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Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold–small molecule interactions

Abstract

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif–small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between ‘undruggable’ biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

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Fig. 1: Chemoinformatics and bioinformatics analysis of small molecules that bind RNA and the RNAs that bind small molecules.
Fig. 2: SMIRNA strategy and method validation in vitro and in HUVECs.
Fig. 3: Overview of Vegfa mRNA regulation, miR-377 pathway and targeting strategy.
Fig. 4: Activity of heterodimer TGP-377 in HUVECs.
Fig. 5: Proteomics analysis of HUVECs treated with TGP-377.
Fig. 6: TGP-377 stimulates angiogenesis.

Data availability

All data supporting this manuscript are contained within the main text, source data and Supplementary figures. Specific data are freely available upon reasonable request from the corresponding author. The Inforna database9 can be accessed via the following URL https://disney.florida.scripps.edu/software/. Users wishing to obtain access must complete a software license agreement with TSRI, upon which login credentials will be provided after approval.

Code availability

No unique code was used in the described data analyses.

References

  1. Angelbello, A. J. et al. Using genome sequence to enable the design of medicines and chemical probes. Chem. Rev. 118, 1599–1663 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Disney, M. D. & Angelbello, A. J. Rational design of small molecules targeting oncogenic noncoding RNAs from sequence. Acc. Chem. Res. 49, 2698–2704 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Stein, C. A. & Castanotto, D. FDA-approved oligonucleotide therapies in 2017. Mol. Ther. 25, 1069–1075 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Stojic, L. et al. Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis. Nucleic Acids Res. 46, 5950–5966 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Watters, K. E., Abbott, T. R. & Lucks, J. B. Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq. Nucleic Acids Res. 44, e12 (2016).

    PubMed  Google Scholar 

  6. Strobel, E. J., Yu, A. M. & Lucks, J. B. High-throughput determination of RNA structures. Nat. Rev. Genet. 19, 615–634 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Velagapudi, S. P., Gallo, S. M. & Disney, M. D. Sequence-based design of bioactive small molecules that target precursor microRNAs. Nat. Chem. Biol. 10, 291–297 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Tran, T. & Disney, M. D. Identifying the preferred RNA motifs and chemotypes that interact by probing millions of combinations. Nat. Commun. 3, 1125 (2012).

    PubMed  Google Scholar 

  9. Disney, M. D. et al. Inforna 2.0: a platform for the sequence-based design of small molecules targeting structured RNAs. ACS Chem. Biol. 11, 1720–1728 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Ferrara, N., Gerber, H.-P. & LeCouter, J. The biology of VEGF and its receptors. Nat. Med. 9, 669–676 (2003).

    CAS  PubMed  Google Scholar 

  11. Sun, N. et al. Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing. Sci. Rep. 8, 17509–17519 (2018).

    PubMed  PubMed Central  Google Scholar 

  12. Ylä-Herttuala, S., Rissanen, T. T., Vajanto, I. & Hartikainen, J. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J. Am. Coll. Cardiol. 49, 1015–1026 (2007).

    PubMed  Google Scholar 

  13. Taimeh, Z., Loughran, J., Birks, E. J. & Bolli, R. Vascular endothelial growth factor in heart failure. Nat. Rev. Cardiol. 10, 519–530 (2013).

    CAS  PubMed  Google Scholar 

  14. Velagapudi, S. P. et al. Approved anti-cancer drugs target oncogenic non-coding RNAs. Cell Chem. Biol. 25, 1086–1094 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Velagapudi, S. P. et al. Defining RNA–small molecule affinity landscapes enables design of a small molecule inhibitor of an oncogenic noncoding RNA. ACS Cent. Sci. 3, 205–216 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Morgan, B. S., Forte, J. E., Culver, R. N., Zhang, Y. & Hargrove, A. E. Discovery of key physicochemical, structural, and spatial properties of RNA-targeted bioactive ligands. Angew. Chem. Int. Ed. 56, 13498–13502 (2017).

    CAS  Google Scholar 

  17. Mehta, A. et al. SMMRNA: a database of small molecule modulators of RNA. Nucleic Acids Res. 42, D132–D141 (2014).

    CAS  PubMed  Google Scholar 

  18. Cordella, L. P., Foggia, P., Sansone, C. & Vento, M. A (sub)graph isomorphism algorithm for matching large graphs. IEEE Trans. Pattern Anal. Mac. Intell. 26, 1367–1372 (2004).

