Antiviral activity of 2,3-dihydro-1H-imidazo[1,2-b]pyrazole in herpes simplex virus type 1-infected mammalian cells.

2,3-Dihydro-lH-imidazo[l,2-blpyrazole (IMPY), a known inhibitor of DNA synthesis, has been shown to be a useful drug for the synchronization of mammalian cells in culture. Recent studies in our laboratory indicate that IMPY may possess significant antiviral activity against herpes simplex virus (HSV) type 1. IMPY. at a concentration of 80 pg/ml or 0.73 mM, reduced syncytia formation approximately 80 per cent. A 50 per cent inhibitory dose was calculated for each drug in order to compare potency in syncytia reduction of IMPY with that of arabinosyladenine (ara-A) and arabinosylhypoxanthine (ara-H). Our results indicated that the antiviral potencies of the three drugs were ranked in the order ara-A > ara-H > IMPY, the 50 per cent inhibitory doses being 22, 195 and 309 PM respectively. Utilizing the microplate procedure of Sidwell and Huffman [Appl. Microbial. 22, 797 (197111, inhibition of viral cytopathic effect was rated against drug cytotoxicity and a virus rating (VR) established. A virus rating of 0.68 was calculated for IMPY. In comparison, VR values of 0.84 and 0.66 were obtained for ara-A and ara-H respectively. In contrast to the syncytia reduction studies, IMPY appeared to possess antiviral activity equivalent to that of ara-H according to the criteria of the virus rating assay. A technique was developed for evaluating the degree of selectivity (‘S) of a drug with respect to its differential effect on viral and cellular DNA synthesis. IMPY was found to possess a negative selectivity at all concentrations studied, reflecting the fact that it inhibited cellular DNA synthesis more than viral DNA synthesis. In contrast, ara-A and ara-H both expressed positive degrees of selectivity in that they inhibited viral DNA synthesis more extensively than cellular DNA synthesis. The antiviral and antitumor activities of many agents depend, at least in part, on their ability to inhibit DNA synthesis. Studies in L cells have shown that 2,3-dihydro-lH-imidazo[l,2-blpyrazole (IMPY) reversibly inhibited DNA synthesis without significantly affecting RNA or protein synthesis [ 11. Although the exact mode of action of this drug is unknown, it was postulated that IMPY may be interfering with deoxyguanosine triphosphate synthesis. The inhibition of DNA synthesis seen with IMPY was prevented and reversed by deoxyguanosine and potentiated by deoxycytidine. More recently, it has been demonstrated that both monolayer and suspension cultures of HeLa cells could be synchronized at the point of entry into the synthetic (S) phase of the cell cycle by IMPY [2] ; addition of deoxynucleosides, however, failed to reverse or prevent the inhibition of DNA synthesis by IMPY. It was suggested that this limitation may be due to the poor capacity of HeLa cells to phosphorylate and utilize deoxyguanosine. The first indication that IMPY possessed antiviral activity appeared during studies with the nucleoside antibiotic 9-/?-D-arabinofuranosyladenine (ara-A), a *Requests for reprints: The University of Michigan. School of Dentistry. Department of Oral Biology, Ann Arbor, Mich. 48109. known antiviral compound [3]. IMPY was used in our laboratory as an agent for synchronizing suspension cultures of KB cells. After synchronization was achieved, the cells were infected with herpes simplex virus type 1 (HSV-1) and treated with ara-A. The intracellular concentration of IMPY remaining after washing appeared to exert antiviral activity against HSV-1 even in infected cell cultures not treated with ara-A. In this paper, we describe studies of IMPY and its action in HSV-infected mammalian cells. Our studies of syncytia reduction and microplate assays indicate that IMPY possesses a significant antiviral action against HSV-1. However, biochemical studies involving the separation and quantitation of viral and cellular DNA from HSV-infected KB cells revealed that IMPY inhibited cellular DNA synthesis to a slightly greater extent than the synthesis of viral DNA. This lack of selectivity is in direct contrast to the selective inhibition of viral DNA synthesis seen with ara-A and its deaminated catabolite arabinosylhypoxanthine (ara-H) [4,5]. MATERIALS AND METHODS Chemicals. IMPY was generously provided by Dr. Herbert L. Ennis, Roche Institute of Molecular Biology (Nutley, N.J.), and Drs. P. Schmidt and K. Scheibli, Ciba-Geigy Ltd. (Basel, Switzerland). Ara-A