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Structural basis and biological consequences for JNK2/3 isoform selective aminopyrazoles.

Park H, Iqbal S, Hernandez P, Mora R, Zheng K, Feng Y, LoGrasso P - Sci Rep (2015)

Bottom Line: It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit.These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity.Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, 130 Scripps Way #2A2, Jupiter, Florida 33458.

ABSTRACT
Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.

No MeSH data available.


Related in: MedlinePlus

Overlay poses for five JNK3 selective aminopyrazole inhibitors.(A) A superposition of four crystal structures and one computational model of JNK3 39–402 with aminopyrazole complexes. SR-12103 is a computational model (starred) and the other four complexes were derived from the crystal structures. L144 is colored red for the crystal structures and pink for the model structure. The Cα RMSD of 351 residues that are superimposed was between 0.16 and 0.24 Å. The distances d1 and d2 in the insert table represent distances between C5 of the inhibitor phenyl ring and Cα of I92, and C4 of the inhibitor phenyl ring and Cβ of L144, respectively. (B) The crystal structure of JNK3/SR-12326 was chosen as a representative example for all five compounds bound to JNK3 to illustrate the van der Waals distances differences between Leu 144 and Ile144. The C4 of the phenyl ring of the compound, and Cβ of leucine are shown in Van der Waals spheres, and distances between the carbon atoms are also shown. The optimum distances are predicted with metadynamics calculations. (C) A computational model of SR-12326 bound to JNK3 that is mutated to isoleucine in residue 144 (L144I). The C4 of the phenyl ring of the compound, and Cβ and Cγ of isoleucine are shown in Van der Waals spheres, and distances between the C atoms are also shown.
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f3: Overlay poses for five JNK3 selective aminopyrazole inhibitors.(A) A superposition of four crystal structures and one computational model of JNK3 39–402 with aminopyrazole complexes. SR-12103 is a computational model (starred) and the other four complexes were derived from the crystal structures. L144 is colored red for the crystal structures and pink for the model structure. The Cα RMSD of 351 residues that are superimposed was between 0.16 and 0.24 Å. The distances d1 and d2 in the insert table represent distances between C5 of the inhibitor phenyl ring and Cα of I92, and C4 of the inhibitor phenyl ring and Cβ of L144, respectively. (B) The crystal structure of JNK3/SR-12326 was chosen as a representative example for all five compounds bound to JNK3 to illustrate the van der Waals distances differences between Leu 144 and Ile144. The C4 of the phenyl ring of the compound, and Cβ of leucine are shown in Van der Waals spheres, and distances between the carbon atoms are also shown. The optimum distances are predicted with metadynamics calculations. (C) A computational model of SR-12326 bound to JNK3 that is mutated to isoleucine in residue 144 (L144I). The C4 of the phenyl ring of the compound, and Cβ and Cγ of isoleucine are shown in Van der Waals spheres, and distances between the C atoms are also shown.

Mentions: We next superimposed the crystal structures of four of these compounds and a computational model of SR-12103 to see what structural features of the inhibitors and JNK3 were crucial for selectivity and the potency differences that were observed. Figure 3A presents the overlay poses for five JNK3 selective aminopyrazole inhibitors. The Cα RMSD of 351 residues that are superimposed were between 0.16 and 0.24 Å indicating the highly similar structures for all compounds. Optimum binding of inhibitors in the JNK3 pocket were assessed in terms of distances, d1 and d2, which were the distance between C5 position on the phenyl ring of the inhibitors and Cα of I92 (d1), and C4 of phenyl ring and Cβ of Leu144 (d2). Substitution on the para position of the inhibitors caused the two distances to increase resulting in the decreased potency of these compounds compared to the para-unsubstituted analog (SR-12326 shown in orange). The van der Waals distances between d1 and d2 for JNK3 which has Leu at position 144 are shown in Figure 3B where the van der Waals spheres for the phenyl ring in SR-12326 and JNK3 L144 are also shown. For comparison, a computational model of SR-12326 as a representative example of all five compounds bound to JNK3 where Leu was mutated to Ile (L144I) is also shown (Figure 3C). The increased distance (d2) from 3.6 Å (Figure 3B) to 4.0 Å (Figure 3C) for C4 of the phenyl ring of SR-12326 to Cβ and Cγ of isoleucine is indicated. It should be noted that the lowest energy rotameric state of the Ile side chain in the computational model shown in Figure 3C corresponded to the same state determined in the crystal structure for JNK3L144I (Supplemental Figure 2).


