Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes

5-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the prototype drug of a recently discovered novel class of tumor-selective compounds that preferentially inhibit the proliferation of specific tumor cell types (e.g. leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-3-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the mid-nanomolar range with 500- to 1000-fold tumor cell selectivity. The compounds preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3, kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against other tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic activity, already after 4 h of exposure. The unusual tumor selectivity could not be explained by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells. Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the cytoplasm, no visible appearance in the nucleus, and a predominant localization in the endoplasmic reticulum. These observations may be helpful to narrow down the intracellular localization and identification of the molecular target of the 5-substituted thiophene derivatives. publisher: Elsevier articletitle: Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes journaltitle: European Journal of Medicinal Chemistry articlelink: http://dx.doi.org/10.1016/j.ejmech.2017.03.044 content_type: article copyright: © 2017 The Authors. Published by Elsevier Masson SAS. ispartof: European Journal of Medicinal Chemistry vol:132 pages:219-235 ispartof: location:France status: publishe

Synthesis, Computational Analysis, and Antiproliferative Activity of Novel Benzimidazole Acrylonitriles as Tubulin Polymerization Inhibitors: Part 2

We used classical linear and microwave-assisted synthesis methods to prepare novel Nsubstituted, benzimidazole-derived acrylonitriles with antiproliferative activity against several cancer cells in vitro. The most potent systems showed pronounced activity against all tested hematological cancer cell lines, with favorable selectivity towards normal cells. The selection of lead compounds was also tested in vitro for tubulin polymerization inhibition as a possible mechanism of biological action. A combination of docking and molecular dynamics simulations confirmed the suitability of the employed organic skeleton for the design of antitumor drugs and demonstrated that their biological activity relies on binding to the colchicine binding site in tubulin. In addition, it also underlined that higher tubulin affinities are linked with (i) bulkier alkyl and aryl moieties on the benzimidazole nitrogen and (ii) electron-donating substituents on the phenyl group that allow deeper entrance into the hydrophobic pocket within the tubulin’s -subunit, consisting of Leu255, Leu248, Met259, Ala354, and Ile378 residues

An intrabody based on a llama single-domain antibody targeting the N-terminal alpha-helical multimerization domain of HIV-1 REV prevents viral production

The HIV-1-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal alpha-helical multimerization domain. In the presence of this nanobody we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.status: publishe

Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co- activator LEDGF/p75, and Chromatin

Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator involved in targeting human immunodeficiency virus (HIV) integration and the development of MLL fusion-mediated acute leukemia. A previous study revealed that LEDGF/p75 dynamically scans the chromatin, and upon interaction with HIV-1 integrase, their complex is locked on chromatin. At present, it is not known whether LEDGF/p75-mediated chromatin locking is typical for interacting proteins. Here, we employed continuous photobleaching and fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2, a LEDGF/p75- and c-Myc-interacting protein involved in transcriptional regulation. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, whereas the dynamics of JPO2 chromatin binding are decelerated upon interaction with LEDGF/p75, very strong locking of their complex onto chromatin is absent. Similar results were obtained with the domesticated transposase PogZ, another cellular interaction partner of LEDGF/p75. We furthermore show that diffusive JPO2 can oligomerize; that JPO2 and LEDGF/p75 interact directly and specifically in vivo through the specific interaction domain of JPO2 and the C-terminal domain of LEDGF/p75, comprising the integrase-binding domain; and that modulation of JPO2 dynamics requires a functional PWWP domain in LEDGF/p75. Our results suggest that the dynamics of the LEDGF/p75-chromatin interaction depend on the specific partner and that strong chromatin locking is not a property of all LEDGF/p75-binding proteins.status: publishe

Selective inhibition of Human Immunodeficiency Virus type 1 (HIV-1) by a novel family of tricyclic nucleosides

