Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RRx-001 ameliorates inflammatory diseases by acting as a potent covalent NLRP3 inhibitor

Abstract

The NLRP3 inflammasome plays a crucial role in innate immune-mediated inflammation and contributes to the pathogenesis of multiple autoinflammatory, metabolic and neurodegenerative diseases, but medications targeting the NLRP3 inflammasome are not available for clinical use. RRx-001 is a well-tolerated anticancer agent currently being investigated in phase III clinical trials, but its effects on inflammatory diseases are not known. Here, we show that RRx-001 is a highly selective and potent NLRP3 inhibitor that has strong beneficial effects on NLRP3-driven inflammatory diseases. RRx-001 inhibits the activation of the canonical, noncanonical, and alternative NLRP3 inflammasomes but not the AIM2, NLRC4 or Pyrin inflammasomes. Mechanistically, RRx-001 covalently binds to cysteine 409 of NLRP3 via its bromoacetyl group and therefore blocks the NLRP3-NEK7 interaction, which is critical for the assembly and activation of the NLRP3 inflammasome. More importantly, RRx-001 treatment attenuates the symptoms of lipopolysaccharide (LPS)-induced systemic inflammation, dextran sulfate sodium (DSS)-induced colitis and experimental autoimmune encephalomyelitis (EAE) in mice. Thus, our study identifies RRx-001 as a new potential therapeutic agent for NLRP3-driven diseases.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: RRx-001 inhibits NLRP3 inflammasome activation.
Fig. 2: RRx-001 suppresses NLRP3 inflammasome assembly by blocking the NEK7–NLRP3 interaction.
Fig. 3: RRx-001’s activity depends on both the twin NO2 and bromoacetyl groups.
Fig. 4: RRx-001 directly binds to NLRP3.
Fig. 5: RRx-001 binds to cysteine 409 of NLRP3.
Fig. 6: RRx-001 alleviates DSS-induced colitis in mice.
Fig. 7: RRx-001 prevents the development of EAE.

References

  1. 1.

    Martinon, F., Mayor, A. & Tschopp, J. The Inflammasomes: guardians of the Body. Annu. Rev. Immunol. 27, 229–265 (2009).

    CAS  PubMed  Google Scholar 

  2. 2.

    Davis, B. K., Wen, H. T. & Ting, J. P. Y. The inflammasome NLRs in immunity, inflammation, and associated diseases. Annu. Rev. Immunol. 29 29, 707–735 (2011).

    PubMed  Google Scholar 

  3. 3.

    Chen, G., Shaw, M. H., Kim, Y. G. & Nunez, G. NOD-like receptors: role in innate immunity and inflammatory disease. Annu Rev. Pathol.-Mech. 4, 365–398 (2009).

    CAS  Google Scholar 

  4. 4.

    Broz, P. & Dixit, V. M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).

    CAS  PubMed  Google Scholar 

  5. 5.

    Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 (2006).

    CAS  PubMed  Google Scholar 

  6. 6.

    Masters, S. L. et al. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes. Nat. Immunol. 11, 897–904 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Wen, H. et al. Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat. Immunol. 12, 408–415 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Unamuno, X. et al. NLRP3 inflammasome blockade reduces adipose tissue inflammation and extracellular matrix remodeling. Cell Mol. Immunol. 18, 1045–1057 (2021).

  9. 9.

    He, Y. et al. Immunopathobiology and therapeutic targets related to cytokines in liver diseases. Cell Mol. Immunol. 18, 18–37 (2021).

  10. 10.

    Heneka, M. T. et al. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 493, 674–678 (2013).

    CAS  PubMed  Google Scholar 

  11. 11.

    Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Bauer, C. et al. Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome. Gut 59, 1192–1199 (2010).

    CAS  PubMed  Google Scholar 

  13. 13.

    Zaki, M. H. et al. The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 32, 379–391 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Lamkanfi, M. & Dixit, V. M. Inflammasomes and their roles in health and disease. Annu Rev. Cell Dev. Biol. 28, 137–161 (2012).

    CAS  PubMed  Google Scholar 

  15. 15.

    He, Y. et al. 3,4-Methylenedioxy-beta-nitrostyrene Inhibits NLRP3 Inflammasome Activation by Blocking Assembly of the Inflammasome. J. Biol. Chem. 289, 1142–1150 (2014).

    CAS  PubMed  Google Scholar 

  16. 16.

    Coll, R. C. et al. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat. Med. 21, 248–255 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Jiang, H. et al. Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders. J. Exp. Med. 214, 3219–3238 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Huang, Y. et al. Tranilast directly targets NLRP3 to treat inflammasome-driven diseases. EMBO Mol. Med. 10, e8689 (2018).

