BCL6 is a context-dependent mediator of the glioblastoma response to irradiation therapy.

Glioblastoma is a rapidly fatal brain cancer that does not respond to therapy. Previous research showed that the transcriptional repressor protein BCL6 is upregulated by chemo and radiotherapy in glioblastoma, and inhibition of BCL6 enhances the effectiveness of these therapies. Therefore, BCL6 is a promising target to improve the efficacy of current glioblastoma treatment. BCL6 acts as a transcriptional repressor in germinal centre B cells and as an oncogene in lymphoma and other cancers. However, in glioblastoma, BCL6 induced by therapy may not be able to repress transcription. Using a BCL6 inhibitor, the whole proteome response to irradiation was compared with and without BCL6 activity. Acute high dose irradiation caused BCL6 to switch from repressing the DNA damage response to promoting stress response signalling. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) enabled comparison of BCL6 partner proteins between untreated and irradiated glioblastoma cells. BCL6 was associated with transcriptional coregulators in untreated glioblastoma including the known partner NCOR2. However, this association was lost in response to acute irradiation, where BCL6 unexpectedly associated with synaptic and plasma membrane proteins. These results reveal the activity of BCL6 under therapy-induced stress is context-dependent, and potentially altered by the intensity of that stress.

A Structure-Activity Investigation of the Fungal Metabolite (-)-TAN-2483B: Inhibition of Bruton's Tyrosine Kinase.

The natural product (-)-TAN-2483B is a fungal secondary metabolite which displays promising anti-cancer and immunomodulatory activity. Our previous syntheses of (-)-TAN-2483B and sidechain analogues uncovered inhibitory activity against Bruton's tyrosine kinase (Btk), an established drug target for various leukaemia and immunological diseases. A structure-based computational study using ensemble docking and molecular dynamics was performed to determine plausible binding modes for (-)-TAN-2483B and analogues in the Btk binding site. These hypotheses guided the design of new analogues which were synthesised and their inhibitory activities determined, providing insights into the structural determinants of the furopyranone scaffold that confer both activity and selectivity for Btk. These findings offer new perspectives for generating optimised (-)-TAN-2483B-based kinase inhibitors for the treatment of leukaemia and immunological diseases.

Identification and characterization of protein interactions with the major Niemann-Pick type C disease protein in yeast reveals pathways of therapeutic potential.

Niemann-Pick type C (NP-C) disease is a rare lysosomal storage disease caused by mutations in NPC1 (95% cases) or NPC2 (5% cases). These proteins function together in cholesterol egress from the lysosome, whereby upon mutation, cholesterol and other lipids accumulate causing major pathologies. However, it is not fully understood how cholesterol is transported from NPC1 residing at the lysosomal membrane to the endoplasmic reticulum (ER) and plasma membrane. The yeast ortholog of NPC1, Niemann-Pick type C-related protein-1 (Ncr1), functions similarly to NPC1; when transfected into a mammalian cell lacking NPC1, Ncr1 rescues the diagnostic hallmarks of cholesterol and sphingolipid accumulation. Here, we aimed to identify and characterize protein-protein interactions (PPIs) with the yeast Ncr1 protein. A genome-wide split-ubiquitin membrane yeast two-hybrid (MYTH) protein interaction screen identified 11 ER membrane-localized, full-length proteins interacting with Ncr1 at the lysosomal/vacuolar membrane. These highlight the importance of ER-vacuole membrane interface and include PPIs with the Cyb5/Cbr1 electron transfer system, the ceramide synthase complex, and the Sec61/Sbh1 protein translocation complex. These PPIs were not detected in a sterol auxotrophy condition and thus depend on normal sterol metabolism. To provide biological context for the Ncr1-Cyb5 PPI, a yeast strain lacking this PPI (via gene deletions) exhibited altered levels of sterols and sphingolipids including increased levels of glucosylceramide that mimic NP-C disease. Overall, the results herein provide new physical and genetic interaction models to further use the yeast model of NP-C disease to better understand human NP-C disease.

Mechanistic Studies on the Base-Promoted Ring Opening of Glycal-Derived gem-Dibromocyclopropanes.

