Publications

Publications


Fostamatinib & related projects:

Design, synthesis of diaminopyrimidine inhibitors targeting IgE- and IgG-mediated activation of Fc receptor signaling.
Argade A, Bhamidipati S, Li H, Sylvain C, Clough J, Carroll D, Keim H, Braselmann S, Taylor V, Zhao H, Herlaar E, Issakani SD, Wong BR, Masuda ES, Payan DG, Singh R.

Bioorg Med Chem Lett. 2015 May 15;25(10):2122-8.

Abstract
Using cultured human mast cells (CHMC) the optimization of 2,4-diaminopyrimidine compounds leading to 22, R406 is described. Compound 22 is a potent upstream inhibitor of mast cell degranulation and its mechanism of action is via inhibition of Syk kinase. Compound 22 has significant activity in inhibiting both IgE- and IgG-mediated activation of Fc receptor (FcR) in mast cells and basophils, and in addition inhibits Syk kinase-dependent activity of FcR-mediated activation of monocytes, macrophages, neutrophils, and B cell receptor (BCR)-mediated activation of B lymphocytes. Overall, the biological activity of 22 suggests that it has potential for application as a novel therapeutic for the treatment of an array of autoimmune maladies and hematological malignancies.

PMID: 25891105
Application of cultured human mast cells (CHMC) for the design and structure-activity relationship of IgE-mediated mast cell activation inhibitors.
Argade A, Bhamidipati S, Li H, Carroll D, Clough J, Keim H, Sylvain C, Rossi AB, Coquilla C, Issakani SD, Masuda ES, Payan DG, Singh R.

Bioorg Med Chem Lett. 2015 May 15;25(10):2117-21.

Abstract
Here we report the optimization of small molecule inhibitors of human mast cell degranulation via anti-IgE-mediated tryptase release following cross-linking and activation of IgE-loaded FcεR1 receptors. The compounds are selective upstream inhibitors of FcεR1-dependent human mast cell degranulation and proved to be devoid of activity in downstream ionomycin mediated degranulation. Structure-activity relationship (SAR) leading to compound 26 is outlined.

PMID: 25872982
Spleen Tyrosine Kinase Inhibition Attenuates Autoantibody Production and Reverses Experimental Autoimmune GN
McAdoo SP, Reynolds J, Bhangal G, Smith J, McDaid JP, Tanna A, Jackson WD, Masuda ES, Cook HT, Pusey CD, Tam FW.

J Am Soc Nephrol. 2014 Oct;25(10):2291-2302.

Abstract
Spleen tyrosine kinase (SYK) has an important role in immunoreceptor signaling, and SYK inhibition has accordingly attenuated immune-mediated injury in several in vivo models. However, the effect of SYK inhibition on autoantibody production remains unclear, and SYK inhibition has not been studied in an autoimmune model of renal disease. We, therefore, studied the effect of SYK inhibition in experimental autoimmune GN, a rodent model of antiglomerular basement membrane disease. We show glomerular SYK expression and activation by immunohistochemistry in both experimental and clinical disease, and we show that treatment with fostamatinib, a small molecule kinase inhibitor selective for SYK, completely prevents the induction of experimental autoimmune GN. In established experimental disease, introduction of fostamatinib treatment led to cessation of autoantibody production, reversal of renal injury, preservation of biochemical renal function, and complete protection from lung hemorrhage. B cell ELISpot and flow cytometric analysis suggest that short-term fostamatinib treatment inhibits the generation and activity of antigen-specific B cells without affecting overall B-cell survival. Additionally, fostamatinib inhibited proinflammatory cytokine production by nephritic glomeruli ex vivo and cultured bone marrow-derived macrophages in vitro, suggesting additional therapeutic effects independent of effects on autoantibody production that are likely related to inhibited Fc receptor signaling within macrophages in diseased glomeruli. Given these encouraging results in an in vivo model that is highly applicable to human disease, we believe clinical studies targeting SYK in GN are now warranted.

PMID: 24700868
Targeting autoimmunity: strategies and tactics.
Masuda ES, Payan DG.

Drug Discov Today. 2014 Aug;19(8):1193-4.

PMID: 24886764
Pharmacokinetics of fostamatinib, a spleen tyrosine kinase (SYK) inhibitor, in healthy human subjects following single and multiple oral dosing in three phase I studies
Baluom M, Grossbard EB, Mant T, Lau DT.

Br J Clin Pharmacol. 2013 Jul;76(1):78-88.

Abstract
AIM: Fostamatinib (R788) is an orally dosed prodrug designed to deliver the active metabolite R940406 (R406), a spleen tyrosine kinase (SYK) inhibitor, for the treatment of rheumatoid arthritis. The objectives were to evaluate the human pharmacokinetic properties of fostamatinib and R406. METHOD: Three clinical studies were conducted in healthy subjects: (A) A single ascending dose study for R406 with doses ranging from 80-600 mg, (B) a single- and multiple-dose study of fostamatinib in aqueous suspension, with single doses ranging from 80-400 mg and multiple doses at 160 mg twice daily and (C) a study comparing suspension and tablet of fostamatinib, with the latter tested in both fed and fasted states. RESULTS:These studies demonstrated that when administered as a solution, R406 was rapidly absorbed. Increases in exposure were observed with doses up to 400 mg. A terminal half-life of 12-21 h was observed. Similar R406 exposure could be achieved with fostamatinib suspension and steady-state was achieved after 3-4 days following twice daily administration. Fostamatinib tablet and suspension exhibited similar R406 exposure. Upon co-administration with food, a delay in peak time and lower peak concentrations of R406 were observed but at the same time the overall exposure did not change. CONCLUSION: Fostamatinib demonstrates rapid and extensive conversion to R406, an inhibitor of SYK. Solid dosage forms of fostamatinib overcome the challenge of low aqueous solubility of R406. The PK profile of R406 could potentially allow once daily or twice daily oral administration of fostamatinib.

PMID: 23190017
Effects of Fostamatinib (R788), an Oral Spleen Tyrosine Kinase Inhibitor, on Health-related Quality of Life in Patients with Active Rheumatoid Arthritis: Analyses of Patient-reported Outcomes from a Randomized, Double-blind, Placebo-controlled Trial.
Weinblatt ME, Kavanaugh A, Genovese MC, Jones DA, Musser TK, Grossbard EB, Magilavy DB.

J Rheumatol. 2013 Apr;40(4):369-78.

Abstract
OBJECTIVE: To assess the influence of fostamatinib on patient-reported outcomes (PRO) in patients with active rheumatoid arthritis and an inadequate response to methotrexate (MTX). METHODS: Patients taking background MTX (N = 457) were enrolled in a phase II clinical trial (NCT00665925) and randomized equally to placebo, fostamatinib 100 mg twice daily (bid), or fostamatinib 150 mg once daily (qd) for 24 weeks. Self-administered PRO measures included pain, patient's global assessment (PtGA) of disease activity, physical function, health-related quality of life (HRQOL), and fatigue. Mean change from baseline and a responder analysis of the proportion of patients achieving a minimal clinically important difference were determined. RESULTS: At Week 24, there were statistically significant improvements in pain, PtGA, physical function, fatigue, and the physical component summary of the Medical Outcomes Study Short Form-36 (SF-36) for fostamatinib 100 mg bid compared with placebo. Mean (standard error) changes from baseline in the fostamatinib 100 mg bid group versus the placebo group were -31.3 (2.45) versus -17.8 (2.45), p < 0.001 for pain; -29.1 (2.26) versus -16.7 (2.42), p < 0.001 for PtGA; -0.647 (0.064) versus -0.343 (0.062), p < 0.001 for physical function; 7.40 (1.00) versus 4.50 (0.94), p < 0.05 for fatigue; 8.52 (0.77) versus 4.90 (0.78), p < 0.01 for SF-36 physical component score; and 3.99 (0.93) versus 3.71 (0.99), p = 0.83 for SF-36 mental component score. Patients receiving fostamatinib 150 mg qd showed improvements in some PRO, including physical function. CONCLUSION: Patients treated with fostamatinib 100 mg bid showed significant improvements in HRQOL outcomes.

PMID: 23378467
Spleen Tyrosine Kinase Is Important in the Production of Proinflammatory Cytokines and Cell Proliferation in Human Mesangial Cells following Stimulation with IgA1 Isolated from IgA Nephropathy Patients.
Kim MJ, McDaid JP, McAdoo SP, Barratt J, Molyneux K, Masuda ES, Pusey CD, Tam FW. Source

J Immunol. 2012 Oct 1;189(7):3751-8.

Abstract
IgA immune complexes are capable of inducing human mesangial cell (HMC) activation, resulting in release of proinflammatory and profibrogenic mediators. The subsequent inflammation, cellular proliferation, and synthesis of extracellular matrix lead to the progression of IgA nephropathy (IgAN). Spleen tyrosine kinase (SYK) is an intracellular protein tyrosine kinase involved in cell signaling downstream of immunoreceptors. In this study, we determined whether SYK is involved in the downstream signaling of IgA1 stimulation in HMC, leading to production of proinflammatory cytokines/chemokines and cell proliferation. Incubation of HMC with IgA1 purified from IgAN patients significantly increased the synthesis of MCP-1 in a dose-dependent manner. There was also significantly increased production of IL-6, IL-8, IFN-γ-inducible protein-10, RANTES, and platelet-derived growth factor-BB. Stimulation of HMC with heat-aggregated IgA1 purified from IgAN patients induced significantly increased HMC proliferation. Both pharmacological inhibition of SYK and knockdown of SYK by small interfering RNA significantly reduced the synthesis of these mediators and inhibited HMC proliferation. Moreover, positive immunostaining for total and phospho-SYK in glomeruli of kidney biopsies from IgAN patients strongly suggests the involvement of SYK in the pathogenesis of IgAN. To our knowledge, we demonstrate, for the first time, the involvement of SYK in the downstream signaling of IgA1 stimulation in HMC and in the pathogenesis of IgAN. Hence, SYK represents a potential therapeutic target for IgAN.

