Real-time monitoring of 3T3-L1 preadipocyte differentiation using a commercially available electric cell-substrate impedance sensor system

Real-time analysis offers multiple benefits over traditional end point assays. Here, we present a method of monitoring the optimisation of the growth and differentiation of murine 3T3-L1 preadipocytes to adipocytes using the commercially available ACEA xCELLigence Real-Time Cell Analyser Single Plate (RTCA SP) system. Our findings indicate that the ACEA xCELLigence RTCA SP can reproducibly monitor the primary morphological changes in pre- and post-confluent 3T3-L1 fibroblasts induced to differentiate using insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone; and may be a viable primary method of screening compounds for adipogenic factors

Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition

Contemporary medicinal chemistry considers fragment‐based drug discovery (FBDD) and inhibition of protein‐protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano‐ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow‐up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C‐terminal domain. An in‐silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose‐dependent inhibitor of the target PPI

Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis

Semi-synthesis and evaluation of sargahydroquinoic acid derivatives as potential antimalarial agents:

Malaria continues to present a major health problem, especially in developing countries. The development of new antimalarial drugs to counter drug resistance and ensure a steady supply of new treatment options is therefore an important area of research. Meroditerpenes have previously been shown to exhibit antiplasmodial activity against a chloroquinone sensitive strain of Plasmodium falciparum (D10). In this study we explored the antiplasmodial activity of several semi-synthetic analogs of sargahydroquinoic acid

Coumarin-Annulated Ferrocenyl 1,3-Oxazine Derivatives Possessing In Vitro Antimalarial and Antitrypanosomal Potency

A tailored series of coumarin-based ferrocenyl 1,3-oxazine hybrid compounds was synthesized and investigated for potential antiparasitic activity, drawing inspiration from the established biological efficacy of the constituent chemical motifs. The structural identity of the synthesized compounds was confirmed by common spectroscopic techniques: NMR, HRMS and IR. Biological evaluation studies reveal that the compounds exhibit higher in vitro antiparasitic potency against the chemosensitive malarial strain (3D7 P. falciparum) over the investigated trypanosomiasis causal agent (T. b. brucei 427) with mostly single digit micromolar IC50 values. When read in tandem with the biological performance of previously reported structurally similar non-coumarin, phenyl derivatives (i.e., ferrocenyl 1,3-benzoxazines and α-aminocresols), structure-activity relationship analyses suggest that the presence of the coumarin nucleus is tolerated for biological activity though this may lead to reduced efficacy. Preliminary mechanistic studies with the most promising compound (11b) support hemozoin inhibition and DNA interaction as likely mechanistic modalities by which this class of compounds may act to produce plasmocidal and antitrypanosomal effects

Halogenated oxindole and indoles from the South African marine ascidian Distaplia skoogi:

The known 3,6-dibromoindole (1), 6-bromo-3-chloroindole (2) and 6-bromo-2-oxindole (3) were isolated from the marine ascidian (sea squirt) Distapia skoogi collected from Algoa Bay, South Africa. Standard spectroscopic techniques were used to elucidate the structures of 1-3. All three compounds were found to be moderately cytotoxic to metastatic MDA-MB-231 breast cancer cells

Detection of the in vitro modulation of Plasmodium falciparum Arf1 by Sec7 and ArfGAP domains using a colorimetric plate-based assay:

The regulation of human Arf1 GTPase activity by ArfGEFs that stimulate GDP/GTP exchange and ArfGAPs that mediate GTP hydrolysis has attracted attention for the discovery of Arf1 inhibitors as potential anti-cancer agents. The malaria parasite Plasmodium falciparum encodes a Sec7 domain-containing protein - presumably an ArfGEF - and two putative ArfGAPs, as well as an Arf1 homologue (PfArf1) that is essential for blood-stage parasite viability. However, ArfGEF and ArfGAP-mediated activation/deactivation of PfArf1 has not been demonstrated

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay:

The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents

Reaction of Perrhenate with Phthalocyanine Derivatives in the Presence of Reducing Agents and Rhenium Oxide Nanoparticles in Biomedical Applications

A novel alternative route to access rhenium(V)−phthalocyanine complexes through direct metalation of metal-free phthalocyanines (H2Pcs) with a rhenium(VII) salt in the presence of various two-electron reducing agents is presented. Direct ion metalation of tetraamino- or tetranitrophthalocyanine with perrhenate (ReO4−) in the presence of triphenylphosphine led to oxidative decomposition of the H2Pcs, giving their respective phthalonitriles. Conversely, treatment of H2Pcs with ReO4− employing sodium metabisulfite yielded the desired ReVO−Pc complex. Finally, reaction of H2Pcs with ReO4− and NaBH4 as reducing agent led to the formation of rhenium oxide (RexOy) nanoparticles (NPs). The NP synthesis was optimised, and the RexOy NPs were capped with folic acid (FA) conjugated with tetraaminophthalocyanine (TAPc) to enhance their cancer cell targeting ability. The cytotoxicity profile of the resultant RexOy−TAPc−FA NPs was assessed and found to be greater than 80 % viability in four cell lines, namely, MDA−MB-231, HCC7, HCC1806 and HEK293T. Non-cytotoxic concentrations were determined and employed in cancer cell localization studies. The particle size effect on localization of NPs was also investigated using confocal fluorescence and transmission electron microscopy. The smaller NPs (≈10 nm) were found to exhibit stronger fluorescence properties than the ≈50 nm NPs and exhibited better cell localization ability than the ≈50 nm NPs