Use of Selective Trp Side Chain Labeling To Characterize Protein−Protein and Protein−Ligand Interactions by NMR Spectroscopy

Recent studies on amino acid occurrence in protein binding sites suggest that only a reduced number of residues are responsible for most interaction energy in protein−protein and protein−ligand interactions. Above all, tryptophan (Trp) seems to be the most frequent residue in protein's hot spots. Here we report a novel, efficient, and cost-effective method to selectively incorporate specific isotope labels into the side chains of Trp residues in recombinant proteins. We show that the method proposed allows selective NMR observation of Trp side chains that enables studies of ligand binding, protein−protein interactions, hydrogen binding, protein folding, and side chain dynamics. Examples with the protein BIR3 will be given

Determination of the Dihedral Angle ψ Based on <i>J</i> Coupling Measurements in ¹⁵N/¹³C-Labeled Proteins

Determination of the Dihedral Angle ψ Based on J Coupling Measurements in 15N/13C-Labeled Protein

NMR Studies of a Heterotypic Sam−Sam Domain Association: The Interaction between the Lipid Phosphatase Ship2 and the EphA2 Receptor^,

Sterile alpha motif (Sam) domains are protein interaction modules that are implicated in many biological processes mainly via homo- and heterodimerization. It has been recently reported that the lipid phosphatase Ship2 regulates endocytosis of the EphA2 receptor, a process that has been investigated as a possible route to reduce tumor malignancy. A heterotypic Sam−Sam domain interaction is mediating this process. Here, we report NMR and ITC (isothermal titration calorimetry) studies on the Sam domain of Ship2 revealing its three-dimensional structure and its possible mode of interaction with the Sam domain from the EphA2 receptor. These studies have also resulted in the identification of a minimal peptide region of Ship2 that retains binding affinity for the Sam domain of the EphA2 receptor. Hence, this peptide and the detection of key structural elements important for EphA2 receptor endocytosis provide possible ways for the development of novel small molecule antagonists with potential anticancer activity

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl‑1

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein–protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein–protein interactions

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl‑1

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein–protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein–protein interactions

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl‑1

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein–protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein–protein interactions

Targefrin: A Potent Agent Targeting the Ligand Binding Domain of EphA2

Overexpression of the receptor tyrosine kinase EphA2 is invariably associated with poor prognosis and development of aggressive metastatic cancers. Guided by our recently solved X-ray structure of the complex between an agonistic peptide and EphA2-LBD, we report on a novel agent, targefrin, that binds to EphA2-LBD with a 21 nM dissociation constant by isothermal titration calorimetry and presents an IC50 value of 10.8 nM in a biochemical assay. In cell-based assays, a dimeric version of the agent is as effective as the natural dimeric ligands (ephrinA1-Fc) in inducing cellular receptor internalization and degradation in several pancreatic cancer cell lines. When conjugated with chemotherapy, the agents can effectively deliver paclitaxel to pancreatic cancers in a mouse xenograft study. Given the pivotal role of EphA2 in tumor progression, we are confident that the agents reported could be further developed into innovative EphA2-targeting therapeutics

Targefrin: A Potent Agent Targeting the Ligand Binding Domain of EphA2

Overexpression of the receptor tyrosine kinase EphA2 is invariably associated with poor prognosis and development of aggressive metastatic cancers. Guided by our recently solved X-ray structure of the complex between an agonistic peptide and EphA2-LBD, we report on a novel agent, targefrin, that binds to EphA2-LBD with a 21 nM dissociation constant by isothermal titration calorimetry and presents an IC50 value of 10.8 nM in a biochemical assay. In cell-based assays, a dimeric version of the agent is as effective as the natural dimeric ligands (ephrinA1-Fc) in inducing cellular receptor internalization and degradation in several pancreatic cancer cell lines. When conjugated with chemotherapy, the agents can effectively deliver paclitaxel to pancreatic cancers in a mouse xenograft study. Given the pivotal role of EphA2 in tumor progression, we are confident that the agents reported could be further developed into innovative EphA2-targeting therapeutics

Targefrin: A Potent Agent Targeting the Ligand Binding Domain of EphA2

Overexpression of the receptor tyrosine kinase EphA2 is invariably associated with poor prognosis and development of aggressive metastatic cancers. Guided by our recently solved X-ray structure of the complex between an agonistic peptide and EphA2-LBD, we report on a novel agent, targefrin, that binds to EphA2-LBD with a 21 nM dissociation constant by isothermal titration calorimetry and presents an IC50 value of 10.8 nM in a biochemical assay. In cell-based assays, a dimeric version of the agent is as effective as the natural dimeric ligands (ephrinA1-Fc) in inducing cellular receptor internalization and degradation in several pancreatic cancer cell lines. When conjugated with chemotherapy, the agents can effectively deliver paclitaxel to pancreatic cancers in a mouse xenograft study. Given the pivotal role of EphA2 in tumor progression, we are confident that the agents reported could be further developed into innovative EphA2-targeting therapeutics

Lysine Covalent Antagonists of Melanoma Inhibitors of Apoptosis Protein

We have recently reported on Lys-covalent agents that, based on aryl-sulfonyl fluorides, were designed to target binding site Lys 311 in the X-linked inhibitor of apoptosis protein (XIAP). Similar to XIAP, melanoma-IAP (ML-IAP), a less well-characterized IAP family protein, also presents a lysine residue (Lys 135), which is in a position equivalent to that of Lys 311 of XIAP. On the contrary, two other members of the IAP family, namely, cellular-IAPs (cIAP1 and cIAP2), present a glutamic acid residue in that position. Hence, in the present work, we describe the derivation and characterization of the very first potent ML-IAP Lys-covalent inhibitor with cellular activity. The agent can be used as a pharmacological tool to further validate ML-IAP as a drug target and eventually for the development of ML-IAP-targeted therapeutics