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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 of complex crystal structures of JNK3:SR-12326 (pink) and JNK3:SR-3562 (blue, PDB ID: 3KVX).The inhibitor binding modes between SR-12326 and SR-3562, an aminopyrzaole and an aminopyrimidine, respectively are compared. The SR-3562 binding site of JNK3 is located under the β-strands β3/4 and G-rich loop, whereas the SR-12326 binding site was located deeper into the pocket under N-lobe β-sheet β3/4/5/7. The arrow in the insert figure indicates viewing direction in the main figure. The inhibitor binding pocket of JNK3 in JNK3:SR-12326 complex is shown as a grey surface.
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f5: Overlay of complex crystal structures of JNK3:SR-12326 (pink) and JNK3:SR-3562 (blue, PDB ID: 3KVX).The inhibitor binding modes between SR-12326 and SR-3562, an aminopyrzaole and an aminopyrimidine, respectively are compared. The SR-3562 binding site of JNK3 is located under the β-strands β3/4 and G-rich loop, whereas the SR-12326 binding site was located deeper into the pocket under N-lobe β-sheet β3/4/5/7. The arrow in the insert figure indicates viewing direction in the main figure. The inhibitor binding pocket of JNK3 in JNK3:SR-12326 complex is shown as a grey surface.

Mentions: The mutagenesis results showed that Leu144 in hydrophobic pocket I was the key structural feature within JNK3 that gave rise to the selectivity of the aminopyrazoles for JNK3 over JNK1. The greater than 22-fold increase in IC50 value for four of the five compounds tested in this study upon mutagenesis of Leu144 to Ile supports this conclusion as does the shift of L144I mutant IC50 values in the range for that of WT JNK1. In an attempt to understand why this class had such selectivity we overlayed the crystal structure of SR-12326 with that of compound 9l (SR-3562) from our previously reported aminopyrimidine class of pan JNK inhibitors27 (Figure 5). The overlay showed that the phenyl ring of SR-12326 projected into hydrophobic pocket I where the aminopyrimidines represented by SR-3562 did not. Thus, it is likely that the interaction of the hydrophobic pocket residues with the phenyl ring of SR-12326 was largely responsible for the selectivity of this class for JNK3 over JNK1. Indeed, any class of compounds that exploits this pocket, and in particular the Leu144 residue, is likely to demonstrate high selectivity for JNK3 over JNK1. This conclusion is supported by the findings of Swahn et al. who in 2006 reported the only other known JNK3 isoform selective class of inhibitors19. Like the aminopyrazoles presented in this work, the anilino-bipyridines reported by Swahn et al. showed 55-fold selectivity for JNK3 over JNK1. Furthermore the crystal structure for one of their compounds showed that the pyridyl group was in hydrophobic pocket I of JNK3 having contacts with Leu144. It is important to note that while the anilino-bipyridines reported by Swahn et al.19 had selectivity for JNK3 over JNK1 in the same magnitude range as our compounds, they did not have high selectivity over p38 thereby making them unsuitable for biological evaluation in cell-based assays to assess the role of JNK in mitochondrial function and cell death. Moreover, no cell-based inhibition data was reported so it is unclear if these compounds were cell permeable or potent in vitro JNK inhibitors.


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 of complex crystal structures of JNK3:SR-12326 (pink) and JNK3:SR-3562 (blue, PDB ID: 3KVX).The inhibitor binding modes between SR-12326 and SR-3562, an aminopyrzaole and an aminopyrimidine, respectively are compared. The SR-3562 binding site of JNK3 is located under the β-strands β3/4 and G-rich loop, whereas the SR-12326 binding site was located deeper into the pocket under N-lobe β-sheet β3/4/5/7. The arrow in the insert figure indicates viewing direction in the main figure. The inhibitor binding pocket of JNK3 in JNK3:SR-12326 complex is shown as a grey surface.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Overlay of complex crystal structures of JNK3:SR-12326 (pink) and JNK3:SR-3562 (blue, PDB ID: 3KVX).The inhibitor binding modes between SR-12326 and SR-3562, an aminopyrzaole and an aminopyrimidine, respectively are compared. The SR-3562 binding site of JNK3 is located under the β-strands β3/4 and G-rich loop, whereas the SR-12326 binding site was located deeper into the pocket under N-lobe β-sheet β3/4/5/7. The arrow in the insert figure indicates viewing direction in the main figure. The inhibitor binding pocket of JNK3 in JNK3:SR-12326 complex is shown as a grey surface.
Mentions: The mutagenesis results showed that Leu144 in hydrophobic pocket I was the key structural feature within JNK3 that gave rise to the selectivity of the aminopyrazoles for JNK3 over JNK1. The greater than 22-fold increase in IC50 value for four of the five compounds tested in this study upon mutagenesis of Leu144 to Ile supports this conclusion as does the shift of L144I mutant IC50 values in the range for that of WT JNK1. In an attempt to understand why this class had such selectivity we overlayed the crystal structure of SR-12326 with that of compound 9l (SR-3562) from our previously reported aminopyrimidine class of pan JNK inhibitors27 (Figure 5). The overlay showed that the phenyl ring of SR-12326 projected into hydrophobic pocket I where the aminopyrimidines represented by SR-3562 did not. Thus, it is likely that the interaction of the hydrophobic pocket residues with the phenyl ring of SR-12326 was largely responsible for the selectivity of this class for JNK3 over JNK1. Indeed, any class of compounds that exploits this pocket, and in particular the Leu144 residue, is likely to demonstrate high selectivity for JNK3 over JNK1. This conclusion is supported by the findings of Swahn et al. who in 2006 reported the only other known JNK3 isoform selective class of inhibitors19. Like the aminopyrazoles presented in this work, the anilino-bipyridines reported by Swahn et al. showed 55-fold selectivity for JNK3 over JNK1. Furthermore the crystal structure for one of their compounds showed that the pyridyl group was in hydrophobic pocket I of JNK3 having contacts with Leu144. It is important to note that while the anilino-bipyridines reported by Swahn et al.19 had selectivity for JNK3 over JNK1 in the same magnitude range as our compounds, they did not have high selectivity over p38 thereby making them unsuitable for biological evaluation in cell-based assays to assess the role of JNK in mitochondrial function and cell death. Moreover, no cell-based inhibition data was reported so it is unclear if these compounds were cell permeable or potent in vitro JNK inhibitors.

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