Candida albicans' key mediators in the PKC pathway as novel targets of bisindolylmaleimides

Zahlreiche Infektionen in unserem Körper werden heutzutage von Pilzen hervorgerufen. Unter einer großen Vielfalt verschiedener Pilzarten befindet sich Candida albicans, der als einer der Haupterreger gilt. Welweit wird er gefürchtet, da er sich durch eine hohe Todesrate auszeichnet. Besonders immunsuprimierte Patienten sind von der Candidose betroffen, in deren Körper sich der Erreger sehr schnell ausbreitet. Unter normalen physiologischen Bedingungen ist dieser Pilz ein harmloser Bewohner unserer Flora, doch minimale Abweichungen dieser Lebensverhältnisse können eine Infektion auslösen. Die herkömmlichen Behandlungen mit antimykotischen Therapeutika stoßen meist auf eine Blockade, da der Erreger ausgeklügelte Mechanismen entwickelt hat, um eine Resistenz aufzubauen. Die Behandlung von Candida albicans mit herkömmlichen Antimykotika induziert die Bildung von reaktiven Sauerstoffradikalen, welche parallel zahlreiche Signalwege aktivieren. Besonders der PKC- und Calcineurin Signalweg scheinen maßgeblich an Candida albicans Abwehrsystem beteiligt zu sein. Dabei treten die Schlüsselenzyme (Hsp90, PKC und GSK3) in den Vordergrund, die eine Reihe von Transkriptionen im Körper codieren. Die Erforschung der verschiedenen Proteinexpressionen dieses Hefepilzes bestätigte das Vorhandensein dieser Schlüsselenzyme. Nun musste ein passender Inhibitor gefunden werden, der diese Signaltransduktionskette zu unterbrechen vermochte. Von der Gruppe der Bisindolymaleimides war bereits bekannt, dass sie PKC angreifen und inhibieren. Jedoch wurden diese Studien nur mit krebsbefallenen menschlichen Zellen durchgeführt und nicht mit Candida albicans. Mit Hilfe der Micro-Affinitäts-Studie wurden die Interaktionen zwischen PKCƞ und BisIII beobachtet, wobei sich die Tatsache zu bewahrheiten erschien , dass BisIII genau auf den PKC Signalweg wirkt. Diese Erkenntnis könnte nun als Ansatzpunkt für weitere Forschungen mit BisIII verwendet werden, um so die Resistenz von Candida albicans gegen Antimykotika effizient zu eliminieren und dabei den Erfolg einer antimykotischen Therapie zu erhöhen.Nowadays fungal pathogens are causal agents of various infections in the human body. Among this life threatening fungi, Candida albicans stands out due to high mortality worldwide. Especially for immunocompromised patients candiasis has turned out to be a serious health concern. Under normal physiological conditions they are innocuous inhabitants of the human’s normal flora, but only minimal variations from normal conditions can precipitate an attack. The widespread usage of antifungal therapeutics has lost efficacy because of the resistance that Candida albicans has developed by various mechanisms. The exposure of Candida albicans to antifungal drugs creates stress responses which activate parallel signal pathways. Two major pathways (PKC and Calcineurin p.) display a major contribution to Candida albicans’ barrier. Thereby the key regulators of these pathways (Hsp90, GSK3 and PKC) show a link between the transcriptional events which make Candida albicans invulnerable against therapeutic treatment. The investigation of different expressions of these key regulators confirms their presence in Candida albicans. An attractive inhibitor would have the capability to stop the cascade which contributes to the resistance of Candida. We expected this attribute from the group of bisindolylmaleimides which has already been described to have an effect on PKC. The inhibition of PKC by BisIII was studied in carcinogenic cells but still unknown in Candida albicans. The affinity micro assay should help to elucidate a possible interaction between PKCƞ and BisIII. Finally the affinity micro assay has confirmed an effect that BisIII exercises on Candida albicans’ mechanism of resistance by interfering with the PKC pathway. The knowledge about a possible interaction could be used as a starting point for further investigations with BisIII which may restore the sensitivity to azoles and furthermore increase the efficacy of the antifungal treatment

Marine Pyrrolocarbazoles and Analogues: Synthesis and Kinase Inhibition

Granulatimide and isogranulatimide are alkaloids obtained from marine sources which have been shown to inhibit cell-cycle G2-checkpoint, targeting more particularly checkpoint 1 kinase (Chk1). At a structural level, they possess a characteristic pyrrolocarbazole framework also shared by the well-known rebeccamycin and staurosporine microbial metabolites which have been described to inhibit topoisomerase I and diverse kinases, respectively. This review reports precisely on the synthesis and kinase inhibitory activities of pyrrolocarbazole-based analogues of granulatimide

