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HM71224, a novel Bruton's tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis.

Park JK, Byun JY, Park JA, Kim YY, Lee YJ, Oh JI, Jang SY, Kim YH, Song YW, Son J, Suh KH, Lee YM, Lee EB - Arthritis Res. Ther. (2016)

Bottom Line: The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation.Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β by human monocytes, and osteoclast formation by human monocytes.Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehank-no Chongno-gu, Seoul, 03080, Korea.

ABSTRACT

Background: Bruton's tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis.

Methods: The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA).

Results: HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA.

Conclusions: HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

No MeSH data available.


Related in: MedlinePlus

Effect of HM71224 on Bruton’s tyrosine kinase (Btk) signaling. a The inhibitory effects of HM71224 on B cell receptor (BCR) signaling in the Ramos cell line. Ramos cells were stimulated with anti-IgM F(ab’)2 fragments in the presence of the indicated doses of HM71224. b Time-dependent restoration of Btk phosphorylation in Ramos cells after HM71224 washout. c Btk occupancy by HM71224 on Ramos cells. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control (crtl). ERK extracellular signal-regulated kinase
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Fig2: Effect of HM71224 on Bruton’s tyrosine kinase (Btk) signaling. a The inhibitory effects of HM71224 on B cell receptor (BCR) signaling in the Ramos cell line. Ramos cells were stimulated with anti-IgM F(ab’)2 fragments in the presence of the indicated doses of HM71224. b Time-dependent restoration of Btk phosphorylation in Ramos cells after HM71224 washout. c Btk occupancy by HM71224 on Ramos cells. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control (crtl). ERK extracellular signal-regulated kinase

Mentions: The Ramos B cell lymphoma cell line was chosen to study the inhibitory effects of HM71224 on the BCR signaling cascade as it endogenously expresses Btk [14]. Crosslinking of BCRs with anti-IgM F(ab’)2 induced phosphorylation of Btk and PLCγ2. However, phosphorylation of Btk and PLCγ2 decreased markedly in the presence of HM71224 (Fig. 2a). Next, we investigated whether HM71224 irreversibly inhibited Btk. Ramos cells were incubated with 100 nM HM71224 for 1 h. After washing, the cells were stimulated with anti-IgM F(ab’)2. The inhibitory effect of BTK phosphorylation by HM71224 treatment lasted at least 24 h (Fig. 2b). Biotinylated probe bound to free Btk in a dose-dependent manner; approximately 90 % of Btk molecules were occupied by HM71224 at 10 nM, and close to 100 % at 100 nM (Fig. 2c).Fig. 2


HM71224, a novel Bruton's tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis.

Park JK, Byun JY, Park JA, Kim YY, Lee YJ, Oh JI, Jang SY, Kim YH, Song YW, Son J, Suh KH, Lee YM, Lee EB - Arthritis Res. Ther. (2016)

Effect of HM71224 on Bruton’s tyrosine kinase (Btk) signaling. a The inhibitory effects of HM71224 on B cell receptor (BCR) signaling in the Ramos cell line. Ramos cells were stimulated with anti-IgM F(ab’)2 fragments in the presence of the indicated doses of HM71224. b Time-dependent restoration of Btk phosphorylation in Ramos cells after HM71224 washout. c Btk occupancy by HM71224 on Ramos cells. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control (crtl). ERK extracellular signal-regulated kinase
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835877&req=5

Fig2: Effect of HM71224 on Bruton’s tyrosine kinase (Btk) signaling. a The inhibitory effects of HM71224 on B cell receptor (BCR) signaling in the Ramos cell line. Ramos cells were stimulated with anti-IgM F(ab’)2 fragments in the presence of the indicated doses of HM71224. b Time-dependent restoration of Btk phosphorylation in Ramos cells after HM71224 washout. c Btk occupancy by HM71224 on Ramos cells. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control (crtl). ERK extracellular signal-regulated kinase
Mentions: The Ramos B cell lymphoma cell line was chosen to study the inhibitory effects of HM71224 on the BCR signaling cascade as it endogenously expresses Btk [14]. Crosslinking of BCRs with anti-IgM F(ab’)2 induced phosphorylation of Btk and PLCγ2. However, phosphorylation of Btk and PLCγ2 decreased markedly in the presence of HM71224 (Fig. 2a). Next, we investigated whether HM71224 irreversibly inhibited Btk. Ramos cells were incubated with 100 nM HM71224 for 1 h. After washing, the cells were stimulated with anti-IgM F(ab’)2. The inhibitory effect of BTK phosphorylation by HM71224 treatment lasted at least 24 h (Fig. 2b). Biotinylated probe bound to free Btk in a dose-dependent manner; approximately 90 % of Btk molecules were occupied by HM71224 at 10 nM, and close to 100 % at 100 nM (Fig. 2c).Fig. 2

Bottom Line: The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation.Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β by human monocytes, and osteoclast formation by human monocytes.Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehank-no Chongno-gu, Seoul, 03080, Korea.

ABSTRACT

Background: Bruton's tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis.

Methods: The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA).

Results: HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA.

Conclusions: HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

No MeSH data available.


Related in: MedlinePlus