| This dissertation work is a continuation of efforts in this lab to explore broad spectrum antiviral agents containing ring-expanded ("fat") heterocyclic bases, nucleosides, and nucleotides. The specific viral targets of my work are (a) the Hepatitis B Virus (HBV), (b) the Human Immunodeficiency Virus (HIV), (c) the West Nile Virus (WNV) and (d) the Hepatitis C Virus (HCV), the latter two of which belong to the same family of viruses called Flaviviridae. The specific ring systems that are targeted in this work include (a) imidazo[4,5-e][1,3]diazepine-4,8-dione (I), (b) imidazo[4,5-e][1,2,4]triazepine-5,8-dione (II), and (c) imidazo[4,5-e][1,3]diazepine-4-one (III), containing a variety of halogen, alkyl, aryl, aralkyl, ribosyl or 2'-deoxyribosyl substituents at 1-, 2-, and 6- (I & III) or 1-, 2-, and 7- (II) positions of the heterocyclic rings as shown: *; The entire work is divided into two parts: Part A: Medicinal Chemistry. This includes organic synthesis, antiviral screening, and structure-activity relationship (SAR) studies. Based on the preliminary antiviral activity of a few heterocycles and nucleosides belonging to ring systems I and II, part of my work under Part A involved enhancement of their antiviral potency through analogue synthesis and SAR studies against HBV, HCV, HIV and WNV. In this regard, I mainly focused on the little-explored 2-position of ring systems I and II. The potential biological rewards of ring system III, on the other hand, are yet uncertain. The research with this new ring system has nevertheless thrown more light on its little-known chemistry and physicochemical properties.; Part B: Biochemistry. This part deals with biochemical investigations of the mechanism of activity of a few potent antiviral compounds discovered in Part A. In particular, we focused on some key enzymes involved in the life cycle and replication of the mentioned viruses, including: (a) Flaviviridae NTPase/helicase, (b) Flaviviridae RNA-dependent RNA polymerase (RdRp), and (c) HIV integrase. These enzyme activities, which are mostly based on cell-based assays, were explored through collaborative arrangements with the Berhard-Nocht Institute of Tropical Medicine, Hamburg, Germany (Dr. Peter Borowski, for Flaviviridae) and the National Institutes of Health (Dr. Yves Pommier, for HIV Intergrase). My own research in this regard is more general in that it involves nucleic acid incorporation studies of a couple of selected triphosphate analogues of ring-expanded (or "fat") nucleosides (RENs or fNs), employing a polymerase. A number of fNs containing ring structures I and II were found to be potent inhibitors of NTPase/helicase and/or RNA-dependent RNA Polymerase (RdRp) of Flaviviridae, both of which use nucleoside triphosphates (NTP) for their catalytic activities, the former as an energy cofactor while the latter as a substrate. Therefore, I synthesized a few 5'-triphosphate derivatives of fNs for biochemical investigations. Specifically, my work is focused on investigating the effect of introducing fNs into developing nucleic acid chains during transcription catalyzed by a polymerase, and exploring whether or not the chain elongation would proceed past the insertion point, and if so, with what efficiency as compared to the natural nucleotides. Employing various synthetic oligos incorporated with "fat" nucleotides and a polymerase (Klenow exo+/exo- fragments), we discovered that dATP, dGTP, or dCTP can be incorporated in a complementary strand against an fN in a reference strand, but the incorporation was not as efficient as with a reference strand containing natural nucleotides.; In order to explore the stability of DNA duplexes incorporated with "fat" nucleotides, we also performed DNA melting experiments using different oligonucleotides incorporated with fNs. The highest duplex stability Tm was observed with a C in the complementary strand against an fN in the reference strand. This result is in sharp contrast to that obtaine... |