Structural basis and biological consequences for JNK2/3 isoform selective aminopyrazoles.

Park H, Iqbal S, Hernandez P, Mora R, Zheng K, Feng Y, LoGrasso P - Sci Rep (2015)

Overlay poses for five JNK3 selective aminopyrazole inhibitors.(A) A superposition of four crystal structures and one computational model of JNK3 39–402 with aminopyrazole complexes. SR-12103 is a computational model (starred) and the other four complexes were derived from the crystal structures. L144 is colored red for the crystal structures and pink for the model structure. The Cα RMSD of 351 residues that are superimposed was between 0.16 and 0.24 Å. The distances d1 and d2 in the insert table represent distances between C5 of the inhibitor phenyl ring and Cα of I92, and C4 of the inhibitor phenyl ring and Cβ of L144, respectively. (B) The crystal structure of JNK3/SR-12326 was chosen as a representative example for all five compounds bound to JNK3 to illustrate the van der Waals distances differences between Leu 144 and Ile144. The C4 of the phenyl ring of the compound, and Cβ of leucine are shown in Van der Waals spheres, and distances between the carbon atoms are also shown. The optimum distances are predicted with metadynamics calculations. (C) A computational model of SR-12326 bound to JNK3 that is mutated to isoleucine in residue 144 (L144I). The C4 of the phenyl ring of the compound, and Cβ and Cγ of isoleucine are shown in Van der Waals spheres, and distances between the C atoms are also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306959&req=5

f3: Overlay poses for five JNK3 selective aminopyrazole inhibitors.(A) A superposition of four crystal structures and one computational model of JNK3 39–402 with aminopyrazole complexes. SR-12103 is a computational model (starred) and the other four complexes were derived from the crystal structures. L144 is colored red for the crystal structures and pink for the model structure. The Cα RMSD of 351 residues that are superimposed was between 0.16 and 0.24 Å. The distances d1 and d2 in the insert table represent distances between C5 of the inhibitor phenyl ring and Cα of I92, and C4 of the inhibitor phenyl ring and Cβ of L144, respectively. (B) The crystal structure of JNK3/SR-12326 was chosen as a representative example for all five compounds bound to JNK3 to illustrate the van der Waals distances differences between Leu 144 and Ile144. The C4 of the phenyl ring of the compound, and Cβ of leucine are shown in Van der Waals spheres, and distances between the carbon atoms are also shown. The optimum distances are predicted with metadynamics calculations. (C) A computational model of SR-12326 bound to JNK3 that is mutated to isoleucine in residue 144 (L144I). The C4 of the phenyl ring of the compound, and Cβ and Cγ of isoleucine are shown in Van der Waals spheres, and distances between the C atoms are also shown.
Mentions: We next superimposed the crystal structures of four of these compounds and a computational model of SR-12103 to see what structural features of the inhibitors and JNK3 were crucial for selectivity and the potency differences that were observed. Figure 3A presents the overlay poses for five JNK3 selective aminopyrazole inhibitors. The Cα RMSD of 351 residues that are superimposed were between 0.16 and 0.24 Å indicating the highly similar structures for all compounds. Optimum binding of inhibitors in the JNK3 pocket were assessed in terms of distances, d1 and d2, which were the distance between C5 position on the phenyl ring of the inhibitors and Cα of I92 (d1), and C4 of phenyl ring and Cβ of Leu144 (d2). Substitution on the para position of the inhibitors caused the two distances to increase resulting in the decreased potency of these compounds compared to the para-unsubstituted analog (SR-12326 shown in orange). The van der Waals distances between d1 and d2 for JNK3 which has Leu at position 144 are shown in Figure 3B where the van der Waals spheres for the phenyl ring in SR-12326 and JNK3 L144 are also shown. For comparison, a computational model of SR-12326 as a representative example of all five compounds bound to JNK3 where Leu was mutated to Ile (L144I) is also shown (Figure 3C). The increased distance (d2) from 3.6 Å (Figure 3B) to 4.0 Å (Figure 3C) for C4 of the phenyl ring of SR-12326 to Cβ and Cγ of isoleucine is indicated. It should be noted that the lowest energy rotameric state of the Ile side chain in the computational model shown in Figure 3C corresponded to the same state determined in the crystal structure for JNK3L144I (Supplemental Figure 2).

Bottom Line: It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit.These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity.Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, 130 Scripps Way #2A2, Jupiter, Florida 33458.

ABSTRACT
Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.

No MeSH data available.


Related in: MedlinePlus