Abstract
Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1–2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 μM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 μM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 μM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.
Footnotes
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- Syk
- spleen tyrosine kinase
- pSyk
- phosphorylated Syk
- BCR
- B cell antigen receptor
- CAIA
- collagen antibody-induced arthritis
- CIA
- collagen-induced arthritis
- FRET
- fluorescence resonance energy transfer
- PBS
- phosphate-buffered saline
- PMA
- phorbol 12-myristate 13-acetate
- P505-15
- (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate
- RA
- rheumatoid arthritis
- JAK
- Janus tyrosine kinase
- BLNK
- B cell linker
- pBLNK
- phosphorylated BLNK
- ERK
- extracellular signal-regulated kinase
- pERK
- phosphorylated ERK
- PE
- phycoerythrin
- PerCP
- peridinin-chlorophyll-protein complex
- APC
- allophycocyanin
- BSA
- bovine serum albumin
- TEL
- translocated ETS leukemia
- H&E
- hematoxylin and eosin
- MFI
- mean fluorescence intensity
- CP-690,550
- (3R,4R)-4methyl-3-(methyl-1H-pyrrolo[2,3-d]pyrimidine-4-ylamino]-ß-oxo-1-piperidinepropanenitrile.
- Received September 26, 2011.
- Accepted October 25, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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