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. 2016 Oct;12(10):838-44.
doi: 10.1038/nchembio.2151. Epub 2016 Aug 22.

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

Lavoisier Ramos-Espiritu  1   2 , Silke Kleinboelting  3 , Felipe A Navarrete  4 , Antonio Alvau  4 , Pablo E Visconti  4 , Federica Valsecchi  5 , Anatoly Starkov  5 , Giovanni Manfredi  5 , Hannes Buck  1 , Carolina Adura  2 , Jonathan H Zippin  6 , Joop van den Heuvel  7 , J Fraser Glickman  2 , Clemens Steegborn  3 , Lonny R Levin  1 , Jochen Buck  1
Affiliations

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

Lavoisier Ramos-Espiritu et al. Nat Chem Biol. 2016 Oct.

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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Conflict of interest statement

Drs. Buck, Levin and Zippin own equity interest in CEP Biotech which has licensed commercialization of a panel of monoclonal antibodies directed against sAC. All other authors declare that they have no conflicts of interest with the contents of this article.

Figures

Fig. 1
Fig. 1. Validation of RapidFire Mass Spectrometry System (RF-MSS) cyclase assay and high throughput screening conditions
An example of a RF-MS chromatogram showing (a) the product cAMP as extracted ion intensity (EIC) × 103, and (b) substrate ATP as EIC × 104. Peaks derive from increasing input ATP (in mM) recorded at time “0” (black) and after 180 minutes incubation with sAC (red). (c) Human sAC activity measured using RF-MSS as a function of substrate of ATP-Mg2+ for 120 min in the presence of excess MgCl2 (20 mM). Shown are curves for Mg2+ alone (red dots), Mg2+/40 mM HCO3− (green squares), Mg2+/10 mM Ca2+ (blue triangles), and Mg2+/40 mM HCO3−/10 mM Ca2+ (purple triangles). Determinations are representative of at least two independent experiments and curves are nonlinear fits generated by Prism. (d–f) RF-MSS screen assaying sAC in the presence of 1 mM ATP/5 mM MgCl2/5 mM CaCl2/40 mM NaHCO3. (d) Comparison of active (black dots) and denatured (open squares) sAC enzyme: Z’ score = 0.7. (e) LOPAC library (1280 compounds) pilot screen (black); DMSO control (red); denatured sAC protein (blue). (f) Results of screening LOPAC library twice. LOPAC compounds (gray); DMSO control (green square); denatured sAC protein (red). (R2 = 0.81).
Fig. 2
Fig. 2. LRE1 is a potent and selective inhibitor of sAC in vitro and in cells
(a) Concentration-response curve of LRE1 on sAC protein in the presence of 1 mM ATP/5 mM MgCl2/5 mM CaCl2/40 mM NaHCO3. Values are averages of triplicate determinations with SEM. (b) AC activities of 293 cells overexpressing each of the indicated tmACs in the absence (black bars) or presence of 50 µM LRE1 (red bars). Shown are representative assays repeated at least three times; values represent averages of quintuple determinations of tmAC specific activity with SEM. (c) Concentration-response of LRE1 (red squares) and KH7 (black circles) on cellular accumulation of cAMP in sAC overexpressing 4-4 cells. (d) Concentration-response of LRE1 on cellular accumulation of cAMP in forskolin-stimulated sAC knockout fibroblasts. (c,d) Representative assays repeated at least three times; values are means ± SEM of triplicate determinations normalized to activity in the absence of any compound. (e) Concentration-response of LRE1 on glucose-induced cAMP production in INS-1E cells in 2.5 mM (low) or 16 mM (high) glucose with 0 (black), 10 (blue), 30 (cyan), 50 (red), or 100 (brown) µM LRE1. Shown are means ± SEM of triplicate determinations of at least three independent experiments. (f) The glucose-induced cAMP, calculated from panel (e) by subtracting the cAMP accumulated in low glucose from the cAMP accumulated in high glucose, in the presence of the indicated concentration of LRE1.
