By the use of recombinant baculoviruses, the transcleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein.Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also fou ad that the partially purified NS3 serine proteinase prepared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained will provide basic knowledge on processing of the HCV polyprotein.

BE-23372M, a novel protein tyrosine kinase inhibitor, was isolated from the culture broth of a fungus. The producing strain, F23372, was identified as Rhizoctonia solani, based on the cultural and morphological characteristics. The active principle was extracted from the myceliumwith acetone and purified by solvent extraction, silica gel column chromatography and Sephadex LH-20 column chromatography.BE-23372Mshowed strong inhibitory activity against EGF receptor kinase with IC50 values of 0.02 and 0.03/im on two different substrates, whereas IC50 values against protein kinase C and CAMP-dependent protein kinase were 4.5 and >20 /jM, respectively. The compound inhibited the growth of A431 human epidermoid carcinoma and MKN-7human stomach cancer cell lines with IC50 values of 8 and 24/xm, respectively.

The fungal metabolite BE‐23372M is a structurally novel protein kinase inhibitor. Its IC50 for epidermal growth factor (EGF) receptor kinase was 0.03 μM. IC50 values of BE‐23372M for other protein tyrosine kinases, erbB‐2, p43v‐abl, insulin receptor kinase, and p60c‐src were 0.42, 1.0, 3.3, and 4.5 μM, respectively, and the IC50 for protein kinase C, a serine/threonine kinase, was 4.1 μM. Cdc2 kinase, casein kinases I and II and cAMP‐dependent protein kinase were not inhibited by 20 μM BE23372M. A kinetic study showed that BE‐23372M was competitive with respect to the substrate peptide and to ATP. Autophosphorylation of solubilized EGF receptor kinase was clearly inhibited by 0.1 μM BE‐23372M. Autophosphorylation of EGP receptor in A431 cells was also inhibited. These results show that BE‐23372M is a potent and specific EGF receptor kinase inhibitor. It should be a valuable tool for EGF receptor kinase research.

The serine proteinase of hepatitis C virus (HCV) non-structural protein NS3 was efficiently expressed in an active form as a fused protein with oligohistidine in Escherichia coli. The recombinant fusion protein was purified to near homogeneity by affinity chromatography on a metal chelation column. Trans-cleavage activity of this protein was investigated by using the substrate NS5 protein expressed in insect cells. The purified serine proteinase trans-cleaved the partially purified NS5 protein. In contrast, the NS3 proteins with mutations at the proposed catalytic site, Ser1165 or His1083, lost the trans-cleavage activity. Analysis of the authentic enzyme and variants with site-directed mutations provides a useful tool for understanding the structure-function relationship of the NS3 serine proteinase. We then developed an in vivo trans-cleavage assay system by coexpression of the NS3 proteinase and the NS5 substrate in E coli, and examined the effect of known inhibitors of serine proteinase. Inhibition of its proteolytic activity by N-p-tosyl-L-lysine chloromethyl ketone (TLCK) was observed, but only at high concentrations. The in vitro and in vivo trans-cleavage assays for NS3 serine proteinase will facilitate efficient testing for inhibitors of the replication of HCV and specific treatment for hepatitis C.