Ro 09-0179 (4',5-dihydroxy-3,3',7-trimethoxyflavone), isolated from a Chinese medicinal herb, was found to have potent antiviral activity. It selectively inhibited the replication of human picomaviruses, such as rhinoviruses and coxsackieviruses in tissue culture, but not other DNA and RNA viruses. Ro 09-0298 (4',5-diacetyloxy-3,3',7-trimethoxyflavone), an orally active derivative of Ro 09-0179, prevented coxsackievirus (Bi) infection in mice. The critical time for the inhibition of rhinovirus replication by Ro 09-0179 was 2 to 4 h after virus adsorption, i.e., in the early stages of virus replication. It markedly inhibited coxsackievirus and rhinovirus RNA synthesis in infected HeLa cells, but not in a cell-free system using the RNA polymerase complex isolated from the infected cells. In the infected cells, the RNA polymerase complex was not formed in the presence of Ro 09-0179. Therefore, it is suggested that Ro 09-0179 interferes with some process of viral replication which occurs between viral uncoating and the initiation of viral RNA synthesis.
Studies of various analogs related to the antipicornavirus agent, 4',5-dihydroxy-3,3',7-trimethoxyflavone (Ro 09-0179), led to the identification of 4'-ethoxy-2'-hydroxy4,6'-dimethoxychalcone (Ro 09-0410), a new and different type of antiviral agent. Ro 09-0410 had a high activity against rhinoviruses but no activity against other picorraviruses. Of 53 rhinovirus serotypes so far tested, 46 were susceptible to Ro 09-0410 in HeLa cell cultures. The concentration of Ro 09-0410 inhibiting 50% of the types of rhinovirus was about 0.03 ,ug/ml, whereas the 50%o cytotoxic concentration was 30 ,Ig/thl. Ro 09-0410 inactivated rhinoviruses in direct dose-, time-, and temperture-dependent fashion. Since infectivity, reduced by exposure to the agent, completely regained the original level by extraction of the agent with chloroform, the inactivation may be associated with the binding of the agent to some specific site of the rhinovirus capsid.Ro 09-0179 (4',5-dihydroxy-3,3',7-trimethoxyflavone), a potent antipicornavirus agent, was isolated from a Chinese medicinal herb, Agastache Folium (Agastache rugosa Kuntze) (3). The discovery of this agent led to the synthesis of a series of derivatives and testing of their antiviral activities in tissue culture. Among hundreds of compounds prepared and evaluated, Ro 09-0410 (4'-ethoxy-2'-hydroxy-4,6'-dimethoxychalcone) (Fig. 1) emerged as an agent exclusively active against rhinoviruses.The mode of action of Ro 09-0410 differed significantly from that of Ro 09-0179. The latter appeared to inhibit the replication of picornavirus at a stage between uncoating and the initiation of RNA synthesis in infected cells (3). On the other hand, Ro 09-0410 directly inactivated rhinoviruses. Subsequent studies showed that the infectivity of rhinoviruses was reduced by exposure to Ro 09-0410 but was regained completely by the extraction of the agent with chloroform, to which rhinovirus is resistant (1). While bound to the agent, rhinovirus was inactive. In this report, we describe the results of tissue culture studies on the antirhinovirus activity of ( All rhinoviruses used in this study were purchased from the American Type Culture Collection, Rockville, Md., except type 2 (HGP), which was kindly supplied by R. Kohno, the National Institute of Health of Japan. Viruses were propagated in HeLa and WI-38 cells at 33°C. Poliovirus, echoviruses, coxsackieviruses, mengovirus, vesicular stomatitis virus, respiratory syncytial virus, herpes simplex virus, vacciia virus, and influenza virus used in this study were propagated as described in the accompanying paper (3).Asy of rhino titer and ePt"mation of MIC5..The rhinovirus titer was assayed, and the 509o minimal inhibitory concentration (MIC50) was estimated as described in the accompanying paper (3). The virus titer was expressed as plaque-forming units per milliliter. The MIC50 was expressed as the concentration at which the viral cytopathic effect was inhibited by about 50%6 as compared with the control.