  19. 19.

    He, H. et al. Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity. Nat. Commun. 9, 2550 (2018).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Marchetti, C. et al. OLT1177, a beta-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation. Proc. Natl Acad. Sci. USA 115, E1530–E1539 (2018).

    CAS  PubMed  Google Scholar 

  21. 21.

    Coll, R. C. et al. MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat. Chem. Biol. 15, 556–559 (2019).

    CAS  PubMed  Google Scholar 

  22. 22.

    Mullard, A. NLRP3 inhibitors stoke anti-inflammatory ambitions. Nat. Rev. Drug Disco. 18, 405–407 (2019).

    CAS  Google Scholar 

  23. 23.

    Kluck, V. et al. Dapansutrile, an oral selective NLRP3 inflammasome inhibitor, for treatment of gout flares: an open-label, dose-adaptive, proof-of-concept, phase 2a trial. Lancet Rheumatol. 2, e270–e280 (2020).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Oronsky, B. et al. Rockets, radiosensitizers, and RRx-001: an origin story part I. Disco. Med 21, 173–180 (2016).

    Google Scholar 

  25. 25.

    Kim, M. M. et al. Whole brain radiotherapy and RRx-001: two partial responses in radioresistant melanoma brain metastases from a phase I/II clinical trial: a TITE-CRM phase I/II clinical trial. Transl. Oncol. 9, 108–113 (2016).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Carter, C. et al. Early results: “Rocket” a Phase II Study of Rrx-001, a novel triple epigenetic inhibitor, resensitization to irinotecan in colorectal cancer. Ann. Oncol. 26, ii3 (2015).

  27. 27.

    Morgensztern, D. et al. RRx-001 followed by platinum plus etoposide in patients with previously treated small-cell lung cancer. Br. J. Cancer 121, 211–217 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Oronsky, B. et al. REPLATINUM Phase III randomized study: RRx-001 + platinum doublet versus platinum doublet in third-line small cell lung cancer. Future Oncol. 15, 3427–3433 (2019).

    CAS  PubMed  Google Scholar 

  29. 29.

    Carter, C. A. et al. Partial response to platinum doublets in refractory EGFR-positive non-small cell lung cancer patients after RRx-001: evidence of episensitization. Case Rep. Oncol. 9, 62–67 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Oronsky, B. et al. RRx-001: a systemically non-toxic M2-to-M1 macrophage stimulating and prosensitizing agent in Phase II clinical trials. Expert Opin. Investig. Drugs 26, 109–119 (2017).

    CAS  PubMed  Google Scholar 

  31. 31.

    Oronsky, B. et al. RRx-001, a novel dinitroazetidine radiosensitizer. Invest N. Drugs 34, 371–377 (2016).

    CAS  Google Scholar 

  32. 32.

    Scicinski, J. et al. Preclinical evaluation of the metabolism and disposition of RRx-001, a novel investigative anticancer agent. Drug Metab. Dispos. 40, 1810–1816 (2012).

    CAS  PubMed  Google Scholar 

  33. 33.

    Zhao, H. et al. Epigenetic effects of RRx-001: a possible unifying mechanism of anticancer activity. Oncotarget 6, 43172–43181 (2015).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Oronsky, B., Scicinski, J., Cabrales, P. & Minchinton, A. RRx-001, an epigenetic-based radio- and chemosensitizer, has vascular normalizing effects on SCCVII and U87 tumors. Clin. Epigenetics 8, 53 (2016).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Cabrales, P. RRx-001 acts as a dual small molecule checkpoint inhibitor by downregulating CD47 on cancer cells and SIRP-alpha on monocytes/macrophages. Transl. Oncol. 12, 626–632 (2019).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Oronsky, B., Scribner, C., Aggarwal, R. & Cabrales, P. RRx-001 protects normal tissues but not tumors via Nrf2 induction and Bcl-2 inhibition. J. Cancer Res Clin. Oncol. 145, 2045–2050 (2019).

    CAS  PubMed  Google Scholar 

  37. 37.

    Reid, T. et al. Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study. Lancet Oncol. 16, 1133–1142 (2015).

    CAS  PubMed  Google Scholar 

  38. 38.

    Gross, C. J. et al. K+ efflux-independent NLRP3 inflammasome activation by small molecules targeting mitochondria. Immunity 45, 761–773 (2016).

    CAS  PubMed  Google Scholar 

  39. 39.

    Yu, W. et al. One-carbon metabolism supports S-adenosylmethionine and histone methylation to drive inflammatory macrophages. Mol. Cell 75, 1147–1160 e1145 (2019).

