Precise druggability of the PTH type 1 receptor

Abstract

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

Extended Data Fig. 1. Close-up view of the pocket at the EC vestibule.

The pocket is lined by TM1 (red) and TM2 (cyan). Essential residues R181^1.33b and Y245^2.72b, whose interaction with ligands is predicted to affect the global dynamics of the overall PTHR, are shown in yellow sticks. Two additional residues, D252^ECL and F184^1.36b are also shown, which may help attract or coordinate the ligands through electrostatic interactions. The role of F184 in complementing R181^1.33b and Y245^2.72b to line a druggable pocket is further corroborated by druggability simulations.

Extended Data Fig. 2. Probe molecules used in druggability simulations.

(a) Seven probe molecules used in PTHR druggability simulations. The structure of each probe is shown along with its 4-letter acronym, its name and main chemical properties. Also shown is the percentage of drugs containing those fragments based on the SMILES data of 2,453 approved drugs in DrugBank. (b) An example of a common drug, benzyl-penicillin, containing four types of fragments represented by the probe molecules. (c) Binding score profile for PTHR residues near Site 1 (labeled along the abscissa), evaluated for different probe molecules (bars in different colors). The binding score is defined as Σ (1/d_ki) where k is frame/snapshot index and d_ki is corresponding distance between the probe and the residue i summed over all snapshots n (12,000 compiled from six independent runs), provided that they make atom-atom contacts of d_ki < 4.0 Å. Residues with score above 1500/Å (dashed line) are selected as high affinity residues for each probe type: R181^1.33b for acetate, and F184^1.36b and Y245^2.72b for isopropanol. (d) Close-up view of site 1 preferentially sampled by three probes (two isopropanol and an acetic acid, shown in yellow and red sticks respectively), and associated residues R1811.33b, F184^1.36b and Y245^2.72b (right). (e, f) Construction of pharmacophore model (PM) composed of a hydrogen bond acceptor, a negatively charged region, and two hydrophobic sites (spheres), based on the preferential positions observed in (c), and overlay of a hit compound Pitt12 (aquamarine sticks, extracted from the ZINC database) and the PM. The box on the left in panel (f) shows the partial charges for the carboxylate group atoms O, C, O’, and H in Pitt12 in its protonated and deprotonated forms. In line with the use of acetate probe molecule to construct the PM, we used the deprotonated form in further analyses and simulations.

Extended Data Fig. 3. Actions of Pitt8 on PTH signaling.

(a, b) Binding isotherms showing competitive inhibition of radio-labeled peptide ligand binding to PTHR by PTH or Pitt8, using plasma membrane extracts from HEK293 cells expressing recombinant PTHR. Inhibition of [¹²⁵I]-LAPTH binding to G protein-independent conformational R₀ state of PTHR (i.e., in the absence of G proteins) by PTH_1-34 (black) or Pitt8 (pink) (a). Inhibition of [¹²⁵I]-M-PTH_1-15 binding to G protein-dependent R_G conformational state of PTHR (i.e., in the presence of G proteins) by M-PTH_1-15 (black) or Pitt8 (pink). Mean ± s.d. of N = 6 experiments. (c) Concentration-response curves for cAMP production by PTH alone or together with Pitt8. Data are mean ± s.e.m. of N = 3 independent experiments. (d, e) Averaged cAMP time-courses following brief stimulation with 1 nM PTH without (Ctrl, black) or with 10 μM Pitt12 measured by FRET changes from HEK293 cells stably expressing PTHR and a FRET-based cAMP sensor Epac^CFP/YFP in the absence (d) or presence of the dominant-negative dynamin mutant (DynK44A) tagged with RFP (e). (f) Time course of β-arrestin 2 interaction with PTHR measured by FRET in HEK293 cells transiently expressing PTHR^CFP and βarr-2^YFP following brief stimulation with 10 nM PTH without (Ctrl) or with 10 μM Pitt8. Cells were continuously perfused with control buffer or PTH alone or together with Pitt8 (horizontal bar). Data are the mean ± s.e.m. of N = 3 experiments with n = 52 (DMSO) and 47 (Pitt8) cells examined. (i) Time courses of Ca²⁺ release in response to PTH (100 nM) with or without Pitt8 (10 μM) in live HEK-293 cells expressing recombinant PTHR. Data are the mean ± s.e.m. of N = 3 experiments with n = 32 (DMSO) and 32 (Pitt8) cells examined.

Extended Data Fig. 4. Statistics for integrated responses from Figure 3 and ED Figure 3.

