Potent Cytotoxin Research Articles

Total Synthesis and Biological Evaluation of Pederin, Psymberin, and Highly Potent Analogs

The potent cytotoxins pederin and psymberin have been prepared through concise synthetic routes (10 and 14 steps in the longest linear sequences, respectively) that proceed via a late-stage multicomponent approach to construct the N-acyl aminal linkages. This route allowed for the facile preparation of a number of analogs that were designed to explore the importance of the alkoxy group in the N-acyl aminal and functional groups in the two major subunits on biological activity. These analogs, including a pederin/psymberin chimera, were analyzed for their growth inhibitory effects, revealing several new potent cytotoxins and leading to postulates regarding the molecular conformational and hydrogen bonding patterns that are required for biological activity. Second generation analogs have been prepared based on the results of the initial assays and a structure-based model for the binding of these compounds to the ribosome. The growth inhibitory properties of these compounds are reported. These studies show the profound role that organic chemistry in general and specifically late-stage multicomponent reactions can play in the development of unique and potent effectors for biological responses.

Cylindrospermopsin production and release by the potentially invasive cyanobacterium Aphanizomenon ovalisporum under temperature and light gradients

The growth rates, production and release of the potent cytotoxin cylindrospermopsin (CYN) were studied in batch and semi-continuous cultures of Aphanizomenon ovalisporum (Cyanobacteria; Nostocaceae) strains UAM 289 and UAM 290 from Spain, over a gradient of temperatures (10–40 °C) and irradiances (15–340 μE m −2 s −1). This species grew in temperatures ranging from 15 °C to 35 °C as well as under all irradiances assayed. The growth rates ranged from 0.08 d −1 to 0.35 d −1, and the maximum growth was recorded above 30 °C and at 60 μE m −2 s −1. CYN was produced under all conditions where net growth occurred. Total CYN reached up to 6.4 μg mg −1 dry weight, 2.4 μg mm −3 biovolume, 190.6 fg cell −1 and 0.5 μg μg −1 chlorophyll a. Although CYN concentrations varied only 1.9-fold within the 15–30 °C range, a drastic 25-fold decrease was observed at 35 °C. The irradiance induced up to 4-fold variations, with maximum total CYN measured at 60 μE m −2 s −1. An elevated extracellular CYN share ranging from 20% to 35% was observed during the exponential growth phase in most experiments, with extreme temperatures (15 and 35 °C) being related to the highest release (63% and 58%, respectively) and without remarkable influence of irradiance. Growth did not have a direct influence on either CYN production or release throughout the entire range of experimental conditions. Our study demonstrates a strong and stable production and release of CYN by A. ovalisporum along field-realistic gradients of temperature and light, thus becoming a predictive tool useful for the management of water bodies potentially affected by this ecologically plastic cyanobacterium.

