Identification of Inhibitors of Integrin Cytoplasmic Domain Interactions With Syk.

Deenadayalan Bakthavatsalam,Asra Aslam,Amy R Caivano,Hema D Parthiban,Safa Ali,Shashikant Gupta,Peter Vanderslice,John W Craft,Clifford C Stephan,C William Gundlach,Nghi Nguyen,Anna Kazansky,Goeun Bae,Darren G Woodside,Sophie Y Lin

doi:10.3389/fimmu.2020.575085

Deenadayalan Bakthavatsalam, Asra Aslam + Show 13 more

Open Access

https://doi.org/10.3389/fimmu.2020.575085

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Abstract

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin β-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the β-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin β-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02–4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1β and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.

Highlights

Integrin cell adhesion molecules are expressed on all cell types and are essential for proper organism development and tissue homeostasis [1, 2]
The binding between the integrin b3 cytoplasmic domain peptide and spleen tyrosine kinase (Syk) juxtaposed the donor and acceptor beads, resulting in a fluorescent signal after donor beads are activated at 680 nm. b3 cytoplasmic domain interaction with GST-Syk[6-370] was dose dependent and selective, as no signal was generated when purified GST was conjugated to the acceptor beads (Figure 1H)
Integrin expression on THP-1 cells was analyzed by flow cytometry to determine expression levels of integrins a4b1, b2, and avb3, and the immune response receptor FcgRI; we wanted to identify integrins that were expressed at similar levels as FcgRI because our goal was to determine if antagonists of integrin:Syk interactions would affect the canonical activation of Syk that occurs via interaction with dually phosphorylated immunoreceptor tyrosinebased activation motif (ITAM) found within transmembrane adaptor molecules associated with immune response receptors such as FcgRI [42]

Summary

Introduction

Integrin cell adhesion molecules are expressed on all cell types and are essential for proper organism development and tissue homeostasis [1, 2]. Integrins play a significant role in the pathophysiology of numerous diseases including those of the immune system [3,4,5,6,7,8]. Because they regulate key steps in leukocyte recruitment into tissue, including leukocyte tethering, firm adhesion, diapedesis, and migration [9], integrins have been directly implicated in inflammation and autoimmunity. Identified as a key signaling component of the B-cell antigen receptor and Fc-receptors [12], the non-receptor spleen tyrosine kinase (Syk) is recognized as an essential integrin signaling component in inflammation [13, 14]. Integrin signaling via Syk is involved in neutrophil spreading, respiratory burst and degranulation [11], costimulation of the expression of interleukin (IL)-1b in monocytes [18], and extracellular signal-regulated protein kinase activation in macrophages [10, 19]

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