Nucleoside 1, with an unusual tricyclic carbohydrate moiety, specifically inhibits HIV-1 replication while being inactive against HIV-2 or other (retro) viruses. In an attempt to increase the inhibitory efficacy against HIV-1, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on this prototype compound. These include substitution of the ethoxy group at the C-400 position by alkoxy groups of different length, branching, conformational freedom or functionalization. In addition, the 400-ethoxy group has been removed or substituted by other functional groups. The role of the tert-butyldimethylsilyl (TBDMS) group at the 20 position has also been studied by preparing the corresponding 20-deprotected derivative or by replacing it by other silyl (terthexyldimethylsilyl) or acyl (acetyl) moieties. Finally, the thymine of the prototype compound has been replaced by N-3-methylthymine, uracil or thiophenyl. Some of these compounds were endowed with a 6- to 7-fold higher selectivity than the prototype 1. The tricyclic nucleosides here described represent a novel type of selective anti HIV-1 inhibitors, targeted at the HIV-1-encoded reverse transcriptase.We thank Susana Ruiz, Ann Absillis and Leen Ingels for excellent technical assistance. The Spanish MEC/MCINN (Project SAF 2009- 13914-C02-01), the Spanish CSIC (Project PIF 08-019-2) and the Concerted Research Actions of the K.U. Leuven (GOA 10/014) are also acknowledged for financial support. The Spanish CSIC (JAEDoc Program) and European Science Foundation (ESF) are also acknowledged for a JAE-Doc contract to M.-C. Bonache.Peer reviewe

Understanding the molecular mechanism of host-based statin resistance in hepatitis C virus replicon containing cells

A number of statins, the cholesterol-lowering drugs, inhibit the in vitro replication of hepatitis C virus (HCV). In HCV-infected patients, addition of statins to the earlier standard of care therapy (pegIFN-α and ribavirin) resulted in increased sustained virological response rates. The mechanism by which statins inhibit HCV replication has not yet been elucidated. In an attempt to gain insight in the underlying mechanism, hepatoma cells carrying an HCV replicon were passaged in the presence of increasing concentrations of fluvastatin. Fluvastatin-resistant replicon containing cells could be generated and proved ∼8-fold less susceptible to fluvastatin than wild-type cultures. The growth efficiency of the resistant replicon containing cells was comparable to that of wild-type replicon cells. The fluvastatin-resistant phenotype was not conferred by mutations in the viral genome but is caused by cellular changes. The resistant cell line had a markedly increased HMG-CoA reductase expression upon statin treatment. Furthermore, the expression of the efflux transporter P-gp was increased in fluvastatin-resistant replicon cells (determined by qRT-PCR and flow cytometry). This increased expression resulted also in an increased functional transport activity as measured by the P-gp mediated efflux of calcein AM. In conclusion, we demonstrate that statin resistance in HCV replicon containing hepatoma cells is conferred by changes in the cellular environment.publisher: Elsevier articletitle: Understanding the molecular mechanism of host-based statin resistance in hepatitis C virus replicon containing cells journaltitle: Biochemical Pharmacology articlelink: http://dx.doi.org/10.1016/j.bcp.2015.06.003 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe

3D printing of plant tissue for innovative food manufacturing: encapsulation of alive plant cells into pectin based bio-ink

3D food printing allows creation of foods by depositing food material according to computer aided designs. However, the number of printable materials for food is still low which limits the possibilities of creating specific structures and textures. A novel approach is tested of using food printing materials incorporating plant cells in order to print foods that resemble plant tissues in various ways. A 3D printing method was developed based on the extrusion of bio-inks composed of a low-methoxylated pectin gel and embedded lettuce leaf cells. Bovine serum albumin was added in order to increase the air fraction in the printed gel matrix. Objects containing up to 5 × 106 cells/mL were successfully 3D printed. The mechanical strength increased by the pectin concentration and decreased with the increase of air fraction and concentration of encapsulated cells. The viability of the encapsulated plant cells depended on the pectin concentration and varied from 50 to 60 %.status: accepte

Heterozygous mutation of cysteine528 in XPO1 is sufficient for resistance to selective inhibitors of nuclear export

