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

Abstract

Esitetään Jyväskylän yliopiston matemaattis-luonnontieteellisen tiedekunnan suostumuksella julkisesti tarkastettavaksi yliopiston Ylistönrinteellä, salissa YAA303 tammikuun 24. päivänä 2014 kello 12.Academic dissertation to be publicly discussed, by permission of the Faculty of Mathematics and Science of the University of Jyväskylä, in Ylistönrinne, hall YAA303, on January 24, 2014 at 12 o'clock noon. Ligand-binding in a specific manner is vital to all cellular actions and can have a major effect on the activity and conformation of proteins. In addition to experimental techniques, computational methods, such as molecular dynamics (MD) simulations, have become an integral part in studies related to proteinligand interactions. The computational approach introduces a dynamic view of molecular interactions, and enables detailed structure-function studies. In addition, it may facilitate the estimation of binding affinities and help in identifying ligand-binding sites in proteins. In this thesis three pharmacologically important protein targets were studied with both computational and experimental methods: 1) ionotropic glutamate receptors (iGluRs), 2) filamin A (FLNa), and 3) T-cell protein tyrosine phosphatase (TCPTP). iGluRs mediate synaptic transmission in the nervous system and are linked to many neurological disorders. Partial agonism of the ligand binding domains (LBD) for these receptors was studied with MD simulations and the molecular mechanics generalised Born surface area (MM-GBSA) method. A previously unobserved intermediate closure stage for the GluN1 receptor subtype was identified, and new information about the closure mechanism was obtained. FLNa is an actin cross-linking protein linked to many cellular functions via its numerous binding partners. MM-GBSA calculations of binding free energy were shown to correlate well with the experimental data. Thus, ligands could be ranked based on their binding affinity, suggesting that also the rational design of FLNa-binding peptide mimetics would be conceivable. TCPTP is a ubiquitously expressed non-transmembrane phosphatase that negatively regulates many cancer-related kinases. TCPTP is normally autoinhibited, but several agonist molecules are known to activate it. Here, a putative binding site for TCPTP activators was identified and important structural determinants for novel activators were recognised. As TCPTP is not down-regulated in cancer cells, it is a promising target for tumor suppression. UNIVERSITY

Activation of Engineered Protein Tyrosine Phosphatases with the Biarsenical Compound AsCy3‐EDT2

“…We also sought to compare the level of act TCPTP activation with that induced by the previously identified activator of wild-type TCPTP, mitoxantrone. [17] Under the conditions of our enzymatic assays, neither mitoxantrone (10 μM) nor AsCy3 (1 μM) induced any detectable increase in activity of wild-type TCPTP (Figure 6A). By contrast, AsCy3 treatment of act TCPTP gave rise to a strong and dose-dependent activity increase that plateaued at approximately 500 nM (Figure 6A).…”

“…[16] Therefore TCPTP activators could be useful, both for probing the role of TCPTP activity in pathogenesis and for validating TCPTP as a potential activation target for therapeutic intervention. [6,17] Moreover, TCPTP is one of the few PTPs for which an activator of the wild-type enzyme has been reported. [6] The precedent compound, mitoxantrone, therefore provides a useful comparison point for the evaluation of novel TCPTP activation strategies.…”

“…[6] The precedent compound, mitoxantrone, therefore provides a useful comparison point for the evaluation of novel TCPTP activation strategies. [17] …”

Regulation of platelet-activating factor-mediated interleukin-6 promoter activation by the 48 kDa but not the 45 kDa isoform of protein tyrosine phosphatase non-receptor type 2

“…Indeed, post-translational modifications such as serine phosphorylation by CDK [35] or PKR [47] modulate its ability to dephosphorylate its substrates in vivo without affecting the catalytic activity. On the other hand, the phosphatase activity levels of the 45 kDa isoform have been shown to be lowered by an intramolecular interaction between the non-catalytic C-terminal domain and the PTP domain; an interaction that can be disrupted by the binding of proteins, such as the cytoplasmic tail of the α-1 integrin, or compounds, such as the chemotherapeutic agent mitoxantrone [43, 44, 48, 49]. The presence of such an intramolecular interaction in the 48 kDa isoform and the possible impacts of interacting proteins such as C3G or syntaxin 17 on its phosphatase activity are still unknown [50–52].…”

Soft TCPTP Agonism—Novel Target to Rescue Airway Epithelial Integrity by Exogenous Spermidine

“…Recently, a high-throughput assay aimed at selecting novel TCPTP agonists screened as many as 64,280 small molecules to identify finally two leads, mitoxantrone and spermidine, both active at low micromolar levels (Mattila et al, 2010 ). Modalities of action were elucidated: the former directly binds to TCPTP catalytic domain, whereas spermidine agonism does not require interference with TCPTP active center (Ylilauri et al, 2013 ).…”