PMID: 22956578
Inhibition of Syk activity by R788 in platelets prevents remote lung tissue damage after mesenteric ischemia-reperfusion injury.
Lapchak PH, Kannan L, Rani P, Pamuk ON, Ioannou A, Dalle Lucca JJ, Pine P, Tsokos GC.

Am J Physiol Gastrointest Liver Physiol. 2012 Jun;302(12):G1416-22.

Abstract
Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.

PMID: 22492694
Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors.
Singh R, Masuda ES, Payan DG.

J Med Chem. 2012 Apr 26;55(8):3614-43.

Introduction
Allergic and autoimmune disorders share significant functional overlap in the biologic pathways responsible for the activation of signal transduction events leading to production of numerous proinflammatory factors involved in disease initiation and progression. Given the reciprocal connections in these mechanistic pathways, it would be advantageous to target strategic master regulators with novel therapeutics to treat allergic and autoimmune diseases. One such crucial regulator is spleen tyrosine kinase (SYK), a member of the cytoplasmic protein tyrosine kinase (PTK) family.

PMID: 22257213
Fostamatinib, a syk-kinase inhibitor, does not affect methotrexate pharmacokinetics in patients with rheumatoid arthritis.
Baluom M, Samara E, Grossbard EB, Lau DT.

J Clin Pharmacol. 2011 Sep;51(9):1310-8.

Abstract
Fostamatinib (R788) is being investigated as an add-on therapy for the treatment of rheumatoid arthritis (RA) in patients with inadequate response to methotrexate (MTX). This study evaluated the potential pharmacokinetic interaction between R788 and MTX. Sixteen RA subjects on a stable weekly MTX regimen were enrolled and received MTX on days 1 and 8. Twelve subjects received 100 mg of R788 orally, and 4 subjects received a matching placebo twice daily from days 4 to 8 and once daily on days 3 and 9. Blood samples were collected on days 1 and 8 for MTX and 7-hydroxymethotrexate (7-OH-MTX), and days 3 and 9 for R788 and its active metabolite, R406. MTX and 7-OH-MTX pharmacokinetic parameters were similar on days 1 and 8. In the R788 group, the mean day 8 to day 1 ratios (90% confidence intervals) of maximum concentration and area under the plasma concentration-time curve estimates were 1.01 (0.85-1.20) and 1.12 (0.90-1.40) for MTX and 1.06 (0.82-1.35) and 1.06 (0.83-1.36) for 7-OH-MTX, respectively. Urinary excretion of MTX and 7-OH-MTX was also similar with or without R788, averaging 58% to 69% and 4% to 5% of the MTX dose, respectively. The data suggest that there is no clinically significant pharmacokinetic interaction of R788 and MTX in RA patients.

PMID: 21209239
The oral spleen tyrosine kinase inhibitor fostamatinib attenuates inflammation and atherogenesis in low-density lipoprotein receptor-deficient mice
Hilgendorf I, Eisele S, Remer I, Schmitz J, Zeschky K, Colberg C, Stachon P, Wolf D, Willecke F, Buchner M, Zirlik K, Ortiz-Rodriguez A, Lozhkin A, Hoppe N, von Zur Muhlen C, Zur Hausen A, Bode C, Zirlik A.

Arterioscler Thromb Vasc Biol. 2011 Sep;31(9):1991-9.

Abstract
OBJECTIVE: Spleen tyrosine kinase (SYK) has come into focus as a potential therapeutic target in chronic inflammatory diseases, such as rheumatoid arthritis and asthma, as well as in B-cell lymphomas. SYK has also been involved in the signaling of immunoreceptors, cytokine receptors, and integrins. We therefore hypothesized that inhibition of SYK attenuates the inflammatory process underlying atherosclerosis and reduces plaque development.

METHODS AND RESULTS: Low-density lipoprotein receptor-deficient mice consuming a high-cholesterol diet supplemented with 2 doses of the orally available SYK inhibitor fostamatinib for 16 weeks showed a dose-dependent reduction in atherosclerotic lesion size by up to 59±6% compared with the respective controls. Lesions of fostamatinib-treated animals contained fewer macrophages but more smooth muscle cells and collagen-characteristics associated with more stable plaques in humans. Mechanistically, fostamatinib attenuated adhesion and migration of inflammatory cells and limited macrophage survival. Furthermore, fostamatinib normalized high-cholesterol diet -induced monocytosis and inflammatory gene expression.

CONCLUSIONS: We present the novel finding that the SYK inhibitor fostamatinib attenuates atherogenesis in mice. Our data identify SYK inhibition as a potentially fruitful antiinflammatory therapeutic strategy in atherosclerosis.

PMID: 21700926
An oral Syk kinase inhibitor in the treatment of rheumatoid arthritis: A three-month randomized, placebo-controlled, phase II study in patients with active rheumatoid arthritis that did not respond to biologic agents. (TASKi3)
Genovese MC, Kavanaugh A, Weinblatt ME, Peterfy C, Dicarlo J, White ML, O'Brien M, Grossbard EB, Magilavy DB.

Arthritis Rheum. 2011 Feb;63(2):337-45.

OBJECTIVE: To assess the efficacy and safety of R788 (fostamatinib disodium), an inhibitor of spleen tyrosine kinase (Syk), in patients with active rheumatoid arthritis (RA) that did not respond to biologic therapies.

METHODS: A total of 219 patients with active RA in whom treatment with biologic agents had failed were enrolled in a 3-month multicenter, randomized, double-blind, placebo-controlled trial of R788. The primary end point was the percentage of patients who met the American College of Rheumatology 20% improvement criteria (achieved an ACR20 response) at month 3. Secondary end points included changes in inflammation and damage, as assessed by magnetic resonance imaging (MRI), and changes in the Disease Activity Score.

RESULTS: The ACR20 response in the R788 100 mg twice daily group was 38%, versus 37% in the placebo group, at month 3. No significant differences were achieved in the ACR20, ACR50, or ACR70 response levels at 3 months. There were differences between the groups from baseline to month 3 in the secondary end points C-reactive protein (CRP) level and synovitis score on MRI. There were baseline differences in steroid use, prior biologic use, and synovitis score on MRI between the R788 group and the placebo group that may have affected the outcomes. A high placebo response rate was seen in this trial, and exploratory analysis suggested that this may in part have been driven by patients who entered the trial with an elevated erythrocyte sedimentation rate but normal CRP level.

CONCLUSION: Our findings indicate that there were no differences in the primary end point between the R788 and placebo groups. Differences were observed between the R788 and placebo groups in secondary end points, particularly in those patients who entered the study with an elevated CRP level.

PMID: 21279990
An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis. (TASKi2)
Weinblatt ME, Kavanaugh A, Genovese MC, Musser TK, Grossbard EB, Magilavy DB.

N Engl J Med. 2010 Sep 30;363(14):1303-12.

Comment in: * N Engl J Med. 2010 Sep 30;363(14):1362-4.

Abstract

BACKGROUND: Spleen tyrosine kinase (Syk) is an important modulator of immune signaling. The objective of this phase 2 study was to evaluate the efficacy and safety of R788, an oral inhibitor of Syk, in patients with active rheumatoid arthritis despite methotrexate therapy.

METHODS: We enrolled 457 patients who had active rheumatoid arthritis despite long-term methotrexate therapy in a 6-month, double-blind, placebo-controlled trial. The primary outcome was the American College of Rheumatology (ACR) 20 response (which indicates at least a 20% reduction in the number of both tender and swollen joints and improvement in at least three of five other criteria) at month 6.

RESULTS: R788, at a dose of 100 mg twice daily and at a dose of 150 mg once daily, was significantly superior to placebo at month 6 (ACR 20 response rates of 67% and 57%, respectively, vs. 35%; P<0.001 for the comparison of both doses with placebo). It was also significantly superior with respect to ACR 50, which indicates at least a 50% improvement (43% and 32% vs. 19%; P<0.001 for the comparison of the 100-mg dose with placebo, P=0.007 for the comparison of the 150-mg dose with placebo) and ACR 70 (28% and 14% vs. 10%; P<0.001 for the comparison of the 100-mg dose with placebo, P=0.34 for the comparison of the 150-mg dose with placebo). A clinically significant effect was noted by the end of the first week of treatment. Adverse effects included diarrhea (in 19% of subjects taking the 100-mg dose of R788 vs. 3% of those taking placebo), upper respiratory infections (14% vs. 7%), and neutropenia (6% vs. 1%). R788 was associated with an increase in systolic blood pressure of approximately 3 mm Hg between baseline and month 1, as compared with a decrease of 2 mm Hg with placebo; 23% of the patients taking R788 vs. 7% of the patients receiving placebo required the initiation of or a change in antihypertensive therapy.

