scholarly journals SAT0016 DEVELOPMENT OF FIBROBLAST-LIKE SYNOVIOCYTE ASSAYS FOR TARGET DISCOVERY IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 938.1-938
Author(s):  
D. Ruelas ◽  
C. Chen ◽  
H. Truong ◽  
V. Lira ◽  
Y. Moazami ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Growth Factor ◽  
Synovial Fluid ◽  
Real Time ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Complete Inhibition ◽  
Cytokine Secretion ◽  
Target Discovery ◽  
Secretion Assay

Background:The rheumatoid arthritis (RA) synovium is characterized by an overabundance of fibroblast-like synoviocytes (FLS), which play a central role in the initiation and perpetuation of disease via multiple mechanisms.1FLS promote disease progression by producing high levels of proinflammatory factors, migrating to and invading cartilage and bone, and promoting self-proliferation and resistance to apoptosis. Our current understanding of the molecular mechanisms that govern FLS-mediated pathology in the synovial joint remains incomplete. Importantly, almost 30% of treatment-naïve early RA patients exhibit a strong fibroid phenotype that correlates with relatively poor response to disease-modifying anti-rheumatic drugs.2Yet, current therapies in RA are not directly aimed at FLS pathology, creating an opportunity for novel therapeutic target discovery.Objectives:Our aim is to develop a broad suite of screening-amenable assays in RA patient-derived FLS for the discovery of target pathways that control multiple pathological properties, including cytokine secretion, migration, and invasion.Methods:A sensitive high-throughput RA-FLS secretion assay was developed to examine the ability of small-molecule inhibitors to block the production of interleukin (IL)-6 and matrix metalloproteinase (MMP)-3 in response to stimuli. To create a physiologically relevant stimulus, a surrogate synovial fluid cocktail (composed of 12 factors) was defined and titrated for optimal concentration selection. Small-molecule inhibitors (N=170) of diverse biological pathways were screened using the full cocktail or individual stimulation (TNFα, IL-1α, or IL-17) to characterize assay performance. In addition, an FLS platelet-derived growth factor (PDGF)-mediated migration screening assay was developed using a live cell imaging system (IncuCyte) to quantify real-time FLS migration.Results:Due to the variability and limited volume of synovial fluid, we developed a surrogate synovial fluid cocktail to mimic the relevant stimulation of RA-FLS in the inflamed joint. The surrogate cocktail was composed of 12 factors: TNFα, IL-1α, IL-17, IFNγ, OSM, LIF, GM-CSF, IP-10, VEGF, PDGF, AREG, and FGF2. Individual titration of these factors demonstrated that only 3 stimulatory factors (TNFα, IL-1α, and IL-17) resulted in a robust increase of IL-6 production. Importantly, when all 12 factors were combined, a synergistic increase in IL-6 and MMP-3 production by FLS was observed. Screening results identified several reference compounds, including an inhibitor of transforming growth factor-b–activated kinase 1 (TAK1), that was previously reported to block cytokine secretion in FLS.3Treatment with this compound showed complete inhibition of IL-6 and MMP-3 secretion. In addition to the cytokine secretion assay, treatment of FLS with this TAK1 inhibitor resulted in almost complete inhibition of migration (Fig. 1).Conclusion:Novel FLS assays were developed to discover new targets and interrogate pathways involved in multiple disease-driving mechanisms of FLS in RA. In order to mimic the inflammatory environment present in the RA synovium, we developed a 12-factor surrogate synovial fluid cocktail. A synergistic release of both IL-6 and MMP-3 was demonstrated following cocktail stimulation compared to individual cytokines. This points to the important contribution that multiple factors play in the FLS pathogenic processes and will allow us to uncover pathway interactions that may not be captured with single stimuli. In addition, the development of a real-time, 96-well, imaging-based assay to interrogate FLS migration will allow us to identify targets that control this critical pathological function of FLS.References:[1] Bartok B, et al.J Immunol. 2014;192(5):2063-2070.[2] Humby F, et al.Ann Rheum Dis. 2019;78(6):761-772.[3] Jones DS, et al.Nat Chem Bio. 2017;13(1):38-45.Disclosure of Interests:Debbie Ruelas Employee of: Gilead, Chen Chen Employee of: Gilead, Hoa Truong Employee of: Gilead, Victor Lira Employee of: Gilead, Yasamin Moazami Employee of: Gilead, Kevin Currie Employee of: Gilead, Julie A. Di Paolo Employee of: Gilead, Helen Yu Employee of: Gilead, Gundula Min-Oo Employee of: Gilead

scholarly journals Small molecule inhibitors of Hsp90 potently affect inflammatory disease pathways and exhibit activity in models of rheumatoid arthritis

