Abstract
Endothelin receptors (ET A and ET B) are G-protein coupled receptors activated by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a potent ET B-selective antagonist, which has preventive effects against brain edema. Here, we report the crystal structure of the human ET B receptor in complex with IRL2500 at 2.7 A-resolution. The structure revealed the different binding modes between IRL2500 and ET-1, and provides structural insights into its ET B-selectivity. Notably, the biphenyl group of IRL2500 penetrates into the transmembrane core proximal to D2.50, stabilizing the inactive conformation. Using the newly-established constitutively active mutant, we clearly demonstrate that IRL2500 functions as an inverse agonist for the ET B receptor. The current findings will expand the chemical space of ETR antagonists and facilitate the design of inverse agonists for other class A GPCRs.
Introduction
Endothelin receptors (ETR) are G-protein coupled receptors activated by vaso active peptide, endothelins 1. Two endothelin receptor subtypes (ET A and ET B) are widely expressed in the vascular endothelium, brain, and other circulatory organs 2, 3. Endothelin-1 (ET-1) activates the endothelin receptors (ETRs) with sub-nanomolar affinities. The activation of the ET A receptor leads to potent and long-lasting vasoconstriction, whereas that of the ET B receptor induces nitric oxide-mediated vasorelaxation. Therefore, the up-regulation of ET-1 is significantly related to circulatory-system diseases, including pulmonary arterial hypertension (PAH) 4-7. Moreover, the autocrine and paracrine signaling functions of ET-1 through the ET A receptor play a critical role in tumor growth and survival 8. Therefore, ETR antagonists have been developed for the treatment of circulatory-system diseases and cancers 6, 7. Bosentan is the first orally-active ETR antagonist 9, 10, and is used to treat PAH. The ET B receptor is the prominent ET receptor subtype in the brain, with high expression levels in astrocytes 11. Stimulation of the ET B receptor modulates astrocytic responses, indicating its important roles in regulating astrocytic functions 12. The up-regulation of the astrocytic ET B receptor by ET-1 increases the vascular permeability and reduces the AQP4 levels, thereby aggravating vasogenic brain edema 11. The application of ET B-selective antagonists may provide preventive effects against brain edema in the acute phase of brain insults 13-16.
To date, most endothelin receptor antagonists have been developed based on bosentan 17, 18. The ETR antagonists that have been developed till now are mostly N-heterocyclic sulfonamides with similar structures and molecular weights, and nonsulfonamide antagonists (atrasentan, ambrisentan, darusentan, and enrasentan) still retain high similarities with each other and with the sulfonamides 7. Therefore, the ETR agents are chemically very similar, and expanded chemical space should be exploited. IRL2500 is a peptide ETR antagonist developed based on the partial structure of ET-1 19, rather than bosentan. IRL2500 has been characterized as an ET B-selective antagonist with an IC 50value of 1.2 nM 20, which shows higher affinity than that of bosentan. In an animal model, the intracerebroventricular administration of IRL2500 attenuated cold injury-mediated brain edema and disruption of the blood-brain barrier, indicating the neuroprotective effect of IRL2500 14, 15. An understanding of the IRL2500 binding mode would facilitate the expansion of the chemical space of ET agents.
We previously reported the crystal structures of the ET B receptor bound to ET-1 21and bosentan 22; however, both the binding mode and ET B-selectivity of IRL2500 remained to be elucidated. Here, we present the crystal structure of the ET B receptor in complex with IRL2500. This structure revealed the unique binding mode of IRL2500, which differs from those of ET-1 and bosentan. Structure-guided functional analyses clearly demonstrate that IRL2500 functions as an inverse agonist for the ET B receptor, and thus will provide the basis for design of inverse agonists for other class A GPCRs.
