Identification of Novel Extracellular Signal-Regulated Kinase Docking Domain Inhibitors

Hancock, Chad N.; Macías, A.; Lee, Eun Kyoung; Yu, Su-Yeon; MacKerell, Alexander D.; Shapiro, Paul

doi:10.1021/jm0501174

Cited by 108 publications

(118 citation statements)

References 44 publications

(81 reference statements)

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“…CADD screening was performed as described previously (42)(43)(44) to identify small molecule inhibitors of TLR2 signaling. Briefly, CADD analysis required the following steps: (i) visual identification of a putative "pocket" in the 3D structure of the TLR2 TIR domain (Fig.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Mistry¹,

Laird²,

Schwarz³

et al. 2015

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

135

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Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C 16 H 15 NO 4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.small molecule inhibitor | BB loop | TLR2 pocket | CADD T oll-like receptors (TLRs) are type I transmembrane receptors that detect conserved "pathogen-associated molecular patterns" from microbes, as well as host-derived "danger-associated molecular patterns" (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4-9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11)(12)(13)(14). Several inhibitors of TLR2 signaling have been developed (15-18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions ...

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Section: Methodsmentioning

confidence: 99%

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Mistry¹,

Laird²,

Schwarz³

et al. 2015

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

135

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“…These structural differences could be exploited to design small-molecule inhibitors that specifically target the docking site as novel reagents to modulate MAP kinase signal pathways and as starting points for potential therapeutic development (31). Finally, it is important to note that interactions between the docking site and the KIM sequence alone may be only partially responsible for recognition of effector molecules by the MAP kinases (12,16).…”

Section: Map Kinases Share a Common Docking Sitementioning

confidence: 99%

Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3

Liu

Sun

Zhou

et al. 2006

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

127

113

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Mitogen-activated protein (MAP) kinases are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. Signaling efficiency and specificity are modulated in large part by docking interactions between individual MAP kinase and the kinase interaction motif (KIM), (R͞K) 2-3-X1-6-⌽A-X-⌽B, in its cognate kinases, phosphatases, scaffolding proteins, and substrates. We have determined the crystal structure of extracellular signal-regulated protein kinase 2 bound to the KIM peptide from MAP kinase phosphatase 3, an extracellular signal-regulated protein kinase 2-specific phosphatase. The structure reveals that the KIM docking site, situated in a noncatalytic region opposite of the kinase catalytic pocket, is comprised of a highly acidic patch and a hydrophobic groove, which engage the basic and ⌽A-X-⌽B residues, respectively, in the KIM sequence. The specific docking interactions observed in the structure consolidate all known biochemical data. In addition, structural comparison indicates that the KIM docking site is conserved in all MAP kinases. The results establish a structural model for understanding how MAP kinases interact with their regulators and substrates and provide new insights into how MAP kinase docking specificity can be achieved.mitogen-activated protein kinase specificity

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“…Thus, we and others propose that selective ATP-independent inhibition of substrates involved in the disease processes, such as cancer cell proliferation, but not normal cell functions may be a more rationale approach for developing new chemotherapeutic agents. [15][16][17] The approach to selectively block ERK interactions with substrate proteins involves targeting specific docking domains that have been identified within the C-terminal lobe of MAP kinases. 18-21 The first ERK docking domains identified include the common docking (CD) and ED domain, which are located opposite the activation loop in the 3D crystallographic structure.…”

mentioning

confidence: 99%

“…The CADD screening approach using the 3D structure of ERK2 in the unphosphorylated state has been previously described. 15 In the current studies, the 3D structure of ERK2 in the phosphorylated state 26,27 was retrieved from the Protein DataBank, deposition number 1ERK. 28 Charges and hydrogens were added using SYBYL6.4 (Tripos, Inc.).…”

mentioning

confidence: 99%

Characterization of ATP-independent ERK inhibitors identified through in silico analysis of the active ERK2 structure

Chen

Hancock

Macías

et al. 2006

Bioorganic & Medicinal Chemistry Letters

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The extracellular signal regulated kinases (ERK1 and ERK2) are important mediators of cell proliferation. Constitutive activation of the ERK proteins plays a critical role in the proliferation of many human cancers. Taking advantage of recently identified substrate docking domains on ERK2, we have used computer-aided drug design (CADD) to identify novel low molecular weight compounds that interact with ERK2 in an ATP-independent manner and disrupt substrate specific interactions. In the current study, a CADD screen of the 3D structure of active phosphorylated ERK2 protein was used to identify inhibitory compounds. We tested 13 compounds identified by the CADD screen in ERK-specific phosphorylation, cell proliferation, and binding assays. Of the 13 compounds tested, 4 compounds strongly inhibited ERK-mediated phosphorylation of ribosomal S6 kinase-1 (Rsk-1) and/or the transcription factor Elk-1 and inhibited the proliferation of HeLa cervical carcinoma cells with IC 50 values in the 2-10 μM range. These studies demonstrate that CADD can be used identify lead compounds for development of novel non-ATP dependent inhibitors selective for active ERK and its interactions with substrates involved in cancer cell proliferation.The mitogen activated protein (MAP) kinase family of enzymes regulates most biological processes including cell growth, proliferation, differentiation, inflammatory responses, and programmed cell death. Changes in MAP kinase activity have been implicated in the pathophysiology of cancer, inflammatory diseases, and neurodegenerative disorders. 1-4The three main members of MAP kinases include the extracellular signal regulated kinases (ERK), the c-Jun N-terminal kinases (JNK), and p38 MAP kinases. 5 Currently, there is much interest in understanding the role of MAP kinases in disease as these proteins may be promising targets of new chemotherapy and anti-inflammatory agents. 6The ERK proteins consist of 2 isoforms (ERK1 and ERK2; referred to as ERK1/2) that are linked to the proliferation and survival of cancer cells. 7 The ERK1/2 pathway is commonly activated by extracellular ligands, which stimulate plasma membrane receptors and the sequential activation of Ras G-protein isoforms (H, K, and N-Ras), Raf isoforms (A, B, and C-Raf), and the MAP or ERK kinases-1 and 2 (MEK1/2), which are currently the only known Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ERK1/2 regulation of dozens of different proteins underscores the importance of these proteins in regulating a variety of cellular functions associated with normal and diseased tissue. Thus, we and ...

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Identification of Novel Extracellular Signal-Regulated Kinase Docking Domain Inhibitors

Cited by 108 publications

References 44 publications

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3

Characterization of ATP-independent ERK inhibitors identified through in silico analysis of the active ERK2 structure

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