Explore these ideas and more!G protein-coupled receptors Dibal k-335 manual are central to many physiological processes. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, Dibal k-335 manual, a library of hybrid inhibitors was developed. Four dibal k-335 manual the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors. Best way to inject steroids failure is characterized by an inability of the heart to produce a myocardial contraction strong enough to effectively pump blood throughout the body. To compensate for the reduced output, the sympathetic nervous system increases the levels of catecholamines such as norepinephrine and epinephrine. Inhibition of GRKs is another route by which heart failure might be addressed.
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G protein-coupled receptors GPCRs are central to many physiological processes. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, TakedaA, a library of hybrid inhibitors was developed.
Four of the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors. Heart failure is characterized by an inability of the heart to produce a myocardial contraction strong enough to effectively pump blood throughout the body. To compensate for the reduced output, the sympathetic nervous system increases the levels of catecholamines such as norepinephrine and epinephrine. Inhibition of GRKs is another route by which heart failure might be addressed.
GRK2, a member of the GRK2 subfamily, and GRK5, a member of the GRK4 subfamily, are known to play significant roles in the heart and have shown promise as alternative therapeutic targets for heart failure. When targeting GRKs, it is imperative to identify potent chemical probes that are highly selective against other AGC kinases as they play key roles in regulating many vital physiological processes, including inotropy.
There is, however, precedence for both potent and selective chemical probes. Despite their impressive in vitro profile, these compounds never advanced to clinical trials, presumably due to poor bioavailability.
The A, B, C and D rings pack in the adenine, ribose, polyphosphate, and hydrophobic subsites of the kinase domain, respectively. The selective serotonin reuptake inhibitor paroxetine Figure 1 was later identified as a modestly potent inhibitor of GRK2 with an IC 50 of 1.
However, unlike compound 1 and balanol, 2 does not have a substituent that can occupy the hydrophobic subsite. Crystal structures of four representative compounds in complex with GRK2 give insights into the molecular basis for the improved potency and selectivity of these compounds. The hydrophobic subsite is unexploited in the GRK2 inhibitor 2 complex.
Hydrophobic surfaces are colored yellow. The D-ring of balanol Figure 1 also extends into the hydrophobic subsite. Hybrid analogs 12a-r and 13a-c were prepared as described in Scheme 1. Synthesis began with Fischer esterification of 4-fluoromethylbenzoic acid 3 followed by benzylic bromination under radical conditions to give the methyl ester 4. After reduction of the ester, displacement of the bromine using sodium cyanide afforded the nitrile 5. The nitrile was hydrolyzed under basic conditions to yield the carboxylic acid 6.
Oxidation of the benzylic alcohol of 6 using 2-iodoxybenzoic acid or under Swern conditions proved unsuccessful. Fortunately, Parikh Doering oxidation yielded the aldehyde 7 cleanly and in high yield.
In a converging pathway, treatment of 5-aminoindazole 8 with 2,2,6-trimethyl-4 H -1,3-dioxinone resulted in the acetylated compound 9. All IC 50 measurements are an average of three separate experiments run in duplicate. Synthesis of the N-methylated hybrid molecule 20 commenced with acylation of 5-aminoindazole 8 using trifluoroacetic anhydride to give Therefore, protection of the indazole nitrogen was necessary, and was accomplished using Boc-anhydride to give two regioisomers of Methyl iodide in the presence of sodium hydride then furnished the methylated amide Removal of the trifluoroacetate and Boc under refluxing basic conditions afforded the monomethylated amine 17 which was then acylated with 2,2,6- trimethyl-4 H -1,3-dioxinone to give Final analogue 20 was completed using Biginelli cyclization followed by amide coupling as described previously in Scheme 1.
Incorporation of the benzodioxole ring was undertaken as shown in Scheme 3. Synthesis began with alkylation of 3,4- methylenedioxy aniline through heating with ethyl acetoacetate and pyridine to give ketoamide Biginelli cyclization followed by HATU-mediated amidation furnished the hybrid compound Our hybrid design utilized an amide linker between the template of 2 and the new substituents intended to fill the hydrophobic subsite of GRK2.
Although it was hypothesized that addition of a methyl amide group alone would establish an extra hydrogen bond interaction with the P-loop of GRK2 similar to that observed in the crystal complex with 1 , prototype compound 12a exhibited a 5-fold loss in potency against GRK2. It is possible that the additional hydrogen bond formed by the amide with the enzyme cannot overcome a desolvation penalty or an entropic penalty for fixing the conformation of the P-loop.
