characterization of novel dual a2a/a2b adenosine receptor ......2b adenosine receptors inhibitors....
TRANSCRIPT
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Characterization of novel dual A2A/A2B adenosine receptor antagonists for cancer immunotherapy
Michał Gałęzowski, Paulina Węgrzyn, Aneta Bobowska, Katarzyna Dziedzic, Joanna Szeremeta-Spisak, Marcin Nowogródzki, Grzegorz Satała, Alicja Obara, Iwona Łozińska-Raj, Przemysław Wyrębek,
Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Magdalena Zastawna, Grzegorz Statkiewicz, Maciej Rogacki, Mateusz Świrski, Jakub Woyciechowski, Magdalena Ziembik, Karolina
Grycuk, Kinga Michalik, Agnieszka Adamus, Karolina Wiatrowska, Natalia Lewandowska, Urszula Sierańska, Agata Stachowicz, Aniela Gołas, Olga Pierzchała, Krzysztof Brzózka, Mateusz Nowak
Selvita S.A., Bobrzyńskiego 14, 30-348 Kraków, Poland
Corresponding author: [email protected]
B cells CD3 cells CD4 cells CD8 cells
12 h
INTRODUCTION
Project supported by the European Regional Development Fundsunder the Measure 1.1.1. Operational Program - Smart Growth (POIR.01.01.01-00- 0987/16-
00)
SUMMARY
Adenosine is the key immunometabolite responsible for immune tolerance in tumors. It is present in normal tissue inlow concentrations, having various physiological functions. In the tumor, its concentration increases rapidly, as aresult of overexpression of enzymes producing adenosine, additionally enhanced by hypoxia and inflammation.Adenosine inhibits the biological functions of T lymphocytes infiltrating the cancer tissue by binding to the A2A
receptor. The affinity to A2B receptor is believed to attenuate the response of dendritic cells and other parts of innatesystem. Thus blocking simultaneously the effects mediated by both receptor subtypes with dual inhibitor seems to bea viable approach for cancer immunotherapy in comparison to A2A inhibitors currently tested in clinical trials.
We have discovered a novel series of potent and dual A2A/A2B adenosine receptors inhibitors. Our best compounds present high in vitro activity not only in low adenosine conditions but also in tumor like adenosine rich environment. We have observedrescue of adenosine-suppressed cytotoxicity of NK cells and reversal of NECA-induced CREB phosphorylation in in vivo murine model (up to 24 hours). Additionally the A2B activity is key factor in restoring of the adenosine agonist-impaired functionalactivity of moDC (cytokine release assays). To sum up, presented compounds show improved pharmacological profile in comparison to A2A inhibitors currently tested in clinical trials.
SEL330-639 OUTPERFORMS COMPETITORS IN DOWNREGULATIONIN VIVO OF CREB PHOSPHORYLATION
SEL330-584 AND SEL330-639 RESTORE NK CELLS CYTOTOXIC FUNCTION
SEL330-584 AND SEL330-639 ARE HIGHLY POTENT IN ADENOSINE-RICH TUMOR MICROENVIRONMENT
A2A EC50 [nM]c
0.1 mM ADOA2A EC50 [nM]c
100 mM ADOA2B EC50 [nM]d
0.1 mM ADOA2B EC50 [nM]d
100 mM ADO
SEL330-584 0.26 92 3.6 634
SEL330-639 0.25 4.2 6.4 1618
AZD4635e,f 57.6 > 10000 188 >10000
CPI-444e,f 13.8 >10000 138 >10000
Arcus Ex. I e,g 1.4 1962 1.9 120
Preladenante,f 3.2 9222 >4000 >4000
Fig 2. SLV A2A/A2B antagonists outperform competitors in a) adenosine-low and b) adenosine-rich environment; c) assays used PC12 cell line; d) assays used A2B
overexpressing HEK293 cell line; e) data generated in Selvita with compound samples synthesized by Selvita; f) CPI‐444: Structure from AACR, April 2017 (#CT119);AZD4635: Structure from AACR, April 2017 (#2641); Preladenant purchased from MedChemExpress g) Example I from WO2018/136700A1 (synthesized and tested inSelvita)
Fig. 3. 10 mM NECA (potent pan-adenosine agonist) simulatesHi-Ado conditions and suppresses the cytotoxicity of NKdelivered cell line against target tumor cells. SEL330-475outperforms competitors in restoration of NK cytotoxicity.Representative data show. CPI‐444: Structure from AACR, April2017 (#CT119), synthesized by Selvita; AZD4635: Structurefrom AACR, April 2017 (#2641) synthesized by Selvita.
Fig 5. a) SEL-330-639 and Arcus Example I fromWO2018/136700A1 (synthesized in Selvita) dualantagonists inhibit CREB phosphorylation insurrogate PK/PD assay. Antagonists wereadministrated to C57BL/6 mice (50mg/kg for SEL-330-639 and 100mg/kg for Arcus Ex. I). Blood wascollected at three timepoints 30min, 12h and 24h.NECA (10 mM) was used as CREB activator via theadenosine receptors. The level of CREBphosphorylation was detected in different immunecells via flow cytometry analysis, and comparedwith control group (no antagonist). The mostsignificant changes are visible in B cells and CD8+cells subpopulations; b) Disconnection between PKand PD plots shows unsurmountable mode ofaction of SEL330-639.
a)
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SEL330-584
SEL330-639
CPI-444
AZD4635
Fig 1. Immunosuppressive activities of adenosine in cancer’ adapted from: Allard et al.; Curr. Opin. Pharmacol. 29:7-16
SEL330-584 AND SEL330-639 RESTORE moDC CELLS FUNCTIONBY A2B ACTIVITY
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TNFa secretion by mature moDC
IL-12 secretion by mature moDC
Concentration [mM]
b)a)
SEL330-584SEL330-639CPI-444AZD-4635PreladenantArcus Ex. I
Low-Ado (0.1mM) Hi-Ado (100mM)
Fig. 4. Restoration of adenosine suppression of a) TNFa and b) IL-12 production in mature moDCs is observed only for A2B antagonists (Selvita dual antagonists andLAS101057 – selective A2B antagonist) neither for selective A2A inhibitors (Preladenant, CPI-444, AZD-4635). Human monocyte derived dendritic cells (moDCs) weredifferentiated in culture with GM-CSF and IL-4 for 6 days. Then cells were pretreated with A2A/A2B selective or dual receptor antagonists and maturated by LPS stimulation inpresence of 10 mM NECA (Hi-Ado conditions). The effect was measured by TNFa and IL-12 production after 24h.Modulation of A2A and A2B receptors expression during maturation of dendritic cells. After maturation the level of A2A receptor is downregulated (c), while the A2B level isslightly upregulated (d). CPI‐444: Structure from AACR, April 2017 (#CT119), synthesized by Selvita; AZD4635: Structure from AACR, April 2017 (#2641) synthesized bySelvita. Preladenant (selective A2A antagonist) and LAS101057 (selective A2B antagonist) were purchased from MedChemExpress.
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