Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors

Abstract

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson's disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

Figure 1

Small molecule inhibitors of GRKs, ROCK, and IGF-1R. Compounds identified in a DSF screen were classified into three classes: (a) an indazole class of compounds, (b) a pyrrolopyrimidine class, and (c) other compounds. The previously identified inhibitor paroxetine is structurally related to the indazole compounds and is included in panel a for comparison. Shaded boxes illustrate where molecules are different from the reference GSK180736A or GSK2163632A compounds. (d) ROCK inhibitors CHEMBL225282 and CHEMBL1082820, related to the indazole class. (e) IGF-1R inhibitors CHEMBL464552 and CHEMBL507625, related to the pyrrolopyrimidine class.

Figure 2

GRK2 and 5 inhibitor potency correlates with ΔT_m and buried surface area. Compounds with undetermined IC₅₀ values in Table 2 are omitted. (a) ΔT_m of GRK2 is strongly correlated (Pearson’s r = −0.833, P = 0.0004) with potency. (b) ΔT_m of GRK5 is also significantly correlated (r = −0.6309, P = 0.0156). (c) ΔT_m of GRK1 does not, however, correlate (r = −0.091). (d) Buried surface area of small molecules crystallized in complex with GRK2 is significantly correlated with their potency (r = −0.787, P = 0.0316). In each panel, paroxetine is denoted by a square.

Figure 3

Comparison of cocrystal structures of GSK inhibitors with those of paroxetine in complex with GRK1 and GRK2. (a) GRK2·GSK180736A (stick model with dark gray carbons) is well-ordered in the active site. Three σ |F_o| – |F_c| omit map density is shown as a light gray mesh in panels a and c. (b) GSK180736A binds in a manner that is nearly superimposable with paroxetine (black carbons, PDB entry 3V5W). (c) Crystal structure of the GRK1·GSK2163632A complex. (d) Occupation of the AST subsite by GSK2163632A alters the conformation of the AST loop (magenta) compared to that in the GRK1·paroxetine complex (PDB entry 4L9I). Orange dashed lines indicate hydrogen bonds or salt bridges.

Figure 4

Comparison of GRK·inhibitor complexes with ROCK1 and IR bound to related compounds. (a) Superposition of the GRK2·GSK180736A structure (yellow carbons for residues in small lobe and green carbons for large lobe, GRK2 numbering) with that of ROCK1·CHEMBL1082820 (transparent black carbons) reveals similar hydrogen bonds (orange dashed lines) formed with the hinge in the adenine subsite and similar docking of the halogen-substituted phenyl in the polyphosphate subsite. Numbering corresponds to human GRK2. (b) Superposition of the GRK1·GSK2163632A structure with that of the IR kinase·CHEMBL464552 complex (transparent black carbons) shows excellent alignment with the exception of the P-loop. Numbering corresponds to bovine GRK1.