In the presence of a nucleophilic base, ring-fused gem-dibromocyclopropanes derived from d-glycals undergo ring opening to give 2-deoxy-2-(E-bromomethylene)glycosides. Such cleavage of an exocyclic cyclopropane bond contrasts with the more usual silver-promoted ring-expansion reactions in which endocyclic bond cleavage occurs. Experimental and theoretical studies are reported which provide insights into the reaction mechanism and the origin of its kinetic selectivity for E-configured bromoalkene products. Density functional theory computations (M06-2X) predict that the reaction commences with alkoxide-induced HBr elimination from the dibromocyclopropane to form a bromocyclopropene. Ring opening then gives a configurationally stable zwitterionic (oxocarbenium cation/vinyl carbanion) intermediate, which undergoes nucleophilic addition and protonation to give the bromoalkene. There are two competing sources of the proton in the final step: One is the alcohol (co)solvent, and the other is the molecule of alcohol produced during the initial deprotonation step. The roles of the formed alcohol molecule and the bulk (co)solvent are demonstrated by isotope-labeling studies performed with deuterated solvents. The acid-promoted isomerization of the E-bromoalkene product into the corresponding Z-bromoalkene is also described. The mechanistic knowledge gained in this investigation sheds light on the unusual chemistry of this system and facilitates its future application in new settings.

The Big Picture of Glioblastoma Malignancy: A Meta-Analysis of Glioblastoma Proteomics to Identify Altered Biological Pathways.

Glioblastoma is a highly malignant cancer with no effective treatment. It is vital to elucidate the mechanisms which drive glioblastoma in order to identify therapeutic targets. The differences in protein expression between glioblastoma, grade I-III glioma, and normal brain tissue reflect the functional alterations driving malignancy. However, proteomic analysis of glioblastoma has been hampered by the heterogeneity of glioblastoma and the variety of methodology used in its study. To reduce these inconsistencies, we performed a meta-analysis of the literature published since 2015, including 14 datasets from eight papers comparing the whole proteome of glioblastoma to normal brain or grade I-III glioma. We found that 154 proteins were commonly upregulated and 116 proteins were commonly downregulated in glioblastoma compared to normal brain. Meanwhile, 240 proteins were commonly upregulated and 125 proteins were commonly downregulated in glioblastoma compared to grade I-III glioma. Functional enrichment analysis revealed upregulation of proteins involved in mRNA splicing and the immune system and downregulation of proteins involved in synaptic signaling and glucose and glutamine metabolism. The identification of these altered biological pathways provides a basis for deeper investigation in the pursuit of an effective treatment for glioblastoma.

Gadolinium Complexes Attached to Poly Ethoxy Ethyl Glycinamide (PEE-G) Dendrons: Magnetic Resonance Imaging Contrast Agents with Increased Relaxivity.

A range of poly ethoxy ethyl glycinamide (PEE-G) dendron scaffolds with gadolinium (III) complexes attached were synthesized with a focus on product purity and high Gd(III) loading. The nuclear magnetic resonance relaxivity of these products was measured and compared with commercially available low-molecular-weight magnetic resonance imaging contrast agents. Over twice the relaxivity based on Gd(III) concentration, and up to 20-fold increase in relaxivity were observed based on molecular concentration. Relaxivity properties were observed to increase with both increasing molecular weight and number of Gd(III) complexes attached, however a plateau was reached for molecular weight increase. T1 and T2 relaxivity properties were also investigated at two different magnetic fields. Transverse relaxivity is unaffected by magnetic field strength whereas increase in longitudinal relaxivity was not as pronounced at the higher field.

The Synthesis and Anti-tumour Properties of Poly Ethoxy Ethyl Glycinamide (PEE-G) Scaffolds with Multiple PD-1 Peptides Attached.

Multivalent structures can provide multiple interactions at a target site and improve binding affinity. The multivalent presentation of the anti-tumour heptapeptide, SNTSESF, was investigated. This peptide's activity has been attributed to blockade of the PD-1 receptor-mediated signalling pathway. Two and four peptide units were conjugated to poly ethoxy ethyl glycinamide (PEE-G) scaffolds to prepare high-purity products. These conjugates and the peptide were examined in a mouse model implanted with GL261 tumours that indicated that presenting more than two copies of peptide SNTSESF on the dendritic scaffold does not increase anti-tumour activity per peptide. The fluorescent labelled peptide and most active multivalent peptide conjugate were therefore screened for their interaction with the human PD-L1 protein in a fluorescence polarisation assay. No indication of a specific SNTSESF peptide/PD-L1 interaction was observed. This finding was further supported by a molecular modelling binding study.

Synthesis of Bioactive Side-Chain Analogues of TAN-2483B.

The fungal metabolite TAN-2483B has a 2,6-trans-relationship across the pyran ring of its furo[3,4-b]pyran-5-one core, which has thwarted previous attempts at its synthesis. We have now developed a chiral pool approach to this core and prepared side-chain analogues of TAN-2483B. The synthesis relies on ring expansion of a reactive furan ring-fused dibromocyclopropane and alkynylation of the resulting pyran. The furan ring is constructed by palladium-catalysed carbonylative lactonisation. Various side-chains are appended through Wittig-type chemistry. The prepared analogues showed micromolar activity towards cancer cell lines HL-60, 1A9 and MCF-7 and certain human disease-relevant kinases, including Bruton's tyrosine kinase (Btk).

