Studies on the mode of inhibition of avian myeloblastosis virus multipulication by cupric isonicotinic acid hydrazide complex

Abstract

The primary objectives of cancer chemotherapy programs include the screening and evaluation of new chemicals and natural products in experimental systems for their anticancer activity and the recommendation of new anticancer agents for clinical trial. The ideal chemotherapeutic agent should be selectively toxic to cancer cells or have the property to revert cancer cells to normal cells. In contrast, most of the antitumor drugs available have significant host toxicity with a narrow spectrum of activity. The possibility of using metal complexes as alternative anticancer agents was stimulated by the discovery of certain platinum coordination compounds having potent anticancer activity. Subsequently, several metal complexes have been studied for their possible antitumor activity. Copper complexes of thiosemicarbazones and isonicotinic acid hydrazide (INH) have been shown to inhibit the reverse transcriptase activity of Rous sarcoma virus and its transforming ability of chick embryo fibroblasts. In the present investigation, emphasis has been given to understand the mode of inhibition of avian myeloblastosis virus (AMV) multiplication by cupric-INH complex. The thesis consists of 5 chapters: Chapter I gives a general introduction. Chapter II describes the following aspects: preparation and characterization of cupric-INH complex, purification of AMV reverse transcriptase, studies on inhibition of reverse transcriptase activity by cupric-INH complex, the kinetics and mode of inhibition, and effect of storage and proteolytic cleavage of reverse transcriptase on enzyme inhibition by cupric-INH complex. The chemical analysis of the complex showed a 1:1 Cu(II) complex formation with INH. The structure arrived from the data showed the molecular weight of the complex to be 332.8. The complex inhibited the reverse transcriptase to an extent of 95% in a poly(rA)·(dT)?? and poly(dA)·(dT)?? dependent reaction at 50 ?M concentration. The complex inhibited the polynucleotide synthesis even after the reaction was initiated. The nature of inhibition was found to be noncompetitive with respect to dTTP and the Ki was calculated to be approximately 2 ?M. The stored or proteolytically cleaved enzyme containing mainly the ?-polypeptide was inhibited to a lesser extent by the complex as compared to the ?? holoenzyme. The ribonuclease H activity associated with AMV reverse transcriptase was also found to be inhibited by cupric-INH complex (74% and 96% inhibition at 50 and 100 ?M concentration respectively). The cupric-INH complex cleaves pBR322 form I DNA into smaller molecules irrespective of the presence or absence of reverse transcriptase-associated endonuclease in the assay mixture. However, in the presence of the enzyme, the DNA was cleaved to a greater extent. At concentrations where AMV reverse transcriptase was effectively inhibited, the cupric-INH complex did not show any inhibition of chick embryo DNA polymerases. The results of these experiments are discussed in Chapter III. The results of the effect of cupric-INH complex on normal and AMV-infected avian cells are presented in Chapter IV. The complex was found to be nontoxic to normal yolk sac macrophages up to a concentration of 100 ?M. It did not significantly inhibit DNA, RNA, or protein synthesis in these cells. The interesting observation was that the complex inhibited AMV multiplication in yolk sac macrophages when added 0 to 4 h post-infection as demonstrated by the inhibition of both focus formation and expression of viral-specific antigens. This inhibition was not observed when the complex was added at 8 and 16 h after AMV infection. Similar studies carried out on AMV-transformed myeloblasts indicated that the complex had no effect on the cellular macromolecular synthesis, the colony (foci) formation, or the viral-specific antigens. It was therefore suggested that the complex inhibits AMV multiplication by interfering in an early event of the viral growth cycle, possibly the process of reverse transcription. In the last chapter, an attempt has been made to study the in vivo antiviral activity of the cupric-INH complex. The complex was found to be nontoxic to chicks up to a dose of 500 ?g/bird. In the experiments designed to study the in vivo distribution of the labelled complex, it was observed that the complex remained in the blood circulation for 2 h after an intravenous injection and an appreciable level of the complex was reached in bone marrow—the target tissue for AMV multiplication. The levels of the complex in various tissues were higher when the complex encapsulated in liposomes was injected as compared to the free complex. The encapsulation also seemed to help in maintaining the stability of the complex. It was observed that the complex (150 ?g) inactivated the AMV when it was preincubated with the virus for 15 min either at 4°C or 37°C. The complex did not show any demonstrable chemotherapeutic activity. In order to enhance the efficacy of the complex, it was encapsulated in liposomes before injecting into chicks. The complex encapsulated in positively charged liposomes showed partial improvement in its chemotherapeutic activity against AMV infection.