    CAS  PubMed  Google Scholar 

  40. 40.

    Oronsky, B. et al. RRx-001, a novel clinical-stage chemosensitizer, radiosensitizer, and immunosensitizer, inhibits glucose 6-phosphate dehydrogenase in human tumor cells. Disco. Med 21, 251–265 (2016).

    Google Scholar 

  41. 41.

    Petrilli, V. et al. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14, 1583–1589 (2007).

    CAS  PubMed  Google Scholar 

  42. 42.

    Munoz-Planillo, R. et al. K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38, 1142–1153 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Daniels, M. J. et al. Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer’s disease in rodent models. Nat. Commun. 7, 12504 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Tang, T. et al. CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation. Nat. Commun. 8, 202 (2017).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Zhou, R., Yazdi, A. S., Menu, P. & Tschopp, J. A role for mitochondria in NLRP3 inflammasome activation. Nature 469, 221–225 (2011).

    CAS  Google Scholar 

  46. 46.

    Xie, J. H., Li, Y. Y. & Jin, J. The essential functions of mitochondrial dynamics in immune cells. Cell Mol. Immunol. 17, 712–721 (2020).

    CAS  PubMed  Google Scholar 

  47. 47.

    Lu, A. et al. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156, 1193–1206 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Dick, M. S., Sborgi, L., Ruhl, S., Hiller, S. & Broz, P. ASC filament formation serves as a signal amplification mechanism for inflammasomes. Nat. Commun. 7, 11929 (2016).

  49. 49.

    He, Y., Zeng, M. Y., Yang, D., Motro, B. & Nunez, G. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 530, 354–357 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Shi, H. X. et al. NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component. Nat. Immunol. 17, 250–258 (2016).

    CAS  PubMed  Google Scholar 

  51. 51.

    Schmid-Burgk, J. L. et al. A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. J. Biol. Chem. 291, 103–109 (2016).

    CAS  PubMed  Google Scholar 

  52. 52.

    Haq, T. et al. Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization. Nat. Commun. 6, 8771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Lomenick, B. et al. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl Acad. Sci. USA 106, 21984–21989 (2009).

    CAS  PubMed  Google Scholar 

  54. 54.

    Scicinski, J. et al. NO to cancer: the complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001. Redox Biol. 6, 1–8 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    He, Y., Franchi, L. & Nunez, G. TLR agonists stimulate Nlrp3-dependent IL-1beta production independently of the purinergic P2X7 receptor in dendritic cells and in vivo. J. Immunol. 190, 334–339 (2013).

    CAS  PubMed  Google Scholar 

  56. 56.

    Gris, D. et al. NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses. J. Immunol. 185, 974–981 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Inoue, M., Williams, K. L., Gunn, M. D. & Shinohara, M. L. NLRP3 inflammasome induces chemotactic immune cell migration to the CNS in experimental autoimmune encephalomyelitis. Proc. Natl Acad. Sci. USA 109, 10480–10485 (2012).

    CAS  PubMed  Google Scholar 

  58. 58.

    Sharif, H. et al. Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome. Nature 570, 338–343 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Feng Shao (National Institute of Biological Sciences, Beijing, China) for providing the TcdB toxin. This research was supported by the National Key Research and Development Program of China (grant numbers 2019YFA0508503 and 2020YFA0509101), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDB29030102), the National Natural Science Foundation of China (grant numbers 82003765, 81821001, 31770991, and 91742202), the Fundamental Research Funds for the Central Universities and the University Synergy Innovation Program of Anhui Province (GXXT-2019-026), the Natural Science Foundation of Anhui Province (1908085QC99) .

Author information

Affiliations

Authors

Contributions

Y.C., H.H. and B.L performed the experiments of this work; X.D., W.J. and R.Z. designed the research. Y.C., W.J. and R.Z. wrote the manuscript. W.J. and R.Z. supervised the project.

Corresponding authors

Correspondence to Wei Jiang or Rongbin Zhou.

Ethics declarations

Competing interests

R.Z., W.J. and Y.C. are named as inventors on China National Intellectual Property Administration Application Serial No. 202011472140.0 related to RRx-001.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, Y., He, H., Lin, B. et al. RRx-001 ameliorates inflammatory diseases by acting as a potent covalent NLRP3 inhibitor. Cell Mol Immunol 18, 1425–1436 (2021). https://doi.org/10.1038/s41423-021-00683-y

Download citation

Keywords

  • RRx-001
  • NLRP3 inflammasome
  • inflammatory diseases

Search

Quick links