Identification of a potent inhibitor of human dual-specific phosphatase, VHR, from computer-aided and NMR-based screening to cellular effects

Human vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) that plays an important role in the mitogen-activated protein (MAP) kinase cascade regulation. It is also a potential drug target for diseases that are related to immune response. By combining a virtual and NMR-based ligand-screening strategy, we successfully identified four VHR inhibitors, of which GATPT ((glucosamine-aminoethoxy)triphenyltin) can bind to VHR with a K(i) value of 2.54 muM. The putative binding mode of GATPT was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs) and block the G(1)-S phase transition in the cell cycle. Therefore, GATPT is a promising lead structure for designing more effective inhibitors of VHR.     

Organometallic compounds with biological activity: a very selective and highly potent cellular inhibitor for glycogen synthase kinase 3

A chiral second-generation organoruthenium half-sandwich compound is disclosed that shows a remarkable selectivity and cellular potency for the inhibition of glycogen synthase kinase 3 (GSK-3). The selectivity was evaluated against a panel of 57 protein kinases, in which no other kinase was inhibited to the same extent, with a selectivity window of at least tenfold to more than 1000-fold at 100 microM ATP. Furthermore, a comparison with organic GSK-3 inhibitors demonstrated the superior cellular activity of this ruthenium compound: wnt signaling was fully induced at concentrations down to 30 nM. For comparison, the well-established organic GSK-3 inhibitors 6-bromoindirubin-3'-oxime (BIO) and kenpaullone activate the wnt pathway at concentrations that are higher by around 30-fold and 100-fold, respectively. The treatment of zebrafish embryos with the organometallic inhibitor resulted in a phenotype that is typical for the inhibition of GSK-3. No phenotypic change was observed with the mirror-imaged ruthenium complex. The latter does not, in fact, show any of the pharmacological properties for the inhibition of GSK-3. Overall, these results demonstrate the potential usefulness of organometallic compounds as molecular probes in cultured cells and whole organisms.     

Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic substrate enhancement is described targeting the secondary binding sites of PTPs. By screening four different PTPs from bacterial (MptpA) and human origin (PTP1B, HePtp, Shp2) with this assay, specific fragments were identified. One highly specific fragment that binds to the secondary site of Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) was characterized in order to validate the assay concept. Finally by covalently linking the secondary fragment to a phosphotyrosine mimetic, a moderately active but highly specific inhibitor of MptpA was obtained.     

Synthesis and characterization of amphiphilic functional polyesters by ring-opening polymerization and click reaction

During this work we have prepared novel amphiphilic graft-block (PαN₃CL-g-alkyne)-b-PCL functional polyesters, comprising poly(α-azido-ε-caprolactone-graft-alkyne) (PαN₃CL-g-alkyne) as the hydrophilic segment and poly(ε-caprolactone) (PCL) as the hydrophobic segment, by ring-opening polymerization of ε-caprolactone (ε-CL) with hydroxyl-terminated macroinitiator PαClCL, substituting pendent chloride with sodium azide. The copolymers were subsequently used for grafting of 2-propynyl-terminal alkyne moieties by the Cu(I)-catalyzed Huisgen’s 1,3-dipolar cycloaddition, thus producing a “click” reaction. ¹H NMR, FT-IR, GPC, and differential scanning calorimetry (DSC) examined the characteristics of the copolymers. Grafting of PMEs or PMPEGs onto the PαN₃CL-b-PCL caused these amphiphilic copolymers to self-assemble into micelles in the aqueous phase. Fluorescence, dynamic light scattering (DLS) and transmission electron microscopy (TEM) then examined these micelles. The critical micelle concentration (CMC) ranged from 8.2 mg L⁻¹ to 39.8 mg L⁻¹ at 25 °C and the average micelle size ranged from 140 to 230 nm. The hydrophilicity and length of the hydrophilic segment influenced micelle stability. The current study describes the drug entrapment efficiency and drug loading content of the micelles, dependent on the composition of graft-block polymers. The results from in vitro cell viability assays indicated that (PαN₃CL-g-alkyne)-b-PCL shows low cytotoxicity.     

Hyrtiosal, a PTP1B inhibitor from the marine sponge Hyrtios erectus, shows extensive cellular effects on PI3K/AKT activation, glucose transport, and TGFbeta/Smad2 signaling

Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling, and PTP1B inhibitors have been seen as promising therapeutic agents against obesity and type 2 diabetes. Here we report that the marine natural product hyrtiosal, from the marine sponge Hyrtios erectus, has been discovered to act as a PTP1B inhibitor and to show extensive cellular effects on PI3K/AKT activation, glucose transport, and TGFbeta/Smad2 signaling. This inhibitor wad able to inhibit PTP1B activity in dose-dependent fashion, with an IC(50) value of 42 microM in a noncompetitive inhibition mode. Further study with an IN Cell Analyzer 1000 cellular fluorescence imaging instrument showed that hyrtiosal displayed potent activity in abolishing the retardation of AKT membrane translocation caused by PTP1B overexpression in CHO cells. Moreover, it was found that this newly identified PTP1B inhibitor could dramatically enhance the membrane translocation of the key glucose transporter Glut4 in PTP1B-overexpressed CHO cells. Additionally, in view of our recent finding that PTP1B was able to modulate insulin-mediated inhibition of Smad2 activation, hyrtiosal was also tested for its capabilities in the regulation of Smad2 activity through the PI3K/AKT pathway. The results showed that hyrtiosal could effectively facilitate insulin inhibition of Smad2 activation. Our current study is expected to supply new clues for the discovery of PTP1B inhibitors from marine natural products, while the newly identified PTP1B inhibitor hyrtiosal might serve as a potential lead compound for further research.     

