Swinholide J, a Potent Cytotoxin from the Marine Sponge Theonella swinhoei

In our ongoing search for new pharmacologically active leads from Solomon organisms, we have examined the sponge Theonella swinhoei. Herein we report the isolation and structure elucidation of swinholide A (1) and one new macrolide, swinholide J (2). Swinholide J is an unprecedented asymmetric 44-membered dilactone with an epoxide functionality in half of the molecule. The structural determination was based on extensive interpretation of high-field NMR spectra and HRESIMS data. Swinholide J displayed potent in vitro cytotoxicity against KB cells (human nasopharynx cancer) with an IC50 value of 6 nM

Pharmacogénétique du cytochrome P450 humain CYP3A5

Les CYP3A sont les P450 les plus abondants dans le foie où ils métabolisent plus de 50 % des médicaments. Le CYP3A5 n'est exprimé que dans 25 % de la population caucasienne. Une mutation (CYP3A5*3) entraînant un épissage alternatif a été associée à un faible niveau de CYP3A5. Un niveau d'ARNm CYP3A5 plus élevé chez les individus *1/*3 par rapport aux homozygotes *3/*3 nous a amené à rechercher les causes de cette variation. L'ARN *3 est plus instable et sa dégradation est réalisée par la NMD (nonsense mediated mRNA decay). Le génotype CYP3A5 a des conséquences en clinique. Nous avons mis au point une technique de génotypage informationnel basé sur la PCR quantitative permettant de déterminer en une seule réaction le génotype et le phénotype CYP3A5. Cette technique permettrait d'ajuster les doses de molécules de faible index thérapeutique lorsque leur pharmacocinétique dépend du CYP3A5. Cette technique peut être généralisée à l'étude des gènes épissés alternativement.CYP3A are the most abundant P450 in the human liver where they metabolize more than 50% of drugs. CYP3A5 is found in only 25% of the Caucasian population. A SNP (CYP3A5*3) yielding to alternative splicing was associated with low CYP3A5 protein content. A higher CYP3A5 mRNA level found in *1/*3 as compared to *3/*3 individuals led us to investigate the mechanism underlying this difference. CYP3A5*3 mRNA was unstable and its degradation was mediated by nonsense mediated mRNA decay (NMD). CYP3A5 genotype has consequences in clinics. We describe a new informative genotyping assay based on quantitative real-time PCR, allowing the determination of CYP3A5 genotype and phenotype in one single step. This assay could be used to improve the dosage of drugs metabolized by CYP3A5 having a narrow therapeutic window. This assay can be generalized to the study of alternatively spliced genesPARIS-BIUP (751062107) / SudocSudocFranceF

Synthesis and biological investigation of the β-thiolactone and β-lactam analogs of tetrahydrolipstatin.

International audienceThe synthesis of β-thiolactone and β-lactam analogs of tetrahydrolipstatin is described from a common late-stage β-lactone derivative. These analogs, and a cis-disubstituted β-lactone analog of tetrahydrolipstatin, were screened for activity against porcine pancreatic lipase and for inhibition of cell growth of a panel of four human cancer lines

IN VITRO METABOLISM STUDY OF BUPRENORPHINE: EVIDENCE FOR NEW METABOLIC PATHWAYS

Buprenorphine (BUP) is a synthetic derivative of the morphine alkaloid thebaine. BUP is metabolized by N-dealkylation to form the active metabolite nor-buprenorphine (Nor-BUP), and both un-dergo subsequent glucuronidation. Although BUP has been used clinically for years, its metabolism has still not been fully eluci-dated. The aim of this study was to clarify the identity of the human hepatic cytochromes P450 (P450s) involved in BUP metabolism and to investigate other potential metabolites. The metabolism of BUP was examined using human liver microsomes (HLM) and Ad293 P450-transfected cell lines, as well as CYP 3A4 and 2C8 recombinant isoforms. The kinetic parameters of metabolite for-mation were calculated for HLM and competent isoforms. Individ-ual contribution of P450 isoforms in BUP metabolism as well as Nor-BUP production was evaluated using chemical inhibition ex-periments, as well as the relative activity factor approach. Th

The Cytosolic Chaperonin CCT/TRiC and Cancer Cell Proliferation

<div><p>The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis as it mediates the folding of the major cytoskeletal proteins tubulins and actins. CCT/TRiC is also involved in the oncoprotein cyclin E, the Von Hippel-Lindau tumour suppressor protein, cyclin B and p21^ras folding which strongly suggests that it is involved in cell proliferation and tumor genesis. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsc/Hsp70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.</p></div

Characteristics of the cell lines and cell extracts used throughout this study.

<p>Characteristics of the cell lines and cell extracts used throughout this study.</p

CCT/TriC, Hop/p60 or PhLP3 immunodepletion affects actin refolding.

<p>CCT/TRiC folding activity in (<b>A</b>) MIA-PaCa-2 and (<b>C</b>) OVCAR-8<b> </b>cell extracts before and after CCT/TriC, Hop/p60 or PhLP3 immunodepletion was assayed using [³⁵S]-labeled, denatured β-actin. The protein concentration of the MIA-PaCa-2 and OVCAR-8 cell extracts was 2.5 and 15 mg/ml, respectively. Native β-actin band intensity was measured using a PhosphorImager. The amount of native β-actin measured after immunodepletion is expressed as a fraction of the amount measured for the cell extracts before immunodepletion in (<b>B</b>) MIA-PaCa-2 and (<b>D</b>) OVCAR-8 cell extracts.</p

CCT/TriC activity modulators in cancer cell lines extracts.

<p>The amount of (<b>A</b>) Hop/p60, (<b>B</b>) PhLP3 and (<b>C</b>) PFD in 18 cancer and a normal (MRC5-SV2) cell lines extracts, a non-cancer liver homogenate (HNCL) and rabbit reticulocyte lysate (RRL) was determined by comparing the chemiluminescence signal recorded for Hop/p60, PhLP3 and PFD in cell extracts diluted 4 and 16 fold to that of known amounts of the proteins on the same Western blots.</p

Hsp/c70 immunodepletion affects CCT/TRiC-mediated β-actin folding.

<p>CCT/TRiC-mediated labeled, denatured β-actin folding in (<b>A</b>) MIA-PaCa-2 and OVCAR-8 cell extracts before and after Hsc/p70 immunodepletion. The protein concentration of the MIA-PaCa-2 and OVCAR-8 cell extracts was 2.5 and 15 mg/ml, respectively. Native β-actin band intensity was recoded using a PhosphorImager. The amount of native β-actin measured after immunodepletion is expressed as a fraction of the amount measured for the cell extracts before immunodepletion in (<b>B</b>) MIA-PaCa-2 and (<b>C</b>) OVCAR-8 cell extracts.</p