CONCLUSIONS: In this phase 2 study, a Syk inhibitor reduced disease activity in patients with rheumatoid arthritis; adverse events included diarrhea, hypertension, and neutropenia. Additional studies will be needed to further assess the safety and efficacy of Syk-inhibition therapy in patients with rheumatoid arthritis. (Funded by Rigel; ClinicalTrials.gov number, NCT00665925.)

PMID: 20879879

Spleen tyrosine kinase inhibition prevents tissue damage after ischemia-reperfusion.
Pamuk ON, Lapchak PH, Rani P, Pine P, Dalle Lucca JJ, Tsokos GC.

Am J Physiol Gastrointest Liver Physiol. 2010 Aug;299(2):G391-9. Epub 2010 Jun 3.

Reperfusion injury to tissue following an ischemic event occurs as a consequence of an acute inflammatory response that can cause significant morbidity and mortality. Components of both the innate (complement, immunoglobulin, monocytes, and neutrophils) and adaptive (B and T lymphocytes) immune systems have been demonstrated to mediate tissue injury. Spleen tyrosine kinase (Syk) is responsible for membrane-mediated signaling in various cell types including B lymphocytes, macrophages, and T cells. We investigated the ability of a small drug Syk inhibitor, R788, to protect mice against mesenteric ischemia-reperfusion (I/R)-induced local (intestine) and remote lung injury. Mice were fed with chow containing a Syk inhibitor for 6 days before the performance of intestinal I/R, which resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local and remote lung injury. The beneficial effect was associated with reduced IgM and complement 3 deposition in the tissues and significant reduction of polymorphonuclear cell infiltration. Our data place Syk upstream of events leading to the binding of natural antibodies to the ischemia-conditioned tissues and urge the consideration of the use of Syk inhibitors in the prevention or improvement of tissue injury of organs exposed to ischemia or hypoperfusion.

PMID: 20522642
Therapeutic Targeting of Syk in Autoimmune Diabetes.
Colonna L, Catalano G, Chew C, D'Agati V, Thomas JW, Wong FS, Schmitz J, Masuda ES, Reizis B, Tarakhovsky A, Clynes R.

J Immunol. 2010 Aug 1;185(3):1532-43.

In APCs, the protein tyrosine kinase Syk is required for signaling of several immunoreceptors, including the BCR and FcR. We show that conditional ablation of the syk gene in dendritic cells (DCs) abrogates FcgammaR-mediated cross priming of diabetogenic T cells in RIP-mOVA mice, a situation phenocopied in wild-type RIP-mOVA mice treated with the selective Syk inhibitor R788. In addition to blocking FcgammaR-mediated events, R788 also blocked BCR-mediated Ag presentation, thus broadly interrupting the humoral contributions to T cell-driven autoimmunity. Indeed, oral administration of R788 significantly delayed spontaneous diabetes onset in NOD mice and successfully delayed progression of early-established diabetes even when treatment was initiated after the development of glucose intolerance. At the DC level, R788 treatment was associated with reduced insulin-specific CD8 priming and decreased DC numbers. At the B cell level, R788 reduced total B cell numbers and total Ig concentrations. Interestingly, R788 increased the number of IL-10-producing B cells, thus inducing a tolerogenic B cell population with immunomodulatory activity. Taken together, we show by genetic and pharmacologic approaches that Syk in APCs is an attractive target in T cell-mediated autoimmune diseases such as type 1 diabetes.

PMID: 20601600
Suppression of skin and kidney disease by inhibition of spleen tyrosine kinase in lupus-prone mice.
Deng GM, Liu L, Bahjat FR, Pine PR, Tsokos GC.

Arthritis Rheum. 2010 Jul;62(7):2086-2092.

OBJECTIVE: Spleen tyrosine kinase (Syk) is involved in membrane-mediated signaling in various cells, including immune cells. It is overexpressed in T cells from patients with systemic lupus erythematosus (SLE), and its inhibition has been shown to improve T cell function as well as to improve disease manifestations in (NZB x NZW)F(1) lupus-prone mice and in patients with rheumatoid arthritis. While clinical trials examining Syk inhibition in patients with SLE are being considered, the aim of our experiments was to determine whether the therapeutic effects of Syk inhibition extend to other strains of lupus-prone mice and whether they result in improvement in skin disease and modification of established disease. METHODS: Female MRL/lpr or BAK/BAX mice were studied. Starting either at age 4 weeks (before disease) or at age 16 weeks (after established disease) and continuing for up to 16 weeks, mice were fed chow containing the Syk inhibitor R788 or control chow. RESULTS: We found that inhibition of Syk in MRL/lpr and BAK/BAX mice prevented the development of skin disease and significantly reduced established skin disease. Similarly, Syk inhibition reduced the size of the spleen and lymph nodes, suppressed the development of renal disease, and suppressed established renal disease. Discontinuation of treatment resulted in extended suppression of skin disease for at least 8 weeks and suppression of renal disease for 4 weeks. CONCLUSION: Syk inhibition suppresses the development of lupus skin and kidney disease in lupus-prone mice, suppresses established disease in lupus-prone mice, and may represent a valuable treatment for patients with SLE.

PMID: 20222110
Metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleen tyrosine kinase inhibitor R406 in humans: contribution of hepatic and gut bacterial processes to the overall biotransformation
Sweeny DJ, Li W, Clough J, Bhamidipati S, Singh R, Park G, Baluom M, Grossbard E, Lau DT.

Drug Metab Dispos. 2010 Jul;38(7):1166-76. Epub 2010 Apr 6.

The metabolism of the spleen tyrosine kinase inhibitor N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine (R406) and its oral prodrug N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine disodium hexahydrate (R788, fostamatinib) was determined in vitro and in humans. R788 was rapidly converted to R406 by human intestinal microsomes, and only low levels of R788 were observed in plasma of human subjects after oral administration of (14)C-R788. R406 was the major drug-related compound in plasma from human subjects, and only low levels of metabolites were observed in plasma. The plasma metabolites of R406 were identified as a sulfate conjugate and glucuronide conjugate of the para-O-demethylated metabolite of R406 (R529) and a direct N-glucuronide conjugate of R406. Elimination of drug-related material into the urine accounted for 19% of the administered dose, and the major metabolite in urine from all the human subjects was the lactam N-glucuronide of R406. On average, 80% of the total drug was recovered in feces. Two drug-related peaks were observed; one peak was identified as R406, and the other peak was identified as a unique 3,5-benzene diol metabolite of R406. The 3,5-benzene diol metabolite appeared to result from the subsequent O-demethylations and dehydroxylation of R529 by anaerobic gut bacteria because only R529 was converted to this metabolite after the in vitro incubation with human fecal samples. These data indicate that the major fecal metabolite of R406 observed in humans is a product of a hepatic cytochrome P450-mediated O-demethylation and subsequent O-demethylations and dehydroxylation by gut bacteria.


PMID: 20371637
Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia.

Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, Lacasce A, Schaefer-Cutillo J, De Vos S, Sinha R, Leonard JP, Cripe LD, Gregory SA, Sterba MP, Lowe AM, Levy R, Shipp MA.

Blood. 2010 Apr 1;115(13):2578-85. Epub 2009 Nov 17.

Comment in: Blood. 2010 Apr 1;115(13):2561-2.

Certain malignant B cells rely on B-cell receptor (BCR)-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the BCR signal. In in vivo analyses of B-cell lymphoma cell lines and primary tumors, Syk inhibition induces apoptosis. These data prompted a phase 1/2 clinical trial of fostamatinib disodium, the first clinically available oral Syk inhibitor, in patients with recurrent B-cell non-Hodgkin lymphoma (B-NHL). Dose-limiting toxicity in the phase 1 portion was neutropenia, diarrhea, and thrombocytopenia, and 200 mg twice daily was chosen for phase 2 testing. Sixty-eight patients with recurrent B-NHL were then enrolled in 3 cohorts: (1) diffuse large B-cell lymphoma (DLBCL), (2) follicular lymphoma (FL), and (3) other NHL, including mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma, lymphoplasmacytic lymphomas, and small lymphocytic leukemia/chronic lymphocytic leukemia (SLL/CLL). Common toxicities included diarrhea, fatigue, cytopenias, hypertension, and nausea. Objective response rates were 22% (5 of 23) for DLBCL, 10% (2 of 21) for FL, 55% (6 of 11) for SLL/CLL, and 11% (1/9) for MCL. Median progression-free survival was 4.2 months. Disrupting BCR-induced signaling by inhibiting Syk represents a novel and active therapeutic approach for NHL and SLL/CLL. This trial was registered at www.clinicaltrials.gov as #NCT00446095.

PMID: 19965662

A Spleen Tyrosine Kinase Inhibitor Reduces the Severity of Established Glomerulonephritis.

Smith J, McDaid JP, Bhangal G, Chawanasuntorapoj R, Masuda ES, Cook HT, Pusey CD, Tam FW.

