Sir: The binding of epidermal growth factor (EGF) to its receptor, which has an associated tyrosine protein kinase (TPK) activity, stimulates the phosphorylation of tyrosine residue in EGF receptor, and the phosphorylated receptor transfers the terminal phosphate of ATP to the tyrosine residue of other proteins1~ 3). Recently, receptors for platelet-derived growth factor (PDGF) 4, 5), insulin'6, 7), and insulin-like growth factor (IGF-I) 3) were found to have tyrosinspecific protein kinase activity, suggesting the involvement of tyrosine phosphorylation in triggering cell proliferation. Moreover, it has been observed that the protein products of malignant genes of most retroviruses have tyrosine protein kinase activity9). Furthermore, the amino acid sequence of v-erb-B protein of avian erythroblastosis virus (AEV) has been confirmed to be closely related to that of EGF receptor10). We have undertaken the screening of culture filtrates of actinomycetes for inhibitors of tyrosin protein kinase, using the membrane fraction of human epidermoid carcinoma cell line A-431 as the source of this enzyme, and we found two strains capable of producing inhibitors. One of these strains (MD88-A6), as previously reported11), has been classified as Streptornyces neyagawaensis var. orobolere, produced an isoflavon compound which was identified to be oroborl. The concentration of orobol exhibiting 50% inhibition of tyrosine protein kinase was 3 yg/ml. It inhibited the growth of rat kidney cells transformed by a temperature sensitive mutant (srcts-NRK) of Rous sarcoma virus: 50% inhibition concentrations were 4 µg/ml at 33 C and 16 ug/ml at 39 C. The inhibitor produced by the other strain (No. MH435-hF3) is a novel compound, and we named it erbstatin.

Taxonomic studies indicated that the strain producing erbstatin was closely related to S. viridosporus. In this paper, we report on production, isolation and properties of erbstatin. The study of the structure (Fig. 1) determination will be reported in another paper12). The strain MH435-hF3 was cultured in a 500-ml Erlenmeyer flask containing 110 ml of the medium consisted of glycerol 3%, fish meal 2 and CaCO3 0.2%, pH 7.4 (before sterilization), on a rotary shaker at 27 C for 48 hours, and 3.0 ml of the cultured broth thus prepared was inoculated in to a 500-m1 flask containing 110 ml of the medium described above. The fermentation was carried out at 27 C for 4 days. The production of erbstatin was followed by testing for inhibition of tyrosine protein kinase activity using the method of CARPENTER et al. 13) Erbstatin in culture filtrates (5 liters) was extracted with 5 liters of butyl acetate, and the extract was concentrated under reduced pressure to give a yellowish powder (370 mg). The dried material was dissolved in CHC13i and subjected to a silicic acid column chromatography. After washing with CHC13-MeOH (100: 2), the active fraction was eluted with CHCl3-MeOH (100: 5) and concentrated in vacuo to give a yellow powder (110 mg). It was dissolved in MeOH-CHCl3