Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

Extending recognition by peptide nucleic acids (PNAs): binding to duplex DNA and inhibition of transcription by tail-clamp PNA-peptide conjugates.

Peptide nucleic acids (PNAs) are a powerful tool for recognition of double-stranded DNA. Strand invasion is most efficient when pyrimidine PNAs are linked to form a bisPNA in which one strand binds by Watson-Crick base pairing while the other binds by Hoogsteen base pairing to the newly formed PNA-DNA duplex. Within many genes, however, polypyrimidine target sequences may not be located in optimal positions relative to transcription factor binding sites, and this deficiency may complicate attempts to identify potent antigene PNAs. To increase the versatility of strand invasion by PNAs, we have synthesized bisPNAs and bisPNA-peptide conjugates containing a mixed base extension of the Watson-Crick polypyrimidine strand. We find that these tail-clamp PNAs (TC-PNAs) bind duplex DNA and inhibit transcription. DNA recognition occurs with single-stranded or TC-bisPNAs and requires attachment of positively charged amino acids. Association rate constants, k(a), for binding to DNA by TC-PNAs are as high as 35000 M(-1) s(-1) and are usually only a fewfold lower than for analogous PNAs that lack mixed base extensions. The ability to bind duplex DNA is not always necessary for inhibition of transcription, possibly because PNAs can bind to accessible DNA within the transcription bubble created by RNA polymerase. These results, together with similar findings independently obtained by Nielsen and colleagues [Bentin, T., Larsen, H. J., and Nielsen, P. E. (2003) Biochemistry 42, 13987-13995], expand the range of sequences within duplex DNA that are accessible to PNAs and suggest that TC-PNA-peptide conjugates are good candidates for further testing as antigene agents.

Cross-linking of 2-deoxyribonolactone and its beta-elimination product by base excision repair enzymes.

2-Deoxyribonolactone (3) is produced in DNA as a result of reaction with a variety of DNA damaging agents. The lesion undergoes beta-elimination to form a second metastable electrophilic product (4). In this study, DNA containing 2-deoxyribonolactone (3) and its beta-elimination product (4) are generated at specific sites using a photolabile nucleotide precursor. 2-Deoxyribonolactone is not incised by any of the 8 AP lyases tested. One enzyme, Escherichia coli endonuclease III, cross-links to 3, and the lesion strongly inhibits excision of typical abasic sites by this enzyme. Two of the enzymes, FPG and NEIL1 known to cleave normal abasic sites (1) by effecting beta,delta-elimination form cross-links to the butenolide lesion (4). The observed results are ascribable to characteristics of the enzymes and the lesions. These enzymes are also important for the removal of oxidative base lesions. These results suggest that high concentrations of 3 and 4 may exert significant effects on the repair of normal AP site and oxidative base lesions in cells by reducing the cellular activity of these BER enzymes either via cross-linking or competing with binding to the BER enzymes.

L-methionine inhibits reaction of DNA with anticancer cis-diamminedichloroplatinum(II).

Sufficient evidence has accumulated to identify DNA as the relevant pharmacological target of antitumor cisplatin [cis-diamminedichloroplatinum(II)]. This drug is administered intravenously so that before it reaches DNA in the nucleus of tumor cells it may interact with various compounds including sulfur-containing molecules such as L-methionine or the compounds containing these residues. L-Methionine increases the rate of reaction of cisplatin with monomeric guanosine 5'-monophosphate, and it was suggested on the basis of these results previously obtained by other authors that methionine residues could mediate the transfer of platinum onto DNA. We studied in the present work the reactions of the 1:1 complex formed between cisplatin and L-methionine or N-acetyl-L-methionine with synthetic, single- and double-stranded oligodeoxyribonucleotides and natural, high molecular mass DNA by using high-pressure liquid chromatography and flameless atomic absorption spectrophotometry. The results demonstrate that both L-methionine and N-acetyl-L-methionine decrease the rate of reaction of cisplatin with base residues in natural, high molecular mass DNA. Thus, the possibility that cisplatin bound to methionine residues serves as a drug reservoir available for platination of DNA in the nucleus of tumor cells appears unlikely.

Inhibition of unwinding of G-quadruplex structures by Sgs1 helicase in the presence of N,N'-bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide, a G-quadruplex-interactive ligand.

N,N'-Bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide (PIPER), a perylene derivative, is a very potent and selective G-quadruplex DNA-interactive agent. It has been shown to inhibit DNA polymerase and telomerase by stacking externally to the G-tetrads in the G-quadruplex structures. Recently, we have demonstrated that this small molecule greatly accelerates the assembly of G-quadruplex structures in a cell-free system. In this report, we present data demonstrating that PIPER prevents the unwinding of G-quadruplex structures by yeast Sgs1 helicase. Sgs1 belongs to the RecQ DNA helicase family whose members include other G-quadruplex DNA unwinding helicases, such as human Bloom's syndrome and human Werner's syndrome helicases. PIPER specifically prevents the unwinding of G-quadruplex DNA but not duplex DNA by Sgs1. Competition experiments indicate that this inhibitory activity is due to the interaction of PIPER with G-quadruplex structures rather than the helicase itself. These results combined with previous studies suggest a possible mechanism of action for these G-quadruplex-interactive agents inside cells: they might induce G-quadruplex formation in G-rich regions on genomic DNA, stabilize these structures, and prevent them from being cleared by enzymes such as helicases. The G-quadruplex structures may, in turn, disrupt some critical cellular events such as DNA replication, transcription regulation, and telomere maintenance.