Exploring the chemical space of the lysine-binding pocket of the first kringle domain of hepatocyte growth factor/scatter factor (HGF/SF) yields a new class of inhibitors of HGF/SF-MET binding.

The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activity and we show that selected fragments inhibit MET downstream signalling. Thus we demonstrate that targeting the lysine-binding pocket of NK1 is an effective strategy to generate MET receptor antagonists and we offer proof of concept that the HGF/SF-MET interface may be successfully targeted with small molecules. These studies have broad implications for the development of HGF/SF-MET therapeutics and cancer treatment.

SKOV-3 and Me45 cell response to cisplatin-based chemotherapy: an in vitro study.

We studied malignant melanoma cell line Me45 and human ovarian carcinoma cell line SKOV-3 (resistant to cisplatin, adriamycin and diphtheria toxin), assessing their expression level of p53, HSP70 and glutathione S-transferase GST-π before and after chemotherapy with cisplatin. These proteins may be responsible for the occurrence of chemoresistance in cancer patients. To assess protein expression we used the immunocytochemical Avidin-Biotin-peroxidase Complex (ABC) method. Before application of chemotherapy, proteins p53, HSP70 and GST-π were present in 100 % of the examined melanoma cells. After the treatment, the intensity of the immunocytochemical reaction for p53 increased, whereas the intensity of immunocytochemical staining for HSP70 and GST-π decreased. In SKOV-3 cells, p53 and HSP70 were present in 100 % of the examined cells both prior to chemotherapy and after it. However, the intensity of the immunocytochemical reaction for p53 decreased, while that of HSP70 increased. As regards GST-π, only 5 % of all examined SKOV-3 cells revealed its expression before chemotherapy. Incubation with cisplatin caused an elevation in the number of ovarian cancer cells expressing GST-π up to 50 %. Moreover, the intensity of the immunocytochemical reaction for GST-π significantly increased.