Nitrogen Balance in the Ecosystem of the Cystic Fibrosis Lung

In this issue of the Journal, Grasemann and coworkers (pp. 1363–1368) report that levels of asymmetric dimethyl arginine (ADMA) are increased in cystic fibrosis (CF) airways (1). ADMA inhibits cellular arginine uptake and nitric oxide (NO) synthase (NOS) activity. Levels of ADMA decrease during antibiotic therapy in association with improved lung function. This observation may prove to have therapeutic relevance. However, it is important to note that increased ADMA in CF airways may be both beneficial (through inhibition of NO production) and harmful (through inhibition of S-nitrosothiol production). Nitric oxide in the concentrations measured in exhaled air (parts per billion) is generally irrelevant to normal lung physiology (2). However, NO can be relevant to lung pathology. It interacts with oxygen, superoxide, and other molecules to injure airway epithelium. Products of these reactions, such as nitrous acid and peroxynitrous acid, are potent cytotoxins downstream of inducible NOS activity (3). Nitric oxide can also affect bacteria in the CF airway (4, 5). Indeed, the CF airway is a complex ecosystem in which nitrogen oxides, oxygen, protons, and more complex chemical species are exchanged between prokaryotic and eukaryotic cells (Figure 1; 4–7). For example, levels of NO are lower than normal in the CF airway, in part because NO is consumed by Pseudomonas, Aspergillus, and other denitrifying organisms (4). NO can serve both as an electron acceptor (dissimilatory denitrification, ultimately forming ammonia) and as a precursor for amino acid formation (assimilatory denitrification). Airway NO levels rise and NH3 levels fall with antibiotic therapy in CF (4). Levels of oxidized NO in the form of nitrate are high in the CF airway (6); and nitrate, like NO, can feed denitrifying organisms. Together, the effects of NOS products to cause cytotoxicity and promote prokaryotic growth suggest that the high levels of ADMA should be advantageous for patients with CF, and that a decrease in ADMA levels with antibiotic therapy might be disadvantageous. Figure 1. Cystic fibrosis airway nitrogen redox ecology. Nitric oxide (NO) synthase (NOS) isoforms in the cystic fibrosis (CF) lung produce both NO and S-nitrosothiols. NO is oxidized to nitrite (NO2−) and nitrate (NO3−). In this issue of the Journal ... However, NOS also produces S-nitrosothiols in concentrations two log orders higher than NO (8–10). S-nitrosothiols are antimicrobial. They augment ciliary beat frequency (2). They relax human airway smooth muscle and prevent tachyphylaxis to β2-adrenergic agonists (2, 10, 11). They inhibit amiloride-sensitive sodium transport (12). They augment expression, maturation and function of delF508 CF transmembrane conductance regulator through inhibition of the expression of Hsp70/Hsp90-organizing protein (13). S-nitrosothiol levels are decreased in the CF airway (14), consistent with high ADMA levels (1); indeed, S-nitrosothiol replacement therapy improves oxygenation in CF (9). Prevention of S-nitrosothiol formation is therefore likely to be an important disadvantage of having high levels of ADMA in the CF airway (1). We do not know why ADMA levels are high in CF. One possible mechanism is based on what we know about the CF airway ecosystem. Anti-pseudomonal therapy decreases ADMA levels in patients with CF (1). Biochemically, ADMA levels are decreased by dimethylarginine dimethylaminohydrolases (DDAHs). DDAHs are inhibited by S-nitrosylation, or physiological protein modification by NO (15). S-nitrosylation is driven both by NOS activation and—in relatively acidic conditions in the CF airway ecosystem (5)—by nitrite protonation; at baseline, this should increase ADMA levels. Antimicrobial therapy can decrease bacterial conversion of abundant airway nitrate to nitrite. Nitrite depletion would decrease DDAH S-nitrosylation, thereby increasing ADMA breakdown during the course of therapy. However, this proposed mechanism is speculative. Much work remains to be done. The CF airway is dark, damp, and largely anaerobic (7). It is also surprisingly well-suited to denitrifying prokaryotic species, given that NOS expression is decreased and ADMA levels are increased. To understand why—and to learn how to use nitrogen oxide redox ecology to therapeutic advantage—we need to get beyond simply modeling NO radical diffusion (2, 3). The Cystic Fibrosis Foundation has shown the benefit of promoting interactions among scientists across disciplines. The work of Grasemann and coworkers suggests that there might be much to gain by organizing a small working group to bring together nitrogen balance ecologists, biochemists, CF airway epithelial biologists, and mathematicians experienced in biochemical modeling. This group could model prokaryotic and eukaryotic outcomes of specific CF interventions to identify strategies to optimize therapeutic development in CF. The elegant insights of Grasemann and coworkers serve to alert us that both the benefits and toxicities of airway nitrogen oxides need to be better understood to improve clinical outcomes in CF.