Materials for stem cell factories of the future

The materials community is now identifying polymeric substrates that could permit translation of human pluripotent stem cells (hPSCs) from lab-based research to industrial scale biomedicine. Well defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large scale drug screening programs and clinical use, wherein >1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells for one patient is challenging and a rethink is needed to scalable technology with the potential to meet the needs of millions of patients a year. Here we consider the role of materials discovery, an emerging area of materials chemistry that is in a large part driven by the challenges posed by biologists to materials scientists1-4

Synthesis and evaluation of novel functionalised indoles as anticancer agents

This thesis outlines the design and application of new routes towards a range of novel bisindolylmaleimide and indolo[2,3-a]carbazole derivatives, and evaluation of their biological effects and their chemotherapeutic potential. A key part of this work focussed on utilising a hydroxymaleimide as a replacement for the prevalent lactam/maleimide functionality and forming a series of novel derivatives through substitution on the indole nitrogens. To achieve this, a robust synthetic strategy was developed which allowed access to key maleic anhydride intermediates using Perkin-type methodology. These hydroxymaleimides were further modified via a Lossen rearrangement to furnish a series of analogues containing a 6-membered F-ring. The theme of F-ring modulation was further expanded through the utilisation of a second route involving the design and synthesis of β-keto ester intermediates, which afforded novel derivatives containing pyrazolone and isocytosine headgroups, and various N-substituents. Work on a further route involving a dione intermediate resulted in the isolation of a bisindolyl derivative with a novel imidazole F-ring. Following the synthesis of 42 novel compounds, extensive screening was undertaken using the NCI-60 cell line screen, with twelve candidates progressing to evaluation via the five dose assay. This led to the identification of several lead compounds with high cytotoxicity and excellent selectivity profiles, which included derivatives with low nanomolar GI50 values against specific cancer cell lines, and also derivatives with selective cytotoxicity. Preliminary results from a kinase screen indicated noteworthy selectivity towards GSK3α/β and PIM1 kinases, with low micromolar IC50 values being observed for these enzymes

Activation of phospholipase D by the LHRH receptor and associated mechanisms

The αT3 -1 gonadotroph- derived cell line was used as a model system to study intracellular signalling systems associated with the LHRH receptor, with particular interest in phospholipase D (PLD) and mechanisms involved in its regulation, such as protein kinase C (PKC) and non -receptor tyrosine kinases.Phospholipase D activation in aT3 -1 cells proceeded after a delay when stimulated by LHRH, or by the phorbol ester, phorbol 12,13 -dibutyrate (PDBu). LHRH -stimulated PLD activity was fully resistant to desensitisation over a 40 min time course. The Cat+ ionophore, ionomycin was unable to stimulate PLD activity. LHRH -stimulated PLD activity was inhibited by PKC downregulation or by bisindolylmaleimide PKC inhibitors at an approximately 8 -fold higher concentration than that seen for the PDBu- stimulated PLD activity. Another PKC inhibitor, H7, inhibited PDBu- stimulated PLD activity in a manner that indicated multiple components to the inhibition and inhibited LHRH -stimulated PLD activity with a low potency. This is consistent with the possibility that one mediator of the LHRH response is a form of PKC which is relatively resistant to certain PKC inhibitors and insensitive to phorbol esters, or that some other unknown kinase is involved. The tyrosine kinase inhibitors, lavendustin A and genistein were able to selectively inhibit the LHRH -stimulated PLD activity. Phospholipase D activation stimulated by LHRH was insensitive to pertussis toxin pretreatment, indicating that the heterotrimeric G- proteins, Gi and Go are not involved.LHRH induced tyrosine phosphorylation of a number of proteins in aT3 -1 cells; a similar phosphorylation profile was also produced by stimulation with PDBu. Tyrosine phosphorylation of proteins in aT3 -1 cells 4 was reduced by ionomycin. Two members of the src- family of non -receptor tyrosine kinases, src and fyn, were detected at significant levels in these cells by immunoblot. Using immunoprecipitation techniques we have shown that the enzymic activity of fyn is increased within 10 min of LHRH stimulation.The LHRH -stimulated PLD activity could not be detectably enhanced by a conditioning preincubation with LHRH, such as brings about the priming effect of LHRH ( a phenomenon of cellular sensitisation elicited by LHRH receptor activation). Removal of steroids from the growth medium severely impaired the PLD activation due to LHRH; this response was largely restored by addition of oestradiol -1713 but not progesterone. This may have implications for the LHRH priming effect in vivo, which is dependent upon the level of circulating steroid hormones.The murine LHRH receptor was transiently expressed in COS 7 cells from the cDNA in a pcDNA vector using a DEAE dextran transfection procedure. It was demonstrated that LHRH -stimulated PLD activation displayed similar sensitivity to the bisindolylmaleimide PKC inhibitor, Ro 31 -8220, to that seen in aT3 -1 cells. It was not however sensitive to tyrosine kinase inhibitors or pertussis toxin. Using site -directed mutagenesis of residues 87 and 318 in the transmembrane 2 and 7 domains of the LHRH receptor (which represents an inversion of the consensus motif in the G- protein coupled receptor family), it was found that mutation of Asp 318 to Asn was alone sufficient to produce a rapidly desensitising PLD response to LHRH, in contrast to the non -desensitising wild type