    Google Scholar 

  19. Ehrlich, H.-C. & Rarey, M. Systematic benchmark of substructure search in molecular graphs - from Ullmann to VF2. J. Cheminform. 4, 13 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Haniff, H. S., Graves, A. & Disney, M. D. Selective small molecule recognition of RNA base pairs. ACS Comb. Sci. 20, 482–491 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Schneider, T. D. & Stephens, R. M. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18, 6097–6100 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Stelzer, A. C., Kratz, J. D., Zhang, Q. & Al-Hashimi, H. M. RNA dynamics by design: biasing ensembles towards the ligand-bound state. Angew. Chem. Int. Ed. 49, 5731–5733 (2010).

    CAS  Google Scholar 

  23. Stelzer, A. C. et al. Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. Nat. Chem. Biol. 7, 553–559 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Chen, J. L., VanEtten, D. M., Fountain, M. A., Yildirim, I. & Disney, M. D. Structure and dynamics of RNA repeat expansions that cause Huntington’s disease and myotonic dystrophy type 1. Biochemistry 56, 3463–3474 (2017).

    CAS  PubMed  Google Scholar 

  25. Childs-Disney, J. L. et al. A massively parallel selection of small molecule-RNA motif binding partners informs design of an antiviral from sequence. Chem 4, 2384–2404 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Costales, M. G. et al. Small molecule inhibition of microRNA-210 reprograms an oncogenic hypoxic circuit. J. Am. Chem. Soc. 139, 3446–3455 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Velagapudi, S. P. et al. Design of a small molecule against an oncogenic noncoding RNA. Proc. Natl Acad. Sci. USA 113, 5898–5903 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Wen, Z. et al. MicroRNA-377 regulates mesenchymal stem cell-induced angiogenesis in ischemic hearts by targeting VEGF. PLoS ONE 9, e104666 (2014).

    PubMed  PubMed Central  Google Scholar 

  29. el Azzouzi, H. et al. The hypoxia-inducible microRNA cluster miR-199a214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation. Cell Metab. 18, 341–354 (2013).

    CAS  PubMed  Google Scholar 

  30. Duan, Q. et al. MicroRNA-214 is upregulated in heart failure patients and suppresses XBP1-mediated endothelial cells angiogenesis. J. Cell. Physiol. 230, 1964–1973 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang, R., Dong, L.-D., Meng, X.-B., Shi, Q. & Sun, W.-Y. Unique microRNA signatures associated with early coronary atherosclerotic plaques. Biochem. Biophys. Res. Commun. 464, 574–579 (2015).

    CAS  PubMed  Google Scholar 

  32. Yan, X. C. et al. MiR-342-5p is a notch downstream molecule and regulates multiple angiogenic pathways including Notch, vascular endothelial growth factor and transforming growth factor β signaling. J. Am. Heart Assoc. 5, e003042 (2016).

    PubMed  PubMed Central  Google Scholar 

  33. DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Renton, A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Brčić, J. & Plavec, J. NMR structure of a G-quadruplex formed by four d(G4C2) repeats: insights into structural polymorphism. Nucleic Acids Res. 46, 11605–11617 (2018).

    PubMed  PubMed Central  Google Scholar 

  36. Su, Z. et al. Discovery of a biomarker and lead small molecules to target r(GGGGCC)-associated defects in c9FTD/ALS. Neuron 84, 1043–1050 (2014).

    Google Scholar 

  37. Wang, Z. F. et al. The hairpin form of r(G4C2)exp in c9ALS/FTD is repeat-associated non-ATG translated and a target for bioactive small molecules. Cell Chem. Biol. 26, 179–190 (2019).

    CAS  PubMed  Google Scholar 

  38. Balendra, R. & Isaacs, A. M. C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat. Rev. Neurol. 14, 544–558 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ferrara, N., Hillan, K. J. & Novotny, W. Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem. Biophys. Res. Commun. 333, 328–335 (2005).

