Janice R. Sufrin

S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei.

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10-250 microM the Km for methionine was 20 microM, and this changed to 200 microM for the range 0.5-5.0 mM. In the range 10-250 microM the Km for ATP was 53 microM, and this changed to 1.75 mM for the range 0.5-5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 microM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-alpha-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration approximately 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.

Microwave synthesis and evaluation of phenacylhomoserine lactones as anticancer compounds that minimally activate quorum sensing pathways in Pseudomonas Aeruginosa

The bacterial quorum sensing (QS) signal molecule 3-oxo-dodecanoyl-L-homoserine lactone (OdDHL) is produced by the opportunistic pathogen Pseudomonas aeruginosa and controls expression of virulence factors associated with life threatening infections in immune compromised individuals. OdDHL has also demonstrated anticancer activity, yet its ability to enhance pathogenicity of P. aeruginosa compromises further consideration as a potential anticancer agent. In search of acylhomoserine lactones that selectively inhibit cancer cell growth, a library of phenacylhomoserine lactone analogues has been prepared by microwave synthesis and evaluated for cancer growth inhibition and quorum sensing activation. Comparative SAR analysis demonstrates that both anticancer and QS signaling systems require long acyl side chains with a 3-oxo substitution for maximum activity. Compound 12b, 3-oxo-12-phenyldodecanoyl-L-homoserine lactone, was identified as a lead compound with strong cancer growth inhibitory activity that minimizes activation of QS signaling pathways in a P. aeruginosa reporter assay.

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.

A Novel Function for the rTS gene

The rTS gene codes for a naturally occurring antisense RNA to thymidylate synthase (TS) mRNA and two proteins (rTSa and rTSβ). The role of the major protein product of rTS, rTβ has been linked to alterations in TS protein expression, but the precise function of rTSβ is unknown. In this report we demonstrate that increased expression of rTSβ is associated with the decrease in TS protein expression due to production of novel, diffusible signal molecules. These signal molecules are produced more abundantly when rTSβ amounts are elevated. This hypothesis is supported by the demonstration that the rTSβ-overproducing cell line H630-1 can down-regulate TS protein in other cells without direct cellular contact. These cells are shown to secrete significant amounts of lipophilic metabolites derived from methionine, in contrast to cells that do not overproduce rTSβ. In support of the hypothesis that rTSβ is essential for the generation of these compounds, we demonstrate that rTSβ can catalyze the transfer of the carboxyl carbon of methionine from S-adenosylmethionine to a lipophilic acceptor molecule in vitro. We propose rTS is involved in regulation of TS through a novel methionine-based signaling pathway.

Enhancement of 5-fluorouracil sensitivity by an rTS signaling mimic in H630 colon cancer cells.

The rTSbeta protein has been hypothesized to synthesize signaling molecules that can down-regulate thymidylate synthase. These molecules share biological and chemical properties with acyl-homoserine lactones (AHL), suggesting some AHLs might act as rTS signaling mimics and down-regulate thymidylate synthase. We have determined that the AHL, 3-oxododecanoyl homoserine lactone (3-oxo-C12-(L)-HSL) can down-regulate thymidylate synthase protein at 10 micromol/L and reduce H630 (human colorectal cancer) growth by 50% at 23 micromol/L (IC50) in cell culture. At its IC50 concentration, 3-oxo-C12-(L)-HSL reduces the apparent IC50 of 5-fluorouracil (5-FU) from 1 micromol/L to 80 nmol/L (12-fold) in a colony formation assay. 3-Oxo-C12-(L)-HSL enhances the activity of 5-fluorodeoxyuridine, tomudex, and taxol but not the activity of 5-fluorouridine, methotrexate or Adriamycin. The unexpected interaction with taxol probably results from effects of the AHL on tubulin expression. Differences in taxol sensitivity, tubulin, and cellular morphology between H630 and the thymidylate synthase and rTSbeta-overproducing, 5-FU-resistant H630-1 cell line as determined by colony formation assays, Western analysis of one-dimensional and two-dimensional gels, and photomicroscopy confirm that cytoskeletal changes are induced by the AHL or by rTS signaling. Isozyme differences in thymidylate synthase and rTSbeta also exist in the two cell lines. Phosphorylation of rTSbeta amino acid S121 is shown to occur and is decreased at least 10-fold in the drug-resistant cells. The data presented provide support for further investigations of rTS signaling mimics as enhancers to thymidylate synthase-directed chemotherapy, evidence that the phosphorylation state of rTSbeta may be a marker for 5-FU resistance and a previously unrealized relationship between rTS signaling and the cytoskeleton.