A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation

Ricin is a potent plant cytotoxin composed of an A-chain [RTA (ricin A-chain)] connected by a disulfide bond to a cell binding lectin B-chain [RTB (ricin B-chain)]. After endocytic uptake, the toxin is transported retrogradely to the ER (endoplasmic reticulum) from where enzymatically active RTA is translocated to the cytosol. This transport is promoted by the EDEM1 (ER degradation-enhancing α-mannosidase I-like protein 1), which is also responsible for directing aberrant proteins for ERAD (ER-associated protein degradation). RTA contains a 12-residue hydrophobic C-terminal region that becomes exposed after reduction of ricin in the ER. This region, especially Pro250, plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro250 with alanine)] in the hydrophobic region of RTA to study the intracellular transport of the modified toxin. The introduced mutation alters the secondary structure of RTA into a more helical structure. Mutation P250A increases endosomal-lysosomal degradation of the toxin, as well as reducing its transport from the ER to the cytosol. Transport of modified RTA to the cytosol, in contrast to wild-type RTA, appears to be EDEM1-independent. Importantly, the interaction between EDEM1 and RTA(P250A) is reduced. This is the first reported evidence that EDEM1 protein recognition might be determined by the structure of the ERAD substrate.

Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

Synthesis and hydrolytic evaluation of acid-labile imine-linked cytotoxic isatin model systems

In this study a series of isatin-based, pH-sensitive aryl imine derivatives with differing aromatic substituents and substitution patterns were synthesised and their acid-catalysed hydrolysis evaluated. These derivatives were functionalised at the C3 carbonyl group of a potent N-substituted isatin cytotoxin and were stable at physiological pH but readily cleaved at pH 4.5. Observed rates of hydrolysis for the embedded imine-acid moiety were in the order para-phenylpropionic acid>phenylacetic acid (para>meta)>benzoic acid (meta>para). The ability to fine-tune hydrolysis rates in this way has potential implications for optimising imine linked, tumour targeting cytotoxin–protein conjugates.

Unique Dynamic Properties of DNA Duplexes Containing Interstrand Cross-Links

Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.

Mouse models of Escherichia coli O157:H7 infection and shiga toxin injection.

Escherichia coli O157:H7 has been responsible for multiple food- and waterborne outbreaks of diarrhea and/or hemorrhagic colitis (HC) worldwide. More importantly, a portion of E. coli O157:H7-infected individuals, particularly young children, develop a life-threatening sequela of infection called hemolytic uremic syndrome (HUS). Shiga toxin (Stx), a potent cytotoxin, is the major virulence factor linked to the presentation of both HC and HUS. Currently, treatment of E. coli O157:H7 and other Stx-producing E. coli (STEC) infections is limited to supportive care. To facilitate development of therapeutic strategies and vaccines for humans against these agents, animal models that mimic one or more aspect of STEC infection and disease are needed. In this paper, we focus on the characteristics of various mouse models that have been developed and that can be used to monitor STEC colonization, disease, pathology, or combinations of these features as well as the impact of Stx alone.

Crystal structure of RNase A tandem enzymes and their interaction with the cytosolic ribonuclease inhibitor

Because of their ability to degrade RNA, RNases are potent cytotoxins. The cytotoxic activity of most members of the RNase A superfamily, however, is abolished by the cytosolic ribonuclease inhibitor (RI). RNase A tandem enzymes, in which two RNase A molecules are artificially connected by a peptide linker, and thus have a pseudodimeric structure, exhibit remarkable cytotoxic activity. In vitro, however, these enzymes are still inhibited by RI. Here, we present the crystal structures of three tandem enzymes with the linker sequences GPPG, SGSGSG, and SGRSGRSG, which allowed us to analyze the mode of binding of RI to the RNase A tandem enzymes. Modeling studies with the crystal structures of the RI-RNase A complex and the SGRSGRSG-RNase A tandem enzyme as templates suggested a 1 : 1 binding stoichiometry for the RI-RNase A tandem enzyme complex, with binding of the RI molecule to the N-terminal RNase A entity. These results were experimentally verified by analytical ultracentrifugation, quantitative electrophoresis, and proteolysis studies with trypsin. As other dimeric RNases, which are comparably cytotoxic, either evade RI binding or potentially even bind two RI molecules, inactivation by RI cannot be the crucial limitation to the cytotoxicity of dimeric RNases.