Fig. 3
Fig. 3. Crystal structure of sAC/LRE1 complex
(a) Overall structure of the sAC/LRE1 complex, with C1 in grey and C2 in cyan. LRE1 is shown in stick representation, and active site and BBS are indicated. (b) Close-up of the BBS with ligand and interacting residues as sticks colored according to atom type. LRE1 is overlaid with 2Fo-Fc electron density (blue) contoured at 1σ. (c) Interaction scheme for sAC and LRE1. Interactions of side chains are indicated by black dots and by arcs for aromatic residues, and backbone interactions by dashed lines.
Fig. 4
Fig. 4. Mechanistic characterization of sAC inhibition by LRE1
(a) LRE1 is competitive with HCO3−. sAC activity, measured by RF-MSS in the presence of the indicated concentration of LRE1 and 1 mM ATP/5 mM MgCl2/5 mM CaCl2 with 0 (red); 10 (purple squares); 20 (blue triangles); or 80 (orange diamonds) mM NaHCO3. Data represent means of triplicate determinations of an experiment repeated twice. (b) Overlay of sAC/LRE1 (grey) with a sAC/bicarbonate complex (cyan, RMSD 0.3 Å5 for 352 Cα atoms). The ligands and the two key residues for bicarbonate binding are shown as sticks colored according to atom type. (c) LRE1 does not compete with ATP. sAC activity, measured by RF-MSS in the presence of the indicated concentration of LRE1 and either 0.6 (pink diamonds), 0.9 (cyan triangles), 1.3 (blue squares), or 2.0 (red dots) mM ATP and 5 mM MgCl2/5 mM CaCl2/40 mM NaHCO3. Data represent individual points of a serial dilution of an experiment repeated three times. (d) Crystal structure of a sAC/ApCpp/LRE1 complex. The substrate analog ApCpp in the active site and the inhibitor in the BBS are shown as sticks colored according to atom type and overlaid with 2Fo-Fc electron density contoured at 1σ (blue). Ca2+ is shown as yellow sphere. (e) Overlay of sAC/ApCpp/LRE1 (green) with a sAC/ApCpp complex (blue, RMSD 0.31 Å2 for Cα atoms). Ligands and key interacting residues are shown as sticks, and Ca2+ as a yellow sphere.
Fig. 5
Fig. 5. LRE1 inhibits sAC dependent processes in sperm and mitochondria
(a–d) Sperm experiments. (a) Western blot using anti-PKA substrates antibodies of mouse cauda sperm activated by incubation in capacitation (Cap) media in the presence of the shown amounts of LRE1 in presence (+) or absence (−) of dibutyryl cAMP (1 mM) and IBMX (100 µM). N.Cap = non capacitated negative control. (b) Western blot using anti-phospho tyrosine antibodies of the same blot as in (a). For a,b, shown are representative Western blots of experiments repeated three times using independent sperm preparations from different mice. The complete gels used for these images are shown in Supplementary Figures 4 and 5, respectively. (c) The percentage of hyperactivated motility in sperm capacitated in the presence of the indicated concentration of LRE1 (black). Non capacitated sperm (red). Values are averages (± S.E.M.) of three independent sperm preparations from three different mice collected and analyzed on separate days. (d) Percentage of fertilized eggs from sperm capacitated in the presence of the indicated concentration of LRE1 (black bars) or sperm capacitated in the presence of 100 µM LRE1 + 1 mM dibutyryl cAMP (red bar). Values are averages (± S.E.M.) of four independent sperm preparations from four different mice collected and analyzed on separate days. (e) Cytochrome c oxidase (COX) activities in cells from WT and sAC KO MEFs treated with DMSO or 50 µM LRE1 for 30 min. Values are averages (± S.D.); N=5. COX activity was statistically different in WT cells ± LRE1 (P=0.037) by t-test.
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