Hsu-Tso Ho

Discovery of the first antibacterial small molecule inhibitors of MurB

A series of imidazolinone analogues was synthesized and shown to possess potent MurB inhibitory as well as good antibacterial activity.

Biochemical Pharmacology of Acyclic Nucleotide Analogues

Our studies have shown that the acyclic nucleotide analogues PMEA and HPMPC are able to penetrate into cells and are then activated to mono- and diphosphate derivatives. The latter correspond to triphosphate analogues and presumably serve an important role in the biological activity exerted by these antiviral agents. In support of this idea, the inhibitory effect of PMEApp on HIV reverse transcriptase has been demonstrated with both RNA and DNA template-primer systems. Further studies will be undertaken to determine the effect of HPMPCpp on viral DNA polymerases. Whereas the metabolism of PMEA in CEM cells gives rise to only PMEAp and PMEApp, additional metabolites were obtained in MRC-5 cells; the identity of these metabolites remains to be determined. In the case of HPMPC, a third metabolite was obtained in addition to HPMPCp and HPMPCpp, which has been tentatively assigned as a phosphate-choline adduct by analogy with activation of cytosine-based nucleoside derivatives. The metabolism of HPMPC was unchanged between uninfected and infected cells, indicating that viral enzymes are not necessary for the activation of HPMPC. The long intracellular half-lives of the HPMPC metabolites may have implications for the antiviral efficacy of this compound. The persistence of activated metabolites suggests that infrequent dosing may be possible due to a prolonged antiviral effect. Our results on the effectiveness of infrequent dosing schedules with HPMPC in the treatment of HSV 2 infections in mice support this hypothesis. It is also possible that HPMPCp-choline may serve as a reservoir for HPMPC and therefore for the presumed active metabolite HPMPCpp.

(S)-1-(3-hydroxy-2-(phosphonylmethoxy)propyl)cytosine (HPMPC): a potent antiherpesvirus agent.

(S)-1-(3-Hydroxy-2-(phosphonylmethoxy)propyl)cytosine (HPMPC; 1, fig. 1, left) has been identified from a series of phosphonate nucleotide analogues as having the greatest therapeutic index against lethal herpes simplex virus (HSV) infections in mice (1, 2). The prototype compound of the series is the adenine analogue 2 (HPMPA; 2, fig. 1, right), which was first described by De Clercq et al. in 1986 as a broad-spectrum antiviral agent (3). HPMPA was shown to have a mechanism of action that involves, at least in part, two intracellular phosphorylations to give a diphosphate, which then selectively inhibits the viral polymerase. Since the discovery of HPMPA, additional studies have shown that many related phosphonylmethoxy nucleotide analogues have broad-spectrum in vitro activity (4, 5). Of this class of nucleotide analogues, HPMPC has been described as having the best in vitro therapeutic index against cytomegalovirus (CMV) (2, 6).

Biochemical Pharmacology of 2′-Fluoro-2′,3′-Dideoxyarabinosyladenine, an Inhibitor of HIV with Improved Metabolic and Chemical Stability over 2′,3′-Dideoxyadenosine

We have investigated some properties of the acid stable, 2′-arafluoro-substituted congeners of dideoxyadenosine (ddA) and dideoxyinosine (ddl) in order to understand their potential for in vivo utility as anti-HIV agents. Activities of 2′-fluoro-2′,3′-dideoxyarabinosyl-adenine (FddA) and hypoxanthine (Fddl) against human immunodeficiency virus (HIV) in cells are similar and are about an order of magnitude lower than those of ddA and ddl. FddA is a much poorer substrate than ddA for adenosine deaminase, and is deaminated about 50-fold slower at pharmacological concentrations. The product Fddl is (in contrast to ddl) resistant to cleavage by purine nucleoside phosphorylase. As for ddl, Fddl is phosphorylated by a 5′-nucleotidase, using inosine monophosphate (IMP) as the phosphate donor. In cells, both FddA and Fddl are phosphorylated to the triphosphate of FddA (FddATP). However, the concentration of FddATP produced from FddA is about two-to five-fold higher than from Fddl. FddATP is extremely stable in cells and persists with a half-life of 20 h after removal of FddA from the medium. Using HIV reverse transcriptase, FddATP is a less efficient inhibitor than ddATP by about 20-fold. Thus the activity of these compounds against HIV in cells correlates well with the intracellular concentration and potency of their triphosphates. If the efficacy of ddl in clinical trials is the result of its activity in certain cell types, the fluoro congeners could be active in similar cells since they use the same enzymes for activation to the active triphosphorylated forms. The reduction in potency may, to some extent, be offset by the greater stability to acid and enzyme degradation. Thus further evaluation of FddA and Fddl as anti-HIV agents is warranted.

Cellular Metabolism and Enzymatic Phosphorylation of 9- (2-Phosphonylmethoxyethyl) Guanine (PMEG),a Potent Antiviral Agent

PMEG (9-(2-Phosphonylmethoxyethyl)guanine) is a potent, broad spectrum antiviral agent in the nucleoside phosphonate class (Figure 1). In vitro, PMEG is active against HSV-1, HSV-2, VZV, HCMV and Rauscher murine leukemia virus with IC50s (the concentrations of drug required to reduce the plaque formation by 50%) less than 1 ug/ml (1,2,3). Similar antiviral activity was demonstrated with TK-minus mutants of HSV-1. However, the toxic effect exerted by PMEG on CEM and Vero cells in culture (TC50 5-10 μg/ml, the concentrations of drug affecting the viability of uninfected cells by 50%) limits the utility of PMEG as an antiviral agent. On the other hand, PMEG has antitumor activity (4) against intraperitoneal P388 leukemia and subcutaneously implanted B16 melanoma in mice. PMEG, also supresses human condylomas from papillomavirus (HPV-11) infected human foreskin in transplanted mice (5). We have investigated the cellular metabolism of PMEG and the viral and cellular target enzymes involved in the mode of action to provide rationale for the low antiviral selectivity and to assist with future design of more selective analogs of PMEG.