Sequential cytotoxicity: A theory examined using a series of 3,5-bis(benzylidene)-1-diethylphosphono-4-oxopiperidines and related phosphonic acids

The concept of sequential cytotoxicity, which states that successive chemical attacks on cellular constituents can be more deleterious to neoplasms than normal cells, was evaluated using a series of 3,5-bis(benzylidene)-1-diethylphosphono-4-oxopiperidines 1 and related phosphonic acids 2, which were screened against a panel of malignant and normal cell lines. The compounds proved to be not only potent cytotoxins (71% of the CC50 figures are submicromolar) but to display greater cytotoxicity to the neoplastic cells. QSAR revealed that both cytotoxic potencies and selective toxicity were increased by a rise in the electron-withdrawing properties and a decrease in the hydrophobicity of the aryl substituents. Utilisation of the PL10 concept and evaluation of druglike properties revealed 1c as the lead tumour-specific cytotoxin. This molecule activated caspase-3 in HL-60 cells but not in the HSC-2 cell line. While 1c caused internucleosomal DNA fragmentation in HL-60 cells, it did not elicit this effect in either HSC-2 and HSC-4 cells. Clearly 1c exerts its cytotoxic potencies by different mechanisms and such pleiotropy is likely the principal reason for the remarkable display of preferential toxicity towards malignant cells of the compounds in series 1 and 2.

Structural modifications on the phenazine N, N′-dioxide-scaffold looking for new selective hypoxic cytotoxins

We have identified phenazine 5,10-dioxides as prodrugs for antitumour therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here, we investigated some structural modifications in order to find new selective hypoxic cytotoxins and to establish the structural requirements for adequate activity. Three different chemical-series were prepared and the clonogenic survival of V79 cells on aerobic and anaerobic conditions was determined. Electrochemical- and DNA-interaction studies were done for the most relevant derivatives. The new fluoro-derivative 7-fluoro-2-aminophenazine 5,10-dioxide displayed selective toxicity towards hypoxic V79 cells having adequate hypoxic cytotoxicity ratio (HCR = 6.8) and being the most potent hypoxic cytotoxins ( P = 2.5 μM) described for this family of bioreductive agents. The reduction potential of the N-oxide moiety in this new fluoro-derivative was in the range for adequate bioreduction property. According to the fluorescence studies, the DNA-interaction mechanism was especially operative in the phenazine drugs more than in the corresponding prodrugs, phenazine dioxides.

Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus α-hemolysin–mediated cellular injury

Staphylococcus aureus alpha-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, beta-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla-ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions.

Cathepsin H Is an Additional Convertase of Pro-granzyme B

The serine protease granzyme B (GrB) is the most potent proapoptotic cytotoxin of the granule exocytosis pathway of cytotoxic lymphocytes. GrB is synthesized as a zymogen (proGrB) and activated in cytotoxic granules by the lysosomal cysteine protease cathepsin C (CatC) which removes the N-terminal dipeptide Gly-Glu. It has been shown recently that mice lacking CatC nonetheless express significant residual GrB activity, indicating the presence of additional proGrB convertases. Here, we describe an assay to assess activation of proGrB and show that the amino-peptidase cathepsin H (CatH) has proGrB convertase activity in vitro, whereas dipeptidylpeptidase II does not. We generated mice lacking both CatC and CatH expression (CatCH(-/-)) and found that their lymphocytes have reduced convertase activity compared with those from CatC-deficient mice. Despite this, cytotoxic lymphocytes from CatCH(-/-) mice retain cytotoxic activity and some residual GrB activity. We conclude that CatH can act as an additional proGrB convertase and that other protease/s (apart from dipeptidylpeptidase II) must also possess convertase activity. This indicates a great deal of functional redundancy in GrB maturation, which would prevent pathogen-mediated immune suppression by via convertase inhibition.

Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

Abstract 5424: Combination of decitabine and the tumor-selective CYP450-activated compound ICT2700 produces a synergistic effect in HeLa cells

Abstract Over the past decade, much research has been conducted in assessing the role of epigenetics in cancer. Epigenetics refers to transmissible modifications in gene activity, which does not involve any alterations in the DNA sequence. DNA methylation occurs predominantly at CpG sites in the mammalian genome by the DNA methyltransferase (DNMT) enzymes. The interaction of methylated DNA with proteins that detect methylated DNA and other chromatin remodeling proteins render an altered chromatin configuration that prevents the expression of a gene. DNA methylation plays an important role in the expression of oncogenes, tumour suppressor genes and more recently it has also been shown that drug metabolising enzymes are affected by the DNA methylation process. Aberrant alterations in the methylation status of CpG dinucleotides, target sites for DNMT, have been shown in the promoter regions of extrahepatic cytochromes P450 including CYP1A1, CYP1B1 and CYP2W1. We are investigating extra-hepatic CYPs (CYP1A1, 1B1, 2J2, 2S1, 2W1, 4Z1 and 4F11) as targets for tumor-selective prodrug activation. Tumors expressing high levels of active CYP450s could induce their own demise via the conversion of otherwise inert agents into potent cytotoxins. We have identified the aryl-ring-fused chloromethylindolines as a class of agent that possess a pharmacophore capable of undergoing oxidation by CYPs. We have previously shown ICT2700 to be activated by CYP1A1 in vitro and in vivo. Here we present data that suggest that epigenetic silencing of CYP1A1 affect the cytotoxicity of ICT2700. We have pre-treated MCF-7 and HeLa cell lines with either the demethylating agent decitabine (either 200 nM, 1 or 2 µM, on day 1 or day 1 + 2) or the HDAC inhibitor trichostatin A (100 nM, after day 1) and subsequently exposed the cell lines to ICT2700 (concentration range: 1nM - 200 µM, on days 3, 6 or 9). A higher level of cytotoxic kill by the prodrug ICT2700 in the HeLa cells but not in the MCF-7 cells were observed in combination with decitabine. Combination with trichostatin A had no apparent synergistic effect. In all experiments, a higher level of CYP1A1 mRNA was observed after treatment with decitabine and trichostatin A. Regardless of the pharmacoepigenetic status, ICT2700 exhibited low nM IC50 in the MCF-7 but not in the HeLa cell line, indicating that low levels of CYPs in the former cell line are able to convert the prodrug into a potent cytotoxin. In conclusion, we have demonstrated that the conversion of the prodrug ICT2700 to some extent is affected by epigenetic silencing and that combination with decitabine can sensitise cells prior to treatment with ICT2700. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5424.

Abstract 3715: Potent cytotoxins and their prodrugs

Abstract Epothilones, dolastatins (auristatins), tubulysins, duocarmycins, and enediynes are among the most potent anticancer agents, which inhibit cell growth of cancer cells with a low or subnanomolar activity. Epothilones inhibit tumor growth by stabilizing the microtubules in a manner similar to Taxol. There are hundreds of natural and synthetic epothilones known, and some of them are undergoing clinical trials. One semisynthetic analog, ixabepilone, has also been approved for the treatment of metastatic breat cancer. Similarly, there are numerous dolastatins, their synthetic analogs - auristatins, and tubulysins known, of which auristatins are currently being developed by Seattle Genetics, Inc. These compounds also inhibit microtubules, however unlike epothilones and Taxol, they function by destabilizing the microtubules. The other compounds, duocarmycins and enediynes, cause toxicity by interacting with DNA; duocarmycins alkylate DNA whereas enediynes cleave. At least one enediyne antibody conjugate, Mylotarg (or Gemtuzumab ozogamycin), prepared from calicheamicin and targeted to CD33, is an approved therapy for the treatment of acute myeloid leukemia. Not surprisingly, most of these naturally occurring compounds or their synthetic analogs also cause nonselective toxicities to the rapidly dividing normal cells, including those from hair follicles, bone marrow, and gastrointestinal lining. To minimize such indiscriminate toxicity, these compounds can be conjugated to tumor-targeting antibodies, as the case is with auristatins and calicheamicin. While we are developing antibody conjugates, we have also been focusing on a prodrug approach, in that the prodrugs can be activated selectively in tumor and tumor microenvironments by the endogenous tumor-associated proteases. Alternatively, an exogenous enzyme, such as aldolase antibody, 38C2, that are directed using low molecular weight inhibitors of the cell surface receptors overexpressed in tumor cells and tumor microenvironments, can also catalyze the prodrug activation. In this presentation, we will be discussing our studies toward the synthesis and evaluation of many of these cytotoxins and/or their prodrugs, especially those belonging to the auristatins. The prodrugs are targeting to integrins and/or proteases, and activated by the aldolase antibody or the tumor-associated proteases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3715.