An Efficient Synthesis of Phenanthroindolizidine Core via Hetero Diels-Alder Reaction of In Situ Generated α-Allenylchalcogenoketenes With Cyclic Imines

Synthesis of phenanthroindolizidine core was efficiently achieved through a pathway involving hetero Diels-Alder reaction of α-allenylchalcogenoketenes, generated in situ by thermal [3,3] sigmatropic rearrangement of alkynyl propargyl sulfides or selenides, with cyclic imines and the subsequent iodine-assisted photochemical cyclization.journal articl

Structures and photophysical properties of 3,4-diaryl-1H-pyrrol-2,5-diimines and 2,3-diarylmaleimides

Structural features of 3,4-diaryl-1H-pyrrol-2,5-diimines and their derivatives have been studied by molecular spectroscopy techniques, single-crystal X-ray diffraction, and DFT calculations. According to the theoretical calculations, the diimino tautomeric form of 3,4-diaryl-1H-pyrrol-2,5-diimines is more stable in solution than the imino-enamino form. We also found that the structurally related 2,3 exist in the solid state in the dimeric diketo form. 3,4-Diary1-1H-pyrrol-2,5-diimines and 2,3-diarylmaleimides exhibit fluorescence in the blue region of the visible spectrum. The fluorescence spectra have large Stokes shifts. Aryl substituents at the 3,4-positions of 1H-pyrrol-2,5-diimine do not significantly affect fluorescence properties. The insertion of donor substituents into 2,3diarylmaleimides leads to bathochromic shift of emission bands with hyperchromic effect. (C) 2017 Elsevier B.V. All rights reserved

Synthesis and evaluation of novel azaindolocarbazole derivatives as cancer chemotherapeutics

This thesis details the design and implementation of novel chemical routes towards a series of highly propitious 7-azaindolyl derivatives of the indolocarbazole (ICZ) and bisindolylmaleimide (BIM) families, with subsequent evaluation for use as cancer chemotherapeutic agents. A robust synthetic strategy was devised to allow the introduction of a 7-azaindolyl moiety into our molecular template. This approach allowed access to a wide range of β-keto ester and β-keto nitrile intermediates. Critical analysis identified F-ring modulation as a major theme towards the advancement of ICZ and BIM derivatives in drug therapy. Thus, the employment of cyclocondensation methodology furnished a number of novel aminopyrazole, isoxazolone, pyrazolone and pyrimidinone analogues, considerably widening the scope of the prevalent maleimide functionality. Photochemical cyclisation provided for the first reported aza ICZ containing a six-membered F-ring. Another method towards achieving the aza ICZ core involved use of a Perkin-type condensation approach, with chemical elaboration of the headgroup instigated post-aromatisation. Subsequent use of a modified Lossen rearrangement allowed access to further analogues containing a six-membered F-ring. Extensive screening of the novel aza ICZ and BIM derivatives was carried out against the NCI-60 cancer cell array, with nine prospective candidates selected for continued biological evaluation. From these assays, a number of compounds were shown to inhibit cancer cell growth at concentrations of below 10 nM. Indeed, the most active aza ICZ tested is currently under assessment by the Biological Evaluation Committee of the NCI due to excellent antiproliferative activity demonstrated across the panel of cell lines, with a mean GI50 of 34 nM, a mean total growth inhibition (TGI) of 4.6 μM and a mean cytotoxicity (LC50) of 63.1 μM. Correlation to known topoisomerase I (topo I) inhibitors was revealed by COMPARE analysis, and subsequent topo I-mediated DNA cleavage assays showed inhibitory activity below 1 μM for several derivatives