Characterisation of the biological response of Saccharomyces cerevisiae to the loss of an allele of the eukaryotic initiation factor 4A.

The translation initiation machinery is emerging as an important target for therapeutic intervention, with potential in the treatment of cancer, viral infections, and muscle wasting. Amongst the targets for pharmacological control of translation initiation is the eukaryotic initiation factor 4A (eIF4A), an RNA helicase that is essential for cap-dependent translation initiation. We set out to explore the system-wide impact of a reduction of functional eIF4A. To this end, we investigated the effect of deletion of TIF1, one of the duplicate genes that produce eIF4A in yeast, through synthetic genetic array interactions and system-wide changes in GFP-tagged protein abundances. We show that there is a biological response to deletion of the TIF1 gene that extends through the proteostasis network. Effects of the deletion are apparent in processes as distributed as chromatin remodelling, ribosome biogenesis, amino acid metabolism, and protein trafficking. The results from this study identify protein complexes and pathways that will make ideal targets for combination therapies with eIF4A inhibitors.

Synthesis of a simplified triazole analogue of pateamine A.

Pateamine A is a naturally occurring metabolite extracted from the marine sponge Mycale hentscheli. It exhibits potent cytotoxicity towards cancer cell lines and has been shown to target protein translation initiation via inhibition of the function of eukaryotic initiation factor 4A proteins. We have synthesised a simplified analogue of pateamine A, consisting of the skeletal core of the natural product but with the thiazole heterocycle replaced by a triazole. The convergent design of the synthesis features a base-induced opening of a δ-valerolactone to access the Z,E-dienoate moiety, Julia-Kocienski olefination and copper-catalysed azide-alkyne cycloaddition. Bioactivity testing of the simplified pateamine A analogue (3) indicated a significant reduction in cytotoxicity, compared to natural pateamine A. We propose that this reduced activity is due mainly to the substitution of the thiazole for the triazole heterocycle. This supports the hypothesis that the thiazole of pateamine A is important for binding to its biological target.

ßI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug-tubulin interactions and microtubule stability.

Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to ß-tubulin at a site that is different from the classical taxoid site. Multiple ßI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M ßI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian ßI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug-tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on ß-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.

Synthesis of mycothiol conjugate analogues and evaluation of their antimycobacterial activity.

Drug-resistant Mycobacterium tuberculosis is a growing health problem. As proof of principle that the bacterial-specific metabolite mycothiol could be used as a delivery agent for antimycobacterial agents, simplified analogues of mycothiol were synthesised containing an S-trichloroethenyl substituted cysteine residue. It was envisaged that uptake of the mycothiol analogue would be followed by release of the known cytotoxin S-trichloroethenyl cysteine by the action of mycothiol S-conjugate amidase or its paralog, mycothiol deacetylase MshB. Promising activity was displayed against model Mycobacteria, although further development will be required to improve selectivity.

Characterizing the laulimalide-peloruside binding site using site-directed mutagenesis of TUB2 in S. cerevisiae.

Baker's yeast, Saccharomyces cerevisiae, has significant sequence conservation with a core subset of mammalian proteins and can serve as a model for disease processes. The aim of this study was to determine whether yeast could be used as a model system to identify new agents that interact with the laulimalide-peloruside binding site on ß-tubulin. Agents that bind to this site cause stabilization of microtubules and interfere with cell division. Based on the location of the proposed laulimalide-peloruside binding site and of previously identified mutations shown to cause resistance in mammalian cells, we made the corresponding mutations in yeast and tested whether they conferred resistance to laulimalide and peloruside. Mutations A296T and R306H, which cause 6-fold and 40-fold increased resistance in human 1A9 ovarian carcinoma cells, respectively, also led to resistance in yeast to these compounds. Similarly, other mutations led to resistance or, in one case, increased sensitivity. Thus, we conclude that yeast is an appropriate model to screen for small molecule drugs that may be efficacious in cancer therapy in humans through the newly characterised laulimalide-peloruside binding site.

Cytoskeletal alterations that confer resistance to anti-tubulin chemotherapeutics.

Drugs that target microtubules are a successful class of anti-cancer agents that have been in clinical use for over two decades. Acquired resistance to these drugs, however, remains a serious problem. Microtubule alterations, such as tubulin mutations and altered ß- tubulin isotype expression, are prominent factors in development of resistance. Changes in actin and intermediate filament proteins can also mediate sensitivity to microtubule-targeting drugs. This review focuses on the mechanisms by which alterations in cytoskeletal proteins lead to drug resistance. This information will be helpful for improving the targeting of microtubule toxins.

