Synthesis of (-)-dactylolide and 13-desmethylene-(-)-dactylolide and their effects on tubulin

An efficient new synthesis has been elaborated for non-natural (-)-dactylolide ((-)-2) and its 13-desmethylene analogue 4, employing a HWE-based macrocyclization approach with beta-keto-phosphonate/aldehyde 19 and the respective 13-desmethylene derivative as the key intermediates. Both (-)-2 and 4 as well as the corresponding C20 alcohols inhibit human cancer cell proliferation with IC(50) values in the sub-micromolar range and induce the polymerization of tubulin in vitro.

Stereoselective synthesis of a monocyclic peloruside a analogue

The stereoselective synthesis of the monocyclic peloruside A analogue 4 has been achieved, following a new efficient approach for the introduction of the side chain, involving a late-stage addition of vinyl lithium species 7a to aldehyde 8. Further key steps are a highly diastereoselective allyltitanation reaction and a RCM-based macrocyclization.

Fine structure of hippocampal mossy fiber synapses following rapid high-pressure freezing

Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.

Total synthesis of (-)-zampanolide and structure-activity relationship studies on (-)-dactylolide derivatives

A new total synthesis of the marine macrolide (-)-zampanolide (1) and the structurally and stereochemically related non-natural levorotatory enantiomer of (+)-dactylolide (2), that is, ent-2, has been developed. The synthesis features a high-yielding, selective intramolecular Horner-Wadsworth-Emmons (HWE) reaction to close the 20-membered macrolactone ring of 1 and ent-2. The β-keto phosphonate/aldehyde precursor for the ring-closure reaction was obtained by esterification of a ω-diethylphosphono carboxylic acid fragment and a secondary alcohol fragment incorporating the THP ring that is embedded in the macrocyclic core structure of 1 and ent-2. THP ring formation was accomplished through a segment coupling Prins-type cyclization. Employing the same overall strategy, 13-desmethylene-ent-2 as well as the monocyclic desTHP derivatives of 1 and ent-2 were prepared. Synthetic 1 inhibited human cancer cell growth in vitro with nM IC(50) values, while ent-2, which lacks the diene-containing hemiaminal-linked side chain of 1, is 25- to 260-fold less active. 13-Desmethylene-ent-2 as well as the reduced versions of ent-2 and 13-desmethylene-ent-2 all showed similar cellular activity as ent-2 itself. The same activity level was attained by the monocyclic desTHP derivative of 1. Oxidation of the aldehyde functionality of ent-2 gave a carboxylic acid that was converted into the corresponding N-hexyl amide. The latter showed only μM antiproliferative activity, thus being several hundred-fold less potent than 1.

The glycine deportation system and its pharmacological consequences

The glycine deportation system is an essential component of glycine catabolism in man whereby 400 to 800mg glycine per day are deported into urine as hippuric acid. The molecular escort for this deportation is benzoic acid, which derives from the diet and from gut microbiota metabolism of dietary precursors. Three components of this system, involving hepatic and renal metabolism, and renal active tubular secretion help regulate systemic and central nervous system levels of glycine. When glycine levels are pathologically high, as in congenital nonketotic hyperglycinemia, the glycine deportation system can be upregulated with pharmacological doses of benzoic acid to assist in normalization of glycine homeostasis. In congenital urea cycle enzymopathies, similar activation of the glycine deportation system with benzoic acid is useful for the excretion of excess nitrogen in the form of glycine. Drugs which can substitute for benzoic acid as substrates for the glycine deportation system have adverse reactions that may involve perturbations of glycine homeostasis. The cancer chemotherapeutic agent ifosfamide has an unacceptably high incidence of encephalopathy. This would appear to arise as a result of the production of toxic aldehyde metabolites which deplete ATP production and sequester NADH in the mitochondrial matrix, thereby inhibiting the glycine deportation system and causing de novo glycine synthesis by the glycine cleavage system. We hypothesize that this would result in hyperglycinemia and encephalopathy. This understanding may lead to novel prophylactic strategies for ifosfamide encephalopathy. Thus, the glycine deportation system plays multiple key roles in physiological and neurotoxicological processes involving glycine.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing, CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, pre-existing SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SC-like BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (AR(low)) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The AR(low) seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

The laulimalide family: total synthesis and biological evaluation of neolaulimalide, isolaulimalide, laulimalide and a nonnatural analogue