J Am Soc Nephrol. 2010 Feb;21(2):231-236

Antibody-mediated glomerulonephritis, including that resulting from immune complexes, is an important cause of renal failure and is in need of more specific and effective treatment. Binding of antibody or immune complexes to Fc receptors activates intracellular signal transduction pathways, including spleen tyrosine kinase (Syk), leading to the production of inflammatory cytokines. We examined the effect of R788 (fostamatinib disodium), an oral prodrug of the selective Syk inhibitor R406, in nephrotoxic nephritis in Wistar-Kyoto rats. Treatment with R788 reduced proteinuria, tissue injury, glomerular macrophage and CD8+ cell numbers, and renal monocyte chemoattractant protein-1 (MCP-1) and IL-1beta, even when we started treatment after the onset of glomerulonephritis. When we administered R788 from days 4 to 10, glomerular crescents reduced by 100% (P < 0.01) compared with the vehicle group. When we administered R788 treatment from days 7 to 14, established glomerular crescents reversed (reduced by 21%, P < 0.001), and renal function was better than the vehicle group (P < 0.001). In vitro, R406 downregulated MCP-1 production from mesangial cells and macrophages stimulated with aggregated IgG. These results suggest that Syk is an important therapeutic target for the treatment of glomerulonephritis.

PMID: 19959716

Of mice and men: an open label pilot study for treatment of immune thrombocytopenic purpura (ITP) by an inhibitor of Syk.

Podolanczuk A, Lazarus AH, Crow AR, Grossbard E, Bussel JB.

Blood. 2009 Apr 2;113(14):3154-60. Epub 2008 Dec 18.
Comment in: Blood. 2009 Apr 2;113(14):3133-4.

To determine if inhibition of Syk would be useful in FcgammaR-dependent cytopenias such as immune thrombocytopenic purpura (ITP) or autoimmune hemolytic anemia (AHA), mouse models were employed to evaluate efficacy of R406, an inhibitor of Syk function, in treating cytopenia. Both disease models responded favorably to treatment, with amelioration of ITP being more dramatic of the two. Thus, Phase II clinical trial was initiated to study the effects of Syk inhibition in humans with ITP. Sixteen adults with chronic ITP were entered into an open label, single arm, cohort, dose escalation trial beginning with 75mg and escalating as high as 175mg twice daily. Doses were increased until either a persistent response was seen, toxicity occurred, or 175mg twice daily was reached. Eight patients achieved persistent responses with counts >50K on >67% (actually 95%) of their study visits, including three who had not persistently responded to thrombopoietic agents. Four others had non-sustained responses. The mean peak platelet count was >100,000/mm(3) in these 12 patients. Toxicity was primarily GI-related with diarrhea (urgency) and vomiting; 2 patients had transaminitis. In conclusion, inhibition of Syk was an efficacious means of increasing and maintaining the platelet count in half the patients with chronic refractory ITP. This trial is registered with ClinicalTrials.gov under Identifier NCT00706342.

PMID: 19096013

Mouse models of non-Hodgkins lymphoma reveal Syk as an important therapeutic target.

Young RM, Hardy IR, Clarke RL, Lundy N, Pine P, Turner BC, Potter TA, Refaeli Y.

Blood. 2009 Mar 12;113(11):2508-16. Epub 2008 Nov 3.
Comment in: Blood. 2009 Mar 12;113(11):2371.

We have generated mouse models of Non-Hodgkin's Lymphoma (NHL) that rely on the cooperation between MYC overexpression and B-cell antigen receptor (BCR) signaling for the initiation and maintenance of B-cell lymphomas. Using these mouse models of NHL, we have focused on the identification of BCR-derived signal effectors that are important for the maintenance of NHL tumors. In the present study we concentrate on Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase required to transduce BCR-dependent signals. Using a genetic approach, we show that Syk expression is required for the survival of murine NHL-like tumors in vitro and that tumor cells deficient in Syk fail to expand in vivo. In addition, a pharmacological inhibitor of Syk was able to induce apoptosis of transformed B-cells in vitro and led to tumor regression in vivo. Finally, we show that genetic or pharmacological inhibition of Syk activity in human NHL cell lines are generally consistent with results found in the mouse models, suggesting that targeting Syk may be a viable therapeutic strategy.

PMID: 18981293
Syk inhibitors as treatment for allergic rhinitis.

Masuda ES, Schmitz J.

Pulm Pharmacol Ther. 2008;21(3):461-7

Allergic rhinitis is characterized by a hypersensitive immune response in the upper airways to seasonal or perennial allergens leading to episodes of sneezing, itching, runny nose and nasal congestion. These symptoms are mainly the manifestations of a large number of mediators released by mast cells and basophils localized in the nasal mucosa, following their activation via allergen-specific immunoglubulin E (IgE) receptors. Current medications antagonize the action of distinct mediators such as histamine and leukotrienes for symptom relief, or block the production of pro-inflammatory cytokines to suppress allergic inflammation. Notably, rather than neutralizing individual mediators, Syk kinase inhibitors can block the allergen-induced release of all mast cell mediators and the production of most eicosanoids and cytokines. Thus, Syk kinase represents an attractive therapeutic target for acute and chronic allergic inflammation. Syk kinase inhibitors are now entering clinical trials. Using cell-based structure-activity relationships with primary human mast cells, a series of 2,4-diaminopyrimidine Syk kinase inhibitors was developed. One of these compounds, referred to as R112, exhibited suitable characteristics for intranasal delivery and was tested for safety and efficacy in allergic rhinitis patients. In a park environment, R112 showed remarkable amelioration of acute allergic rhinitis symptoms with rapid onset of action. These results demonstrate the clinical significance of inhibiting Syk in allergic upper airway disorders.

PMID: 17669674

Differential expression and molecular associations of Syk in systemic lupus erythematosus T cells.

Krishnan S, Juang YT, Chowdhury B, Magilavy A, Fisher CU, Nguyen H, Nambiar MP, Kyttaris V, Weinstein A, Bahjat R, Pine P, Rus V, Tsokos GC.

J Immunol. 2008 Dec 1;181(11):8145-52

Diminished expression of TCR zeta and reciprocal up-regulation and association of FcRgamma with the TCR/CD3 complex is a hallmark of systemic lupus erythematosus (SLE) T cells. In this study we explored whether differential molecular associations of the spleen tyrosine kinase Syk that preferentially binds to FcRgamma contribute to pathological amplification of signals downstream of this "rewired TCR" in SLE. We detected higher amounts of Syk expression and activity in SLE compared with normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, phospholipase C-gamma1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.

PMID: 19018007

Treatment of rheumatoid arthritis with a syk kinase inhibitor: A twelve-week, randomized, placebo-controlled trial.

Weinblatt ME, Kavanaugh A, Burgos-Vargas R, Dikranian AH, Medrano-Ramirez G, Morales-Torres JL, Murphy FT, Musser TK, Straniero N, Vicente-Gonzales AV, Grossbard E.


Arthritis Rheum. 2008 Nov;58(11):3309-18

OBJECTIVE: Spleen tyrosine kinase (Syk) has been identified as an important modulator of immune signaling in B cells and cells bearing Fcgamma-activating receptors. R788, a prodrug of active metabolite R406, has been shown to be an inhibitor of Syk kinase, active in a variety of in vitro and in vivo models, suggesting potential activity in the treatment of rheumatoid arthritis (RA). METHODS: We enrolled 189 patients with active RA despite methotrexate therapy in a 3-month, multicenter, ascending-dose, double-blind, placebo-controlled trial. The primary end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12. RESULTS: Twice-daily oral doses of 100 mg and 150 mg of R788 were significantly superior to placebo or twice-daily oral doses of 50 mg at week 12 (ACR20 achieved in 65% and 72% versus 38% and 32% of patients, respectively [P < 0.01]). ACR50 (achieved in 49% and 57% versus 19% and 17% of patients, respectively) and ACR70 (achieved in 33% and 40% versus 4% and 2% of patients, respectively) scores showed a similar pattern. Clinical effect was noted as early as 1 week after initiation of therapy. Reductions in serum interleukin-6 and matrix metalloproteinase 3 levels also occurred as early as week 1 in the groups receiving 100 mg and 150 mg R788. The major adverse effects were gastrointestinal side effects (predominantly diarrhea) and neutropenia (<1,500/mm(3)), both of which were dose related. CONCLUSION: These results indicate that an inhibitor of Syk kinase produces significant clinical benefits at 12 weeks in a population of patients with active RA receiving methotrexate therapy. Syk kinase may be an important new therapeutic target in RA and related autoimmune conditions.

PMID: 18975322
An orally bioavailable spleen tyrosine kinase inhibitor delays disease progression and prolongs survival in murine lupus.

Bahjat FR, Pine PR, Reitsma A, Cassafer G, Baluom M, Grillo S, Chang B, Zhao FF, Payan DG, Grossbard EB, Daikh DI.

