PMC

Reagents and conditions: a) ethyl acetoacetate, pyridine, 100°C, b) 2-fluoro-5-formylbenzoic acid, Yb(OTf)₃, Urea, CH₃CN, 100°C c) HATU, DIEA, 2-(aminomethyl)pyridine, DMF

Comparison of GRK2 and GRK5 hydrophobic binding pockets when bound to 12h (yellow) (a) GRK2 has a much wider and shallower binding pocket (non-polar residues highlighted in purple) and (b) GRK5 has a deeper, narrower, and overall smaller binding pocket (non-polar residues are highlighted in teal).

The hydrophobic subsite is unexploited in the GRK2 inhibitor 2 complex. Shown is a superposition of the small lobes of GRK2 in complex with 2 (salmon) and 1 (purple) (PDB entries 4PNK and 3PVW, respectively). Hydrophobic surfaces are colored yellow. The D-ring of balanol () also extends into the hydrophobic subsite.

Reagents and conditions: a) TFAA, Et₃N, Toluene, 0°C, b) Boc₂O, DIEA, DMAP, THF, c) NaH, MeI, DMF, 0°C to rt, d) K₂CO₃, H₂O, MeOH, reflux, e) 2,2,6-trimethyl-4H-1,3-dioxin-4-one, CH₃CN, 100°C, f) 2-fluoro-5-formylbenzoic acid, Yb(OTf)₃, urea, CH₃CN, 100°C, j) HATU, DIEA, 2,6 – dimethyl benzylamine, DMF

Reagents and conditions: a) cat. H₂SO₄, MeOH, b) (BzO)₂, NBS, CCl₄, 80°C, 25 min, c) DIBAL, toluene, d) NaCN, DMSO, e) NaOH, reflux, f) SO₃·pyridine, DMSO, Et₃N, g) 2,2,6-trimethyl-4H-1,3-dioxin-4-one, CH₃CN, 100°C, h) 2-fluoro-5-formylbenzoic acid or 7, Yb(OTf)₃, urea, CH₃CN, 100°C, i) HATU, DIEA, RNH₂, DMF

Maximum contraction amplitudes of the known GRK2 inhibitor paroxetine, the lead 2, 12h, 12d,12m, and 12n before and after isoproterenol stimulation. Doses shown are the minimum inhibitory concentrations that exhibit a p-value <0.05 versus Control. Values represent the mean ± SEM for 8–10 cardiomyocytes.

Known GRK2 inhibitors. The A, B, C and D rings pack in the adenine, ribose, polyphosphate, and hydrophobic subsites of the kinase domain, respectively. That is, the A ring interacts with the hinge, whereas the D ring interacts in a pocket defined by the P-loop, the αB and αC helices in the small lobe, and by the DFG loop in the large loop.

Adaptive structural changes in the GRK2 P-loop. Compared to the P-loop conformation when bound to compound 2 (green), the Cα carbon of Gly201 shifts away from the binding site by 2.2 Å when bound to compound 12n (blue), 12h or 12r (not shown), and by 3.0 Å when bound to 12k (orange). The magnitude of the shift thus appears to depend on the size of the D-ring.

Structural differences in the hinge regions of PKA, GRK2, and GRK5. Cα traces of PKA bound to AMPPNP (PDB entry 4HPT, red) or balanol (PDB entry 1BX6, orange), superimposed onto GRK2·compound 2 (PDB entry 4PNK, blue). GRK2·12n (purple) and GRK5·12h (green) are also shown for comparison. Hinge residues that form hydrogen bonds with the indazole nitrogens of compound 2 and its derivatives are 1.5–1.7 Å closer to the inhibitor in the structures of GRK2 and GRK5 relative to those of PKA.

Molecular origins of selectivity for compound 12n. Surface representations of GRK2·12n (a), GRK5 (PDB entry 4WNK, (b), and ROCK1 (PDB entry 3V8S, (c) with hydrophobic and polar/charged residues colored green and gray, respectively. 12n is superimposed onto GRK5 and ROCK1 to demonstrate potential clashes. (b) In GRK5, 12n appears to clash with both Met230 from the αC helix (Leu235 in GRK2) and Gly331 from the DFG loop (Gly337 in GRK2). (c) In ROCK1, 12n may clash with Phe120 (Leu-235 in GRK2) and backbone atoms of Gly218 in the DFG loop (Gly337 in GRK2). For this modeled complex, ROCK1-Asp216 was changed to the rotamer of the analogous residue in the GRK2·12r complex (Asp335).

Co-crystal structures reveal that the inhibitors bind in the ATP-binding pocket in a similar conformation as the compound 2 parent structure. 3σ |F_o| – |F_c| omit maps of compounds 12h (a), 12k (b), 12n (c), and a 2σ |F_o| – |F_c| omit map of 12r (d) are represented as magenta wire cages superimposed onto the refined X-ray crystal structures. Hydrogen bonds with the labeled GRK2 residues are shown as black dashed lines. The P-loop and hinge region are indicated for reference.

Inhibitor potency for GRK2 correlates with buried ASA (R²=0.8) but not the number of hydrogen bonds (R²=0.01). K_i values were calculated from experimentally determined IC₅₀ values of the GRK2 inhibitors for which there is a crystal structure () using the Cheng-Prusoff transformation and plotted as a function of both their buried ASA (circles) and the number of hydrogen bonds they form with GRK2 (x’s). Buried ASA for inhibitors from this study are shown in green.