We herein describe in full detail the first total synthesis of the antitumor agents neolaulimalide and isolaulimalide as well as a highly efficient route to laulimalide. A Kulinkovich reaction followed by a cyclopropyl-allyl rearrangement is used to install the exo-methylene group. The C(2)-C(16) aldehyde fragment is coupled with the C(17)-C(28) sulfone fragments by a highly (E)-selective Julia-Lythgoe-Kocienski olefination to deliver the key intermediates of all three syntheses. Various conditions for the Yamaguchi macrolactonization are applied to close the individual macrocycles. Finally a carefully elaborated endgame was developed to solve the problem of acyl migration in the case of neolaulimalide. All compounds were tested against several cell lines. The cytotoxicity of neolaulimalide could be confirmed for the first time since its original isolation and it could be shown that it induces tubulin polymerization as efficiently as laulimalide.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

Synthesis and Biological Activity of 7,8,9-Trideoxy- and 7 R DesTHP-Peloruside A

The stereoselective syntheses of 7,8,9-trideoxypeloruside A (4) and a monocyclic peloruside A analogue lacking the entire tetrahydropyran moiety (3) are described. The syntheses proceeded through the PMB-ether of an ω-hydroxy β-keto aldehyde as a common intermediate which was elaborated into a pair of diastereomeric 1,3-syn and -anti diols by stereoselective Duthaler–Hafner allylations and subsequent 1,3-syn or anti reduction. One of these isomers was further converted into a tetrahydropyran derivative in a high-yielding Prins reaction, to provide the precursor for bicyclic analogue 4. Downstream steps for both syntheses included the substrate-controlled addition of a vinyl lithium intermediate to an aldehyde, thus connecting the peloruside side chain to C15 (C13) of the macrocyclic core structure in a fully stereoselective fashion. In the case of monocyclic 3 macrocyclization was based on ring-closing olefin metathesis (RCM), while bicyclic 4 was cyclized through Yamaguchi-type macrolactonization. The macrolactonization step was surprisingly difficult and was accompanied by extensive cyclic dimer formation. Peloruside A analogues 3 and 4 inhibited the proliferation of human cancer cell lines in vitro with micromolar and sub-micromolar IC50 values, respectively. The higher potency of 4 highlights the importance of the bicyclic core structure of peloruside A for nM biological activity.

Characterization and Clinical Relevance of ALDHbright Populations in Prostate Cancer

PURPOSE High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. RESULTS ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

Pharmacogenetics-based population pharmacokinetic analysis of etravirine in HIV-1 infected individuals

Objectives: Etravirine (ETV) is metabolized by cytochrome P450 (CYP) 3A, 2C9, and 2C19. Metabolites are glucuronidated by uridine diphosphate glucuronosyltransferases (UGT). To identify the potential impact of genetic and non-genetic factors involved in ETV metabolism, we carried out a two-step pharmacogenetics-based population pharmacokinetic study in HIV-1 infected individuals. Materials and methods: The study population included 144 individuals contributing 289 ETV plasma concentrations and four individuals contributing 23 ETV plasma concentrations collected in a rich sampling design. Genetic variants [n=125 single-nucleotide polymorphisms (SNPs)] in 34 genes with a predicted role in ETV metabolism were selected. A first step population pharmacokinetic model included non-genetic and known genetic factors (seven SNPs in CYP2C, one SNP in CYP3A5) as covariates. Post-hoc individual ETV clearance (CL) was used in a second (discovery) step, in which the effect of the remaining 98 SNPs in CYP3A, P450 cytochrome oxidoreductase (POR), nuclear receptor genes, and UGTs was investigated. Results: A one-compartment model with zero-order absorption best characterized ETV pharmacokinetics. The average ETV CL was 41 (l/h) (CV 51.1%), the volume of distribution was 1325 l, and the mean absorption time was 1.2 h. The administration of darunavir/ritonavir or tenofovir was the only non-genetic covariate influencing ETV CL significantly, resulting in a 40% [95% confidence interval (CI): 13–69%] and a 42% (95% CI: 17–68%) increase in ETV CL, respectively. Carriers of rs4244285 (CYP2C19*2) had 23% (8–38%) lower ETV CL. Co-administered antiretroviral agents and genetic factors explained 16% of the variance in ETV concentrations. None of the SNPs in the discovery step influenced ETV CL. Conclusion: ETV concentrations are highly variable, and co-administered antiretroviral agents and genetic factors explained only a modest part of the interindividual variability in ETV elimination. Opposing effects of interacting drugs effectively abrogate genetic influences on ETV CL, and vice-versa.

The Pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.