Arthritis Rheum. 2008 Apr 25;58(5):1433-1444

OBJECTIVE: To assess whether R788, an orally bioavailable small molecule inhibitor of spleen tyrosine kinase (Syk)-dependent signaling, could modulate disease in lupus-prone (NZB x NZW)F(1) (NZB/NZW) mice via inhibition of Fc receptor (FcR) and B cell receptor signaling. METHODS: R788 was administered to NZB/NZW mice before and after disease onset. Proteinuria, blood urea nitrogen levels, and autoantibody titers were examined periodically, and overall survival and renal pathologic features were assessed following long-term treatment (24-34 weeks). The distribution and immunophenotype of various splenic T cell and B cell subpopulations were evaluated at the time of study termination. Arthus responses in NZB/NZW mice pretreated with R788 or Fc-blocking antibody (anti-CD16/32) were also examined. RESULTS: When R788 was administered prior to or after disease onset, it delayed the onset of proteinuria and azotemia, reduced renal pathology and kidney infiltrates, and significantly prolonged survival of lupus-prone NZB/NZW mice; autoantibody titers were minimally affected throughout the study. Dose-dependent reductions in the numbers of CD4+ activated T cells expressing high levels of CD44 or CD69 were apparent in spleens from R788-treated mice. Minimal effects on the numbers of naive T cells expressing CD62 ligand and total CD8+ T cells per spleen were observed following long-term drug treatment. R788 pretreatment resulted in reduced Arthus responses in NZB/NZW mice, similar to results obtained in mice pretreated with FcR-blocking antibody. CONCLUSION: We demonstrate that a novel Syk-selective inhibitor prevents the development of renal disease and treats established murine lupus nephritis. These data suggest that Syk inhibitors may be of therapeutic benefit in human lupus and related disorders.

PMID: 18438845

Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor.

Pine PR, Chang B, Schoettler N, Banquerigo ML, Wang S, Lau A, Zhao F, Grossbard EB, Payan DG, Brahn E.

Clin Immunol. 2007 Sep;124(3):244-57. Epub 2007 May 29.

Spleen tyrosine kinase (Syk), a key mediator of immunoreceptor signaling in inflammatory cells, is essential for immune complex-mediated signal transduction initiated by activated receptors for immunoglobulin G. In collagen-induced arthritis, R788/R406, a novel and potent small molecule Syk inhibitor suppressed clinical arthritis, bone erosions, pannus formation, and synovitis. Serum anti-collagen type II antibody levels were unaltered, while the half-life of exogenous antibody was extended when co-administered with R406. Expression of the targeted kinase (Syk) in synovial tissue correlated with the joint level of inflammatory cell infiltrates and was virtually undetectable in treated rats. Syk inhibition suppressed synovial cytokines and cartilage oligomeric matrix protein (COMP) in serum, suggesting a sensitive and reliable biomarker for R406 activity. These results highlight the role of activating Fcgamma receptors in inflammatory synovitis and suggest that interruption of the signaling cascade with a novel Syk inhibitor may be a useful addition to immunosuppressive disease-modifying anti-rheumatic drugs currently used in the treatment of human autoimmune diseases such as rheumatoid arthritis.

PMID: 17537677

R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation.

Braselmann S, Taylor V, Zhao H, Wang S, Sylvain C, Baluom M, Qu K, Herlaar E, Lau A, Young C, Wong BR, Lovell S, Sun T, Park G, Argade A, Jurcevic S, Pine P, Singh R, Grossbard EB, Payan DG, Masuda ES.

J Pharmacol Exp Ther. 2006 Dec;319(3):998-1008. Epub 2006 Aug 31.

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs).

Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM. Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR).

R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models.

Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

PMID: 16946104

Inhibition of spleen tyrosine kinase prevents mast cell activation and airway hyperresponsiveness.

Matsubara S, Li G, Takeda K, Loader JE, Pine P, Masuda ES, Miyahara N, Miyahara S, Lucas JJ, Dakhama A, Gelfand EW.

Am J Respir Crit Care Med. 2006 Jan 1;173(1):56-63. Epub 2005 Sep 28.

RATIONALE: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling.

OBJECTIVE: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo.

METHODS: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen.

RESULTS: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge.

CONCLUSION: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.

JAK Inhbitors & related projects:

The Selective JAK1/3-Inhibitor R507 Mitigates Obliterative Airway Disease Both With Systemic Administration and Aerosol Inhalation
Deuse T, Hua X, Stubbendorff M, Spin JM, Neofytou E, Taylor V, Chen Y, Park G, Fink JB, Renne T, Kiefmann M, Kiefmann R, Reichenspurner H, Robbins RC, Schrepfer S.

Transplantation. 2016 May;100(5):1022-31.

Abstract
BACKGROUND: The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models. METHODS: Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells. RESULTS: In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1β. CONCLUSIONS: The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.

PMID: 26910327
Janus Kinase Inhibition Prevents Cancer- and Myocardial Infarction-Mediated Diaphragm Muscle Weakness in Mice.
Smith IJ, Roberts B, Beharry A, Godinez GL, Payan DG, Kinsella TM, Judge AR, Ferreira LF.

Am J Physiol Regul Integr Comp Physiol. 2016 Apr 15;310(8):R707-10.

Abstract
Respiratory dysfunction is prevalent in critically ill patients, and can lead to adverse clinical outcomes including respiratory failure and increased mortality. Respiratory muscles, which normally sustain respiration through inspiratory muscle contractions, become weakened during critical illness, and recent studies suggest that respiratory muscle weakness is related to systemic inflammation. Here we investigate the pathophysiological role of the inflammatory JAK 1/3 signaling pathway in diaphragm weakness in two distinct experimental models of critical illness. In the first experiment, mice received subcutaneous injections of PBS or C26 cancer cells, and were fed chow formulated with or without the JAK 1/3 inhibitor R548 for twenty-six days. Diaphragm specific force was significantly reduced in tumor bearing mice receiving standard chow, however treatment with the JAK 1/3 inhibitor completely prevented diaphragm weakness. Diaphragm cross-sectional area was diminished by approximately 25% in tumor bearing mice, but was similar to healthy mice in tumor-bearing animals treated with R548. In the second study, mice received sham surgery or coronary artery ligation leading to myocardial infarction (MI), and were treated with R548 or vehicle one-hour post-surgery, and once daily for three days. Diaphragm specific force was comparable between sham-surgery/vehicle, sham-surgery/R548 and MI/R548 groups, but significantly decreased in the MI/vehicle group. Markers of oxidative damage and activated caspase-3, mechanisms previously identified to reduce muscle contractility, were not elevated in diaphragm extracts. These experiments implicate JAK 1/3 signaling in cancer- and MI-mediated diaphragm weakness in mice, and provide a compelling case for further investigation.

PMID: 26864813
The JAK-STAT Pathway is Critical in Ventilator-induced Diaphragm Dysfunction
Tang H, Smith IJ, Hussain SN, Goldberg P, Lee M, Sugiarto S, Godinez GL, Singh BK, Payan DG, Rando TA, Kinsella TM, Shrager JB.

Mol Med. 2014;20:579-89.

Abstract
Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life-saving in many critically ill patients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongs patients' need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We report here that the JAK-STAT pathway is activated in MV human diaphragm, as evidenced by significantly increased phosphorylation of JAK and STAT. Blockage of the JAK-STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm muscle contractile dysfunction (by ~85%, p<0.01). We further demonstrate that activated STAT3 compromises mitochondrial function and induces oxidative stress in vivo, and interestingly that oxidative stress also activates JAK-STAT. Inhibition of JAK-STAT prevents oxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells. Therefore, in ventilated diaphragm muscle, activation of JAK-STAT is critical in regulating oxidative stress and is thereby central to the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise of JAK-STAT inhibitors in ventilated ICU patients.

PMID: 25286450
Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction
Smith IJ, Godinez GL, Singh BK, McCaughey KM, Alcantara RR, Gururaja T, Ho MS, Nguyen HN, Friera AM, White KA, McLaughlin JR, Hansen D, Romero JM, Baltgalvis KA, Claypool MD, Li W, Lang W, Yam GC, Gelman MS, Ding R, Yung SL, Creger DP, Chen Y, Singh R, Smuder AJ, Wiggs MP, Kwon OS, Sollanek KJ, Powers SK, Masuda ES, Taylor VC, Payan DG, Kinoshita T, Kinsella TM.

FASEB J. 2014 Jul 1;28(7):2790-2803.

Abstract
Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.

PMID: 24671708
Janus kinase 1/3 signaling pathways are key initiators of TH2 differentiation and lung allergic responses.
Ashino S, Takeda K, Li H, Taylor V, Joetham A, Pine PR, Gelfand EW.

J Allergy Clin Immunol. 2014 Apr;133(4):1162-1174.

BACKGROUND:
Janus kinases (JAKs) are regulators of signaling through cytokine receptors. The importance of JAK1/3 signaling on TH2 differentiation and development of lung allergic responses has not been investigated.

OBJECTIVE:
We sought to examine a selective JAK1/3 inhibitor (R256) on differentiation of TH subsets in vitro and on development of ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and inflammation in an experimental model of asthma.

METHODS:
A selective JAK1/3 inhibitor was used to assay the importance of this pathway on induction of TH1, TH2, and TH17 differentiation in vitro. In vivo, the effects of inhibiting JAK1/3 signaling were examined by administering the inhibitor during the sensitization or allergen challenge phases in the primary challenge model or just before provocative challenge in the secondary challenge model. Airway inflammation and AHR were examined after the last airway challenge.

RESULTS:
In vitro, R256 inhibited differentiation of TH2 but not TH1 or TH17 cells, which was associated with downregulation of signal transducer and activator of transcription (STAT) 6 and STAT5 phosphorylation. However, once polarized, TH2 cells were unaffected by the inhibitor. In vivo, R256 administered during the OVA sensitization phase prevented the development of AHR, airway eosinophilia, mucus hypersecretion, and TH2 cytokine production without changes in TH1 and TH17 cytokine levels, indicating that selective blockade of TH2 differentiation was critical. Inhibitor administration after OVA sensitization but during the challenge phases in the primary or secondary challenge models similarly suppressed AHR, airway eosinophilia, and mucus hypersecretion without any reduction in TH2 cytokine production, suggesting the inhibitory effects were downstream of TH2 cytokine receptor signaling pathways.

