Background/Purpose: We have been investigating the possible involvement of BAFF (B cell activating factor of TNF family) in the pathogenesis of primary Sjögren’s syndrome (pSS). We found that soluble BAFF (sBAFF) robustly increased IL-6 production in vitro by peripheral monocytes of patients with pSS, and that the expression level of a BAFF receptor (BR3) was significantly elevated in pSS monocytes compared to that of normal monocytes. Additionally, the proportion of BR3-positive monocytes to total monocytes was positively and significantly correlated with the sBAFF-induced IL-6 production and the serum IgG level of pSS patients. These data collectively suggest that the elevated expression of BR3 on monocytes is involved in the pathogenesis of pSS. Consequently, our findings suggest that BR3 is a therapeutic target to treat pSS. However, no compounds except antibodies were reported to have inhibitory activities against BAFF signaling so far. In this study, we show some of our latest data about drug discovery for pSS aiming at BAFF signaling pathway.

Methods: High-throughput screening (HTS) of a chemical library was carried out to search for compounds that block binding of sBAFF to BR3. To this end, BR3 expressing CHO-K1 cells were established by transfection of a full-length cDNA of human BR3. Transfectants were cultured in the presence of FMAT Blue-labeled sBAFF and each compound in 384-well plates, and the binding of sBAFF to the cells was monitored by an Applied Biosystems 8200 Cellular Detection System.Hit compounds were further screened as follows: IFN-γ-stimulated THP-1, a human acute monocytic leukemia cell line, was cultured in vitro with sBAFF and each compound for 96 hr, and the cumulative amount of IL-6 produced by the cells was measured by ELISA. Cytotoxicities of the compounds were analyzed by measuring LDH in culture supernatants. The expression level of NF-κB in the cells was analyzed by quantitative PCR.

Results: A total of 18,562 compounds were examined for inhibitory activities of sBAFF binding to BR3. To eliminate false positives, inhibitory activities of the HTS-hits against IL-6 production by sBAFF-stimulated THP-1 were measured. Their cytotoxicities, which result in the reduction of IL-6 production, were also examined. As a result, two pyrrolopyrimidine derivatives, BIK12 and BIK13, showed substantial inhibition of sBAFF-binding. IC₅₀ values for BIK12 and 13 were 11 and 6 μM, respectively. sBAFF-induced IL-6 production by THP-1 was significantly suppressed by these compounds in a dose dependent manner, while the compounds had no cytotoxicities. Additionally, the expression of NF-κB in the cells was also repressed by BIK12 and 13. These results collectively suggest that these compounds suppress IL-6 production by THP-1 through an inhibition of binding of sBAFF to its receptor, BR3, and possibly subsequent BAFF signaling pathway.

Conclusion: We have successfully discovered low molecular weight compounds which have inhibitory activities against BAFF signaling. Although IC₅₀ values were not so low, these compounds may provide not only lead compounds for therapeutic drugs of pSS, but also novel tools to explore the pathological mechanism of pSS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pyrrolopyrimidine-derivatives-that-inhibit-binding-of-baff-to-its-receptor-br3-are-drug-candidates-for-primary-sjogrens-syndrome/