OBJECTIVE The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. The objective of this study was to explore the expression of all the PTP genes (PTPome) in FLS. METHODS A comparative screening was conducted of the expression of the PTPome in FLS from patients with RA or osteoarthritis (OA). The functional effect of a PTP up-regulated in RA, SHP-2, was then analyzed by knock-down using cell-permeable antisense oligonucleotides in RA FLS. RESULTS PTPN11 was over-expressed in RA compared to OA FLS. Knock-down of PTPN11, which encodes SHP-2, using a cell-permeable antisense oligonucleotide, decreased the invasion, migration, adhesion, spreading and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by the knock-down of SHP-2. RA FLS deficient in SHP-2 displayed decreased activation of focal adhesion kinase and mitogen-activated protein kinases. CONCLUSION These findings indicate a novel role for SHP-2 in mediating human FLS function, and suggest that SHP-2 promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.

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Objective In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK, promotes RA FLS invasiveness. Methods Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. Results PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. Conclusions By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFβ in RA FLS.

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

Immune activating agents represent a valuable class of therapeutics for the treatment of cancer. An area of active research is expanding the types of these therapeutics that are available to patients via targeting new biological mechanisms. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of immune signaling and a target of high interest for the treatment of cancer. Herein, we present the discovery and optimization of novel amino-6-aryl pyrrolopyrimidine inhibitors of HPK1 starting from hits identified via virtual screening. Key components of this discovery effort were structure-based drug design aided by analyses of normalized B-factors and optimization of lipophilic efficiency.