The carboxylic acid derivative 10 , on the other hand, gave a dramatic fold decrease in potency for GRK2 and only a 2-fold loss against PKA. As both 1 and balanol place an aromatic ring into the hydrophobic pocket of GRK2, we introduced a benzyl amide 12b , which was essentially equipotent with the lead 2 with respect to GRK2 and ROCK1, but gained substantial potency for GRK5.
The results with both 12b and 12c suggest that addition of lipophilicity to the amide appendage is a path to higher potency, but not necessarily GRK2 selectivity. Incorporation of the 2,6-difluorobenyzl amide 12d , the same D ring as in TakedaA, did not improve upon the GRK2 potency of the 3-fluorobenzyl amide, but restored selectivity for GRK2 versus GRK1 and GRK5, providing a clue that the size of the amide substituent may be an important selectivity determinant among GRKs.
Next, the effect of adding polar groups to the D-ring extension was explored as the D-ring of balanol has two polar groups in the 2 and 6 positions. Movement of the methoxy substituent around the benzyl ring to the meta 12f and para 12g positions resulted in a significant loss of potency compared to the 2-methoxy benzyl amide 12e for GRK2 and GRK1, but was more tolerated in GRK5 and ROCK1. Extending the linker between the amide bond and the terminal pyridine ring by one carbon 12i substantially improved selectivity in comparison to 12h against GRK1 and GRK5 but somewhat reduced inhibition of GRK2.
The 4-pyridyl ethyl amide 12j was not well tolerated by any of the GRKs, suggesting the importance of the 2-pyridyl nitrogen to binding. We next investigated the potential of increasing the size of the pyridine of 12h to an isoquinoline 12k , which improved selectivity against GRK5 6. Interestingly, 12k achieved the highest potency and selectivity for GRK1 with an IC 50 of nM — a fold increase in comparison to the 2-pyridylmethyl analog 12h and a greater than fold increase in comparison to the parent compound 2.
As the 2,6-difluorobenzyl 12d showed marginally improved selectivity in comparison to the unsubstituted benzyl 12a , the potential for achieving greater selectivity for GRK2 by further increasing the size of the D-ring amide substituents was explored. Both the 2,6-dichloro 12l and the 2,6-dimethyl 12m resulted in dramatic improvements in GRK2 selectivity. Thus, for the first time we observed that ROCK inhibition can be mitigated via the introduction of steric bulk into the hydrophobic subsite.
Extending this line of reasoning to the larger dimethoxy 12n and di-trifluoromethyl 12o analogs essentially eliminated all kinase inhibitory activity except for GRK2.
The dimethoxy analog 12n retains excellent potency against GRK2 compared to the parent compound 0. Moving the added bulk to the meta positions with the 3,5-bis trifluoromethyl benzyl amide 12p produced a profound loss in GRK2 potency revealing that the 2,6 disubstitution pattern of the D ring is critical for potency and selectivity. The remarkable impact of sterics on GRK2 selectivity is further highlighted by comparing 2-pyridyl amide 12h with the corresponding ortho-methyl analog 12q.
In general, homologating the amide substituent by insertion of a methylene between the amide and the aromatic ring led to decreases in potency and selectivity. This was particularly evident with 13a and 13b.
This interesting pattern was also observed with ethyl homolog 12r , which has decreased GRK2 potency and increased ROCK1 potency versus the shorter 12m. We briefly explored replacing the indazole moiety of 12h , which binds to the hinge of the kinase domain, with the benzodioxole group of paroxetine 24 , Table 2.
Despite the potent pan-kinase inhibition of 12h , the corresponding benzodioxole analog 24 was completely inactive. Several other benzodioxole analogs of other hybrids were similarly inactive data not shown.
In a final attempt to further design out ROCK1 affinity, the nitrogen of the amide bond linking the indazole and the dihydropyrimidone core was methylated. Prior crystallization of a close analog of 2 into ROCK1 revealed that the amide nitrogen forms a water-mediated hydrogen bond to ROCK1-Asp, 24 and therefore, methylation of the amide would be expected to weaken its affinity.
The methylated amide of potent hybrid 12m was thus synthesized. All four complexes crystallized in space group C 1 with nearly identical unit cell constants with resolution ranges of 2. This result is consistent with the idea that the indazole, which occupies the adenine subsite and forms two hydrogen bonds with the hinge of the kinase domain, dictates the overall conformation of the large and small lobes.