Results
Overall structure
For crystallization, we used the previously established, thermostabilized ET B receptor (ET B-Y4) 22, 23. The IC 50value of IRL2500 for ET B-Y4 was similar to that for the wild type receptor in the TGFa shedding assay 24(Fig. 1), suggesting that the themostabilizing mutations minimally affect the IRL2500 binding. In contrast, the IC 50value of IRL2500 for the ET A receptor is over 3 (Fig. 1), indicating that IRL2500 has over 100-fold ET B-selectivity, consistent with the previous pharmacological analysis 20. To facilitate crystallization, we replaced the third intracellular loop (ICL3) of the receptor with minimal T4 Lysozyme 25(ET B-Y4-mT4L). Using in meso crystallization 26, we obtained crystals of ET B-Y4-mT4L in complex with IRL2500 (Supplementary Fig. 1a, b). In total, 58 datasets were collected and merged by the data processing system KAMO 27. Eventually, we determined the ET B structure in complex with IRL2500 at 2.7 A resolution, by molecular replacement using the antagonist-bound ET B structure (PDB code: 5X93) (Table 1).
The overall structure consists of the canonical 7 transmembrane helices (TM), the amphipathic helix 8 at the C-terminus (H8), and two antiparallel P-strands in the extracellular loop 2 (ECL2), as in the previously determined ET B structures (Fig. 2a). The IRL2500-bound structure is similar to the bosentan-bound structure, rather than the ET-1-bound structure (R.M.S.D. values for Ca atoms=1.34 and 1.95 A, respectively), reflecting the inactive conformation. We observed a remarkable difference in the conformation of ECL2. The P strands are opened up by 9 A, as compared with those in the ligand-free structure (Fig. 2b and Supplementary Fig. 2a), and are the widest among the peptide-activated class A GPCRs (Supplementary Fig. 2b). This structural feature indicates the innate flexibility of ECL2, to capture the large peptide ligand endothelin, in the inactive conformation of the ETB receptor.
IRL2500 binding site
We first describe the IRL2500 binding mode. IRL2500 consists of a tryptophan, a 3,5-dimethylbenzoyl group, and a biphenyl group 19, which are connected by two peptide bonds (Fig. 2c). IRL2500 binds to the transmembrane binding cleft exposed to the extracellular side, with a clear electron density in the Fo-Fc omit map (Supplementary Fig. 3a, b). The carboxylate group of the tryptophan moiety in IRL2500 forms salt bridges with K182 3.33 and R343 6.55(superscripts indicate Ballesteros-Weinstein numbers 28) (Fig. 2c, d). The tryptophan side chain of IRL2500 hydrogen bonds with the carbonyl group of the N158 2.61side chain, and forms extensive van der Waals interactions with N158 261, K161 2.64, V17 7 3.28, P178 3.29, and F240 4.64(Fig. 2d). The dimethyl phenyl group of IRL2500 forms van der Waals interactions with the hydrophobic pocket, and is surrounded by V185 3.36, L27 7 5, 42, Y28 1 5, 46, W3 3 6 6, 48, L339 6.51, and H340 6.52The biphenyl group penetrates deeply into the receptor core proximal to D147 2, 50, and forms van der Waals interactions with D147 2, 50, H15 0 2, 53, W336 6.48, and S376 7.43. Overall, the carboxylate of IRL2500 is specifically recognized by the positively charged residues of the ET Breceptor, and the other moieties fill the space within the transmembrane binding pocket.
To elucidate the structural basis for the ET B-selectivity of IRL2500, we compared the residues constituting the IRL2500 binding site between the ET B and ET A receptors (Fig. 3a and Supplementary Fig. 4). Although most of the residues are conserved, three residues are replaced with bulkier residues in the ET A receptor (H150Y, V177F, and S376T). These replacements may cause steric clashes with the aromatic groups of IRL2500 and reduce its affinity. To investigate this hypothesis, we measured the IC 50values of IRL2500 for the H150Y, V177F, and S346T ET B receptor mutants. These mutants showed similar responses for ET-1 in the TGFa shedding assay (Supplementary Fig. 5), and only V177F showed a 4-fold higher IC 50value with IRL2500 (Fig. 3b). These data suggest that the V177F mutation in the ET Areceptor sterically clashes with the tryptophan moiety of IRL2500 and reduces its affinity, thus partially accounting for the ET B-selectivity of IRL2500. This is consistent with the previous study, in which the replacement of the tryptophan moiety with the smaller valine residue in IRL2500 weakened its ET B-selectivity 29.