Molecular biological and biochemical investigations on the biosynthetic enzymes of prenylated indole alkaloids from fungi

Prenylated indole alkaloids are widely distributed in plants, fungi and bacteria, especially in the family of Clavicipitaceae and Trichocomaceae of Ascomycota, and commonly exhibit interesting biological and pharmaceutical activities. In the biosynthetic pathway of prenylated indole alkaloids, prenylation catalyzed by prenyltransferases contributes significantly to the large structure diversity of these compounds in nature. Investigation on indole prenyltransferases would help to understand the construction of prenylated indole alkaloids in nature and also be useful for structural modification of indole derivatives and other substances to produce analogues of prenylated derivatives. Three indole prenyltransferases belonging to the dimethylallyltryptophan synthase (DMATS) superfamily were biochemically identified and characterized in vitro, including CdpC3PT from Neosartorya fischeri (N. fischeri), BrePT from Aspergillus versicolor (A. versicolor) and 5-DMATS from Aspergillus clavatus (A. clavatus). The responsible genes cdpC3PT and brePT were cloned into expression vector and heterologously expressed in Escherichia coli (E. coli). These works were carried out by Dr. Wen-Bing Yin, Suqin Yin and Qing Wang, respectively. In this thesis, CdpC3PT was confirmed to catalyze the formation of C3-prenylated products with a characteristic 6/5/5/6-fused tetracyclic ring system from tryptophan-containing cyclic dipeptides in one-step reaction. The NotF homologue BrePT showed much higher flexibility towards its aromatic substrates than NotF, and was proven to catalyze the highly regiospecific reverse prenylation at C-2 of the indole nucleus. The cloning of 5-dmats was carried out by Yan Liu. Functional proof of this gene was provided within this thesis by heterologous expression in E. coli and subsequent structure elucidation of enzyme products by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. 5-DMATS established high regiospecific activity to catalyze C5-prenylation on indole derivatives. Given the importance of prenylation in structure diversity and bioactivity enhancement, CdpC3PT, BrePT, 5-DMATS and other known prenyltransferases of the DMATS superfamily were applied for the chemoenzymatic synthesis of prenylated compounds. By using AnaPT, CdpC3PT and CdpNPT, eight and six stereoisomers of cis-configured prenylated pyrroloindoline diketopiperazines from cyclo-Trp-Ala and cyclo-Trp-Pro isomers were produced, respectively. The stereospecificity of AnaPT and CdpC3PT depended mainly on the configuration of tryptophanyl moiety in cyclo-Trp-Ala and cyclo-Trp-Pro isomers, while CdpNPT showed lower stereoselectivity, but higher conversion ability towards most tested substrates. 5-DMATS and FgaPT2 from Aspergillus were used for chemoenzymatic synthesis of prenylated indolocarbazoles. Reconstitution of enzyme activity of 5-DMATS and FgaPT2 in vitro revealed that they catalyzed regiospecific prenylation of indolocarbazoles at the para-position of the indole N-atom. This is the first report for prenylated indolocarbazoles. Subsequently, some indole prenyltransferases of the DMATS superfamily were found to accept also hydroxynaphthalenes and flavonoids, which were substrates for enzymes from the CloQ/NphB group and the UbiA superfamily, respectively. Nine prenylated flavonoids and twenty prenylated hydroxynaphthalenes have been isolated, and their structures were elucidated by MS and NMR analyses. It has been shown that, for an accepted hydroxynaphthalene, different enzymes produced usually the same major prenylated product, i.e. with a regular C-prenyl moiety at para- or ortho-position to a hydroxyl group. For flavonoids accepted by 7-DMATS, C-6 between two hydroxyl groups was the favorable prenylation position. The Michaelis-Menten constants (KM) and turnover numbers (kcat) of some prenyltransferases towards selected hydroxynaphthalenes are comparable to those obtained by using indole derivatives. In addition to indole prenyltransferases, other genes in the biosynthetic cluster of prenylated indole alkaloids were also investigated. A putative O-methyltransferase gene hasC and a putative cytochrome P450 gene hasH involved in the biosynthesis of hexadehydroastechrome (HAS) in Aspergillus fumigatus (A. fumigatus) were cloned into pQE60 and pESC-URA, respectively. Soluble His6-HasC was successfully overproduced in E. coli SG13009 and purified to near homogeneity by Ni-NTA. Constructs for co-expression with the reductase gene NFIA_083630 from N. fischeri in pESC-URA and for expression as His6-tagged protein in pESC-URA were also prepared for the putative cytochrome P450 gene hasH