The cellular target specificity of pateamine A.

The natural product pateamine A (pateamine) from the sponge Mycale hentscheli is active against a wide range of dividing cells and has been shown to inhibit the functions of the eukaryotic initiation factor 4A (eIF4A). We have identified that pateamine is additionally able to modulate the formation of actin filaments and microtubules in vitro but at higher concentrations than required for inhibition of eIF4A. Cell cycle analysis confirmed that actin and tubulin are not major mediators of the cellular activity of pateamine. The range of targets identified demonstrates the value of multiple approaches to determining the mode of action of biologically active compounds.

The protein synthesis inhibitors mycalamides A and E have limited susceptibility toward the drug efflux network.

The mycalamides belong to a family of protein synthesis inhibitors noted for antifungal, antitumour, antiviral, immunosuppressive, and nematocidal activities. Here we report a systematic analysis of the role of drug efflux pumps in mycalamide resistance and the first isolation of mycalamide E. In human cell lines, neither P-glycoprotein overexpression nor the use of efflux pump inhibitors significantly modulated mycalamide A toxicity in the systems tested. In Saccharomyces cerevisiae, it appears that mycalamide A is subject to efflux by the principle mediator of xenobiotic efflux, Pdr5p along with the major facilitator superfamily pump Tpo1p. Mycalamide E showed a similar efflux profile. These results suggest that future drugs based on the mycalamides are likely to be valuable in situations where efflux pump-based resistance leads to failure of other chemotherapeutic approaches, although efflux may be a mediator of resistance in antifungal applications.

Alkenylphosphonates: unexpected products from reactions of methyl 2-[(diethoxyphosphoryl)methyl]benzoate under Horner-Wadsworth-Emmons conditions.

Methyl 2-[(diethoxyphosphoryl)methyl]benzoate reacts with several aldehydes to produce an alkenylphosphonate as the major product, together with varying amounts of the expected Horner-Wadsworth-Emmons product, a 1,2-disubstituted E-alkene. Use of a bulky aldehyde or the tert-butyl ester favours the normal HWE product.

Total synthesis of aigialomycin D using a Ramberg-Bäcklund/RCM strategy.

The bioactive resorcylic acid lactone aigialomycin D (1) has been synthesized by a novel combination of ring-closing metathesis (RCM) and Ramberg-Bäcklund reactions. This synthetic strategy enables the C1'-C2' alkene to be masked as a sulfone during formation of the macrocycle by ring closing metathesis at the C7'-C8' olefin, thus avoiding competing formation of a cyclohexene. A subsequent Ramberg-Bäcklund reaction efficiently produces the C1'-C2' E-alkene. This combined RCM/Ramberg-Bäcklund reaction strategy should be widely applicable to the synthesis of macrocyclic dienes.

Synthesis of DNA-directed pyrrolidinyl and piperidinyl confined alkylating chloroalkylaminoanthraquinones: potential for development of tumor-selective N-oxides.

A novel series of 1,4-disubstituted chloroethylaminoanthraquinones, containing alkylating chloroethylamino functionalities as part of a rigid piperidinyl or pyrrolidinyl ring-system, have been prepared. The target compounds were prepared by ipso-displacement of halides of various anthraquinone chromophores by either hydroxylated or chlorinated piperidinyl- or pyrrolidinyl-alkylamino side chains. The chloroethylaminoanthraquinones were shown to alkylate guanine residues of linearized pBR322 (1-20 microM), and two symmetrically 1,4-disubstituted anthraquinones (compounds 14 and 15) were shown to interstrand cross-link DNA in the low nM range. Several 1,4-disubstituted chloroethylaminoanthraquinones were potently cytotoxic (IC50 values:

Synthesis and biological evaluation of novel chloroethylaminoanthraquinones with potent cytotoxic activity against cisplatin-resistant tumor cells.

Novel 1- and 1,4-substituted chloroethylaminoanthraquinones with DNA binding and alkylating properties along with their respective hydroxyethylaminoanthraquinone intermediates were synthesized. Selected chloroethylaminoanthraquinones were shown to cross-link DNA and alkylate guanines (at low nM concentration) with a preference for reaction sites containing 5'-PyG. A compound (Alchemix) with the bis-chloroethyl functionality confined to one side chain alkylated but did not cross-link DNA. All the 1,4-disubstituted chloroethylaminoanthraquinones were potently cytotoxic (nM IC(50)s) against cisplatin-resistant ovarian cancer cell lines.