Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia

Abstract

The adenosine A₁ receptor (A₁R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain^1,2. However, development of analgesic orthosteric A₁R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects³. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A₁R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A₁R co-bound to adenosine, MIPS521 and a G_i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

Extended Data Figure 1.. Physiological effects of VCP171 and MIPS521,

a, Chemical structure of VCP171. b, Time courses of paw withdrawal threshold (PWT) to mechanical stimulus by von Frey filaments in nerve-injured rats post-intrathecal administration of VCP171 (blue) or MIPS521 (red). Significance to vehicle control was determined using Greenhouse-Geisser correction for multiple comparisons, corrected via Dunnett’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean +/− SEM (n=8-10 rats per data group). c, Single trial place preference conditioning with intrathecal VCP171 (30 μg, blue), MIPS521 (10 μg, red) and morphine (10 μg, black) increased the time nerve-injured rats spent in the drug paired chamber, with a corresponding decrease in the vehicle paired chamber. Sham surgery rats showed no chamber preference. Empty circles show individual data points, and bars show mean +SEM (n = 8 per group). Significance was determined using a two-tailed unpaired t test assuming unequal variance, * P < 0.05, ** P < 0.01, compared to vehicle control. d, Rotarod latency in rats following intrathecal administration of VCP171 (blue) or MIPS521 (red) is not significantly different to vehicle controls, whereas intrathecal administration of morphine reduces rotarod latency to fall. Data is shown as mean +/− SEM (n = 3-4 per group). Significance was determined using a two-tailed unpaired t test assuming unequal variance, * P < 0.05, ** P < 0.01, compared to vehicle control. e, Effect of CPA (black; n = 4) or MIPS521 (solid red; n = 6) on rate of atrial contraction. Data represent mean ± SD.

Extended Data Figure 2.

a, Examples of spontaneous excitatory postsynaptic potentials (sEPSCs) recorded from neurons of the superficial laminae of the spinal dorsal horn of nerve-injured rats. b, sEPSC frequency and amplitude were reduced following superfusion of VCP171 or MIPS521, which is reversed by the antagonist, DPCPX (n=8 per group); Data are presented as mean values +/− SEM. Significance compared to baseline was determined using a two-tailed paired t test, * P < 0.05, ** P < 0.01.

Extended Data Figure 3 |. Expression and purification of the MIPS521-ADO–A1R–G_i2 complex.

a, Expression and purification flowchart for the A1R–G_i2 complex. A1R and the G_i2 heterotrimer with Gβ₁γ₂ were expressed separately in insect cell membranes. Addition of ADO (1 mM) and MIP521 (100 nM) initiated complex formation, which was solubilised with 0.5% (w/v) lauryl maltose neopentyl glycol and 0.05% (w/v) cholesteryl hemisuccinate. Solubilised A1R and A1R –G_i2 complex was immobilised on Flag antibody resin. Flag-eluted fractions were purified by size-exclusion chromatography (SEC). b, Representative SDS–PAGE/western blot of the purified A1R–G_i2 complex. An anti-His antibody was used to detect Flag–A1R-His and Gβ₁-His (red) and an anti-G_i2 antibody was used to detect Gα_i2 (green). Experiment was performed three times with similar results c, Representative SDS–PAGE/Coomassie blue stain of the purified complex concentrated from the Superdex 200 Increase 10/30 column. Experiment was performed three times with similar results d, Representative elution profile of Flag-purified complex on Superdex 200 Increase 10/30 SEC. Experiment was performed three times with similar results

Extended Data Figure 4 |. Cryo-EM data processing for the MIPS521-ADO-A1R-G_i2 (a) and ADO-A1R-G_i2 (b) complexes.

Representative cryo-EM micrographs of each of the complexes. Reference-free 2D class averages of the complexes in LMNG and CHS detergent micelles. Gold-standard Fourier shell correlation (FSC) curves, showing the overall nominal resolution of 3.2 Å and 3.3 Å, respectively, at FSC 0.143. Corresponding 3D cryo-EM maps coloured according to local resolution estimation (Å) in Relion. c, Atomic resolution model of representative regions from the MIPS521-ADO-A1R-G_i2 structure of the A1R transmembrane domain, ADO, and MIPS521. The molecular model is shown in ball and stick representation, coloured by heteroatom, and the cryo-EM map displayed in mesh contoured at 0.02.

Extended Data Figure 5 |. Stable hydrogen bonds formed between residue S6.47/L7.41 in A1R and MIPS521 in A1R-G_i2-MIPS521:

(a and c) GaMD and (b and d) cMD simulations. Each simulation trace is displayed in a different colour (black, red, blue). The lines depict the running average over 2 ns.

