Development of Paramagnetic Probes for Molecular Recognition Studies in Protein Kinases

Vázquez, Jesús; De, Surya K.; Chen, Li-Hsing; Riel-Mehan, Megan; Emdadi, Aras; Cellitti, Jason; Stebbins, John L.; Rega, Michele F.; Pellecchia, Maurizio

doi:10.1021/jm800068w

Cited by 38 publications

(55 citation statements)

References 36 publications

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“…To confirm the predicted binding mode of BI-78D3, we used a recently developed chemical probe (21) that, by anchoring in the ATP binding site, brings a paramagnetic spin label into proximity of the JIP binding site of JNK (Fig. 2 A).…”

Section: Resultsmentioning

confidence: 99%

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

Stebbins

Machleidt³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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175

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JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics. JNKs are serine threonine protein kinases and members of the MAPK family (1-3). JNKs can be expressed as 10 different isoforms by mRNA alternative splicing of three highly related genes, JNK1, JNK2, and JNK3 (4). Although JNK1 and JNK2 are ubiquitous, JNK3 is principally present in the brain, cardiac muscle, and testis (4, 5). JNK activation by extracellular stimuli, such as stress or cytokines, leads to the phosphorylation of several transcription factors, and cellular substrates implicated in cell survival and proliferation, insulin receptor signaling, and mRNA stabilization (6-9). Because these pathways are related to the pathogenesis of several diseases, including diabetes, cancer, atherosclerosis, stroke, and Alzheimer's and Parkinson's diseases, JNKs represent valuable targets in the development of new therapies (10).JNKs bind to scaffold proteins and substrates containing a D-domain, consensus sequence of which is R/KXXXXLXL (11, 12). JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases (13). The JNK-JIP1 interaction is mediated by a specific, high affinity D-domain on JIP1, the critical features of which were elucidated by Barr and colleagues (14). Overexpression of either the D-domain of JIP1 or the full-length protein potently inhibits JNK signaling in the cell (15). The minimal region of JIP1, consisting of a single D-domain, has been identified as a JNK inhibitor (14, 16). A peptide corresponding to the D-domain of JIP1 (amino acids 153-163; pepJIP1), inhibits JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 and displays remarkable selectivity with little inhibition of the closely related Erk and p38 MAPKs (17).The mechanism of JNK1 inhibition by pepJIP1 is mainly because of the competition of pepJIP1 with the D-domains of substrat...

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Section: Resultsmentioning

confidence: 99%

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

Stebbins

Machleidt³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

175

158

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“…Applications to bound ligand structure have been limited to qualitative assessment of binding, in the second site screening approach (27, 28). In our case, making multiple measurements of R 2 at different concentrations of peptide - receptor complex enabled us to average the error associated with measurement of an individual R 2 and to obtain a relatively narrowly defined constraint for each proton.…”

Section: Resultsmentioning

confidence: 99%

Paramagnetic Relaxation Assisted Docking of a Small Indole Compound in the HIV-1 gp41 Hydrophobic Pocket

2011

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The hydrophobic pocket contained within the gp41 coiled coil is an important target for small molecules designed to inhibit HIV-1 fusion. While various screening experiments have identified molecules purported to bind in this pocket, few have confirmed details of the interaction, instead relying on computational docking to predict the binding mode. This is made more challenging by the fact that residues lining the hydrophobic pocket are highly flexible, as is typical for a proteinprotein interaction site, limiting the predictive power of computational tools. In this study, we report on an NMR method to define the binding mode of 1-5i, a compound in a series of newly developed indole inhibitors. We show that paramagnetic relaxation enhancement of ligand protons due to an MTSL group positioned close to the binding pocket could be applied quantitatively to distinguish between more than 30 different computational poses, selecting a single pose that agreed with the NMR data. In this pose, important hydrophobic and polar contacts occur with pocket lysine, tryptophan and glutamine residues, including putative hydrogen bonds between the ligand carboxylate and the lysine ε-amino group. A study of the ligand orientation suggests directions for optimization.There has been a great deal of interest in discovering low molecular weight compounds which bind to the hydrophobic pocket of gp41, as a means to inhibit HIV-1 fusion with a small molecule. The hydrophobic pocket is critical for stability of the six-helix bundle that forms during the fusion reaction (1) and it is considered a hotspot for inhibiting the proteinprotein interaction between the N-heptad and C-heptad repeat regions of gp41. While there have been many studies involving screening of small molecule libraries to identify fusion inhibitors in biological or biochemical assays (2-7), there has been very little structural information to confirm that the mechanism of inhibition is hydrophobic pocket binding. Furthermore, the plasticity of the protein -protein interaction surface makes it difficult to rely strictly on computational docking studies (8) as evidence of binding or to provide orientational information.We have recently described a series of indole compounds with confirmed hydrophobic pocket binding activity that is correlated to inhibition of cell-cell fusion (9). The core scaffold, compound 1, called 1-6i in this study, was a small molecule with molecular weight 251, and with good inhibitory potential in the low μM for both hydrophobic pocket binding and cell-cell fusion inhibition. We have continued to investigate the structure-activity relationship properties of this compound and its derivatives, including making an isomer of the compound with a differently substituted indole, 1-5i (Figure 1) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ten-fold reduced affinity for the hydrophobic pocket compared with 1-6i. Modeling predictions do not adequately explain the observed difference, and are quite dependent on the recepto...

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“…The method is very robust because the PRE effect is detectable only if first- and second-site ligands bind to the target simultaneously and close to each other. Successful use of this labeling technique is illustrated in the recent design of protein kinase and tyrosine phosphatase inhibitors (Vazquez et al 2007, Vazquez et al 2008). …”

Section: Generation Of Lead Compoundsmentioning

confidence: 99%

Fragment-based drug discovery using NMR spectroscopy

2013

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Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience.

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Development of Paramagnetic Probes for Molecular Recognition Studies in Protein Kinases

Cited by 38 publications

References 36 publications

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

Paramagnetic Relaxation Assisted Docking of a Small Indole Compound in the HIV-1 gp41 Hydrophobic Pocket

Fragment-based drug discovery using NMR spectroscopy

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