Ilaria Zanardi

Novel Trisubstituted Benzimidazoles, Targeting Mtb FtsZ, As A New Class of Antitubercular Agents

Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM) for their antibacterial activity against Mtb H37Rv strain. Moreover, five of the lead compounds also exhibited excellent activity against clinical Mtb strains with different drug-resistance profiles. All lead compounds did not show appreciable cytotoxicity (IC(50) > 200 μM) against Vero cells, which inhibited Mtb FtsZ assembly in a dose dependent manner. The two lead compounds unexpectedly showed enhancement of the GTPase activity of Mtb FtsZ. The result strongly suggests that the increased GTPase activity destabilizes FtsZ assembly, leading to efficient inhibition of FtsZ polymerization and filament formation. The TEM and SEM analyses of Mtb FtsZ and Mtb cells, respectively, treated with a lead compound strongly suggest that lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb FtsZ assembly and Z-ring formation.

Novel C-seco-taxoids possessing high potency against paclitaxel-resistant cancer cell lines overexpressing class III β-tubulin

Novel C-seco-taxoids were synthesized from 10-deacetylbaccatin III and their potencies evaluated against drug-sensitive and drug-resistant cancer cell lines. The drug-resistant cell lines include ovarian cancer cell lines resistant to cisplatin, topotecan, adriamycin and paclitaxel overexpressing class III beta-tubulin, A2780TC1 and A2780TC3. The last two cell lines were selected through chronic exposure of A2780wt to paclitaxel and Pgp blocker cyclosporine. All novel C-seco-taxoids exhibited remarkable potency against A2780TC1 and A2780TC3 cell lines, and no cross resistance to cisplatin- and topotecan-resistant cell lines, A2780CIS and A2780TOP. Four of those C-seco-taxoids exhibit much higher activities than IDN5390 against paclitaxel-resistant cell lines, A2780ADR, A2780TC1 and A2780TC3. SB-CST-10202 possesses the best all-round high potencies across different drug-resistant cell lines. Molecular modeling studies, including molecular dynamics simulations, on the drug-protein complexes of class I and III beta-tubulins were performed to identify possible cause of the remarkable potency of these C-seco-taxoids against paclitaxel-resistant cell lines overexpressing class III beta-tubulin.

Taxanes with high potency inducing tubulin assembly overcome tumoural cell resistances

We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by P-gp overexpression, by mutations in the β-tubulin binding site or by overexpression of the highly dynamic βIII-tubulin isotype. We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7-S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on P-gp and βIII overexpressing cells and on mutant cells. The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power.

The role of individual caspases in cell death induction by taxanes in breast cancer cells

BackgroundIn previous study we showed that caspase-2 plays the role of an apical caspase in cell death induction by taxanes in breast cancer cells. This study deals with the role of other caspases. We tested breast cancer cell lines SK-BR-3 (functional caspase-3) and MCF-7 (nonfunctional caspase-3).Methods and resultsUsing western blot analysis we demonstrated the activation of initiator caspase-8 and -9 as well as executioner caspase-6 and -7 in both tested cell lines after application of taxanes (paclitaxel, SB-T-1216) at death-inducing concentrations. Caspase-3 activation was also found in SK-BR-3 cells. Employing specific siRNAs after taxane application, suppression of caspase-3 expression significantly increased the number of surviving SK-BR-3 cells. Inhibition of caspase-7 expression also increased the number of surviving SK-BR-3 and MCF-7 cells. On the other hand, suppression of caspase-8 and caspase-9 expression had no significant effect on cell survival. However, caspase-9 seemed to be involved in the activation of caspase-3 and caspase-7. Caspase-3 and caspase-7 appeared to activate mutually. Furthermore, we observed a significant decrease in mitochondrial membrane potential (flow cytometric analysis) and cytochrome c release (confocal microscopy, western blot after cell fractionation) from mitochondria in SK-BR-3 cells. No such changes were observed in MCF-7 cells after taxane treatment.ConclusionWe conclude that the activation of apical caspase-2 results in the activation of caspase-3 and -7 without the involvement of mitochondria. Caspase-9 can be activated directly via caspase-2 or alternatively after cytochrome c release from mitochondria. Subsequently, caspase-9 activation can also lead to caspase-3 and -7 activations. Caspase-3 and caspase-7 activate mutually. It seems that there is also a parallel pathway involving mitochondria that can cooperate in taxane-induced cell death in breast cancer cells.