An RNA Aptamer That Selectively Inhibits the Enzymatic Activity of Protein Tyrosine Phosphatase 1B in vitro

SELEX was used to create an RNA aptamer targeted to protein tyrosine phosphatase 1B (PTP1B), an enzyme implicated in type 2 diabetes, breast cancer and obesity. We found an aptamer that strongly inhibits PTP1B in vitro with a K_i of less than 600 pM . This slow‐binding, high‐affinity inhibitor is also highly selective, with no detectable effect on most other tested phosphatases and approximately 300:1 selectivity over the closely related TC‐PTP. Through controlled synthesis of truncated variants of the aptamer, we isolated shorter forms that inhibit PTP1B. We also investigated various single‐nucleotide modifications to probe their effects on the aptamer's secondary structure and inhibition properties. This family of aptamers represents an exciting option for the development of lead nucleotide‐based compounds in combating several human cancers and metabolic diseases.     

Synthesis of linear polymers containing benzoxazine moieties in the main chain with high molecular design versatility via click reaction

Linear polymers with benzoxazine rings in the main chain have been synthesized applying click chemistry approach. These polymers possess molecular weights significantly higher than the benzoxazine polymers which have been chain extended via Mannich reaction. The number average molecular weight is estimated from size exclusion chromatography (SEC) to be between 20,000 and 40,000 Da. The structure of the polymers is confirmed by ¹H and ¹³C nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). Differential scanning calorimetry (DSC) is used to study crosslinking behavior of the polymers. The nature of the low temperature exotherm DSC peak observed in this work and the previous work of other authors is studied by model reactions. It is due to thermal coupling of the residual propargyl and azide end groups in the absence of active catalyst. In addition, a novel diazide-functional benzoxazine monomer has been prepared, showing a tremendous flexibility for applying click reaction to obtain various polymer architectures. Three types of polymers have been prepared from dipropargyl- and diazide-functional benzoxazine monomers. These polymers have been characterized by dynamic mechanical analysis (DMA) and thermogravimetric analysis (TGA).     

A general method for functionalisation of microgel particles with primary amines using click chemistry

In this study we introduce a general method for functionalising microgel particles with primary amine groups using a one-step copper catalysed azide-alkyne cycloaddition (CuAAC) reaction. Three different families of microgels containing copolymerised propargyl acrylate (PA) were prepared and then reacted with 2-azido-1-ethylamine (AEA) using CuAAC. The microgels contained poly(ethyl acrylate) (PEA), poly(2-vinylpyridine) (PVP) or poly(N-isopropylacrylamide) (PNP). The functionalisation of the microgels containing PA (i.e., PX-PA) by AEA to give primary amine functionalised particles (PX-PA-AEA) was assessed by elemental analysis and FTIR. The reaction of AEA with PA was quantitative for each of the PX-PA-AEA microgels (X = EA, VP and NP). The PX-PA-AEA systems generally showed larger pH-triggered swelling and zeta potentials than the non-clicked PX-PA particles. The results also showed that PA restricted swelling of the PX-PA and PX-PA-AEA particles by acting as a crosslinker. Of the three microgel systems studied, PVP-PA-AEA had the best combination of high AEA incorporation and pH-triggered swelling.     

Design and Structural Analysis of Aromatic Inhibitors of Type II Dehydroquinase from Mycobacterium tuberculosis

3‐Dehydroquinase, the third enzyme in the shikimate pathway, is a potential target for drugs against tuberculosis. Whilst a number of potent inhibitors of the Mycobacterium tuberculosis enzyme based on a 3‐dehydroquinate core have been identified, they generally show little or no in vivo activity, and were synthetically complex to prepare. This report describes studies to develop tractable and drug‐like aromatic analogues of the most potent inhibitors. A range of carbon–carbon linked biaryl analogues were prepared to investigate the effect of hydrogen bond acceptor and donor patterns on inhibition. These exhibited inhibitory activity in the high‐micromolar range. The addition of flexible linkers in the compounds led to the identification of more potent 3‐nitrobenzylgallate‐ and 5‐aminoisophthalate‐based analogues.     

