Paul M. Keller

Enzymatic phosphorylation of acyclic nucleoside analogs and correlations with antiherpetic activities

Abstract The inhibitor and substrate specificities of deoxythymidine (dThd) kinase purified from herpes simplex virus (HSV Type 1) were studied. A number of nucleosides and nucleoside analogs were phosphorylated by the virus coded enzyme. These included several compounds structurally related to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), a potent inhibitor of HSV replication. Some contained guanine with 9-substituents differing from that of acyclovir by methylene additions, methylene and thioether substitutions for the ether oxygen, and branching on the distal side of the ether oxygen. Others were various 2-substituted 6-hydroxypurines with the 9-(2-hydroxyethoxymethyl) substituent. A limitation of the specificity of the enzyme with guanine derivatives was the lack of phosphorylation of any derivative with an acyclic moiety branched on the proximal side of the ether oxygen. Many of the compounds that were phosphorylated were subsequently found to inhibit HSV replication. Such compounds apparently inhibited HSV replication via the same route of activation previously described for acyclovir [G. B. Elion, P. A. Furman, J. A. Fyfe, P. de Miranda, L. Beauchamp and H. J. Schaeffer, Proc. natn. Acad. Sci. U.S.A. 74 , 5716 (1977)]. Moreover, several compounds not phosphorylated by the enzyme did not inhibit replication. However, some other acyclic nucleoside analogs that were phosphorylated were not good antivirals, indicating that phosphorylation catalyzed by the HSV dThd kinase was not sufficient for inhibition of viral replication to occur. These results emphasize the importance of the specificity of cellular kinases and the HSV DNA polymerase to the mechanism of antiviral activity. The dThd kinase from Vero cells was also purified. With this host cell enzyme, kinetic constants of known antiviral compounds were determined and compared to those of dThd (relative V ′ max ; k ′ m ): dThd (100; 1.3 μM), 5-iodo-2′-deoxyuridine (87; 1.8 μM), 5-trifluoromethyl-2′-deoxyuridine (91; 1.2 μM), 5-bromo-2′-deoxycytidine (5; 580 μM), and 9-β- d -arabinofuransoylthymine (23; 2300 μM). None of the purine acyclic nucleoside analogs tested (at 1000 μM) was detectably phosphorylated by the Vero cell enzyme, and all had apparent K i values >300 μM. The phosphorylation catalyzed by host cell dThd kinase correlated with the toxicity of some pyrimidine nucleoside analogs.

Discovery of a Novel and Potent Class of FabI-Directed Antibacterial Agents

ABSTRACT Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.

Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with Herpesvirus genetic information

Abstract Thymidine kinase-deficient LM cells (LMTK−-) biochemically transformed to the TK+ phenotype with herpes simplex virus genetic information showed an increased uptake of and ability to phosphorylate the acyclic nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine, acyclo-Guo). In growth inhibition studies the TK+ transformants were much more sensitive to inhibition with acyclovir than the untransformed cells (13- to 90-fold more sensitive). The synthesis of DNA in the transformed cells was significantly reduced by acyclovir treatment, whereas acyclovir had little effect on the DNA synthesis of the untransformed cells. Alkaline sucrose gradient sedimentation analysis of cellular DNA synthesized in the presence of acyclovir showed that, in contrast to untreated untransformed cells, the DNA newly synthesized by transformed cells was considerably smaller in size. In pulse-chase experiments the small fragments of DNA synthesized in the presence of acyclo-Guo were not chased to high molecular weight DNA. Finally, acyclo-Guo was shown to be incorporated terminally at 3′-ends of growing DNA chains in replicating cells.

Discovery of potent, selective sulfonylfuran urea endothelial lipase inhibitors.

Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and atherogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease. Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selective EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and modifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irreversible inhibitors.

Biochemical and genetic analysis of acyclovir-resistant mutants of herpes simplex virus type 1☆

Abstract Acyclovir-resistant mutants arise under nonselective conditions at relatively high frequencies from recently cloned populations of herpes simplex virus (HSV). The vast majority of mutants isolated following selection for acyclovir-resistance are mutated in the thymidine kinase ( tk ) gene. These mutants vary considerably in their acyclovir-resistance and exhibit TK and acyclovir-phosphorylating activity reduced to varying degrees, indicating that they are mutated at different sites within the gene. Genetically, these mutants behave as alleles of a single locus and map physically to the HSV- tk gene. Two mutants selected for acyclovir-resistance carry mutations in the viral DNA polymerase ( pol ) gene. One, ACG r 4, is mutated in both the tk and pol genes, whereas the acyclovir-resistance phenotype of the other mutant, BW r , can be ascribed to a mutation in the pol gene alone. In addition, mutant PAA r 5—selected for resistance to the antiHSV drug phosphonoacetic acid (PAA)—is acyclovir-resistant due to a mutation in its pol gene. Phenotypically, the partially purified polymerases of these mutants exhibit altered susceptibility to acyclo-GTP compared with their wildtype counterparts. Genetically, these mutants behave as if linked to the PAA r marker of the pol gene. Thus, by both biochemical and genetic criteria, acyclovir-resistant mutants have been shown to be mutated in the HSV tk and/or pol genes. These criteria, generally applicable to studies of mutants resistant to any antiviral drug, are discussed critically. Our results indicate that, although all acyclovir-resistant mutants can be ascribed to mutations in either the tk or pol gene, these mutants display a variety of phenotypes including varying degrees of acyclovir-resistance and coresistance to other antiHSV drugs. The possible implications of these phenotypes for the clinical use of acyclovir are discussed.

A radioimmunoassay for Herpes Simplex Virus (HSV) thymidine kinase

A sensitive RIA for HSV-1 thymidine kinase (TK) has been developed. This assay is based on competition for the binding site of a rabbit antibody against purified HSV-1 TK, between a purified /sup 3/H-labeled HSV-1 TK and a sample containing an unknown amount of viral TK. The assay is capable of detecting 8 ng or more of the HSV enzyme. Purified HSV-1 TK denatured to <1% of its original kinase activity is as effective in binding to the antibody as is native HSV-1 TK. Viral TK is detectable at ranges of 150-460 ng/mg protein of cell extract from infected cells or cells transformed by HSV or HSV genetic material. HSV-2 TK appears highly cross-reactive, VZV TK is slightly less so, and the vaccinia TK shows little or no cross-reactivity. This RIA may serve as a tool for monitoring the expression of the HSV TK during an active herpes virus infection, a latent ganglionic infection, or in neoplastic cells which may have arisen by viral transformation.