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.

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.

A new class of homoserine lactone quorum-sensing signals.

Quorum sensing is a term used to describe cell-to-cell communication that allows cell-density-dependent gene expression. Many bacteria use acyl-homoserine lactone (acyl-HSL) synthases to generate fatty acyl-HSL quorum-sensing signals, which function with signal receptors to control expression of specific genes. The fatty acyl group is derived from fatty acid biosynthesis and provides signal specificity, but the variety of signals is limited. Here we show that the photosynthetic bacterium Rhodopseudomonas palustris uses an acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid rather than fatty acids from cellular pools. The bacterium has a signal receptor with homology to fatty acyl-HSL receptors that responds to p-coumaroyl-HSL to regulate global gene expression. We also found that p-coumaroyl-HSL is made by other bacteria including Bradyrhizobium sp. and Silicibacter pomeroyi. This discovery extends the range of possibilities for acyl-HSL quorum sensing and raises fundamental questions about quorum sensing within the context of environmental signalling.

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases.

5'-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5'-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5'-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 A resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5'-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5'-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK(a) of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.

Novel trypanocidal analogs of 5'-(methylthio)-adenosine.

The purine nucleoside 5'-deoxy-5'-(hydroxyethylthio)-adenosine (HETA) is an analog of the polyamine pathway metabolite 5'-deoxy-5'-(methylthio)-adenosine (MTA). HETA is a lead structure for the ongoing development of selectively targeted trypanocidal agents. Thirteen novel HETA analogs were synthesized and examined for their in vitro trypanocidal activities against bloodstream forms of Trypanosoma brucei brucei LAB 110 EATRO and at least one drug-resistant Trypanosoma brucei rhodesiense clinical isolate. New compounds were also assessed in a cell-free assay for their activities as substrates of trypanosome MTA phosphorylase. The most potent analog in this group was 5'-deoxy-5'-(hydroxyethylthio)-tubercidin, whose in vitro cytotoxicity (50% inhibitory concentration [IC50], 10 nM) is 45 times greater than that of HETA (IC50, 450 nM) against pentamidine-resistant clinical isolate KETRI 269. Structure-activity analyses indicate that the enzymatic cleavage of HETA analogs by trypanosome MTA phosphorylase is not an absolute requirement for trypanocidal activity. This suggests that additional biochemical mechanisms are associated with the trypanocidal effects of HETA and its analogs.

An improved synthesis of psammaplin A.

The marine natural product, psammaplin A, was first isolated from the Psammaplinaplysilla sponge in 1987. Since that time, psammaplin A has shown a wide spectrum of biological activities that include enzyme inhibitory activities resulting in antibacterial and antitumor effects. An improved synthesis of psammaplin A has been developed, making the compound more easily accessible for further biological evaluations. In this context, we find that psammaplin A is an effective DNA methyltransferase inhibitor in vitro but fails to alter genomic DNA methylation levels in treated human cancer cells.

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.

Improved synthesis of beta-D-6-methylpurine riboside and antitumor effects of the beta-D- and alpha-D-anomers.

6-Methylpurine-beta-D-riboside (beta-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the beta-D-anomer exclusively. The in vitro antitumor effects of beta-D-MPR and 6-methyl-purine-alpha-D-riboside (alpha-D-MPR) in five human tumor cell lines showed that beta-D-MPR was highly active (IC(50) values ranging from 6 to 34 nM). alpha-D-MPR, although less active than beta-D-MPR, also exhibited significant antitumor effects (IC50 values ranging from 1.47 to 4.83 microM).

Structural comparison of MTA phosphorylase and MTA/AdoHcy nucleosidase explains substrate preferences and identifies regions exploitable for inhibitor design.

The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. 5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5'-methylthiotubercidin have been solved to 2.0 and 2.1 A resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5'-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5'-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.

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 (rTSalpha and rTSbeta). The role of the major protein product of rTS, rTSbeta has been linked to alterations in TS protein expression, but the precise function of rTSbeta is unknown. In this report we demonstrate that increased expression of rTSbeta 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 rTSbeta amounts are elevated. This hypothesis is supported by the demonstration that the rTSbeta-overproducing cell line H630-1 can downregulate 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 rTSbeta. In support of the hypothesis that rTSbeta is essential for the generation of these compounds, we demonstrate that rTSbeta 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.

Synthesis and evaluation of analogues of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine as inhibitors of tumor cell growth, trypanosomal growth, and HIV-1 infectivity.

A well-defined series of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine analogues was designed and synthesized in order to further ascertain the optimal structural requirements for S-adenosylmethionine decarboxylase inhibition and potentially to augment and perhaps separate their antiproliferative and antitrypanosomal activities. Most structural modifications had a deleterious affect on both the antitrypanosomal and antineoplastic activity of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine. However, di-O-acetylation of the parent compound produced a potential prodrug that caused markedly pronounced inhibition of trypanosomal and neoplastic cell growth and viability. Moreover, the acetylated derivative of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine did inhibit HIV-1 growth and infectivity, whereas the parent compound did not.