Extended Data Figure 6 |. Affinity of orthosteric ligands at mutations of the MIPS521 extrahelical allosteric binding pocket.

a, c, The affinity of (a) [³H]DPCPX and (c) NECA for wildtype and mutant A₁Rs performed in FlpInCHO cells. b, Bmax; determined by [³H]DPCPX radioligand saturation binding studies. Data are the means + S.E.M. of 3-7 independent experiments (shown as circles) performed in duplicate. *P < 0.05 (compared with WT; one-way analysis of variance, Dunnett’s post hoc test).

Extended Data Figure 7 |. Extrahelical binding sites for allosteric modulators of class A GPCRs.

The unique extrahelical binding pose of MIPS521 in the A1R (orange) compared to previously reported extrahelical allosteric binding pockets for class A GPCRs in P2Y1R (BPTU, red; PDB 4XNV), PAR2 (AZ3451, yellow; PDB 5NDZ), CB1 (ORG28569, green; 6KQI), GPR40 (AP8, cyan; PDB 5TZY), C5aR (NDT9513727, blue; PDB 5O9H), D1R (LY3154207, navy; PDB 7LJD), and β2AR (Compound-6FA, pink; PDB 6N48).

Extended Data Figure 8 |. Stability of MIPS521 at the allosteric binding site of A1R is enhanced by G_i2 protein coupling to the receptor.

a, b, RMSD (Å) of MIPS521 relative to the starting cryo-EM conformation obtained from GaMD simulations in the (a) absence and (b) presence of G_i2. c, d, RMSD (Å) of MIPS521 relative to the starting cryo-EM conformation obtained from cMD simulations in the (c) absence and (d) presence of G_i2. Each condition represents three GaMD/cMD simulations, with each simulation trace displayed in a different colour (black, red, blue). Lines depict the running average over 2 ns.

Extended Data Figure 9 |. MIPS521 stabilises the A1R-Gi2 ternary complex.

a-d, RMSD (Å) of ADO from cMD simulations completed in the (a) absence or (b) presence of MIPS521, (c) G_i2, or (d) both G_i2 and MIPS521. e-h, Distance between the intracellular ends of TM3 and TM6 (measured as the distance in Å between Arg105^3.50 and Glu229^6.30) in the (e) absence or (f) presence of MIPS521, (g) G_i2, or (h) both G_i2 and MIPS521. Each condition represents three cMD simulations, with each simulation trace displayed in a different colour (black, red, blue). The lines depict the running average over 2 ns. i,j, Distance between A1R and G_i2 (measured as the distance in Å between the NPxxY motif of A1R and the C terminus of the Gα α5 helix) from GaMD simulations in the (i) absence and (j) presence of MIPS521. k,l, Distance between A1R and G_i2 from cMD simulations in the (k) absence and (l) presence of MIPS521. Each condition represents three GaMD/cMD simulations, with each simulation trace displayed in a different colour (black, red, blue). Thick lines depict the running average over 2 ns. m-p, Flexibility change upon removal of PAM and/or G_i2 protein from the ADO-bound A1R obtained from GaMD simulations. (m) Root-mean-square fluctuations (RMSFs) of the A1R-G_i2-MIPS521. A colour scale of 0.0 Å (blue) to 5.0 Å (red) was used. (n) Change in the RMSFs of the A1R-G_i2 when MIPS521 was removed from A1R-G_i2-MIPS521. (o) Change in the RMSFs of the A1R and MIPS521 when the G_i2 was removed from A1R-G_i2-MIPS521. (p) Change in the RMSFs of the A1R when the G_i2 and MIPS521 were removed from A1R-G_i2-MIPS521 system. A colour scale of −2.0 Å (blue) to 2.0 Å (red) was used for n, o and p.

Extended Data Figure 10 |

Cryo-EM data collection, refinement and validation statistics

Figure 1 |. MIPS521 reduces spinal nociceptive signalling and mechanical allodynia in an animal model of neuropathic pain.

