Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay.     

Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Three-dimensional modeling of cytomegalovirus DNA polymerase and preliminary analysis of drug resistance

Cytomegalovirus (CMV) is the leading cause of congenital infection and a frequent opportunistic agent in immunocompromised hosts such as transplant recipients and AIDS patients. CMV DNA polymerase, a member of the polymerase B family, is the primary target of all available antivirals (ganciclovir, cidofovir, and foscarnet) and certain variations of this enzyme could lead to drug resistance. However, understanding the drug resistance mechanisms at the atomic level is hampered by the lack of its three-dimensional (3D) structure. In the present work, 3D models of two different conformations (closed and open) for CMV DNA polymerase have been built based on the crystal structures of bacteriophage RB69 DNA polymerase (a member of the polymerase B family) by using the 3D-Jury Meta server and the program MODELLER. Most of the variations on CMV DNA polymerase pertinent to ganciclovir/cidofovir and foscarnet resistance can be explained well based on the open and closed conformation models, respectively. These results constitute a first step towards facilitating our understanding of drug resistance mechanisms for CMV and the interpretation of novel viral mutations.     

Identification of a DNA segment containing the human DNA polymerase alpha gene

We isolated a temperature-sensitive mutant from mouse FM3A cells, designated as tsFT20, the DNA polymerase alpha activity of which is heat-labile. A hybrid clone (M6-39 cells) between human cells and tsFT20 cells contained one or two human chromosomes. M6-39 cells (primary hybrid) were exposed to gamma-ray and re-fused with tsFT20, after which we isolated two temperature-resistant secondary hybrids, both of which retained an identical minute portion of the human chromosome, 400-500 kilobase pairs (kbp). Immunological studies demonstrated that this secondary hybrid expressed human DNA polymerase alpha. Thus, the human DNA polymerase alpha gene was located within a DNA region of 400-500 kbp.     

Sequence analysis of human cytomegalovirus US28 gene in low-passage clinical isolates from children and AIDS patients

Human cytomegalovirus (HCMV) is often a dangerous opportunistic pathogen that causes significant morbidity and mortality in newborn children and immunocompromised patients. The different symptoms and tissue tropisms of HCMV infection may result from genetic polymorphism. This study investigated the sequence variability of the HCMV US28 ORF, which shows sequence homology to the G protein-coupled receptor. HCMV isolated from suspected pediatric cases and isolates from AIDS patients were compared in order to examine the possible associations between polymorphisms and pathogenesis. Seventy children with suspected congenital HCMV infection, who suffered from jaundice (47), megacolon (10), and microcephaly (13), and 17 AIDS patients, were studied. Mutation was prevalent among the sequences of US28, with a focus on the two ends of US28. The important functional groups of US28 are highly conserved. An unrooted tree showed that all sequences from suspected congenitally infected infants and AIDS patients were divided into three groups. Comparison showed that most of the sequences (12/17) from pediatric patients were included in the first group (G1), whereas most of the sequences (11/17) from AIDS patients were included in the third group (G3). The specific high mutation sites in US28 from children were located at the C terminus of the protein, whereas those from AIDS patients were located at the N terminus. We demonstrated the existence of polymorphisms among the US28 genes of clinical isolates of HCMV from infants with suspected congenital infection. Comparison of US28 sequences from AIDS patients with those from children showed that both sequences have their own specific high mutation points.     

Inhibition of human cytomegalovirus DNA polymerase by C-terminal peptides from the UL54 subunit

In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially alpha-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.     

Sequence of human DNA polymerase beta mRNA obtained through cDNA cloning

A cDNA library from polyA+ RNA of a human teratocarcinoma cell line in phage lambda gt11 was screened with a fragment of the rat beta-polymerase cDNA, lambda pol beta-10, as probe. Five positive phage were identified and plaque purified. The cDNA of one positive clone selected for detailed study was 1257 bp. This insert was sequenced and found to contain the coding region for beta-polymerase, as well as 163 bp and 137 bp from the 5' and 3' untranslated regions, respectively. The primary structure of human beta-polymerase (318 amino acids, Mr = 36, 133) deduced from the cDNA was similar to rat beta-polymerase (95% matched residues). The greatest difference between the sequences of the human and rat cDNAs was in the 3' untranslated regions (64% matched base residues). These results provide necessary sequence information for study of the human beta-polymerase gene.     

Sequence and analysis of the gene for bacteriophage T3 RNA polymerase.

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.     

Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota

Humans possess four Y-family polymerases: pols eta, iota, kappa and the Rev1 protein. The pivotal role that pol eta plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding pol eta), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols iota, kappa and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of pol iota to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that pol iota can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, pol iota efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of approximately 10(2) over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Pol kappa has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of pol iota and pol kappa result in a significant increase in bypass of T-T CPDs when compared to pol iota alone. Our data suggest that under certain conditions and sequence contexts, pol iota can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochemical activities may, therefore, be of biological significance especially in XP-V cells lacking the primary T-T CPD bypassing enzyme, pol eta.     

Structure-activity relationships of acyloxyamidine cytomegalovirus DNA polymerase inhibitors

This paper describes the structure activity relationships of a new class of cytomegalovirus DNA polymerase inhibitors having two aryl groups joined by an acyloxyamidine linker. Examination of a series of analogues in which the terminal groups are varied revealed a very narrow SAR around the 2,4-dichlorophenyl group of the lead compound, but a variety of replacements for the benzothiazole ring are compatible with activity. The most notable of these is the isoxazole ring of compound 78, which provides a 30-fold enhancement in potency compared to the lead compound. We also describe the design, synthesis and evaluation of 10 analogues in which the acyloxyamidine linker is modified or replaced by an isosteric group. Structure-activity relationship studies identified the linker -NH2 group as a critical pharmacophoric element. Ab initio molecular orbital calculations combined with qualitative estimates of steric interaction energies suggest that the lowest energy conformations of the acyloxyamidine linker are characterized by an extended planar CAr-C=N-O-C arrangement and either a syn-periplanar or anti-periplanar N-O-C-C(Ar') arrangement. Only the anti-periplanar conformation was observed in the crystal structures of three acyloxyamidines. The most active of the linker-modified compounds designed on the basis of these studies is the amidine carbamate 20, which is approximately one-third as potent in the cytomegalovirus DNA polymerase inhibition assay as the comparator acyloxyamidine 53. The activity of 20 suggests that acyloxyamidines may bind to the cytomegalovirus DNA polymerase via an anti-periplanar conformation similar to that observed in the crystal structure of acyloxyamidine 36.