Co-crystal structures reveal that the inhibitors bind in the ATP-binding pocket in a similar conformation as the compound 2 parent structure. Hydrogen bonds with the labeled GRK2 residues are shown as black dashed lines. The P-loop and hinge region are indicated for reference. As expected, the four inhibitors bind in the ATP pocket of GRK2 in essentially the same manner as the parent compound 2 , with the exception that the amide bond connecting the indazole and the dihydropyrimidine is flipped relative to the model of 2 Figure 3.
However, the electron density for this amide in the complex with 2 is ambiguous and hence the linker may adopt multiple configurations in the previous structure.
As noted above, the indazole rings bind in the adenine subsite forming two hydrogen bonds with backbone atoms of the hinge residues Asp and Met, and the dihydropyrimidine and fluorophenyl rings fill the ribose and polyphosphate subsites, respectively. However, the presence of the D-rings, and presumably their interactions in the hydrophobic subsite, seems to alter the conformation of the A-C rings to some extent among the four complexes Figure 4.
As predicted, their variable amide-linked D-rings occupy the hydrophobic subsite of GRK2, and the carbonyls of the amide bond linker ortho to the fluorine atom in the C-ring form a hydrogen bond with the backbone nitrogen of Phe in the P-loop. The D-ring of 12r , however, flips out of the hydrophobic site towards the solvent, and there is no interpretable electron density beyond the amide linker.
Adaptive structural changes in the GRK2 P-loop. The magnitude of the shift thus appears to depend on the size of the D-ring. The largest conformational changes induced by the various inhibitors occur in the P-loop Figure 4. Relative to 2 , each of the four inhibitors causes the P-loop to shift away from the polyphosphate subsite as if to accommodate the terminal aromatic rings.
Compounds 12h , 12n , and 12r each have a maximum P-loop shift of 2. Compound 12k demonstrates the largest P-loop shift of 3. In addition, the benzene ring of Phe rotates to allow space for the terminal aromatic substituents depending on their orientation Figure 3. Notably, AST loop residues —, which are typically ordered in active conformations of AGC kinases, but are disordered in most GRK2 kinase domain structures to date, are visible in the 12n electron density map and pack on top of the P-loop.
The reason for these residues being more ordered in the 12n complex relative to the others is unclear, but the density may simply reflect the higher quality of this particular crystallographic data set Supplemental Table 1. Analysis of these four crystal structures in comparison to structures of other AGC kinase domains provides insight into the molecular basis for their relative potencies and selectivities.
Consistent with previous studies, 30 — 32 the number of hydrogen bonds does not correlate well with inhibitor binding affinity in AGC kinases Figure 5. For example, compound 12r has the second highest affinity of the four crystallized compounds from this study 12h , 12k , 12n , 12r , and the parent compound 2 , but only forms three hydrogen bonds.
Plotting the K i of 10 potent GRK2 inhibitors calculated from the IC 50 values using the Cheng-Prusoff transformation versus both the number of hydrogen bonds and the buried accessible surface area Figure 5 exhibited no correlation between the number of hydrogen bonds and K i but did show a correlation between buried surface area and K i.
The buried accessible surface area is the most consistent determinants of inhibitor potency, as previously noted for GRK2. Buried ASA for inhibitors from this study are shown in green.
However, the hinge in the structures of PKA is shifted 1. This difference may prevent the formation of favorable contacts between hinge backbone atoms and the indazole ring common to 2 and all of its derivatives. Hinge residues that form hydrogen bonds with the indazole nitrogens of compound 2 and its derivatives are 1. 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.
Based on the hydrophobic subsite hypothesis above, the isoquinoline ring of 12k was predicted to select against GRK5 and its close homolog GRK1. This modeling exercise also suggests that the nitrogen of the isoquinoline ring could form an additional hydrogen bond with the active site lysine as 12h does in GRK5. Docking the compound in the active site of GRK5 Figure 8b demonstrates that the 2,6-dimethoxybenzyl substituent of 12n would collide with the DFG loop, which is shifted towards the hydrophobic subsite due to a greater degree of kinase domain closure in GRK5 than in GRK2.
This collision also likely explains the GRK2 selectivity seen with the similar, but not quite as bulky, hybrids 12l and 12m. The packing of 12n is likely mimicked by the analogous potent ortho methoxy hybrid 12e. Molecular origins of selectivity for compound 12n.
The other inhibitors avoid generating this collision but 12n cannot, as a consequence of its two methoxy substituents, which greatly restrict its ability to alter its conformation within the hydrophobic subsite.