Exportin-1 (CRM1/XPO1) is a crucial nuclear export protein that transports a wide variety of proteins from the nucleus to the cytoplasm. These cargo proteins include tumor suppressors and growth-regulatory factors and as such XPO1 is considered a potential anti-cancer target. From this perspective, inhibition of the XPO1-mediated nuclear export by selective inhibitor of nuclear export (SINE) compounds has shown broad-spectrum anti-cancer activity. Furthermore, the clinical candidate SINE, selinexor, is currently in multiple phase I/II/IIb trials for treatment of cancer. Resistance against selinexor has not yet been observed in the clinic, but in vitro selection of resistance did not reveal any mutations in the target protein, XPO1. However, introduction of a homozygous mutation at the drug's target site, the cysteine 528 residue inside the XPO1 cargo-binding pocket, by genetic engineering, confers resistance to selinexor. Here we investigated whether this resistance to selinexor is recessive or dominant. For this purpose we have engineered multiple leukemia cell lines containing heterozygous or homozygous C528S substitutions using CRISPR/Cas9-mediated genome editing. Our findings show that heterozygous mutation confers similar resistance against selinexor as homozygous substitution, demonstrating that SINE resistance can be obtained by a single and dominant mutation of the cysteine528 residue in XPO1.status: publishe

Effect of UV-wavelength on Hardening Process of Porogen-containing and Porogen-free Ultra-low-k PECVD Glasses

The ITRS scaling of ultra-large-scale integrated circuits requires mechanically robust materials with low k-value. Low-k materials recently used in the Cu/low-k integration scheme have k-values between 2.5 and 3.0. One of the limiting factors in further reduction of k-value is mechanical robustness, since major way to decrease k-value is increasing the material porosity. The PECVD low-k deposition of ultra low-k films uses a porogen-based approach. The matrix material is deposited by oxidation of alkylsilanes in a plasma-enhanced chemical vapor deposition (PECVD) process. The porogen molecules, usually cyclic hydrocarbons, are introduced into a SiOCH film by co-deposition with the matrix material. To create porosity, the porogen is removed from the films using UV-assisted-thermal curing. The porogen molecules are photo-dissociated by UV-light with the formation of volatile hydrocarbons and non-volatile carbon-rich residues (porogen residue) [1]. We showed recently that SiOCH glasses with improved mechanical properties and ultra-low-k value could be obtained by controlled decomposition of the porogen molecules prior to the UV-hardening step [2]. The controlled removal of porogen can be performed by H2-based afterglow plasma treatment of PECVD film [1,2]. In this work we study the effect of narrow band 172 nm and broadband >200 nm UV-sources in the new curing scheme of the PECVD dielectrics. The data are compared with the PECVD films fabricated in the conventional UV-curing scheme. The effect of both 172 nm and >200 nm UV-sources is comparable for porogen-containing conventional PECVD films. However, the porogen-free films cured with 172 nm UV-source shows approximately twice as higher Young’s modulus of 6.64 GPa (k100kHz ~ 2.2, 44% open porosity) than those cured with >200 nm UV with YM of 3.38 GPa (k100kHz ~ 2.0, 48% open porosity). The mechanical properties, optical properties 150 nm - 800 nm, dielectric constants at 100 kHz and 4 GHz, porosities and pore size distributions, bonding structure are presented. The impact of porogen on optical characteristic and therefore on photochemical UV-hardening mechanism is discussed. The achieved mechanical properties are explained on a basis of the percolation of rigidity theory and random network concepts. References [1] A. M. Urbanowicz, K. Vanstreels, D. Shamiryan, S. De Gendt and M. Baklanov, Electrochem. Solid State Lett., 12, H292 (2009). [2]Urbanowicz, K. Vanstreels, P. Verdonck, D. Shamiryan, S. De Gendt and M. R. Baklanov, 107 (7), J. Appl. Phys. (2010).http://www2.avs.org/symposium/AVS57/pdfs/techprog.pdfstatus: publishe