RNA viruses cause a plethora of diseases which are difficult to treat due to the high mutation rate of such pathogens. Current therapies for RNA virus infections are severely limited due to the development of antiviral drug resistance. Such viruses exist in nature as a quasispecies resulting from the high error frequency of replication. RNA viruses as thus highly adaptable and also exist on the edge of “error catastrophe.” Thus small increases in the mutation rate can cause a drastic decrease in viral viability. Based on this concept, a new approach to the design of antiviral agents termed “lethal mutagenesis” has emerged whereby drugs that increase the error rate of RNA viruses may be developed to combat viral diseases.
Chapter one provides an overall review of lethal mutagenesis as an antiviral strategy. Ribavirin, a clinically utilized nucleoside antiviral agent, has shown to be a lethal mutagen. Once transported across cellular membranes and phosphorylated to the 5' triphosphate, the base-modified nucleoside triphosphate is a degenerate substrate for the viral RNA-dependent RNA polymerase (RdRP). In the viral genome, misincorporation is templated by the ribavirin pseudo base resulting in an increase in overall frequency of mutations and error catastrophe and decreased infectivity of the virus.
The work described herein explores the efforts to design and synthesize effective antiviral nucleosides and probe their mechanism of action as lethal mutagens. Chapter two describes the investigation of isocarbostyril nucleosides as antiviral agents. Three isocarbostyril ribonucleoside analogs, ICS-R, MICS-R, and SICS-R, were synthesized by the Vorbruggen procedure and evaluated as antivirals. ICS-R was discovered to be the most potent analog followed by SICSR and MICS-R demonstrating antiviral activity greater than clinical agent Ribavirin against poliovirus. Additionally, 5' and 2' modified ICS-R control compounds demonstrated no antiviral effect. The corresponding ICS-R analog triphosphates displayed slow kinetics of incorporation and revealed inhibition of PV viral RdRP as the mode of action. ICS-R(TP) accumulated intracellularly to a greater degree than MICS-R and SICS-R corroborating antiviral and biochemical results.
Chapter three is divided into two sections exploring the mechanism of action of 5-nitroindole nucleoside and the development of 5-halo indole nucleosides. The mechanism of antiviral activity of 5-nitroindole ribonucleoside was investigated though the evaluation of a series of mechanistic probes and biochemical characterization of phosphorylated 5-nitroindole analogs. 5NINDN(DP) and 5NINDN(MP) were synthesized and evaluated as inhibitors of PV viral polymerase in comparison with 5NINDN(TP) and the parent nucleoside 5NINDN. Surprisingly, both triphosphate and diphosphate inhibited the viral polymerase, however reversed-phase HPLC analyses revealed detectable phosphorylation limited only to 5NINDN(MP). Further evaluation of mechanistic probes 5'OMe5NINDN, 5-nitroindole, and N-Eth-OH 5-nitroindole indicated that intracellular phosphorylation was not necessary for antiviral activity and revealed inhibition of cellular translation was proposed as a mediator of antiviral activity. Due to instability of the nitroaromatic nucleobase, stable 5-halo analogs 5CIINDN and 5-BrINDN were synthesized and evaluated as agents more active than the parent 5NINDN.
Chapter four focuses on the application of ribosylation agents with modified protecting groups to optimize access to indole nucleosides. By altering the electronic substituents on ribose protecting groups, a series of ribosylation agents were designed to improve access to biologically active indole nucleosides. Additionally, there was a decrease in an undesired orthoamide byproduct and an increase in the overall yield of the nucleoside.
Chapter five demonstrates the detection of phosphorylation of nucleoside analogs as determinants of antiviral activity. Reversed phase HPLC methods to detect phosphorylation of nucleosides were optimized and applied to evaluation of hydrogen-bonding lethal mutagens. The lack of antiviral activity for nucleoside P is attributed to undetectable conversion to the 5' triphosphate. Additionally, purine analog JA28, is a lethal mutagen which is efficiently phosphorylated to JA28 (TP). Structural comparisons between phosphorylated and non-phosphorylated nucleosides reveal insights into the future design of lethal mutagens.