Antisense derivatives of U7 and other small nuclear RNAs as tools to modify pre-mRNA splicing patterns

The importance of alternative splicing for the diversity of the proteome and the large number of genetic diseases that are due to splicing defects call for methods to modulate alternative splicing decisions. Although splicing can be modulated by antisense oligonucleotides, this approach is confronted with problems of efficient delivery and the need for repeated administrations of large amounts of the oligonucleotides. Therefore we have developed methods allowing us to modulate splicing with the help of modified derivatives of the U7 small nuclear RNA involved in histone RNA 3' end processing. Its nuclear accumulation as a stable ribonucleoprotein particle makes U7 snRNA especially useful for this purpose. In particular, U7 derivatives containing two tandem antisense sequences directed against targets upstream and downstream of an exon can induce the efficient and specific skipping of that exon. U7 expression cassettes have been successfully introduced into a great number of cell lines, primary cells or tissues with the help of lentiviral and adeno-associated viral vectors. Examples of these therapeutic strategies in the fields of β-thalassemia, Duchenne muscular dytrophy and HIV/AIDS are discussed.

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.

Synthesis and SAR of C12-C13-oxazoline derivatives of epothilone A

The SAR of a series of new epothilone A derivatives with a 2-substituted-1,3-oxazoline moiety trans-fused to the C12-C13 bond of the deoxy macrocycle have been investigated with regard to tubulin polymerization induction and cancer cell growth inhibition. Significant differences in antiproliferative activity were observed between different analogs, depending on the nature of the substituent at the 2-position of the oxazoline ring. The most potent compounds showed comparable activity with the natural product epothilone A. Modeling studies provide a preliminary rationale for the observed SAR.

Novel oxazolo-oxazole derivatives of FTY720 reduce endothelial cell permeability, immune cell chemotaxis and symptoms of experimental autoimmune encephalomyelitis in mice

The immunomodulatory FTY720 (fingolimod) is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that acts by modulating sphingosine 1-phosphate (S1P) receptor signaling. In this study, we have developed and characterized two novel oxazolo-oxazole derivatives of FTY720, ST-968 and the oxy analog ST-1071, which require no preceding activating phosphorylation, and proved to be active in intact cells and triggered S1P1 and S1P3, but not S1P2, receptor internalization as a result of receptor activation. Functionally, ST-968 and ST-1071 acted similar to FTY720 to abrogate S1P-triggered chemotaxis of mouse splenocytes, mouse T cells and human U937 cells, and reduced TNFa- and LPS-stimulated endothelial cell permeability. The compounds also reduced TNFα-induced ICAM-1 and VCAM-1 mRNA expression, but restored TNFα-mediated downregulation of PECAM-1 mRNA expression. In an in vivo setting, the application of ST-968 or ST-1071 to mice resulted in a reduction of blood lymphocytes and significantly reduced the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice comparable to FTY720 either by prophylactic or therapeutic treatment. In parallel to the reduced clinical symptoms, infiltration of immune cells in the brain was strongly reduced, and in isolated tissues of brain and spinal cord, the mRNA and protein expressions of ICAM-1 and VCAM-1, as well as of matrix metalloproteinase-9 were reduced by all compounds, whereas PECAM-1 and tissue inhibitor of metalloproteinase TIMP-1 were upregulated. In summary, the data suggest that these novel butterfly derivatives of FTY720 could have considerable implication for future therapies of multiple sclerosis and other autoimmune diseases.

FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P3 and phosphoinositide 3-kinase

Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.

Going live: a comparative analysis of the suitability of the RFP derivatives RedStar, mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.

Concise Synthesis of Pyrrolidine and Indolizidine Alkaloids by a Highly Convergent Three-Component Reaction

The synthesis of pyrrolidine and indolizidine derivatives through radical carboazidation of alkenes with alpha-iodoketones, followed by reductive amination, is described. When properly substituted, further lactamization afforded pyrrolizidinones in good yield. This carboazidation/reductive amination sequence was efficiently applied to the total synthesis of three different simple alkaloids, including (+/-)-monomorine I.

Toxicity of clopidogrel and ticlopidine on human myeloid progenitor cells: importance of metabolites

Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 μM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 μM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 μM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies.

In vitro metacestodicidal activities of genistein and other isoflavones against Echinococcus multilocularis and Echinococcus granulosus

Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.

In vitro efficacy of nitro- and bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoitia besnoiti infection in Vero cells

Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattle.