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SF2312 is a natural phosphonate inhibitor of enolase

Nature Chemical Biology volume 12, pages 1053–1058 (2016)Cite this article

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Abstract

Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.

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Figure 1: SF2312 is a potent inhibitor of enolase with mixed competitive and noncompetitive kinetics.
Figure 2: SF2312 stabilizes ENO2 against thermal denaturation.
Figure 3: PhAH and SF2312 interact with ENO2 through a complex network of electrostatic, metal coordination and hydrogen bond interactions.
Figure 4: SF2312 inhibits enolase activity and is selectively toxic to D423 cells.
Figure 5: SF2312 selectively blocks glycolysis in ENO1-deleted glioma cells.

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Acknowledgements

We thank J. Holton and G. Meigs for their assistance with X-ray diffraction data collection at the Advanced Light Source, beamline 8.3.1. We thank K. Kalhurachi and J. McMurray for assistance with 13C-NMR measurements. We thank S. Millward, J. Marszalek and Y. Sun for critical comments and suggestions. We thank M. Yuan and S. Breitkopf for help with MS experiments. We thank M. Protopopova for assistance with Operetta cell imaging. We thank L. Lebioda, G. Reed and R. Poyner for useful discussions about the catalytic mechanism of enolase. We thank R. Zielinski for assistance with hypoxia experiments. The cell line D423-MG (D423) was kindly shared by D. Bigner (Duke University Medical Center) while the Gli56 cell line was shared by D. Louis (Massachusetts General Hospital) and TS neurospheres were shared by C. Brennan (Memorial Sloan Kettering Cancer Center). Inhibitors were provided by the Pharmaceutical Chemistry Facility at MD Anderson, supported by the US National Institutes of Health (NIH) National Cancer Institute under award number P30CA016672. Financial support was provided by NIH-NCI grant 7P01CA095616-10 (R.A.D.); Cancer Prevention Research Institute of Texas (CPRIT) grant RP140612 (R.A.D.); NCI grants 5P01CA120964 (J.M.A.) and 5P30CA006516 (J.M.A.) and NIH Career Development Project through the Specialized Programs of Research Excellence (SPORE) P50CA127001-07 (F.L.M.). F.L.M. was supported by Research Scholar Grant RSG-15-145-01-CDD from the American Cancer Society.

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Author notes

  1. Paul G Leonard and Nikunj Satani: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genomic Medicine and Core for Biomolecular Structure and Function, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Paul G Leonard, Todd M Link & Gilbert R Lee IV

  2. Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Nikunj Satani, Yu-Hsi Lin, Naima Hammoudi, Federica Pisaneschi & Florian L Muller

  3. Department of Institutional Analytics and Informatics, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    David Maxwell

  4. Cardtronics, Inc., Houston, Texas, USA

    Zhenghong Peng

  5. Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Duoli Sun & Basvoju A Bhanu Prasad

  6. Institute for Applied Cancer Science, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Maria Emilia Di Francesco & Barbara Czako

  7. Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

    John M Asara

  8. Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Y Alan Wang & Ronald A DePinho

  9. Bayou Therapeutics, Inc., Missouri, Texas, USA

    William Bornmann

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Contributions

D.M. performed structural modeling and docking; F.L.M. and D.M. conceived the cyclized inhibitors, which were synthesized by Z.P., D.S., B.A.B.P. and W.B.; P.G.L., G.R.L. and T.M.L. performed isolation of recombinant protein and X-ray crystallography; M.E.D.F., F.P., F.L.M. and B.C. repeated chemical syntheses and wrote synthetic procedures with characterizations. F.P. and F.L.M. performed chiral chromatography. F.L.M. and N.S. performed in vitro enzymatic activity experiments. N.S., N.H. and Y.-H.L. performed cell culture, western blots, 13C-NMR tracing and biochemical profiling experiments. N.S. performed thermal shift assays. J.M.A. performed MS small-molecule metabolite analysis. R.A.D., Y.A.W., F.L.M., N.S. and P.G.L. oversaw overall experimental design and data analysis and wrote the manuscript.

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Correspondence to Florian L Muller.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–26. (PDF 5726 kb)

Supplementary Note 1

Synthetic procedures. (PDF 115 kb)

Supplementary Note 2 (PDF 544 kb)

Source Data

Source data for Figures 1, 2, 4, and 5. (ZIP 142 kb)

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Leonard, P., Satani, N., Maxwell, D. et al. SF2312 is a natural phosphonate inhibitor of enolase. Nat Chem Biol 12, 1053–1058 (2016). https://doi.org/10.1038/nchembio.2195

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