Comparison of the binding modes of IRL2500, ET-1, and bosentan
IRL2500 is designed to mimic the Y13, F14, I19, I20, and W21 residues in ET-1, which play critical roles in ligand binding to the ET B receptor 19. The tryptophan and dimethyl phenyl group of IRL2500 seem to be equivalent to W21 and I20 in ET-1, respectively, while the biphenyl group of IRL2500 seems to be equivalent to F14 and I19 of ET-1. However, a comparison between IRL2500 and ET-1 binding revealed an unexpected difference in their binding interactions (Fig. 4a). The carboxylate of the tryptophan in IRL2500 superimposes well with that of W21 in ET-1, and is coordinated by similar positively charged residues. The tryptophan moiety and dimethyl phenyl group of IRL2500 superimpose well with I20 and W21 of ET-1, respectively. In contrast, the biphenyl group of IRL2500 penetrates into the receptor core, in an opposite manner to the F14 and I19 of ET-1. Overall, the electrostatic interactions between the carboxylates and the positively charged residues are conserved in IRL2500 and ET-1 binding, but the other moieties form totally distinct interactions with the receptor. The volume of the ligand binding pocket in the ligand-free structure is large, thereby allowing the aromatic moieties of IRL2500 to flip.
IRL2500 has distinct chemical moieties as compared with bosentan, because IRL2500 was not developed based on bosentan. To reveal the similarities and differences in their binding modes, we compared the binding modes of IRL2500 and bosentan in detail (Fig. 4b, c). The carboxylate of IRL2500 and the sulfonamide of bosentan are similarly coordinated by the positively charged residue R343655, suggesting that this electrostatic interaction is a common feature of the antagonist binding to the ET B receptor. In addition, like bosentan, the aromatic moieties of IRL2500 fit within the local hydrophobic pockets in the ET B receptor. Overall, IRL2500 has moieties that form similar binding interactions to those of bosentan. However, bosentan lacks the moiety corresponding to the biphenyl group of IRL2500, which deeply penetrates into the receptor core. Thus, IRL2500 fits into the pocket more tightly as compared with bosentan, contributing to its higher affinity.
IRL2500 function an inverse agonist for ETb
To obtain mechanistic insights into the receptor inactivation by IRL2500, we compared the ET B structures bound to ET-1, bosentan, and IRL2500. Previous structural studies showed that ET-1 binding induces the inward moment of the extracellular portion of TM6 including W3 3 6 6, 48, leading to receptor activation on the intracellular side 21(Fig. 5a). Bosentan binding sterically prevents the inward motion of W3 3 6 6, 48with its 2-methoxyphenoxy group, and thus functions as an antagonist 22(Fig. 5b). The dimethylphenyl group of IRL2500 superimposes well with the 2-methoxyphenoxy group of bosentan and similarly prevents the inward motion. Moreover, the dimethyl phenyl and biphenyl groups of IRL2500 sandwich the W3 3 6 6, 48side chain, tightly preventing its inward rotation. These observations suggest that IRL2500 strongly prevents the transition to the active state, as compared with bosentan, thereby possibly working as an inverse agonist that reduces the basal activity.
To investigate the inverse agonist activity of IRL2500 for the ET B receptor, we first measured ligand-induced AP-TGFa release responses. The EC 50value of the agonist ET-1 was 0.11 nM, while IRL2500 and the antagonist bosentan did not change the receptor activation level (Fig. 5c). These data suggested that IRL2500 does not have the inverse agonist activity or that the assay is not sensitive enough to detect inverse agonist activity. Indeed, we observed that the basal activity of the ET B receptor was very low in the assay (Fig. 5d) and thus we could not distinguish whether IRL2500 functions as an antagonist or an inverse agonist by this assay.