(a) pKi values for Pitt12 for the R_G state were: −10.77 ± 0.15 (M-PTH_1-15) and −4.39 ± 0.96 (Pitt12); for the R₀ state: −8.26 ± 0.14 (PTH_1-34) and −5.15 ± 0.63. pKi values for Pitt8 the R_G state were: −10.77 ± 0.15 (M-PTH_1-15) and −5.25 ± 0.48 (Pitt8); for the R₀ state: −8.26 ± 0.14 (PTH_1-34) and −4.79 ± 0.30 (Pitt8). Mean ± s.d. of N = 6 experiments. (b) Quantitation of cAMP responses by measuring the area under the curve (A.U.C.) from 0 to 20 min for figure 3e,f, and ED-figure 3d,e. Data are mean ± s.d. of N = 6 (PTH), 3 (Pitt12), and 3 (Pitt8) experiments for control, and N = 4 (PTH), 4 (Pitt12), and 4 (Pitt8) experiments for Dyn^K44A. NS, not significant, **P < 0.002 and ***P < 0.0002 by two-way ANOVA with Tukey-Kramer post-hoc test. (c, d) Data are from figure 3h and ED-figure 3g. Mean ± s.e.m. of N = 3 independent experiments. P values were assessed by two-tailed Student’s t-test with *P<0.05, **P<0.005.

Figure 1.. ESSA points to an extracellularly-exposed pocket as an essential site that can potentially alter the allosteric dynamics of PTHR upon ligand binding.

(a) Distribution of ESSA z-scores for PTHR active conformer in the presence (red curve) or absence (blue curve) of PTH. Peaks indicate the essential sites. Residue ranges of the PTH, EC domain and TM helices are indicated along the upper abscissa, also delimited by different shades in the graph. TM residues E180, R181, D185, Y191, D241 and Y245 on TM1 and TM2 exhibit z-score above the threshold value of 1.0 (without peptide) (see Methods). Among them, R181 and Y245 exhibit peaks in both structures. Other peaks correspond to loop regions. Thus, R181 and Y245 stand out as essential TM sites that can modulate the global dynamics. (b–c) Same results as in a, illustrated by color-coded diagrams side (top) and cytoplasmic-facing (bottom) views. Panel b shows a global hinge site (yellow dashed line) that corresponds to the blue peaks in A. High-to-low scores are color-coded from red-to-blue. The peptide PTH is shown in magenta ribbon in C. A significant increase in sensitivity is observed at the G protein binding region in the presence of PTH. (d) Two hydrophobic pockets (wheat and gray) determined by Fpocket, surrounded by five of the essential residues detected by ESSA (without the peptide). See also Extended Data Figure 1. (e) Structural elements identified by ESSA to potentially alter the essential dynamics of the receptor, color coded by ESSA score from red (z-score > 3) to blue (z-score close to zero). The analysis was performed for PTH-bound PTHR structure in an active state (PDB id: 6nbf) after modeling the missing loops.

Figure 2.. Identification of druggable sites and drug-like small molecules targeting PTHR.

(a) Summary of a 6-step computational protocol toward identification of small molecule modulators of PTHR activity. The pipeline comprises three major components: druggability simulations using DruGUI (left; blue box); detection of a high-affinity essential site and pharmacophore modeling using Pharmmaker (middle; yellow box), and virtual screening (VS) of one or more pharmacophore models (PM) against libraries of small molecules using Pharmit (right, green box). See text for details. The figure illustrates how two hits, compounds 1 and 2 (thereafter designated as Pitt8 and Pitt12, shown on right) were derived using the PM deduced from DruGUI. (b) Effect of computationally identified small molecules on PTH-induced cAMP production in HEK293 cells stably expressing the recombinant human PTHR. The bars graph represents the area under the curve (AUC) of cAMP time-courses (shown in Supplementary Figure 1) with 1 nM PTH and with or without 10 μM of compound (see Supplementary Tables 1 and 2). The dashed grey line depicts the average level of PTH alone induced cAMP and is shown for guidance. Error bars represent the mean values ± s.d. of N = 3 independent experiments for Pitt8 and Pitt12 (compounds 1 and 2), N = 2 for the rest compounds, and N = 25 for PTH alone. Only Pitt8 and Pitt12 significantly reduced PTH-induced cAMP generation (P values were determined by one-way ANOVA multiple comparisons test, and are 0.0268 for Pitt8, and 0.017 for Pitt12.

Figure 3.. Actions of Pitt12 on PTH signaling.