Marine-Derived Metabolites of S-Adenosylmethionine as Templates for New Anti-Infectives

S-Adenosylmethionine (AdoMet) is a key biochemical co-factor whose proximate metabolites include methylated macromolecules (e.g., nucleic acids, proteins, phospholipids), methylated small molecules (e.g., sterols, biogenic amines), polyamines (e.g., spermidine, spermine), ethylene, and N-acyl-homoserine lactones. Marine organisms produce numerous AdoMet metabolites whose novel structures can be regarded as lead compounds for anti-infective drug design.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth and induces encystment in Acanthamoeba castellanii.

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.

Differential kinetic properties of L-2-amino-4-methylthio-cis-but-3-enoic acid, a methionine analog inhibitor of S-adenosylmethionine synthetase

The L-methionine analog, L-2-amino-4-methylthio-cis-but-3-enoic acid (L-cisAMTB), was examined as a potential inhibitor of the enzyme, S-adenosylmethionine (AdoMet) synthetase. The rational design of L-cisAMTB was based on previously observed potent enzyme inhibitory activity for its closely related structural analog, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB). The kinetic behavior of L-cisAMTB was studied using AdoMet synthetase isozymes I and II fractionated from L1210 murine leukemia cells. L-cisAMTB, which was a competitive inhibitor with respect to L-methionine, gave apparent Ki values of 21 and 5.7 microM for isozymes I and II, respectively. These values indicate that L-cisAMTB was slightly less inhibitory than L-cisAMB. L-cisAMTB was also a substrate for the AdoMet synthetase reaction, with respective Km values of 555 and 33 microM for isozymes I and II. In the absence of added inhibitors, the activity of isozyme II, but not isozyme I, was stimulated 2.5-fold by the presence of 10% DMSO. This preferential stimulation of isozyme II and the highly significant difference in Km values of L-cisAMTB for isozymes I and II point to possible physical differences in these tumor isozymes that were not apparent in earlier studies.

Improved synthetic access to the β,γ-enol ether amino acids, L-2-amino-4-methoxy-trans-but-3-enoic acid and l-2-amino-4-methoxy-cis-but-3-enoic acid

Abstract A synthetic route to L-2-amino-4-methoxy- trans -but-3-enoic acid, exclusive of the cis - isomer, has been developed. The key step is direct formation of a trans -enol ether derivative from the corresponding dimethylacetal, by refluxing in CCl4, in the presence of hexamethyldisilazane and Me3SiI. The isomeric L- cis amino acid could be accessed from this route by isomerizing the intermediate, methyl DL-2-acetamido-2-amino-4-methoxy- trans -but-3-enoate at 217° C to give cis - trans mixture in a 1:10 ratio.

Transient knockdown and overexpression reveal a developmental role for the zebrafish enosf1b gene

BackgroundDespite detailed in vivo knowledge of glycolytic enolases and many bacterial non-enolase members of the superfamily, little is known about the in vivo function of vertebrate non-enolase enolase superfamily members (ENOSF1s). Results of previous studies suggest involvement of the β splice form of ENOSF1 in breast and colon cancers. This study used the zebrafish (Danio rerio) as a vertebrate model of ENOSF1β function.ResultsWhole mount in situ hybridization (WISH) showed that zebrafish ENOSF1β (enosf1b) is zygotic and expressed ubiquitously through the first 24 hours post fertilization (hpf). After 24 hpf, enosf1b expression is restricted to the notochord. Embryos injected with enosf1b-EGFP mRNA grew slower than EGFP mRNA-injected embryos but caught up to the EGFP-injected embryos by 48 hpf. Embryos injected with ATG or exon 10 enosf1b mRNA-targeting morpholinos had kinked notochords, shortened anterior-posterior axes, and circulatory edema. WISH for ntl or pax2a expression showed that embryos injected with either morpholino have deformed notochord and pronephros. TUNEL staining revealed increased apoptosis in the peri-notochord region.ConclusionsThis study is the first report of ENOSF1 function in a vertebrate and shows that ENOSF1 is required for embryonic development. Increased apoptosis following enosf1b knockdown suggests a potential survival advantage for increased ENOSF1β expression in human cancers.