Molecular mechanism of T-cell protein tyrosine phosphatase (TCPTP) activation by mitoxantrone.

T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α1ß1 directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α1-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α1-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α1-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics.

Novel hydrazine molecules as tools to understand the flexibility of vascular adhesion protein-1 ligand-binding site: toward more selective inhibitors.

Vascular adhesion protein-1 (VAP-1) belongs to a family of amine oxidases. It plays a role in leukocyte trafficking and in amine compound metabolism. VAP-1 is linked to various diseases, such as Alzheimer's disease, psoriasis, depression, diabetes, and obesity. Accordingly, selective inhibitors of VAP-1 could potentially be used to treat those diseases. In this study, eight novel VAP-1 hydrazine derivatives were synthesized and their VAP-1 and monoamine oxidase (MAO) inhibition ability was determined in vitro. MD simulations of VAP-1 with these new molecules reveal that the VAP-1 ligand-binding pocket is flexible and capable of fitting substantially larger ligands than was previously believed. The increase in the size of the VAP-1 ligands, together with the methylation of the secondary nitrogen atom of the hydrazine moiety, improves the VAP-1 selectivity over MAO.

Synthesis, in vitro activity, and three-dimensional quantitative structure-activity relationship of novel hydrazine inhibitors of human vascular adhesion protein-1.

Vascular adhesion protein-1 (VAP-1) belongs to the semicarbazide-sensitive amine oxidases (SSAOs) that convert amines into aldehydes. SSAOs are distinct from the mammalian monoamine oxidases (MAOs), but their substrate specificities are partly overlapping. VAP-1 has been proposed as a target for anti-inflammatory drug therapy because of its role in leukocyte adhesion to endothelium. Here, we describe the synthesis and in vitro activities of novel series of VAP-1 selective inhibitors. In addition, the molecular dynamics simulations performed for VAP-1 reveal that the movements of Met211, Ser496, and especially Leu469 can enlarge the ligand-binding pocket, allowing larger ligands than those seen in the crystal structures to bind. Combining the data from molecular dynamics simulations, docking, and in vitro measurements, the three-dimensional quantitative structure-activity relationship (3D QSAR) models for VAP-1 (q(2)(LOO): 0.636; r(2): 0.828) and MAOs (q(2)(LOO): 0.749, r(2): 0.840) were built and employed in the development of selective VAP-1 inhibitors.

Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A.

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa.partner complexes as templates, we generated in silico models of IgFLNa.CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated ( approximately 160 nm) strands, whereas CFTR is compact (6 approximately 8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa.partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.

Beta2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding.

Leukocyte integrins of the beta2 family are essential for immune cell-cell adhesion. In activated cells, beta2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the beta2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of beta2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated beta2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K(d), 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K(d), 0.5 mM). Phosphorylation did not regulate talin binding to beta2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated beta2 integrin. X-ray crystallographic data clearly explained how phosphorylation eliminated filamin binding and induced 14-3-3 protein binding. Filamin knockdown in T cells led to an increase in stimulated cell adhesion to ICAM-1-coated surfaces. Our results suggest that the phosphorylation of beta2 integrins on Thr758 acts as a molecular switch to inhibit filamin binding and allow 14-3-3 protein binding to the integrin cytoplasmic domain, thereby modulating T-cell adhesion.