pathway, within pSS salivary glands. pS6 distribution was found to co-localise with T/B cell markers within pSS aggregates and the CD138+ plasma cells infiltrating the glands. In vivo blockade of PI3Kδ activity with seletalisib, a PI3Kδ-selective inhibitor, in a murine model of focal sialoadenitis decreased accumulation of lymphocytes and plasma cells within the glands of treated mice in the prophylactic and therapeutic regimes. Additionally, production of lymphoid chemokines and cytokines associated with ectopic lymphoneogenesis and, remarkably, saliva flow and autoantibody production, were significantly affected by treatment with seletalisib. CONCLUSION: These data demonstrate activation of PI3Kδ pathway within the glands of patients with pSS and its contribution to disease pathogenesis in a model of disease, supporting the exploration of the therapeutic potential of PI3Kδ pathway inhibition in this condition.

Discovery of a Potent, Orally Bioavailable PI4KIIIß Inhibitor (UCB9608) Able To Significantly Prolong Allogeneic Organ Engraftment in Vivo.

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.

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.

First-in-human studies of seletalisib, an orally bioavailable small-molecule PI3Kδ inhibitor for the treatment of immune and inflammatory diseases.

PURPOSE: PI3Ks are potential therapeutic targets in immune-inflammatory diseases. These studies aimed to investigate the safety, tolerability and PK profile of seletalisib, a selective inhibitor of PI3Kδ in humans. METHODS: These phase I, randomised, double-blind, placebo-controlled, single-centre studies (NCT02303509, NCT02207595) evaluated single and multiple oral doses of seletalisib (5-90 mg QD and 30 mg BID) in healthy adults and subjects with mild-to-moderate psoriasis (Study-1). Pharmacodynamic effects on markers of inflammation were assessed via changes in ex vivo basophil degranulation and histological assessment of psoriatic skin biopsies. RESULTS: Seletalisib was well tolerated at doses ≤15 mg (Study-1) and ≤45 mg QD (Study-2) for 14 days. No safety concerns or dose-limiting toxicities were identified (Study-1). Incidence of gastrointestinal-related AEs was not dose related but higher incidences of rash AEs were associated with higher-dose seletalisib (Study-2 rash AEs: 18 in 12 seletalisib-treated subjects versus 1 in 1 placebo-treated subject). Mean seletalisib plasma concentration-time profiles increased with increasing doses after single and multiple dosing, with no major deviations from dose-proportionality. There was no unexpected accumulation or loss of exposure after multiple dosing (time-independent pharmacokinetic profile). Apparent t 1/2 values were supportive of once-daily dosing (geometric mean t1/2: Study-1, 17.7-21.1 h; Study-2, 18.1-22.4 h). No clinically significant food effect was observed (Study-1). Pharmacodynamic findings demonstrated ex vivo inhibition of basophil degranulation, improvements in histological assessment of skin biopsies and other markers of psoriatic biology and preliminary evidence of target engagement in psoriatic skin tissue. CONCLUSIONS: Seletalisib safety, tolerability and pharmacokinetic/pharmacodynamic profiles support its continued clinical development in immune-inflammatory diseases.

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

PURPOSE: Tumours 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. METHODS: In 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. RESULTS: The 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. CONCLUSION: These 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.

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours.

PURPOSE: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials. EXPERIMENTAL DESIGN: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules. RESULTS: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma. CONCLUSIONS: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly.

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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: The 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.

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.

Fused thiophene derivatives as MEK inhibitors.

A number of novel fused thiophene derivatives have been prepared and identified as potent inhibitors of MEK. The SAR data of selected examples and the in vivo profiling of compound 13 h demonstrates the functional activity of this class of compounds in HT-29 PK/PD models.

Differential involvement of Galpha16 in CC chemokine-induced stimulation of phospholipase Cbeta, 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.

Small molecule receptor agonists and antagonists of CCR3 provide insight into mechanisms of chemokine receptor activation.

Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.

A vascular endothelial growth factor receptor-2 kinase inhibitor potentiates the activity of the conventional chemotherapeutic agents paclitaxel and doxorubicin in tumor xenograft models.

Inhibition of angiogenesis may have wide use in the treatment of cancer; however, this approach alone will not cause tumor regression but may only slow the growth of solid tumors. The clinical potential of antiangiogenic agents may be increased by combining them with conventional chemotherapeutics. 4-[4-(1-Amino-1-methylethyl)phenyl]-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]pyrimidine-5-carbonitrile (JNJ-17029259) represents a novel structural class of 5-cyanopyrimidines that are orally available, selective, nanomolar inhibitors of the vascular endothelial growth factor receptor-2 (VEGF-R2) and other tyrosine kinases involved in angiogenesis, such as platelet-derived growth factor receptor, fibroblast growth factor receptor, VEGF-R1, and VEGF-R3, but have little activity on other kinase families. At nanomolar levels, JNJ-17029259 blocks VEGF-stimulated mitogen-activated protein kinase signaling, proliferation/migration, and VEGF-R2 phosphorylation in human endothelial cells; inhibits the formation of vascular sprouting in the rat aortic ring model of angiogenesis; and interferes with the development of new veins and arteries in the chorioallantoic membrane assay. At higher concentrations of 1 to 3 microM, this compound shows antiproliferative activity on cells that may contribute to its antitumor effects. JNJ-17029259 delays the growth of a wide range of human tumor xenografts in nude mice when administered orally as single-agent therapy. Histological examination revealed that the tumors have evidence of reduced vascularity after treatment. In addition, JNJ-17029259 enhances the effects of the conventional chemotherapeutic drugs doxorubicin and paclitaxel in xenograft models when administered orally in combination therapy. An orally available angiogenesis inhibitor that can be used in conjunction with standard chemotherapeutic agents to augment their activity may have therapeutic benefit in stopping the progression of cancer and preventing metastasis.

Differential chemokine activation of CC chemokine receptor 1-regulated pathways: ligand selective activation of Galpha 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)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta 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 different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 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.

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 zeta-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-gamma 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.