Therefore, we tried the same assay using a constitutively active mutant of the ET B receptor. Constitutively active mutant GPCRs have been employed in pharmacological characterizations of inverse agonists 30, because such mutant GPCRs allow the assay to measure signals in a larger detection window. The substitution of the highly conserved L3.43 to glutamine has been identified as a causative activating mutation in the TSHR 31 and CYSLTR2 32genes, which are related to hyperthyroidism and uveal melanoma, respectively. Therefore, we transferred the L3.43Q mutation into ET B (ET B-L192 3.43Q) and examined its constitutive activity. We found that ET B-L3.43Q induced spontaneous AP-TGFa release in a plasmid volume-dependent manner (Fig. 5d), indicating that L3.43Q works as a constitutive active mutation in the ET B receptor. We evaluated the dose response effects of bosentan and IRL2500, using the constitutive active mutant ET B-L3.43Q (Fig. 5e). Again, the antagonist bosentan did not change the receptor activation from the baseline level, whereas IRL2500 reduced the basal activity (EC so=1.2 nM). These data indicate that IRL2500 works as a potent inverse agonist for the ET Breceptor, consistent with the structural observations. The biphenyl group of IRL2500 prevents the inward motion of W336 6.48and stabilizes the inactive conformation, and thus IRL2500 functions as an inverse agonist.
Discussion
We have determined the crystal structure of the ETB receptor in complex with the peptide compound IRL2500, and thus elucidated the detailed receptor interactions and the structural basis for its ETB selectivity. Although IRL2500 is designed to mimic the partial structure of ET-1, the binding mode is quite different. Moreover, using the constitutively active mutant that we established in the current study, we first revealed that IRL2500 functions as a potent inverse agonist for the ET Breceptor, and provided the structural basis for the inverse agonistic mechanism.
Small-molecule ETR antagonists have been developed over the years; however, most ETR antagonists have been designed based on bosentan. Thus, the presently available ET agents are chemically very similar. IRL2500 was developed based on ET-1 and has totally distinct chemical moieties, as compared with bosentan. However, the comparison of the IRL2500 and bosentan binding modes revealed the unexpected similarity in their binding interactions. This observation suggests that the chargecomplementary interactions in the center of the pocket form the core of the receptor-antagonist interactions, and the other aromatic moieties fit the local hydrophobic pocket. The ligand binding pocket in the inactive ET Bstructures is larger than those in other GPCR structures, and thus aromatic moieties may be necessary to fit well within the pocket.
We revealed that the biphenyl group of IRL2500 penetrates deeply into the receptor core proximal to D147 2, 50, preventing the inward motion of W336 6.48in TM6, and thus IRL2500 functions as an inverse agonist (Fig. 6a). This D2.50 constitutes a sodium binding site adjacent to the orthosteric site, which is highly conserved among the class A GPCRs33. Sodium has negative allosteric effects on ligand binding in most class A GPCRs, by stabilizing the inactive conformations. Therefore, this sodium binding site is the hot spot for the design of allosteric modulators and inverse agonists to fix receptors in the inactive conformations. In the BLT1 structure bound to the inverse agonist BIIL260, the benzamidine group of BIIL260 directly hydrogen bonds with D2.50, stabilizing the inactive conformation instead of the sodium34 (Fig. 6b). The biphenyl group of IRL2500 superimposes well with the benzamidine group of BIIL260 (Fig. 6c). Although the biphenyl group of IRL2500 does not form any hydrogen-bonding interactions with the receptor, it prevents the conformational change around the D2.50 in a similar manner to the benzamidine moiety of BIIL260. For the design of effective inverse agonists, the biphenyl moiety would be also useful as a modulation part along with another moiety that exerts specific and tight binding to the orthosteric site, as well as a benzamidine group.
Materials and methods
Expression and purification
The haemagglutinin signal peptide, followed by the Flag epitope tag (DYKDDDDK) and a nine-amino-acid linker, was added to the N-terminus of the receptor, and a tobacco etch virus (TEV) protease recognition sequence was introduced between G57 and L66, to remove the disordered N-terminus during the purification process. The C-terminus was truncated after S407, and three cysteine residues were mutated to alanine (C396A, C400A, and C405A) to avoid heterogeneous palmitoylation. To improve crystallogenesis, we introduced four thermostabilizing mutations () and inserted minimal T4 lysozyme 25into intracellular loop 3, between L303 5.68and L311 6.23(ET B-Y4-mT4L 22).