a, MIPS521 and VCP171 produce a concentration-dependent potentiation of signalling by the endogenous agonist, adenosine (ADO), in an inhibition of cAMP assay (shown as inhibition of 3μM forskolin mediated cAMP) mediated by the human A1R expressed in CHO cells. An operational model of allosterism and agonism yielded estimated MIPS521 and VCP171 affinities of pK_B = 4.95 ± 0.40 & 5.50 ± 0.29, cooperativity of Logαβ = 1.81 ± 0.53 & 0.76 ± 0.25 and direct allosteric agonism of Logτ_B = 0.96 ± 0.34 & −0.23 ± 0.12, respectively; data shows mean ± SEM n =3, duplicate readings. b, Examples of dorsal root eEPSCs in neurons of the superficial laminae (I-II) of the spinal dorsal horn at baseline and following superfusion of 1 μM VCP171 or MIPS521, which is reversed by the antagonist, DPCPX in nerve-injured rats (partial nerve ligation; PNL). c, MIPS521 is more potent than VCP171 at reducing eEPSC amplitude in dorsal horn neurons. Effects of MIPS521, but not VCP171, were increased in the nerve-injured group compared to the sham surgery control group (n = 4-12 per data point); Data are presented as mean +/− SEM. d, Paw withdrawal threshold (PWT) to mechanical stimulus in nerve-injured rats 1-hour post-intrathecal administration of VCP171 (blue, n = 9 per group) or MIPS521 (red, n = 8-10 per group). Data is displayed as mean +SEM (bars) and individual values (circles). Significant differences were determined using a one-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001. e, The effect of intrathecal VCP171 (30 μg, blue), MIPS521 (10 μg, red) or morphine (10 μg, black) on spontaneous pain was determined using conditioned place preference tests in nerve injured rats (n = 8 per group); Data are presented as mean values +/− SEM.

Figure 2 |. Comparison of the structures of the A1R-G_i2 complex in the presence and absence of the positive allosteric modulator, MIPS521.

a, b, Overlay of the A1R-G_i2 complex in the presence and absence of MIPS521, showing (a) the whole complex and (b) receptor alone (in MIPS521-ADO-A₁R-G_i2 the receptor is blue and the heterotrimeric G_i2 pink, cyan and dark purple for α, β and γ, respectively, ADO in purple and MIPS521 in orange; the ADO-A1R-G_i2 complex is coloured grey, with ADO in plum), c, The orthosteric binding site of the A1R-G_i2 complex in the presence and absence of MIPS521 is highly conserved. Water molecule shown as red sphere. ADO is shown in ball and stick representation and residues of the orthosteric binding pocket in stick representation, coloured by heteroatom.

Figure 3 |. Identification of an extrahelical lipid-facing allosteric binding pocket involving TMs 1,6 & 7 on the A1R.

a, Surface rendering of the MIPS521 binding site located in an extrahelical position between TM6 & 7. b, Extrahelical allosteric binding pocket of the A1R in the presence and absence of MIPS521, with a pivoting of M283^7.48 to accommodate MIPS521 (Pocket residues in stick representation coloured by heteroatom; MIPS521-ADO-A1R-G_i2 complex (blue), MIPS521 (orange); ADO-A1R-G_i2 complex (grey), c, Comparison of the binding pose of MIPS521 from the cryo-EM structure (orange) and in situ docking (yellow), d, Allosteric modulation of orthosteric ligand affinity at the WT A1R demonstrated in a [³H]-DPCPX radioligand interaction binding assay in the presence of the orthosteric agonist NECA and MIPS521. Data are presented as mean +/− SEM, n=6. e, f, Changes in MIPS521 affinity (pK_B) (e) or binding cooperativity (log a) with the orthosteric agonist NECA (f) following mutation of residues proposed to form the allosteric pocket identified in the cryo-EM structure. Parameter estimates are the mean ± SEM determined from n=3-6 (white circles) performed in duplicate. *P < 0.05 (compared with wild type; one-way ANOVA, Dunnett’s post hoc test).

Figure 4 |. MIPS521 stabilises the A1R-Gi2 ternary complex.

a-d, RMSD (Å) of ADO from GaMD simulations completed in the (a) absence or (b) presence of MIPS521, (c) G_i2, or (d) both G_i2 and MIPS521. e-h, Distance between the intracellular ends of TM3 and TM6 (measured as the distance in Å between Arg105^3.50 and Glu229^6.30) in the (e) absence or (f) presence of MIPS521, (g) G_i2, or (h) both G_i2 and MIPS521. Each condition represents three GaMD simulations, with each simulation trace displayed in a different colour (black, red, blue). The lines depict the running average over 2 ns. i, MIPS521 has no effect on the dissociation rate of [³H]DPCPX, promoted by isotopic dilution with excess antagonist, SLV320 (1 μM; Control), at the A1R alone reconstituted in rHDL. Addition of a saturating concentration of G_i results in a retardation of [³H]DPCPX dissociation that is reduced by co-addition of MIPS521. Data is mean ± SEM, n = 3-5 j, Quantification of dissociation rates from traces. Data is mean ± SEM, n ≥ 3 experiments performed in duplicate. *P < 0.05 (compared to control; one-way ANOVA, Tukey’s post hoc test). #P < 0.05 (compared to control in the presence of G_i2; one-way ANOVA, Tukey’s post hoc test).