CONCLUSIONS:
Targeting the TH2-dependent JAK/STAT activation pathway represents a novel therapeutic approach for the treatment of asthma.

PMID: 24365136
Significant Reduction of Acute Cardiac Allograft Rejection by Selective Janus Kinase-1/3 Inhibition Using R507 and R545.
Deuse T, Hua X, Taylor V, Stubbendorff M, Baluom M, Chen Y, Park G, Velden J, Streichert T, Reichenspurner H, Robbins RC, Schrepfer S.

Transplantation. 2012 Oct 15;94(7):695-702.

Abstract
BACKGROUND: Selective inhibition of lymphocyte activation through abrogation of signal 3-cytokine transduction emerges as a new strategy for immunosuppression. This is the first report on the novel Janus kinase (JAK)1/3 inhibitors R507 and R545 for prevention of acute allograft rejection. METHODS: Pharmacokinetic and in vitro enzyme inhibition assays were performed to characterize the drugs. Heterotopic Brown Norway-Lewis heart transplantations were performed to study acute cardiac allograft rejection, graft survival, suppression of cellular host responsiveness, and antibody production. Therapeutic and subtherapeutic doses of R507 (60 and 15 mg/kg 2 times per day) and R545 (20 and 5 mg/kg 2 times per day) were compared with those of tacrolimus (Tac; 4 and 1 mg/kg once per day). RESULTS: Plasma levels of R507 and R545 were sustained high for several hours. Cell-based enzyme assays showed selective inhibition of JAK1/3-dependent pathways with 20-fold or greater selectivity over JAK2 and Tyrosine kinase 2 kinases. After heart transplantation, both JAK1/3 inhibitors reduced early mononuclear graft infiltration, even significantly more potent than Tac. Intragraft interferon-γ release was significantly reduced by R507 and R545, and for interleukin-10 suppression, they were even significantly more potent than Tac. Both JAK1/3 inhibitors and Tac were similarly effective in reducing the host Th1 and Th2, but not Th17, responsiveness and similarly prevented donor-specific immunoglobulin M antibody production. Subtherapeutic and therapeutic R507 and R545 doses prolonged the mean graft survival and were similarly effective as 1 and 4 mg/kg Tac, respectively. In combination regimens, however, only R507 showed highly beneficial synergistic drug interactions with Tac. CONCLUSIONS: Both R507 and R545 are potent novel immunosuppressants with favorable pharmacokinetics and high JAK1/3 selectivity, but only R507 synergistically interacts with Tac.

PMID: 22971540
R723, a selective JAK2 inhibitor, effectively treats JAK2V617F-induced murine myeloproliferative neoplasm.
Shide K, Kameda T, Markovtsov V, Shimoda HK, Tonkin E, Fang S, Liu C, Gelman M, Lang W, Romero J, McLaughlin J, Bhamidipati S, Clough J, Low C, Reitsma A, Siu S, Pine P, Park G, Torneros A, Duan M, Singh R, Payan DG, Matsunaga T, Hitoshi Y, Shimoda K.

Blood. 2011 Jun 23;117(25):6866-75.

Abstract
The activating mutations in the Janus kinase 2 (JAK2), including JAK2V617F, which have been described in patients with myeloproliferative neoplasms (MPNs), link directly to MPN pathogenesis. We have developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% (IC50) at a concentration of 2 nM, while having minimal effects on JAK3, TYK2 and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukaemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival, and decreased tumor burden. In JAK2V617F transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepato-splenomegaly, leukocytosis and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway, rather than JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared to its effects on myeloid cells. Overall, our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F positive MPNs.

PMID: 21531978
JAK3 inhibition significantly attenuates psoriasiform skin inflammation in CD18 mutant PL/J mice.

Chang BY, Zhao F, He X, Ren H, Braselmann S, Taylor V, Wicks J, Payan DG, Grossbard EB, Pine PR, Bullard DC.

J Immunol. 2009 Aug 1;183(3):2183-92

JAK3, a member of the Janus kinase family, is predominantly expressed in hemopoietic cells and binds specifically to the common gamma chain of a subfamily of cytokine receptors that includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies suggest that this tyrosine kinase plays key roles in mediating T cell functions, and inhibition of JAK3 has been shown to prevent graft rejection and decrease the severity of arthritis in rodent models. However, the functions of JAK3 in the development of skin immune responses and diseases such as psoriasis have not been determined. CD18 mutant PL/J mice develop spontaneous T cell-dependent psoriasiform skin disease with several similarities to human psoriasis. In this study, we treated mice with established skin disease with R348, a small molecule inhibitor of JAK3, and observed a marked attenuation of skin lesions following 6 wk of treatment. Histological analyses revealed major reductions of both epidermal and dermal lesion severity scores in R348-treated CD18-deficient PL/J mice compared with vehicle controls, which was associated with decreased CD4(+) T cell infiltration. In addition, systemic levels of IL-17, IL-22, IL-23, and TNF-alpha were significantly lower in mice receiving the compound, and T cells isolated from R348-treated mice also showed reduced phosphorylation of Stat5 after stimulation with IL-2. These findings suggest that small-molecule inhibitors of JAK3 may be useful in the treatment of inflammatory skin diseases such as psoriasis and strongly implicate JAK signaling events as important in the pathogenesis of this disease.

PMID: 19596999

A novel JAK3 inhibitor, R348, attenuates chronic airway allograft rejection.

Velotta JB, Deuse T, Haddad M, Masuda E, Park G, Carroll D, Taylor V, Robbins RC, Schrepfer S.

Transplantation. 2009 Mar 15;87(5):653-9.

BACKGROUND: This study aimed at investigating the role of a novel JAK3 inhibitor, R348, in the prevention of chronic airway allograft rejection. METHODS: The heterotopic rat trachea transplant model was used. Recipients were treated daily with R348 (10, 20, 40, 80 mg/kg) or rapamycin (0.75 or 3 mg/kg). Blood levels of R348 and of its active metabolite R333 were measured. Grafts were harvested after 28 days to analyze epithelial morphology, mononuclear infiltration, and luminal obliteration. Plasma levels of circulating donor strain-reactive IgG antibodies were quantified. CONCLUSIONS: R348 is an effective drug, and it is expected to be introduced into clinical transplant pharmacology soon.


PMID: 19295308

Rigel Oncology:

The Axl receptor tyrosine kinase is an adverse prognostic factor and a therapeutic target in esophageal adenocarcinoma
Alvarez H, Montgomery EA, Karikari C, Canto M, Dunbar KB, Wang JS, Feldmann G, Hong SM, Haffner MC, Meeker AK, Holland SJ, Yu J, Heckrodt TJ, Zhang J, Ding P, Goff D, Singh R, Roa JC, Marimuthu A, Riggins GJ, Eshleman JR, Nelkin BD, Pandey A, Maitra A.

Cancer Biol Ther. 2010 Nov 15;10(10):1013-22.

Abstract
Esophageal adenocarcinoma (EAC) arises in the backdrop of reflux-induced metaplastic phenomenon known as Barrett esophagus. The prognosis of advanced EAC is dismal, and there is an urgent need for identifying molecular targets for therapy. Serial Analysis of Gene Expression (SAGE) was performed on metachronous mucosal biopsies from a patient who underwent progression to EAC during endoscopic surveillance. SAGE confirmed significant upregulation of Axl "tags" during the multistep progression of Barrett esophagus to EAC. In a cohort of 92 surgically resected EACs, Axl overexpression was associated with shortened median survival on both univariate (p < 0.004) and multivariate (p < 0.036) analysis. Genetic knockdown of Axl receptor tyrosine kinase (RTK) function was enabled in two EAC lines (OE33 and JH-EsoAd1) using lentiviral short hairpin RNA (shRNA). Genetic knockdown of Axl in EAC cell lines inhibited invasion, migration and in vivo engraftment, which was accompanied by downregulation in the activity of the Ral GTPase proteins (RalA and RalB). Restoration of Ral activation rescued the transformed phenotype of EAC cell lines, suggesting a novel effector mechanism for Axl in cancer cells. Pharmacological inhibition of Axl was enabled using a small molecule antagonist, R428 (Rigel Pharmaceuticals). Pharmacological inhibition of Axl with R428 in EAC cell lines significantly reduced anchorage-independent growth, invasion and migration. Blockade of Axl function abrogated phosphorylation of ERBB2 (Her-2/neu) at the Tyr877 residue, indicative of receptor crosstalk. Axl RTK is an adverse prognostic factor in EAC. The availability of small molecule inhibitors of Axl function provides a tractable strategy for molecular therapy of established EAC.

PMID: 20818175
Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia.

Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, Lacasce A, Schaefer-Cutillo J, De Vos S, Sinha R, Leonard JP, Cripe LD, Gregory SA, Sterba MP, Lowe AM, Levy R, Shipp MA.

Blood. 2010 Apr 1;115(13):2578-85.

Comment in: Blood. 2010 Apr 1;115(13):2561-2.