The thermostabilized construct ET B-Y4-mT4L was subcloned into a modified pFastBac vector, with the resulting construct encoding a TEV cleavage site followed by a GFP-His 10tag at the C-terminus. The recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression system (Invitrogen). Sf9 insect cells were infected with the virus at a cell density of 4.0 × 10 6cells per millilitre in Sf900 II medium, and grown for 48 h at 27 °C. The harvested cells were disrupted by sonication, in buffer containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The crude membrane fraction was collected by ultracentrifugation at 180,000g for 1 h. The membrane fraction was solubilized in buffer, containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% DDM, 0.2% cholesterol hemisuccinate, 10 gM IRL2500, and 2 mg ml -1iodoacetamide, for 1 h at 4 °C. The supernatant was separated from the insoluble material by ultracentrifugation at 180,000g for 20 min, and incubated with TALON resin (Clontech) for 30 min. The resin was washed with ten column volumes of buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% LMNG, 0.01% CHS, 10 pM IRL2500, and 15 mM imidazole. The receptor was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% LMNG, 0.001% CHS, 10 pM IRL2500, and 200 mM imidazole. The eluate was treated with TEV protease and dialysed against buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 10 pM IRL2500). The cleaved GFP-His 10tag and the TEV protease were removed with Co 2+-NTA resin. The receptor was concentrated and loaded onto a Superdex200 10/300 Increase size-exclusion column, equilibrated in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% LMNG, 0.001% CHS, and 10 pM IRL2500. Peak fractions were pooled, concentrated to 40 mg ml -1using a centrifugal filter device (Millipore 50kDaMW cutoff), and frozen until crystallization. During the concentration, IRL2500 was added to a final concentration of 100 pM.
Crystallization
The purified receptor was reconstituted into molten lipid (monoolein and cholesterol 10:1 by mass) at a weight ratio of 1:1.5 (protein:lipid). The protein-laden mesophase was dispensed into 96-well glass plates in 30 nl drops and overlaid with 800 nl precipitant solution by a Gryphon LCP robot (Art Robbins Instruments) 26. Crystals of ET B-Y4-mT4L bound to IRL2500 were grown at 20°C in precipitant conditions containing 30% PEG300, 100 mM Bis-tris, pH 7.5, 150 mM sodium phosphate monobasic, and 10 mM TCEP hydrochloride. The crystals were harvested directly from the LCP using micromounts (MiTeGen) or LithoLoops (Protein Wave) and frozen in liquid nitrogen, without adding any extra cryoprotectant.
Data collection and structure determination
X-ray diffraction data were collected at the SPring-8 beamline BL32XU, with 10 × 15 2 (width × height) micro-focused beams and an EIGER × 9M detector (Dectris). Various wedge data sets (10°) per crystal were mainly collected with the ZOO system, an automatic data-collection system developed at SPring-8 (K.Y., G.U., K.H., M.Y., and K.H., submitted). The loop-harvested microcrystals were identified by raster scanning and subsequently analyzed by SHIKA35. Each data set was indexed and integrated with XDS36, and the datasets were hierarchically clustered by using the correlation coefficients of the intensities between datasets. After the rejection of outliers, 58 data sets were finally merged with XSCALE36. The IRL2500-bound structure was determined by molecular replacement with PHASER37, using the K8794-bound ET B structure (PDB code: 5X93). Subsequently, the model was rebuilt and refined using COOT38 and PHENIX39, respectively. The final model of IRL2500-bound ET B-Y4-T4L contained residues 91-207, 214-303, and 311-403 of ET B, 1-14 and 22-117 of mT4L, IRL2500, 6 monoolein molecules, two phosphoric acids, and 41 water molecules. The model quality was assessed by MolProbity40. Figures were prepared using CueMol (http://www.cuemol.org/ja/)
TGFa shedding assay
