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Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase

Nature Chemical Biology volume 12, pages 838–844 (2016)Cite this article

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Abstract

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.

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Figure 1: Validation of RF-MSS cyclase assay and high-throughput screening conditions.
Figure 2: LRE1 potently and selectively inhibited sAC in vitro and in cells.
Figure 3: Crystal structure of sAC−LRE1 complex.
Figure 4: Mechanistic characterization of sAC inhibition by LRE1.
Figure 5: LRE1 inhibited sAC-dependent processes in sperm and mitochondria.

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Acknowledgements

We thank D. Warren and The Milstein Synthetic Chemistry Core Facility at Weill Cornell Medical College, and S.S. Gross and Q. Chen. C.S. and S.K. thank the Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY) staff and the Helmholtz Protein Sample Production Facility (PSPF) for technical assistance. L.R.-E. and J.F.G. thank the High-Throughput and Spectroscopy Resource Center staff, the Helmsley Trust for funding the rapid-fire automated solid phase extraction time of flight mass spectrometry instrument, and Agilent staff, especially P. Rye and L. Frick, for technical assistance. This work was supported, in whole or in part, by US National Institutes of Health (NIH) grants GM107442 and EY025810 and by the Weill Cornell Medicine Clinical and Translational Sciences Center, NIH National Center for Advancing Translational Sciences grant UL1TR00457 (to L.R.L. and J.B.); NIH grants HD38082 and HD44044 (to P.E.V.), R01GM088999 (to G.M.), and K08 CA160657 (to J.H.Z.); Melanoma Research Alliance Team Science and Clinique Clinical Scholars Awards (to J.H.Z.); and Deutsche Forschungsgemeinschaft grant STE1701/11 (to C.S.).

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Authors and Affiliations

  1. Department of Pharmacology, Weill Cornell Medical College, New York, New York, USA

    Lavoisier Ramos-Espiritu, Hannes Buck, Lonny R Levin & Jochen Buck

  2. The High-Throughput Screening and Spectroscopy Resource Center, The Rockefeller University, New York, New York, USA

    Lavoisier Ramos-Espiritu, Carolina Adura & J Fraser Glickman

  3. Department of Biochemistry, University of Bayreuth, Bayreuth, Germany

    Silke Kleinboelting & Clemens Steegborn

  4. Department of Veterinary and Animal Science, University of Massachusetts, Amherst, Massachusetts, USA

    Felipe A Navarrete, Antonio Alvau & Pablo E Visconti

  5. Brain and Mind Research Institute, Weill Cornell Medical College, New York, New York, USA

    Federica Valsecchi, Anatoly Starkov & Giovanni Manfredi

  6. Department of Dermatology, Weill Cornell Medical College, New York, New York, USA

    Jonathan H Zippin

  7. Helmholtz Zentrum fur Infektionsforschung, Braunschweig, Germany

    Joop van den Heuvel

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  1. Lavoisier Ramos-Espiritu
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Contributions

L.R.-E., S.K., C.S., J.F.G., L.R.L. and J.B. designed the research project and analyzed data; L.R.-E. and C.A. performed the high-throughput screening; S.K. and C.S. solved the crystal structure; J.v.d.H. expressed protein; L.R.-E. performed the kinetic and cellular inhibition studies; H.B. performed the assays of individual tmACs; F.A.N., A.A., and P.E.V. performed the sperm studies; F.V., A.S., and G.M. performed the mitochondrial studies; and L.R.-E. and J.H.Z. performed the cytotoxicity studies. All authors contributed specific parts of the manuscript, with L.R.-E., L.R.L. and J.B. assuming responsibility for the manuscript in its entirety.

Corresponding author

Correspondence to Lonny R Levin.

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Competing interests

J.B., L.R.L. and J.H.Z. have equity interest in CEP Biotech, which has licensed commercialization of a panel of monoclonal antibodies directed against sAC.

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Ramos-Espiritu, L., Kleinboelting, S., Navarrete, F. et al. Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase. Nat Chem Biol 12, 838–844 (2016). https://doi.org/10.1038/nchembio.2151

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