Certain malignant B cells rely on B-cell receptor (BCR)-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the BCR signal. In in vivo analyses of B-cell lymphoma cell lines and primary tumors, Syk inhibition induces apoptosis. These data prompted a phase 1/2 clinical trial of fostamatinib disodium, the first clinically available oral Syk inhibitor, in patients with recurrent B-cell non-Hodgkin lymphoma (B-NHL). Dose-limiting toxicity in the phase 1 portion was neutropenia, diarrhea, and thrombocytopenia, and 200 mg twice daily was chosen for phase 2 testing. Sixty-eight patients with recurrent B-NHL were then enrolled in 3 cohorts: (1) diffuse large B-cell lymphoma (DLBCL), (2) follicular lymphoma (FL), and (3) other NHL, including mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma, lymphoplasmacytic lymphomas, and small lymphocytic leukemia/chronic lymphocytic leukemia (SLL/CLL). Common toxicities included diarrhea, fatigue, cytopenias, hypertension, and nausea. Objective response rates were 22% (5 of 23) for DLBCL, 10% (2 of 21) for FL, 55% (6 of 11) for SLL/CLL, and 11% (1/9) for MCL. Median progression-free survival was 4.2 months. Disrupting BCR-induced signaling by inhibiting Syk represents a novel and active therapeutic approach for NHL and SLL/CLL. This trial was registered at www.clinicaltrials.gov as #NCT00446095.

PMID: 19965662

R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer.

Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, Duan M, Torneros A, Yu J, Heckrodt TJ, Zhang J, Ding P, Apatira A, Chua J, Brandt R, Pine P, Goff D, Singh R, Payan DG, Hitoshi Y.

Cancer Res. 2010 Feb 15;70(4):1544-54.

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.


PMID: 20145120
Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth.

Mol Biol Cell. 2005 Dec;16(12):5621-9. Epub 2005 Sep 29.

Jenkins Y, Markovtsov V, Lang W, Sharma P, Pearsall D, Warner J, Franci C, Huang B, Huang J, Yam GC, Vistan JP, Pali E, Vialard J, Janicot M, Lorens JB, Payan DG, Hitoshi Y.

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

Multiple roles for the receptor tyrosine kinase axl in tumor formation.

Holland SJ, Powell MJ, Franci C, Chan EW, Friera AM, Atchison RE, McLaughlin J, Swift SE, Pali ES, Yam G, Wong S, Lasaga J, Shen MR, Yu S, Xu W, Hitoshi Y, Bogenberger J, Nor JE, Payan DG, Lorens JB.

Cancer Res. 2005 Oct 15;65(20):9294-303.

A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA-mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.

MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha.

Ge B, Gram H, Di Padova F, Huang B, New L, Ulevitch RJ, Luo Y, Han J.

Science 2002 Feb 15;295(5558):1291-4.

Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.

p15(PAF), a novel PCNA associated factor with increased expression in tumor tissues.

Yu P, Huang B, Shen M, Lau C, Chan E, Michel J, Xiong Y, Payan DG, Luo Y.

Oncogene 2001 Jan 25;20(4):484-9.

Proliferating cell nuclear antigen (PCNA) is an essential protein in both DNA replication and DNA damage repair. A novel 15 kD protein, p15(PAF), was identified as a PCNA-associated factor in a yeast two-hybrid screen using PCNA as the bait. p15(PAF) is localized primarily in the nucleus. p15(PAF) shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21. Overexpression of p15(PAF) competes with p21-PCNA binding. Mutation of this motif in p15(PAF) abolished its PCNA-binding activity. Notably, p15(PAF) expression in several types of tumor tissues was significantly increased, especially in esophageal tumors. Like PCNA, p15(PAF) may possess prognostic significance in a broad array of human cancers.

TNIK, a novel member of the germinal center kinase family that activates the c-Jun N-terminal kinase pathway and regulates the cytoskeleton.

Fu CA, Shen M, Huang BC, Lasaga J, Payan DG, Luo Y.

J Biol Chem 1999 Oct 22;274(43):30729-37.

Germinal center kinases (GCKs) compose a subgroup of the Ste20 family of kinases. Here we describe the cloning and characterization of a novel GCK family kinase, Traf2- and Nck-interacting kinase (TNIK) that interacts with both Traf2 and Nck. TNIK encodes a polypeptide of 1360 amino acids with eight spliced isoforms. It has 90% amino acid identity to the Nck-interacting kinase in both the N-terminal kinase domain and the C-terminal germinal center kinase homology region. The homology drops to 53% in the intermediate region. TNIK specifically activates the c-Jun N-terminal kinase pathway when transfected in Phoenix-A cells (derivatives of 293 cells), similar to many GCKs. However, in contrast to other GCKs, this activation is mediated solely by the GCK homology region of TNIK. In addition, in Phoenix-A, NIH-3T3, and Hela cells, overexpression of wild type TNIK, but not the kinase mutant form of TNIK, results in the disruption of F-actin structure and the inhibition of cell spreading. Furthermore, TNIK can phosphorylate Gelsolin in vitro. This is the first time that a GCK family kinase is shown to be potentially involved in the regulation of cytoskeleton.

Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkB

Curr Biol 1999 May 20;9(10):539-42.

The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death signaling. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2, and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation, but not the NFkB activation induced by TNFa. Therefore, RIP3 appears to function as an intermediary in TNFa-induced apoptosis.

Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells.

Hitoshi Y, Lorens J, Kitada SI, Fisher J, LaBarge M, Ring HZ, Francke U, Reed JC, Kinoshita S, Nolan GP.

Immunity 1998 Apr;8(4):461-71.

Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library-based functional cloning we identified a gene, toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.

PKC inhibitors & related projects:

Pharmacologic inhibition of PKCα and PKCθ prevents GVHD while preserving GVL activity in mice
Haarberg KM, Li J, Heinrichs J, Wang D, Liu C, Bronk CC, Kaosaard K, Owyang AM, Holland S, Masuda E, Tso K, Blazar BR, Anasetti C, Beg AA, Yu XZ.

Blood. 2013 Oct 3;122(14):2500-11.

Abstract
Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell mediated graft-versus-leukemia (GVL) effects, but often leads to severe graft-versus-host disease (GVHD). Given that PKCθ, in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα-/-/θ-/- donor T cells to induce GVHD was further reduced compared to PKCθ-/- T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, chemokine/cytokine production and significantly decreased GVHD in myeloablative pre-clinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.

PMID: 23908466

Rigel Biology:

The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice
Mejia GL, Asiedu MN, Hitoshi Y, Dussor G, Price TJ.

Pain Reports. 2016 Aug;1(1):e562.

Abstract
There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK) can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG) neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF)-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.
The AMPK activator R419 improves exercise capacity and skeletal muscle insulin sensitivity in obese mice
Marcinko K, Bujak AL, Lally JSV, Ford RJ, Wong TH, Smith BK, Kemp BE, Jenkins Y, Li W, Kinsella TM, Hitoshi Y, Steinberg GR.

Mol Metab. 2015 Sept;4(9):643-51.

Abstract
Objective: Skeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects. Methods: Glucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK β1β2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation. Results: There was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice. Conclusions: Treatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity.
Global metabolite profiling of mice with high-fat diet-induced obesity chronically treated with AMPK activators R118 or metformin reveals tissue-selective alterations in metabolic pathways.
Jenkins Y, Sun TQ, Li Y, Markovtsov V, Uy G, Gross L, Goff DA, Shaw SJ, Boralsky L, Singh R, Payan DG, Hitoshi Y.

BMC Res Notes. 2014 Sep 25;7(1):674.

Abstract
BACKGROUND: The novel small molecule R118 and the biguanide metformin, a first-line therapy for type 2 diabetes (T2D), both activate the critical cellular energy sensor 5'-AMP-activated protein kinase (AMPK) via modulation of mitochondrial complex I activity. Activation of AMPK results in both acute responses and chronic adaptations, which serve to restore energy homeostasis. Metformin is thought to elicit its beneficial effects on maintenance of glucose homeostasis primarily though impacting glucose and fat metabolism in the liver. Given the commonalities in their mechanisms of action and that R118 also improves glucose homeostasis in a murine model of T2D, the effects of both R118 and metformin on metabolic pathways in vivo were compared in order to determine whether R118 elicits its beneficial effects through similar mechanisms. RESULTS: Global metabolite profiling of tissues and plasma from mice with diet-induced obesity chronically treated with either R118 or metformin revealed tissue-selective effects of each compound. Whereas metformin treatment resulted in stronger reductions in glucose and lipid metabolites in the liver compared to R118, upregulation of skeletal muscle glycolysis and lipolysis was apparent only in skeletal muscle from R118-treated animals. Both compounds increased β-hydroxybutyrate levels, but this effect was lost after compound washout. Metformin, but not R118, increased plasma levels of metabolites involved in purine metabolism. CONCLUSIONS: R118 treatment but not metformin resulted in increased glycolysis and lipolysis in skeletal muscle. In contrast, metformin had a greater impact than R118 on glucose and fat metabolism in liver tissue.

PMID: 25252968
Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice.
Baltgalvis KA, White K, Li W, Claypool MD, Lang W, Alcantara R, Singh BK, Friera AM, McLaughlin J, Hansen D, McCaughey K, Nguyen H, Smith IJ, Godinez G, Shaw SJ, Goff D, Singh R, Markovtsov V, Sun TQ, Jenkins Y, Uy G, Li Y, Pan A, Gururaja T, Lau D, Park G, Hitoshi Y, Payan DG, Kinsella TM.