Total synthesis of neopeltolide and analogs

Neopeltolide, a potent cytotoxin from a Carribean sponge, was synthesized through a brief sequence that highlights the use of ethers as oxocarbenium ion precursors. Other key steps include an acid-mediated etherification and sequence that features a Sonogashira reaction, an intramolecular alkyne hydrosilylation reaction, and a Tamao oxidation. The alkene that is required for the oxidative cyclization can be hydrogenated to provide access to the natural product or an epimer, or can be epoxidized or dihydroxylated to form polar analogs.

Expression of a truncated form of yeast ribosomal protein L3 in transgenic wheat improves resistance to Fusarium head blight

Fusarium head blight (FHB) is a disease that causes major economic losses in wheat and barley production worldwide. Contamination of food with the trichothecene mycotoxin deoxynivalenol (DON) produced by Fusarium is a major health concern for humans and animals because trichothecenes are potent cytotoxins of eukaryotic cells. Trichothecene mycotoxins inhibit translation by targeting ribosomal protein L3 at the peptidyltransferase center. We previously showed that expression of an N-terminal fragment of yeast L3 (L3Δ) in transgenic tobacco plants reduced the toxicity of DON. Here, we produced transgenic wheat plants that express the same yeast L3 (L3Δ) fragment and evaluated their susceptibility to Fusarium graminearum infection and their ability to accumulate DON. Following F. graminearum infection in greenhouse tests, two transgenic wheat lines expressing the highest levels of L3Δ showed reductions in disease severity and kernel DON levels, compared to non-transformed plants. In a field test, a transgenic wheat line with the highest L3Δ expression controlled by the maize Ubi1 promoter had significant reductions in visually scabby kernels and kernel DON levels. These results demonstrate that expression of a modified form of the ribosomal protein that is the target of DON can improve FHB resistance in wheat.

3,5‐Bis(benzylidene)‐4‐oxo‐1‐phosphonopiperidines and Related Diethyl Esters: Potent Cytotoxins with Multi‐Drug‐Resistance Reverting Properties

A series of 3,5-bis(benzylidene)-4-piperidones 3 were converted into the corresponding 3,5-bis(benzylidene)-1-phosphono-4-piperidones 5 via diethyl esters 4. The analogues in series 4 and 5 displayed marked growth inhibitory properties toward human Molt 4/C8 and CEM T-lymphocytes as well as murine leukemia L1210 cells. In general, the N-phosphono compounds 5, which are more hydrophilic than the analogues in series 3 and 4, were the most potent cluster of cytotoxins, and, in particular, 3,5-bis-(2-nitrobenzylidene)-1-phosphono-4-piperidone 5 g had an average IC(50) value of 34 nM toward the two T-lymphocyte cell lines. Four of the compounds displayed potent cytotoxicity toward a panel of nearly 60 human tumor cell lines, and nanomolar IC(50) values were observed in a number of cases. The mode of action of 5 g includes the induction of apoptosis and inhibition of cellular respiration. Most of the members of series 4 as well as several analogues in series 5 are potent multi-drug resistance (MDR) reverting compounds. Various correlations were noted between certain molecular features of series 4 and 5 and cytotoxic properties, affording some guidelines in expanding this study.

Cytolysins, Superantigens, and Pneumonia Due to Community‐Associated Methicillin‐ResistantStaphylococcus aureus

Cytolysins, Superantigens, and Pneumonia Due to Community‐Associated Methicillin‐Resistant<i>Staphylococcus aureus</i>