Vasiliki Sarli

Targeting the Kinesin Spindle Protein: Basic Principles and Clinical Implications

Kinesin spindle protein (KSP), a member of the kinesin superfamily of microtubule-based motors, plays a critical role in mitosis as it mediates centrosome separation and bipolar spindle assembly and maintenance. Inhibition of KSP function leads to cell cycle arrest at mitosis with the formation of monoastral microtubule arrays, and ultimately, to cell death. Several KSP inhibitors are currently being studied in clinical trials and provide new opportunities for the development of novel anticancer therapeutics alternative from the available microtubule targeting drugs.

Synthesis of cyclopamine using a biomimetic and diastereoselective approach.

 Isolated from corn lily Veratrum californicum.  Ingestion of the corn lily (Veratrum californicum) by pregnant sheep caused the birth of lambs with cyclopia. The Veratrum alkaloid cyclopamine was identified as the causative agent. It is very expensive commercially (~

Inhibition of the Mitotic Kinesin Eg5 Up-regulates Hsp70 through the Phosphatidylinositol 3-Kinase/Akt Pathway in Multiple Myeloma Cells

10/mg), No total synthesis is available in literature after 50 year of its isolation. Inhibition of Hh signaling provides a routeto novel anticancer therapies. Aberrant activation of the Hh pathway  leads to malignancies in number of tissues  play role in other forms of normal and maladaptive growth  Leads to prostate, pancreatic and breast cancers The Veratrum alkaloid cyclopamine was the first known inhibitor of Hh signaling Structural Features  C-nor-D-homo steroid substructure.  A highly substituted furan ring, which is attached through a spiro linkage to the D ring.  A piperidine unit having a basic secondary nitrogen atom. Cyclopamine is quite acid sensitive: at pH<3 or upon treatment of 1 with Lewis acids the furan ring is cleaved and the D ring aromatizes to form the toxic veratramine.

Inhibitors of kinesin Eg5: antiproliferative activity of monastrol analogues against human glioblastoma cells.

The microtubule-dependent motor protein Eg5 plays a critical role in spindle assembly and maintenance in mitosis. Herein we show that the suppression of Eg5 by a specific inhibitor arrested mitosis, induced apoptosis, and up-regulated Hsp70 in human multiple myeloma cells. Mechanistically, Hsp70 induction occurred at the transcriptional level via a cis-regulatory DNA element in Hsp70 promoter and was mediated by the phosphatidylinositol 3-kinase/Akt pathway. Eg5 inhibitor-mediated Hsp70 up-regulation is cytoprotective because blocking Hsp70 induction directly by antisense or small interfering RNA or indirectly by inhibiting the phosphatidylinositol 3-kinase/Akt pathway significantly increased Eg5 inhibitor-induced apoptosis. Furthermore, a farnesyltransferase inhibitor interacted synergistically with the Eg5 inhibitor in inducing apoptosis through disrupting the Akt/Hsp70 signaling axis. These findings provide the first evidence for Eg5 inhibitor activity in hematologic malignancy and identify Hsp70 up-regulation as a critical mechanism responsible for modulating myeloma cell sensitivity to Eg5 inhibitors. In addition, these findings suggest that a combination of Eg5 inhibitors with agents abrogating Hsp70 induction would be useful for myeloma therapy in the clinic.

Synthesis and Biological Evaluation of Novel Eg5 Inhibitors

The inhibition of kinesin Eg5 by small molecules such as monastrol is currently evaluated as an approach to develop a novel class of antiproliferative drugs for the treatment of malignant tumours. Therefore, we studied the effects of the new monastrol analogues enastron, dimethylenastron and vasastrol VS-83 on the proliferation of human glioblastoma cells in the kinetic crystal violet assay. Compared to monastrol, the new cell cycle specific compounds showed an at least one order of magnitude higher anti proliferative activity against U-87 MG, U-118 MG, and U-373 MG glioblastoma cells. The compounds were neither inactivated by hydrolysis nor by binding to serum proteins. Moreover, we demonstrated the characteristic monoaster formation after incubation of cells with the new compounds by confocal laser scanning microscopy. We also showed that the arrangement of β-actin and tubulin, vital components of the cyto-skeleton of mitotic and quiescent cells, were not affected by the new compounds. Due to the necessity of overcoming the blood–brain barrier in the treatment of brain tumours, we investigated if the new monastrol analogues are modulators or substrates of the p-glycoprotein (p-gp) 170 by a flow cytometric calcein-AM efflux assay. The tested compounds showed no modulating effects on the p-gp function. With respect to the treatment of primary and secondary CNS tumours, the results of our experiments suggest that the new monastrol analogues represent an interesting class of potential anticancer drugs, predicted to be less neurotoxic in comparison to classical tubulin inhibitors.

Inhibitors of Mitotic Kinesins: Next-Generation Antimitotics

Human Eg5 is a mitotic kinesin that is essential for bipolar spindle formation and maintenance during mitosis. Recently, the discovery of compounds that inhibit Eg5 and cause mitotic arrest has attracted great interest, due to their potential use as the next generation of antimitotics. Here, we present the synthesis and biological investigation of 3,4‐dihydrophenylquinazoline‐2(1H)‐thiones as selective and potent Eg5 inhibitors.

Synthesis and biological evaluation of SANT-2 and analogues as inhibitors of the hedgehog signaling pathway.

A large number of antimitotics have been proven so far as valuable and effective anticancer agents in clinical oncology. For example, the vinca alkaloids vincristine and vinblastine are used for the treatment of leukemia and Hodgkin’s lymphoma, whereas paclitaxel and docetaxel are approved for the treatment of metastatic breast cancer, as well as for lung and ovarian carcinomas. These drugs inhibit mitosis by targeting the spindle microtubules, which are responsible for cell proliferation and the faithful partitioning of genetic material. However, despite their success, these antimitotics show several serious side effects such as neurotoxicity. These side effects are related to the broad role of tubulin (the fundamental buildingblock protein of microtubules) in important cellular processes such as the maintenance of organelles and cell shape, cell motility, synaptic vesicles, and intracellular transport phenomena. A further limitation to the application of antimitotics is the development of resistance. Resistance mechanisms involve the multidrug-resistant phenotype mediated by the P-glycoprotein efflux pump, tubulin mutations, alterations in the expression of tubulin isotypes, and microtubule-associated proteins. 7] Lately, the revolution in molecular biology combined with advances in high-throughput screening and automated microscopy and imaging has yielded a better understanding of cell division, and has helped in the identification of new and more selective anticancer targets, including members of several kinase families such as Polo, Aurora, and mitotic kinesins. These proteins are exclusively involved in the formation and function of the mitotic spindle and are key regulators for mitotic entry, progression, and cytokinesis.

Synthetic and Biological Studies of Phaeosphaerides

Hedgehog (Hh) signaling plays an important role in cell signaling of embryonic development and adult tissue homeostasis. In vertebrates, the hh gene encodes three different unique proteins: sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh). Disruption of the Hh signaling pathway leads to severe disorders in the development of vertebrates whereas aberrant activation of the Hh pathway has been associated with several malignancies including Gorlin syndrome (a disorder predisposing to basal cell carcinoma, medulloblastoma and rhabdomyosarcoma), prostate, pancreatic and breast cancers. In vivo evidence suggests the antagonism of excessive Hh signaling provides a route to unique mechanism-based anti-cancer therapies. Recently the small molecule SANT-2 was identified as a potent antagonist of Hh-signaling pathway. Here, we describe the synthesis, SAR studies as well as biological evaluation of SANT-2 and its analogues. Fifteen SANT-2 derivatives were synthesized and analyzed for their interference with the expression of the Hh target gene Gli1 in a reporter gene assay. By comparison of structure and activity important molecular descriptors for Gli inhibition could be identified. Furthermore we identified derivative TC-132 that was slightly more potent than the parent compound SANT-2. Selected compounds were tested for Hh related teratogenic effects in the small teleost model medaka. Albeit Gli expression has indicated a 16-fold higher Hh-inhibiting activity than observed for the plant alkaloid cyclopamine, none of the tested compounds were able to induce the cyclopamine-specific phenotype in the medaka assay.

Total Synthesis and Biological Activity of the Proposed Structure of Phaeosphaeride A

The signal transducer and activator of transcription 3 (STAT3) has been validated as a suitable target for cancer therapy. Recent evidence by our group and others has shown that phaeosphaerides act as inhibitors of the STAT3 pathway. An efficient synthetic sequence to phaeosphaeride 1a has been previously disclosed. In this work, the first total synthesis of (±)-phaeosphaeride B (1d) and the unnatural phaeosphaeride 1b is reported. Additionally, the biological activities of 1a and 1b were investigated. (6S,7S,8S)-1a and (6R,7S,8S)-1b inhibited granulocyte colony-stimulating factor (GCSF)-stimulated phosphorylation of STAT1, STAT3, and STAT5 and IL-6-stimulated nuclear translocation of STAT3 alpha. In an SPR-based assay, (6S,7S,8S)-1a and (6R,7S,8S)-1b showed minimal ability to inhibit binding of STAT3 to its immobilized phosphotyrosylpeptide ligand (IC50 > 100 μM). Thus, (6S,7S,8S)-1a and (6R,7S,8S)-1b are likely upstream inhibitors of a kinase in the STAT signaling pathway and do not act through the inhibition of STAT3 dimerization by the blocking of the SH2 binding domain.

RGD-mediated delivery of small-molecule drugs

The total synthesis of the structure assigned to the natural product phaeosphaeride A 1a was accomplished. The key steps involve the addition of vinyllithium reagent 7 to the acetonide-protected aldehyde 8 to access the carbon backbone of 1a, the introduction of the methoxylamino group followed by intramolecular hetero-Michael cyclization, and methanol elimination to form the dihydropyran ring. In this study, both enantiomers of 1a were synthesized and tested for biological activity. Preliminary results showed that (6R,7R,8R)-1a and (6S,7S,8S)-1a inhibit STAT3-dependent transcriptional activity in a dose-dependent manner and exhibit antiproliferative properties in breast (MDA-MB-231) and pancreatic (PANC-1) cancer cells.