Second-generation taxanes effectively suppress subcutaneous rat lymphoma: role of disposition, transport, metabolism, in vitro potency and expression of angiogenesis genes

SummaryThe study investigated possible mechanisms by which second-generation taxanes, established as significantly more effective than paclitaxel in vitro, suppress a rat lymphoma model in vivo. The studied mechanisms included taxane pharmacokinetics, expression of genes dominating their metabolism (Cyp3a1/2) and transport (Abcb1) and genes controlling tumour angiogenesis (growth factors and receptors). SB-T-1214, SB-T-12854 and IDN5109 suppressed rat lymphoma more effectively than paclitaxel, SB-T-12851, SB-T-12852, SB-T-12853 or IDN5390 as well as P388D1 leukaemia cells in vitro. The greater anti-lymphoma effects of SB-T-1214 in rats corresponded to a higher bioavailability than with SB-T-12854, and lower systemic toxicity of SB-T-1214 for rats reflected its lower cytotoxicity for P388D1 cells in vitro. Suppression of Abcb1 and CYP3a1 expression by SB-T-1214 and IDN5109 could partly explain their anti-lymphoma effects, but not that of SB-T-12854. Growth factors genes Egf, Fgf, Pdgf, and Vegf associated with tumour angiogenesis had significantly lower expression following treatment with anti-lymphoma effective IDN5109 and their receptors were unaffected, whereas inefficient IDN5390 increased expression of the most important Vegf. The effective SB-T-12854 inhibited Egf, Egfr, Fgfr and Pdgfr expression, while the ineffective SB-T-12851, SB-T-12852 and SB-T-12853 inhibited only Egf or Egfr expression. Vegfr expression was inhibited significantly by SB-T-12851 and SB-T-12854, but effect of SB-T-12851 was compromised by induced Vegf expression. The very effective SB-T-1214 decreased the expression of Vegf, Egf and all receptors most prominently indicating the possible supporting role of these genes in anti-lymphoma effects. In conclusion, SB-T-1214, SB-T-12854 and IDN5109 are good candidates for further study.

Abstract B9: Role of disposition, transport, in vitro efficiency and expression of relevant genes in the effects of novel taxanes on subcutaneous lymphoma in Sprague‐Dawley/Cub rats

Novel taxoids SB‐T‐1214, SB‐T‐12851, SB‐T‐12852, SB‐T‐12853, SB‐T‐12854 and IDN‐5109 proved to be more effective than paclitaxel in vitro in MDA‐MB‐435 cell line, but especially in NCI/ADR‐RES cell line, highly expressing ABCB1. Thus, their effects in vivo are of considerable interest, because they could overcome drug‐resistance due to high ABCB1 and CYP3A expressions which may limit effects of paclitaxel. Purposes of the study: To investigate if some of the novel taxanes synthesized at Stony Brook exert significant effects on the genetically well defined lymphoma in Sprague‐Dawley/Cub rats, for which paclitaxel is ineffective. Experimental procedures: The rats were kept in standard laboratory conditions and the drugs were administered i.p. under conditions approved by Ethical Committee, Charles University Prague. The intravital area of subcutaneous lymphoma was measured by palpation and their weight during post‐mortem examination. Drug blood levels were determined by HPLC. Quantitative expressions of mRNAs of Abcb1, Cyp3a, Egf, Fgf, Pdgf and Vegf and their receptors Egfr, Fgfr, Pdgfr and Vegfr were made by real‐time PCR system using reaction mixtures of Applied Biosystems. Effects were also compared with in vitro efficiency of the drugs in P388D1 lymphoma line. Summary of new and unpublished data: IDN‐5109, SB‐T‐1214 and SB‐T‐12854 were the most effective drugs against the subcutaneous lymphoma. The higher effects of SB‐T‐1214 than SB‐T‐12854 are ascribed to 3.3‐higher blood levels, as well as to the fact that systemic toxicity of SB‐T‐1214 is significantly lower than that of SB‐T‐12854. Expression of Abcb1 and Cyp3a1 in lymphomas after treatment with the drugs does not correspond to their anti‐lymphoma effects. However, the anti‐lymphoma activities of IDN‐5109, SB‐T‐1214 and SB‐T‐12854 corresponded to their effects on growth factors and their receptors, especially Vegf and Vegfr. The result is consistent with the reported effect of paclitaxel on growth factors and tumor angiogenesis, and these effects could be an important factor for their activities in addition to the proved effects on mitosis and apoptosis. Higher in vitro activities of the novel taxanes than paclitaxel in P388D1 cell line correspond to their higher efficacies on the subcutaneous lymphoma in vivo as compared with paclitaxel. Conclusions: IDN‐5109, already proved effective against various tumors and presently in Phase II clinical studies, as well as novel taxanes SB‐T‐1214 and SB‐T‐12854, are found to be effective against non‐Hodgkin type rat lymphoma. These taxanes could be efficacious against paclitaxel‐resistant tumors, due to the fact that they are not subjected to drug‐resistance caused by high ABCB1 expression and are potentially effective against tumor angiogenesis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B9.