(a–b) Binding isotherms showing competitive inhibition of radio-labelled peptide ligand binding to PTHR by PTH or Pitt12, using plasma membrane extracts from HEK293 cells expressing recombinant PTHR. Inhibition of [¹²⁵I]-LAPTH binding to G protein-independent conformational R₀ state of PTHR (i.e., in the absence of G proteins) by PTH_1-34 (black) or Pitt12 (teal) (a). Inhibition of [¹²⁵I]-M-PTH_1-15 binding to G protein-dependent R_G conformational state of PTHR (i.e., in the presence of G proteins) by M-PTH_1-15 (black) or Pitt12 (teal) (b). Mean ± s.e.m. of N = 6 experiments carried out in duplicate (c). (c, d) Concentration-response curves for cAMP production by PTH alone or together with Pitt12 (c), and effect of a range of Pitt12 concentrations on PTH mediated cAMP (d). Data are mean ± s.e.m. of N = 3 (c) and N = 5 (d) independent experiments. (e, f) Averaged cAMP time-courses following brief stimulation with 1 nM PTH without (Ctrl, black) or with 10 μM Pitt12 measured by FRET changes from HEK293 cells stably expressing PTHR and a FRET-based cAMP sensor Epac^CFP/YFP in the absence (e) or presence of the dominant-negative dynamin mutant (DynK44A) tagged with RFP (f). Data are mean ± s.d. of N = 3 experiments. (g) Time course of β-arrestin 2 interaction with PTHR measured by FRET in HEK293 cells transiently expressing PTHR^CFP and βarr-2^YFP following brief stimulation with 10 nM PTH without (Ctrl) or with 10 μM Pitt12. Cells were continuously perfused with control buffer or PTH alone or together with compound Pitt12 (horizontal bar). Data are the mean ± s.e.m. of N = 3 experiments with n = 71 (DMSO) and n = 71 (Pitt12) cells examined. (h) Time courses of Ca²⁺ release in response to PTH (100 nM) with or without Pitt12 (10 μM) in live HEK-293 cells expressing recombinant PTHR. Data are the mean ± s.e.m. of N = 3 experiments with n = 42 (DMSO) and n = 33 (Pitt12) cells examined.

Figure 4.. Refinement and validation of Pitt12 binding site in PTHR.

(a–c) Results from MD simulations (200 ns) of Pitt12 binding to active state, LA-PTH–bound PTHR (PDB 6NBF) embedded in membrane, showing predictive interactions (a), and time-evolution of Pitt12 engagement with the peptide ligand and PTHR residues (b), as well as the overall view of the simulation system and bound Pitt12 (c). Interaction is defined as existence of any heavy atom contact pairs between PTHR (or LA-PTH) and Pitt12 within distance < 4.0 Å. Inset of panel c shows 2D representations of closely interacting units, where same color circles (orange, magenta, cyan, or green) indicate interacting pairs of atoms between Pitt12 and E180/R181. As the initial pose of Pitt12- and LA-PTH-bound PTHR, we used the cryo-EM structure of LA-PTH-bound PTHR (PDB id: 6NBF), into which we inserted Pitt12 upon structural alignment with our snapshot from druggability simulations (which was adopted in the construction of the pharmacophore model as well as in virtual screening). (d, e) Experimental validation of Pitt12 binding site in PTHR. PTH (1 nM)-induced cAMP generation time-courses (d) and corresponding integrated cAMP responses (e) from wild-type and receptor mutants carrying individual E180A or R181A mutation, in presence or absence of 10 μM Pitt12. Mean ± s.e.m. of N = 2 or 3 experiments with n = 39 (PTH) and n = 45 (PTH+Pitt12) cells examined for PTHR-WT, n = 46 (PTH) and n = 50 (PTH+Pitt12) cells examined for PTHR-E180A, and n = 40 (PTH) and n = 57 (PTH+Pitt12) cells examined for PTHR-R181A. P values were assessed by two-tailed Student’s t-test.

Figure 5.. Conformational change of PTHR due to Pitt12 binding.

(a) Predicted Pitt12 binding-induced conformational changes in PTHR. N-terminal tip of LA-PTH pushes extracellular tip of TM6 further outwards leading to outward displacement of TM5 and TM6 helices. (b) Comparison of the conformations stabilized in the MD runs performed for LA-PTH/PTHR in the presence and absence of Pitt12. Left, the superimposed final (t = 200 ns) snapshots from the two runs. Essential residues coordinating Pitt12 are shown in stick representation and labeled. Right, the time evolution of C_α-C_α distances between E180 of PTHR and R21 of LA-PTH (left panel) and between R181 of PTHR and A18 of LA-PTH (right panel) are presented, for the runs conducted in the presence (black) or in the absence of Pitt12 (green). The space between PTHR and LA-PTH is wider in the absence of Pitt12.

Figure 6.. In vivo action of Pitt12.

(a–c) Serum Ca²⁺ (sCa²⁺) and phosphate (sPi) levels, as well as BUN/creatine ratio were measured 3 hrs after injections of vehicle (Veh), Pitt12 (20 μmole/kg), PTH_1-34 (40 μg/kg), or Pitt12+PTH_1-34. Mean ± s.e.m. of N = 10 (veh), 5 (Pitt12), 9 (PTH_1-34) and 10 (PTH_1-34+Pitt12) mice for sCa²⁺, and N = 10 (veh), 5 (Pitt12), 10 (PTH_1-34) and 10 (PTH_1-34+Pitt12) mice for sPi, and N = 10 (veh), 5 (Pitt12), 9 (PTH_1-34) and 7 (PTH_1-34+Pitt12) mice for BUN/Creatine ratio. P values were assessed by one-way ANOVA with Dunnett test. *P values are indicated.