    CAS  PubMed  Google Scholar 

  40. Ivy, S. P., Wick, J. Y. & Kaufman, B. M. An overview of small-molecule inhibitors of VEGFR signaling. Nat. Rev. Clin. Oncol. 6, 569–579 (2009).

    CAS  PubMed  Google Scholar 

  41. Ahmed, S. I., Thomas, A. L. & Steward, W. P. Vascular endothelial growth factor (VEGF) inhibition by small molecules. J. Chemother. 16, 59–63 (2004).

    CAS  PubMed  Google Scholar 

  42. Arcondéguy, T., Lacazette, E., Millevoi, S., Prats, H. & Touriol, C. VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level. Nucleic Acids Res. 41, 7997–8010 (2013).

    PubMed  PubMed Central  Google Scholar 

  43. Koransky, M. L., Robbins, R. C. & Blau, H. M. VEGF gene delivery for treatment of ischemic cardiovascular disease. Trends Cardiovasc. Med. 12, 108–114 (2002).

    CAS  PubMed  Google Scholar 

  44. Liu, B. et al. Analysis of secondary structural elements in human microRNA hairpin precursors. BMC Bioinf. 17, 112–120 (2016).

    Google Scholar 

  45. Agarwal, V., Bell, G. W., Nam, J. W. & Bartel, D. P. Predicting effective microRNA target sites in mammalian mRNAs. eLife 4, e05005 (2015).

    PubMed Central  Google Scholar 

  46. Holmes, K., Roberts, O. L., Thomas, A. M. & Cross, M. J. Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell. Signal. 19, 2003–2012 (2007).

    CAS  PubMed  Google Scholar 

  47. Mao, Z., Liu, C., Lin, X., Sun, B. & Su, C. PPP2R5A: a multirole protein phosphatase subunit in regulating cancer development. Cancer Lett. 414, 222–229 (2018).

    CAS  PubMed  Google Scholar 

  48. Angelbello, A. J. et al. Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model. Proc. Natl Acad. Sci. USA 116, 7799–7804 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Gan, L. M. et al. Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes. Nat. Commun. 10, 871–879 (2019).

    PubMed  PubMed Central  Google Scholar 

  50. Zangi, L. et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat. Biotechnol. 31, 898–907 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Hassan, M., Brown, R. D., Varma-O’Brien, S. & Rogers, D. Cheminformatics analysis and learning in a data pipelining environment. Mol. Divers. 10, 283–299 (2006).

    CAS  PubMed  Google Scholar 

  52. Costales, M. G., Matsumoto, Y., Velagapudi, S. P. & Disney, M. D. Small molecule targeted recruitment of a nuclease to RNA. J. Am. Chem. Soc. 140, 6741–6744 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was funded by the National Institutes of Health R01 GM097455 and R01 CA249180 (to M.D.D.) and AstraZeneca. We thank J. Childs-Disney and R. Rahaim, Jr. for editing the manuscript. We also thank U. Bauer for supporting the collaboration and K. Jennbacken for useful input on VEGFA biology. Correspondence and request for materials should be addressed to M.D.D.

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Authors

Contributions

M.D.D. conceived and directed the study. L.K., J.B. and M.L. conducted all physicochemical analyses and the library design for the AstraZeneca compound collection; H.S.H. conducted the screening and all in vitro and in cellulis experiments under the guidance of M.D.D.; D.A. and A.A. conducted proteomics analysis of HUVEC samples; E.L. conducted RAN translation analysis for binders to r(G4C2); K.W.W. and I.Y. conducted in silico modelling to assess the binding of the dimer to pre-miR-377; M.D.C. conducted cellular uptake analysis by LC-MS/MS; G.C. conducted all bioinformatic analyses for LOGOS analysis. All authors discussed the results and commented on the manuscript during preparation.

Corresponding author

Correspondence to Matthew D. Disney.

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Competing interests

M.D.D. is a founder of Expansion Therapeutics. M.L., J.B. and L.K. are employees of AstraZeneca.

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Supplementary Information

Supplementary Figs. 1–20, Discussion, Synthetic Methods and Characterization, and Supplementary Methods

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Supporting Tables 1–13

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Haniff, H.S., Knerr, L., Liu, X. et al. Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold–small molecule interactions. Nat. Chem. 12, 952–961 (2020). https://doi.org/10.1038/s41557-020-0514-4

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