The TGFa shedding assay, which measures the activation of Gq and G12 signaling24, was performed as described previously22. Briefly, a plasmid encoding an ET B construct with an internal FLAG epitope tag or an ET A construct was transfected, together with a plasmid encoding alkaline phosphatase (AP)-tagged TGFa (AP-TGFa), into HEK293A cells by using a polyethylenimine (PEI) transfection reagent (1 gg ETR plasmid, 2.5 gg AP-TGFa plasmid, and 25 gl of 1 mg/ml PEI solution per 10-cm culture dish). After a one day culture, the transfected cells were harvested by trypsinization, washed, and resuspended in 30 ml of Hank’s Balanced Salt Solution (HBSS) containing 5 mM HEPES (pH 7.4). The cell suspension was seeded in a 96 well plate (cell plate) at a volume of 80 gl per well and incubated for 30 min in a CO 2incubator. For the measurement of antagonist activity, IRL2500 was diluted in 0.01% bovine serum albumin (BSA) and HEPES-containing HBSS (assay buffer) and added to the cell plate at a volume of 10 gl per well. After 5 min, ET-1, at a final concentration of 0.2 nM, was added to the cell plate at a volume of 10 gl per well. For the measurement of agonistic activity, after adding 10 gl of the assay buffer, serially diluted ET-1 was mixed with the cells at a volume of 10 gl per well. After a 1 h incubation in the CO 2incubator, aliquots of the conditioned media (80 gl) were transferred to an empty 96-well plate (conditioned media (CM) plate). Similarly, for the measurement of inverse agonist activity, the cells were mixed with 10 gl of the assay buffer, followed by the addition of serially diluted IRL2500, and incubated for 4 h before the transfer of the conditioned media. The AP reaction solution (10 mM p-nitrophenylphosphate (p-NPP), 120 mM Tris-HCl (pH 9.5), 40 mM NaCl, and 10 mM MgCh) was dispensed into the cell plates and the CM plates (80 gl per well). The absorbance at 405 nm (Abs 405) of the plates was measured, using a microplate reader (SpectraMax 340 PC384, Molecular Devices), before and after a 1 h incubation at room temperature. AP-TGFa release was calculated as described previously 22. The AP-TGFa release signals were fitted to a four-parameter sigmoidal concentration-response curve, using the Prism 7 software (GraphPad Prism), and the pEC 50(equal to-Log i0EC 50) and E maxvalues were obtained.
To measure the constitutive activity in a plasmid volume-dependent manner, HEK293 cells were seeded in a 96-well plate at a concentration of 4 × 10 5cells per ml in Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific), in a volume of 80 gl per well. A transfection mixture was prepared by mixing the PEI transfection reagent (0.2 gl per well) and plasmids (20 ng AP-TGFa plasmid, titrated ETR plasmid, and an empty vector to balance the total plasmid volume) in Opti-MEM I Reduced Serum Media (20 gl). The mixture was added to the cells, which were then incubated for 24 h before the transfer of the conditioned media. After adding the AP reaction solution, the absorbances of the cells and the CM plates were measured at 20 min intervals. The AP-TGFa release signals were calculated as described above, and the signal in the mock-transfected conditions was set at the baseline.
Author contributions
C.N. expressed, purified, and crystallized the IRL2500-bound ET B receptor, collected data, and refined the structures. W.S. designed all of the experiments, initially crystallized the receptor, and refined the structure. A.I., F.M.N.K., and J.A. performed and oversaw the cell-based assays. The manuscript was prepared by C.N., W.S., A.I., and O.N. W.S. and O.N. supervised the research. Coordinates and structure factors have been deposited in the Protein Data Bank, under the accession number XXXX for the IRL2500-bound structure. The X-ray diffraction images are also available at SBGrid Data Bank (https://data.sbgrid.org/), under the ID YYYY.
Competing interests
The authors declare no competing interests
Acknowledgements
The diffraction experiments were performed at SPring-8 BL32XU (proposal 2017A2527). We thank the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for technical assistance during data collection. We also thank Kouki Kawakami, Takeaki Shibata and Ayumi Inoue (Tohoku University, Japan) for technical assistance in the characterization of the L3.43Q-mutant ET B receptor. This work was supported by grants from the Platform for Drug Discovery, Informatics and Structural Life Science by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), JSPS KAKENHI grants 16H06294 (O.N.), 17J30010 (W.S.), 30809421 (W.S.), 17K08264 (A.I.), and the Japan Agency for Medical Research and Development (AMED) grants: the PRIME JP17gm5910013 (A.I.) and the LEAP JP17gm0010004 (A.I. and J.A.), and the National Institute of Biomedical Innovation.