Trestatin complex which exhibited a potent inhibitory activity on various a-amylases has been isolated from the culture filtrate of Streptomyces dimorphogenes nov. sp. NR-320-OM7HB. Three major components, trestatins A, B and C, have been purified by adsorption and ionexchange chromatography.Their spectral and chemical properties suggested that trestatins were novel basic oligosaccharide homologues each characterized by the possession of a trehalose moiety at the non-reducing end of the molecule.It has been suggested that useful therapy of diabetes and obesity might be achieved by reducing the digestion of dietary starch using inhibitors of a-amylase. This could be expected to decrease hyperglycemia and hyperinsulinemia. From these viewpoints, a number of a-amylase inhibitors have been isolated from the microbial cultures.1~14) In the course of our screening program searching for a-amylase inhibitors, we discovered a novel complex, designated trestatin* in the culture filtrate of a new Streptomyces species to which we have given the name Streptomyces dimorphogenes NR-320-OM7HB. The trestatin complex contained trestatins A, B and C as the major components. They have shown a remarkable inhibitory activity against various a-amylases.The present paper describes the production by fermentation, isolation, physico-chemical characteristics and biological properties of the major components of trestatin. Their structures are reported in the subsequent paper. Mycological characteristics of the producer strain will be described elsewhere. Results and Discussions FermentationThe seed culture was prepared in a medium containing 2.0% potato starch, 2.0% glucose, 2.0 soya meal, 0.5 % yeast extract, 0.25 % NaCl, 0.005 % ZnSO4• 7H2O, 0.0005 % CuSO4 • 5H2O, 0.0005MnCl2.4H2O, 0.32% CaCO3 (pH 7.0) by shaking for 48 hours at 27°C, and transferred into a 50-liter fermentor containing 25 liters of the same medium as the seed culture. Fermentation was carried out at 27°C for 43 hours with an air flow rate of 25 liters per minute under an agitation at 300 rpm. The inhibitory activity of trestatin on porcine pancreatic a-amylase was determined by the method as described in Experimental. A typical trestatin fermentation broth (pH 6.9) had an activity of 3.1 x 104inhibi-tory unit (IU)/ml. The detailed process for the fermentation will be described elsewhere.* Parts of the present work were orally presented at 218th Scientific Meeting
Modes of action of five antirhinovirus agents were compared. Ro 09-0410, 4',6-dichloroflavan, and RMI-15,731 were active preferentially against human rhinovirus. Serotypes of the virus varied in their susceptibility to these three agents, whereas Ro 09-0179 and enviroxime showed activity against all the serotypes of the virus tested to date. Ro 09-0410, RMI-15,731, and 4',6-dichloroflavan inactivated the virus directly, although 4',6-dichloroflavan did so only slightly. Inactivation by 4',731 was associated with the binding of the agents to the virus, since the infectivity, reduced by exposure to the agents, was restored to the original level by extraction of the agents with chloroform. The binding of [3H]Ro 09-0410 to human rhinovirus type 2 was inhibited by unlabeled Ro 09-0410, 4',6-dichloroflavan, and RMI-15,731 but not by Ro 09-0179 or enviroxime. Furthermore, subtypes resistant to both 4',6-dichloroflavan and RMI-15,731 showed cross-resistance to Ro 09-0410 and vice versa. On the other hand, sublines resistant to these three agents were not cross-resistant to Ro 09-0179 or enviroxime. These results indicate (i) that Ro 09-0410, 4',6-dichloroflavan, and RMI-15,731 exert their activities through the same mode of action, namely, binding to or interaction with some specific site on the viral capsid protein, and (ii) that the binding or interaction sites for these three agents are either the same or very close to each other.
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