The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Caenorhabditis elegans as model system for rapid toxicity assessment of pharmaceutical compounds.

INTRODUCTION: The model organism Caenorhabditis elegans is widely used for genetic studies as well as a living biomonitor in ecotoxicology. In this study, we investigated whether C. elegans may represent a suitable model for rapid preliminary toxicity studies of pharmaceutical compounds. METHODS: For this purpose, we used the EGFR kinase inhibitors BIBU1361, BIBX1382, and an inactive chemical analogue BIBU1476. As a first parameter to score for toxicity, we determined lethality of the wild-type C. elegans strain N2 (Bristol) in the presence of the compounds. The transgenic C. elegans strain PC72 (lacZ, heat shock protein-16 [hsp-16] construct) was used as a report organism for toxic effects. PC72 expresses beta-Galactosidase which is induced by hsp-16 in direct response when exposed to toxic compounds. The expression of beta-Galactosidase in cells was subsequently visualized by histochemical staining with X-Gal. RESULTS: A rank order of potency with respect to lethality was established: BIBU1361>BIBX1382>>BIB1476. The induction of beta-Galactosidase was concentration-dependent for each compound and demonstrated the same order of potency as observed for lethality. Furthermore, these compounds showed the same order for lethality in rodents, the first requirement of validation. DISCUSSION: These results indicate that wild-type C. elegans and the transgenic strain PC72 are both suitable models to determine the toxicity of pharmaceutical compounds. This approach allows for an easy and fast ranking of compound toxicity, which may lead to a more rational choice for further in vivo tests.

Inhibition of epidermal growth factor receptor activity by two pyrimidopyrimidine derivatives.

Overexpression of the epidermal growth factor receptors (EGFRs) and human epidermal growth factor receptor 2 occurs frequently in human cancers and is associated with aggressive tumor behavior and poor patient prognosis. We have investigated the effects in vitro and in vivo of a new class of inhibitor molecules on the growth of several human cancer cell lines. BIBX1382 [N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine] and BIBU1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1yl)-pyrimido[5,4-d]pyrimidin-4-yl]-amine] are two new selective EGFR kinase inhibitors that do not block the activity of other tyrosine kinases. BIBU1361 blocked epidermal growth factor-induced phosphorylation of EGFR and also prevented downstream responses such as mitogen-activated protein kinase kinase (MAPK/extracellular signal-regulated kinase kinase) and MAPK activation in cells. In accordance with these observations thymidine incorporation into EGFR-expressing KB cells was selectively and potently inhibited by BIBX1382 and BIBU1361 with half-maximally effective doses in the nanomolar range. Oral administration of these compounds inhibited the growth of established human xenografts in athymic mice, including vulval and head and neck squamous cell carcinomas. Tumor growth inhibition by BIBX1382 coincided with reduced pEGFR and Ki-67 levels in vivo, which is in accordance with the expected effect of EGFR inhibitors. Collectively, these results show that the structural class of pyrimidopyrimidines, exemplified here by BIBX1382 and BIBU1361, represents an interesting scaffold for the design of EGFR inhibitors.