2008 ◽  
Vol 58 (12) ◽  
pp. 3765-3775 ◽  
Author(s):  
John W. Rice ◽  
James M. Veal ◽  
R. Patrick Fadden ◽  
Amy F. Barabasz ◽  
Jeffrey M. Partridge ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Small Molecule ◽  
Inflammatory Disease ◽  
Small Molecule Inhibitors ◽  
Disease Pathways

Cationic Amino Acid Transporter-1 (CAT-1) Promotes Fibroblast-Like Synoviocytes Proliferation and Cytokine Secretion by Taking Up L-Arginine in Rheumatoid Arthritis

2021 ◽  
Author(s):  
Ying Lu ◽  
Chongbo Hao ◽  
Shanshan Yu ◽  
Zuan Ma ◽  
Xuelian Fu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Amino Acid ◽  
Synovial Fluid ◽  
Culture Conditions ◽  
Cytokine Secretion ◽  
Cationic Amino Acid Transporter ◽  
Cationic Amino Acid ◽  
The Relationship ◽  
Fibroblast Like Synoviocytes

Abstract Background: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. Methods: The concentrations of amino acids and cytokines in the synovial fluid of RA (n=9) and osteoarthritis (OA,n=9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of CAT-1 were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo.Results: L-arginine was upregulated in the synovial fluid of RA patients and was positively correlated with elevation of the cytokines IL-1β, IL-6 and IL-8. Further examination demonstrated that cationic amino acid transporter-1 (CAT-1) was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. Conclusion: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

scholarly journals Small Molecule Inhibitors in the Treatment of Rheumatoid Arthritis and Beyond: Latest Updates and Potential Strategy for Fighting COVID-19

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1876
Author(s):  
Magdalena Massalska ◽  
Wlodzimierz Maslinski ◽  
Marzena Ciechomska
Keyword(s):  
Rheumatoid Arthritis ◽  
Small Molecule ◽  
Viral Entry ◽  
Small Molecule Inhibitors ◽  
Large Family ◽  
Downstream Signaling ◽  
Wide Range ◽  
Synthetic Dmards ◽  
Potential Strategy ◽  
Inflammatory Molecules

The development of biological disease-modifying antirheumatic drugs (bDMARDs) and target synthetic DMARDs (tsDMARDs), also known as small molecule inhibitors, represent a breakthrough in rheumatoid arthritis (RA) treatment. The tsDMARDs are a large family of small molecules targeting mostly the several types of kinases, which are essential in downstream signaling of pro-inflammatory molecules. This review highlights current challenges associated with the treatment of RA using small molecule inhibitors targeting intracellular JAKs/MAPKs/NF-κB/SYK-BTK signaling pathways. Indeed, we have provided the latest update on development of small molecule inhibitors, their clinical efficacy and safety as a strategy for RA treatment. On the other hand, we have highlighted the risk and adverse effects of tsDMARDs administration including, among others, infections and thromboembolism. Therefore, performance of blood tests or viral infection screening should be recommended before the tsDMARDs administration. Interestingly, recent events of SARS-CoV-2 outbreak have demonstrated the potential use of small molecule inhibitors not only in RA treatment, but also in fighting COVID-19 via blocking the viral entry, preventing of hyperimmune activation and reducing cytokine storm. Thus, small molecule inhibitors, targeting wide range of pro-inflammatory singling pathways, may find wider implications not only for the management of RA but also in the controlling of COVID-19.

scholarly journals The same site on the integrase-binding domain of lens epithelium–derived growth factor is a therapeutic target for MLL leukemia and HIV

Blood ◽  
2014 ◽  
Vol 124 (25) ◽  
pp. 3730-3737 ◽  
Author(s):  
Marcelo J. Murai ◽  
Jonathan Pollock ◽  
Shihan He ◽  
Hongzhi Miao ◽  
Trupta Purohit ◽  
...  
Keyword(s):  
Growth Factor ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Small Molecule Inhibitors ◽  
Binding Domain ◽  
Lens Epithelium ◽  
New Drugs ◽  
Structural Studies ◽  
Key Points

Key Points Inhibiting LEDGF interaction with a novel fragment of MLL represents an attractive approach to develop new drugs for MLL leukemias. Structural studies reveal a new pocket on the LEDGF IBD suitable for targeting by small-molecule inhibitors.

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