A New Paradigm for KIM-PTP Drug Discovery: Identification of Allosteric Sites with Potential for Selective Inhibition Using Virtual Screening and LEI Analysis

The kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), HePTP, PTPSL and STEP, are involved in the negative regulation of mitogen-activated protein kinase (MAPK) signalling pathways and are important therapeutic targets for a number of diseases. We have used VSpipe, a virtual screening pipeline, to identify a ligand cluster distribution that is unique to this subfamily of PTPs. Several clusters map onto KIM-PTP specific sequence motifs in contrast to the cluster distribution obtained for PTP1B, a classic PTP that mapped to general PTP motifs. Importantly, the ligand clusters coincide with previously reported functional and substrate binding sites in KIM-PTPs. Assessment of the KIM-PTP specific clusters, using ligand efficiency index (LEI) plots generated by the VSpipe, ascertained that the binders in these clusters reside in a more drug-like chemical–biological space than those at the active site. LEI analysis showed differences between clusters across all KIM-PTPs, highlighting a distinct and specific profile for each phosphatase. The most druggable cluster sites are unexplored allosteric functional sites unique to each target. Exploiting these sites may facilitate the delivery of inhibitors with improved drug-like properties, with selectivity amongst the KIM-PTPs and over other classical PTPs.     

Contribution of “click chemistry” to the synthesis of antimicrobial aliphatic copolyester

A straightforward strategy is proposed to impart antimicrobial properties to biodegradable poly(oxepan-2-one) (poly(?-caprolactone) or PCL), which is based on the grafting of pendant ammonium salts by “click” chemistry. First, statistical copolymerization of 3-chlorooxepan-2-one (α-chloro-?-caprolactone or αCl?CL) with oxepan-2-one (?-caprolactone or ?CL) was initiated by 2,2-dibutyl-2-stanna-1,3-dioxepane (DSDOP). In a second step, pendant chlorides were converted into azides by reaction with sodium azide (NaN₃). Finally, quaternary ammonium containing alkynes were quantitatively added to the pendant azide groups of PCL by the copper-catalyzed Huisgen's 1,3-dipolar cycloaddition, which is a typical “click” reaction. An alternative two-step strategy based on the cycloaddition of the amine containing alkyne onto the pendant azides, followed by quaternization turned out to be less efficient. The antimicrobial activity was analyzed by the “shaking flask method” in the presence of Escherichia coli (E. coli).     

Synthesis and characterization of polyhedral oligomeric silsesquioxane (POSS) with multifunctional benzoxazine groups through click chemistry

We prepared a new class of polybenzoxazine-POSS nanocomposites with network structures through thermal curing of multifunctional benzoxazine groups of POSS (OBZ-POSS), which was synthesized from octa-azido functionalized POSS (OVBN₃-POSS) with 3,4-dihydro-3-(prop-2-ynyl)-2H-benzoxazine (P-pa) via a click reaction. Incorporation of the silsesquioxane core into the polybenzoxazine matrix could significantly enhance the thermal stability of these hybrid materials. For these nanocomposites, the POSS nanoparticles in the hybrids were improved their thermal properties with 2,2-bis(3,4-dihydro-3-methyl-2H-1,3-benzoxazine)propane (BA-m) and P-pa polybenzoxazine, analyzed via TGA analyses. In addition, the incorporation of the POSS led to the formation of an inorganic protective layer on the nanocomposite’s surface. Contact angle data provided positive evidence to back up this hypothesis that the incorporation of the POSS units would decrease the surface energy property. In addition, the low glass transition temperature of poly(4-vinyl pyridine) and polycarbonate thin films, which lack liquid resistance, could possess low surface free energy after modification with OBZ-POSS due to low temperature curing of this new compound.     

Small-Molecule Inhibitors of Shp2 Phosphatase as Potential Chemotherapeutic Agents for Glioblastoma: A Minireview

Glioblastoma multiforme (GBM) is a dreadful cancer characterised by poor prognosis, low survival rate and difficult clinical correlations. Several signalling pathways and molecular mediators are known to precipitate GBM, and small-molecular targets of these mediators have become a favoured thrust area for researchers to develop potent anti-GBM drugs. Shp2, an important phosphatase of the nonreceptor type protein tyrosine phosphatase (PTPN) subfamily is responsible for master regulation of several such signalling pathways in normal and glioma cells. Thus, inhibition of Shp2 is a logical strategy for the design and development of anti-neoplastic drugs against GBM. Though tapping the full potential of Shp2 binding sites has been challenging, nevertheless, many synthetic and natural scaffolds have been documented as possessing potent and selective anti-Shp2 activities in biochemical and cellular assays, through either active-site or allosteric binding. Most of these scaffolds share a few common pharmacophoric features, a thorough study of which is useful in paving the way for the design and development of improved Shp2 inhibitors. This minireview summarizes the current scenario of potent small-molecule Shp2 inhibitors and emphasizes the anti-GBM potential of some important scaffolds that have shown promising GBM-specific activity in in vitro and in vivo models, thus proving their efficacy in GBM therapy. This review could guide researchers to design new and improved anti-Shp2 pharmacophores and develop them as anti-GBM agents by employing GBM-centric drug-discovery protocols.