Am J Physiol Heart Circ Physiol. 2014 Apr 15;306(8):H1128-45.

Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary endpoint has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5' AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator increased voluntary wheel running activity, decreased muscle fatigability and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability and decreased circulating levels of the endogenous endothelial nitric oxide synthase (eNOS) inhibitor, asymmetric dimethylarginine (ADMA). These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.

PMID: 24561866
Noninvasive Imaging of In Vivo MuRF1 Expression during Muscle Atrophy.
Li W, Claypool MD, Friera AM, McLaughlin J, Baltgalvis KA, Smith IJ, Kinoshita T, White K, Lang W, Godinez G, Payan DG, Kinsella TM.

PLoS One. 2014 Apr 7;9(4):e94032.

Abstract:
Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3' untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.

PMID: 24710205
AMPK Activation through Mitochondrial Regulation Results in Increased Substrate Oxidation and Improved Metabolic Parameters in Models of Diabetes.
Jenkins Y, Sun TQ, Markovtsov V, Foretz M, Li W, Nguyen H, Li Y, Pan A, Uy G, Gross L, Baltgalvis K, Yung SL, Gururaja T, Kinoshita T, Owyang A, Smith IJ, McCaughey K, White K, Godinez G, Alcantara R, Choy C, Ren H, Basile R, Sweeny DJ, Xu X, Issakani SD, Carroll DC, Goff DA, Shaw SJ, Singh R, Boros LG, Laplante MA, Marcotte B, Kohen R, Viollet B, Marette A, Payan DG, Kinsella TM, Hitoshi Y.

PLoS One. 2013 Dec 5;8(12):e81870. doi:10.1371/journal.pone.0081870.

Abstract
Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both 13C-palmitate and 13C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.

PMID: 24339975

Rigel Technology & Tools:

Synthesis of trans-2-(Trifluoromethyl)cyclopropanes via Suzuki Reactions with an N-Methyliminodiacetic Acid Boronate
Duncton MA, Singh R.

Org Lett. 2013 Sep 6;15(17):4284-7.

Abstract
trans-2-(Trifluoromethyl)cyclopropylboronic acid N-methyliminodiacetic acid (MIDA) ester 5 was synthesized as a pure diastereomer from vinylboronic acid MIDA ester and (trifluoromethyl)diazomethane in a single step. An X-ray study confirmed the trans-stereochemistry around the cyclopropyl ring. Use of 5 in Suzuki reactions, with a variety of aryl or heteroaryl coupling partners, provided trans-2-(trifluoromethyl)cyclopropyl products in moderate to excellent yields (17-90%).

PMID: 23952128
Enantioselective Synthesis of cis-3-Fluoropiperidin-4-ol, a Building Block for Medicinal Chemistry.
Shaw SJ, Goff DA, Boralsky LA, Irving M, Singh R.

J Org Chem. 2013 Sep 6;78(17):8892-7.

Abstract
The first enantioselective route to both enantiomers of cis-1-Boc-3-fluoropiperidin-4-ol, a highly prized building block for medicinal chemistry, is reported. An enantioselective fluorination is employed, taking advantage of the methodology reported by MacMillan, which uses a modified cinchona alkaloid catalyst. In studying the fluorination reaction, we have shown that the catalyst can be replaced by commercially available primary amines, including α-methylbenzylamine, with similar levels of enantioselectivity. The piperidinols are readily crystallized to obtain enantiopure material.

PMID: 23957501
Diverse alkaloid-like structures from a common building block.
Goff DA.

Tetrahedron. 2013 Jan 7;69(1):242-56.

Abstract
A wealth of unique enantiopure polycyclic alkaloid-like scaffolds can be prepared on a multigram scale in only a few steps from a common, commercially available intermediate. The attached nitromethyl group can then be used to construct highly diverse functionalized libraries suitable for screening against biological targets of interest.
Cellular localization and antiproliferative effect of peptides discovered from a functional screen of a retrovirally delivered random peptide library.

Hitoshi Y, Gururaja T, Pearsall DM, Lang W, Sharma P, Huang B, Catalano SM, McLaughlin J, Pali E, Peelle B, Vialard J, Janicot M, Wouters W, Luyten W, Bennett MK, Anderson DC, Payan DG, Lorens JB, Bogenberger J, Demo S.

Chem Biol. 2003 Oct;10(10):975-987.

We have generated a random peptide library fused to GFP in a retroviral vector system and used this library to screen for peptides inhibiting tumor cell growth. Four unique peptide sequences were isolated that exhibited antiproliferative effects and that specifically localized to the plasma membrane and cytoplasmic granular compartments. Mutational analysis revealed critical residues in each peptide sequence and demonstrated a correlation between peptide subcellular localization and antiproliferative activity. Synthetic analogs of the peptides with poly-lysine internalization sequences, but not loss-of-function mutant peptides, competed for subcellular localization of the parent GFP-fused peptides. The synthetic peptides exhibited dose-dependent antiproliferative effects in tumor cells, while mutant peptides had no effect. Our screening approach using retrovirally expressed intracellular peptides enables identification of unique sequences with a specific biological function and with potential as therapeutics.

Retrovirally delivered random cyclic peptide libraries yield inhibitors of interleukin-4 signaling in human B cells.

Kinsella TM, Ohashi CT, Harder AG, Yam GC, Li W, Peelle B, Pali ES, Bennett MK, Molineaux SM, Anderson DA, Masuda ES, Payan DG.

J Biol Chem 2002 Oct 4;277(40):37512-8.

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line epsilon transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

Use of MEDUSA-based data analysis and capillary HPLC-ion-trap mass spectrometry to examine complex immunoaffinity extracts of RBAp48.

Gururaja T, Li W, Bernstein J, Payan DG, Anderson DC.

J Proteome Res 2002 May-Jun;1(3):253-61.

To examine the Jurkat cell interaction partners of RbAp48, we digested entire immunoaffinity extracts with trypsin and identified potential interacting proteins using one- and two-dimensional microcapillary HPLC-ion-trap mass spectrometry. An Oracle-based automated data analysis system (MEDUSA) was used to compare quadruplicate anti-RbAp48 antibody affinity extracts with two sets of quadruplicate control extracts. The anti-RbAp48 extracts contained over 40 difference 1D gel bands. We identified all known proteins of the NuRD/Mi-2 complex including human p66. Three potential homologues of members of this complex were also found, suggesting that there may be more than one variant of this complex. Eleven proteins associated with RNA binding or pre-mRNA splicing were observed. Four other proteins, including a putative tumor suppressor, were identified, as were 18 ribosomal proteins. There was little overlap with RbAp48-interacting proteins defined by yeast two-hybrid methods. These results demonstrate the analysis of a complex immunoaffinity extract and suggest a more complex cellular role for RbAp48 than previously documented.

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

Holland SJ, Liao XC, Mendenhall MK, Zhou X, Pardo J, Chu P, Spencer C, Fu A, Sheng N, Yu P, Pali E, Nagin A, Shen M, Yu S, Chan E, Wu X, Li C, Woisetschlager M, Aversa G, Kolbinger F, Bennett MK, Molineaux S, Luo Y, Payan DG, Mancebo HS, Wu J.

J Exp Med 2001 Nov 5;194(9):1263-76.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Characterization and use of green fluorescent proteins from Renilla mulleri and Ptilosarcus guernyi for the human cell display of functional peptides.

Peelle B, Gururaja TL, Payan DG, Anderson DC.

J Protein Chem 2001 Aug;20(6):507-19.

Green fluorescent protein (GFP) is useful as an intracellular scaffold for the display of random peptide libraries in yeast. GFPs with a different sequence from Aequorea victoria have recently been identified from Renilla mulleri and Ptilosarcus gurneyi. To examine these proteins as intracellular scaffolds for peptide display in human cells, we have determined the expression level of retrovirally delivered human codon-optimized versions in Jurkat-E acute lymphoblastic leukemia cells using fluorescence activated cell sorting and Western blots. Each wild type protein is expressed at 40% higher levels than A. victoria mutants optimized for maximum fluorescence. We have compared the secondary structure and stability of these GFPs with A. victoria GFP using circular dichroism (CD). All three GFPs essentially showed a perfect beta-strand conformation and their melting temperatures (Tm) are very similar, giving an experimental evidence of a similar overall structure. Folded Renilla GFP allows display of an influenza hemagglutinin epitope tag in several internal insertion sites, including one which is not permissive for such display in Aequorea GFP, giving greater flexibility in peptide display options. To test display of a functional peptide, we show that the SV-40 derived nuclear localization sequence PPKKKRKV, when inserted into two different potential loops, results in the complete localization of Renilla GFP to the nucleus of human A549 cells.

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells.

Peelle B, Lorens J, Li W, Bogenberger J, Payan DG, Anderson DC.

Chem Biol 2001 May;8(5):521-34.

BACKGROUND: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes.
RESULTS: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides.
CONCLUSIONS: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.
Dominant effector genetics in mammalian cells.

Xu X, Leo C, Jang Y, Chan E, Padilla D, Huang BC, Lin T, Gururaja T, Hitoshi Y, Lorens JB, Anderson DC, Sikic B, Luo Y, Payan DG, Nolan GP.

Nat Genet 2001 Jan;27(1):23-9

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.