Discovery and Evaluation of Cisatracurium Besylate (51w89) As An Inhibitor of CD73
-
摘要: CD73是一个新兴的肿瘤治疗潜在靶点，与肿瘤的发生、发展、转移及不良预后密切相关。在本研究中，我们通过筛选发现顺苯磺酸阿曲库铵（51w89）可以抑制CD73的酶活性，其在分子水平的IC50为13.30 μmol/L；通过表面等离子共振实验测得51w89与CD73蛋白的结合亲和力KD为20.45 μmol/L；在高表达CD73的人乳腺癌细胞MDA-MB-231中测得51w89抑制细胞中CD73的酶活性IC50为17.70 μmol/L。进一步的研究结果显示，51w89可抑制MDA-MB-231细胞的迁移能力；并且51w89限制AMP对CD8+T细胞分泌IFN-γ的抑制作用。通过以上一系列研究表明，51w89可作为CD73潜在抑制剂用于后续的抗肿瘤研究。Abstract:
Over recent years, immunotherapy has developed rapidly, which has changed the way of cancer treatment. However, most patients cannot benefit from immunotherapy, which may be due to insufficient reprogramming of the immunosuppressive tumor microenvironment (TME) and thus limited reinvigoration of antitumor immunity. In TME, adenosine, a metabolite of ATP, is an effective immunoregulatory factor. Extracellular 5'-nucleotidase (CD73) is the rate-limiting molecule in the process of adenosine production. Overexpression of CD73 on tumor cells and immune cells leads to a higher concentration of adenosine in the TME. The high concentration of adenosine suppresses the anti-tumor immune response, promotes tumor cells proliferate, metastasis and angiogenesis. Therefore, anti-CD73 therapy is expected to become a promising strategy for cancer immunotherapy. Several anti-CD73 mAbs (MEDI9447, BMS986179, SRF373/NZV930, CPI-006/CPX-006, IPH5301, TJ004309) and small molecule CD73 inhibitors ((LY3475070, AB680, CB-708) are being investigated in early phase clinical trials. But so far, there is no CD73 targeted product for the treatment of cancer on the market. In this study, firstly we performed the screening of our compound library containing 876 listed drugs to identify candidate inhibitors targeting CD73. Preliminary experimental results showed that Cisatracurium besylate (51w89) could inhibit the enzyme activity of recombinant CD73 with an IC50 value of 13.30 μmol/L. To verify the interactions between 51w89 and CD73 and evaluate their binding affinities, we performed surface plasmon resonance (SPR) experiments using a Biacore T200 (GE Healthcare). The binding affinity (KD value) of 51w89 binding to CD73 was 20.45 μmol/L. Encouraged by these results at the molecular level, we next evaluated the inhibitory effect of 51w89 against CD73 in MDA-MB-231 cells. The results show that the IC50 of 51w89 against CD73 in MDA-MB-231 cells was 17.70 μmol/L, which was close to the IC50 value at the molecular level. Subsequently, we proved the role of CD73 in the migration of MDA-MB-231 cells through scratch tests, transwell tests and siRNA tests, and found that 51w89 could inhibit the migration of MDA-MB-231 cells. Moreover, we found that 51w89 could limit the inhibitory effect of AMP on CD8+ T cells. Taken together, we speculated that 51w89 could be used as a potent small-molecule inhibitor of CD73 for subsequent antitumor research. -
Key words:
- CD73 /
- inhibitor /
- tumor /
- migration /
- CD8+ T cells
-
图 1 51w89抑制重组蛋白CD73的酶活性
Figure 1. Results for the inhibition activity of 51w89 against recombinant CD73
(a) The primarily screening results against CD73 (red point: 51w89); (b) The IC50 result of 51w89 against recombinant CD73
图 2 51w89与重组CD73蛋白结合的SPR图
Figure 2. SPR results for the binding affinity between 51w89 and CD73
图 3 51w89抑制人乳腺癌细胞MDA-MB-231中CD73的酶活性
Figure 3. Results for the inhibition activity of 51w89 against CD73 in MDA-MB-231 cells
(a) Western blot analysis for the expression of CD73 in different cells; (b) The IC50 result of 51w89 against CD73 in MDA-MB-231 cells
图 4 51w89或siRNA下调CD73表达可抑制MDA-MB-231细胞的迁移能力
Figure 4. Inhibitory effects of 51W89 or siRNA CD73 on the migration of MDA-MB-231 cells by transwell tests
(a) The effect of CD73 siRNA on CD73 expression in MB-MDA-231 cells.; (b) Effects of 51W89 or siRNA CD73 on the migration of MDA-MB-231 cells
图 5 划痕实验表明51w89抑制MDA-MB-231细胞迁移
Figure 5. Inhibitory effects of 51w89 on the migration of MDA-MB-231 cells by scratch tests
-
