This study investigates the immunomodulatory effects of BPA substitutes using in silico and in vitro approaches. A comprehensive in silico screening with Endocrine Disruptome of BPA and its 25 substitutes revealed that BPG, BPS-MAE, BPS-MPE, BPPH and PF201 have the highest potential to bind nuclear receptors. Based on the in silico ranking, 12 compounds were selected and tested in vitro to investigate their effects on THP-1 derived macrophages, Jurkat T cells and LCLs. The metabolic activity tests showed that BPA has IC₅₀ values of 99-182 µM, while the values for BPG, BPPH and BPP are 5-10 times lower. Comparison of the IC₅₀ values with the logP of the tested compounds showed that the BPA analogues affect cell viability in proportion to their lipophilicity (R² = 0.9185). The results of the cytokine release assays showed that the BPA substitutes generally stimulated the proinflammatory response in THP-1 macrophages in a statistically significant manner when exposed to the environmentally relevant concentrations and suppressed it in the micromolar range in all cell lines tested. Further analysis using RamosBlue reporter cells indicated that BPAP, BPG, BPP and BPPH alter the NF-κB/AP-1 signalling pathway. Moreover, BPAP, BPG, BPP, BPPH, BPS-MAE, BPS-MPE, BTUM and PF201 were predicted in silico to be TLR4-MyD88 inhibitors. The overall results of this study identify BPG, BPP, BPPH, BPZ and TCBPA as BPA analogues with the highest immunomodulatory potential. In addition, the immunomodulatory potential of the BPA alternatives Pergafast201 and BTUM was confirmed in vitro for the first time. This study emphasises the need for cautious evaluation of BPA substitutes due to their potential immunomodulatory effect, which could significantly affect public health.

Anti-CD20 monoclonal antibodies (MAbs) have revolutionized the treatment of B-cell leukemia and lymphoma. However, many patients do not respond to such treatment due to either deficiency of the complementary immune response or resistance to apoptosis. Other currently available treatments are often inadequate or induce major side effects. Therefore, there is a constant need for improved therapies. The prostaglandin E2 receptor 4 (EP4) receptor has been identified as a promising therapeutic target for hematologic B-cell malignancies. Herein, we report that EP4 receptor agonists PgE1-OH and L-902688 have exhibited enhanced cytotoxicity when applied together with anti-CD20 MAbs rituximab, ofatumumab and obinutuzumab in vitro in Burkitt lymphoma cells Ramos, as well as in p53-deficient chronic lymphocytic leukemia (CLL) cells MEC-1. Moreover, the enhanced cytotoxic effects of EP4 receptor agonists and MAbs targeting CD20 have been identified ex vivo on primary lymphocytes B obtained from patients diagnosed with CLL. Incubation of cells with PgE1-OH and L-902688 preserved the expression of CD20 molecules, further confirming the anti-leukemic potential of EP4 receptor agonists in combination with anti-CD20 MAbs. Additionally, we demonstrated that the EP4 receptor agonist PgE-1-OH induced apoptosis and inhibited proliferation via the EP4 receptor triggering in CLL. This work has revealed very important findings leading towards the elucidation of the anticancer potential of PgE1-OH and L-902688, either alone or in combination with MAbs. This may contribute to the development of potential therapeutic alternatives for patients with B-cell malignancies.