[1] STRATER N. Ecto-5'-nucleotidase: Structure function relationships[J]. Purinergic Signalling, 2006, 2(2): 343-350. doi: 10.1007/s11302-006-9000-8 [2] KNAPPn K, ZEBISCH M, PIPPEL J, et al. Crystal structure of the human ecto-5′-nucleotidase (CD73): insights into the regulation of purinergic signaling[J]. Structure, 2012, 20(12): 2161-2173. doi: 10.1016/j.str.2012.10.001 [3] ZIMMERMANN H, ZEBISCH M, STRATER N, et al. Cellular function and molecular structure of ecto-nucleotidases[J]. Purinergic Signalling, 2012, 8(3): 437-502. doi: 10.1007/s11302-012-9309-4 [4] GAO Z, DONG K, ZHANG H, et al. The roles of CD73 in cancer[J]. BioMed Research International, 2014: 460654-460654. [5] VIJAYAN D, YOUNG A, TENG M W, et al. Targeting immunosuppressive adenosine in cancer[J]. Nature Reviews Cancer, 2017, 17(12): 709-724. doi: 10.1038/nrc.2017.86 [6] CHALMIN F, MIGNOT G, BRUCHARD M, et al. Stat3 and Gfi-1 transcription factors control Th17 cell immunosuppressive activity via the regulation of ectonucleotidase expression[J]. Immunity, 2012, 36(3): 362-373. doi: 10.1016/j.immuni.2011.12.019 [7] SYNNESTVDT K, FURUTA G T, COMERFORD K M, et al. Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia[J]. The Journal of Clinical Investigation, 2002, 110(7): 993-1002. doi: 10.1172/JCI0215337 [8] REGATEIRO F S, HOWIE D, NOLAN K F, et al. Generation of anti‐inflammatory adenosine byleukocytes is regulated by TGF‐β[J]. European Journal of Immunology, 2011, 41(10): 2955-2965. doi: 10.1002/eji.201141512 [9] NIEMELA J, HENTTINEN T, YEGUTKIN G G, et al. IFN-α induced adenosine production on the endothelium: a mechanism mediated by CD73 (ecto-5′-nucleotidase) up-regulation[J]. The Journal of Immunology, 2004, 172(3): 1646-1653. doi: 10.4049/jimmunol.172.3.1646 [10] SPYCHALA J, KITAJEWSKI J. Wnt and β-catenin signaling target the expression of ecto-5′-nucleotidase and increase extracellular adenosine generation[J]. Experimental Cell Research, 2004, 296(2): 99-108. doi: 10.1016/j.yexcr.2003.11.001 [11] ALLARD D, CHROBAK P, ALLARD B, et al. Targeting the CD73-adenosine axis in immuno-oncology[J]. Immunology Letters, 2019: 31-39. [12] CORBELINI P F, FIGUEIRO F, NEVES G M, et al. Insights into Ecto-5'-Nucleotidase as a New Target for Cancer Therapy: A Medicinal Chemistry Study[J]. Current Medicinal Chemistry, 2015, 22(15): 1776-1792. doi: 10.2174/0929867322666150408112615 [13] BURGER R M, LOWENSTEIN J M. Preparation and Properties of 5'-Nucleotidase from Smooth Muscle of Small Intestine[J]. Journal of Biological Chemistry, 1970, 245(23): 6274-6280. doi: 10.1016/S0021-9258(18)62605-5 [14] BHATTARAI S, FREUNDLIEB M, PIPPEL J, et al. α, β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective ecto-5′-Nucleotidase (CD73) Inhibitors[J]. Journal of Medicinal Chemistry, 2015, 58(15): 6248-6263. doi: 10.1021/acs.jmedchem.5b00802 [15] JUNKER A, RENN C, DOBELMANN C, et al. Structure–Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5′-Nucleotidase (CD73) Inhibitors[J]. Journal of Medicinal Chemistry, 2019, 62(7): 3677-3695. doi: 10.1021/acs.jmedchem.9b00164 [16] BHATTARAI S, PIPPEL J, SCALETTI E et al. 2-Substituted α, β-Methylene-ADP Derivatives: Potent Competitive Ecto-5'-nucleotidase (CD73) Inhibitors with Variable Binding Modes[J]. Journal of Medicinal Chemistry, 2020, 63(6): 2941-2957. doi: 10.1021/acs.jmedchem.9b01611 [17] BOWMAN C E, SILVA R G, PHAM A, et al. An Exceptionally Potent Inhibitor of Human CD73[J]. Biochemistry, 2019, 58(31): 3331-3334. doi: 10.1021/acs.biochem.9b00448 [18] KAVUTCU M, MELZIG M F. In vitro effects of selected flavonoids on the 5'-nucleotidase activity[J]. Pharmazie Die, 1999, 54(6): 457-459. [19] RIPPHAUSEN P, FREUNDLIEB M, BRUNSCHWEIGER A, et al. Virtual screening identifies novel sulfonamide inhibitors of ecto-5′-nucleotidase[J]. Journal of Medicinal Chemistry, 2012, 55(14): 6576-6581. doi: 10.1021/jm300658n [20] BAQI Y, LEE S Y, IQBAL J, et al. Development of potent and selective inhibitors of ecto-5′-nucleotidase based on an anthraquinone scaffold[J]. Journal of Medicinal Chemistry, 2010, 53(5): 2076-2086. doi: 10.1021/jm901851t [21] GONG Y P, WAN R Z, LIU Z P. Evaluation of WO2017098421: GSK’s benzothiazine compounds as CD73 inhibitor filings[J]. Expert Opinion on Therapeutic Patents, 2018, 28(2): 167-171. doi: 10.1080/13543776.2018.1407756 [22] BEATTY J W, LINDSEY E A, THOMAS-TRAN R, et al. Discovery of Potent and Selective Non-Nucleotide Small Molecule Inhibitors of CD73[J]. Journal of Medicinal Chemistry, 2020, 63(8): 3935-3955. doi: 10.1021/acs.jmedchem.9b01713 -
下载:
点击查看大图
图(6)
计量
- 文章访问数: 3
- HTML全文浏览量: 4
- PDF下载量: 0
- 被引次数: 0
