DNA-dependent DNA Polymerase Activity Research Articles

Detection of specific RNA targets by multimerization

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to an essential role of RNA in realization of genetic information and diseases development. Here, we report on an approach for detection of RNA targets based on a particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was approved using genetic material of SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate SARS-CoV-2 RNA-positive samples from SARS-CoV-2 negative samples with high reliability. The proposed technique determines detection of RNA even in samples, which undergone multiple freezing.

Retroviral reverse transcriptase: Structure, function and inhibition.

Reverse transcriptase (RT) is a multifunctional enzyme that has RNA- and DNA-dependent DNA polymerase activity and ribonuclease H (RNase H) activity, and is responsible for the reverse transcription of retroviral single-stranded RNA into double-stranded DNA. The essential role that RT plays in the human immunodeficiency virus (HIV) life cycle is highlighted by the fact that multiple antiviral drugs-which can be classified into two distinct therapeutic classes-are routinely used to treat and/or prevent HIV infection. This book chapter provides detailed insights into the three-dimensional structure of HIV RT, the biochemical mechanisms of DNA polymerization and RNase H activity, and the mechanisms by which nucleoside/nucleotide and nonnucleoside RT inhibitors block reverse transcription.

Chromenone derivatives as a versatile scaffold with dual mode of inhibition of HIV-1 reverse transcriptase-associated Ribonuclease H function and integrase activity

A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1 (HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-hydroxy-2H, 5H-pyrano (3, 2-c) chromene–2, 5–dione derivatives were synthesized. The modeling studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds.

Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time.

Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radio-labeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT's binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 μM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4

Family A DNA polymerase (K4PolI) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4PolI showed thermostable DNA-dependent DNA polymerase activity with 3'-5' exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated by in silico modeling to understand the binding situation between K4PolI and template DNA. Nine amino acids in the 3'-5' exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2' hydroxy group of ribose. To allow K4PolI to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2' hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3'-5' exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3'-5' exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase.

Polymerase-Dependent DNA Synthesis from Phosphoramidate-Activated Nucleotides

Nucleoside triphosphate mimetics, which are substrates for polymerases, can be used in the enzymatic synthesis of nucleic acids. Alternatively, they might also become reversible or irreversible enzyme inhibitors. In order to analyze the effects of 5′-phosphoramidate modification of deoxynucleotide in DNA synthesis, 3-phosphono-L-Ala-dNMP (N = A, T, or G) were evaluated as substrates of HIV-1 RT, Vent (exo−), and Therminator polymerase, respectively. The DNA-dependent DNA polymerase activity is significantly higher for Vent exo− polymerase than for HIV-1 RT, which is reflected by the capacity of Vent exo− polymerase to efficiently synthesize DNA without stalling effects. In addition, Vent (exo−) polymerase proved to be more accurate than Therminator polymerase, based on Watson–Crick base-pairing. The optimal yield (88%–97%) of full-length elongation can be obtained in 60 minutes by Vent (exo−) polymerase at 0.025 U/μL, with the phosphoramidate analogues as substrates. These data led us to conclude that the optimal pyrophosphate mimetic for the enzyme-catalyzed synthesis of DNA is polymerase dependent.

Kinetic Basis of Sugar Selection by a Y-Family DNA Polymerase from Sulfolobus solfataricus P2

DNA polymerases use either a bulky active site residue or a backbone segment to select against ribonucleotides in order to faithfully replicate cellular genomes. Here, we demonstrated that an active site mutation (Y12A) within Sulfolobus solfataricus DNA polymerase IV (Dpo4) caused an average increase of 220-fold in matched ribonucleotide incorporation efficiency and an average decrease of 9-fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000-fold in sugar selectivity. Thus, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation. Other than synthesizing DNA as the wild-type Dpo4, the Y12A Dpo4 mutant incorporated more than 20 consecutive ribonucleotides into primer/template (DNA/DNA) duplexes, suggesting that this mutant protein possesses both a DNA-dependent DNA polymerase activity and a DNA-dependent RNA polymerase activity. Moreover, the binary and ternary crystal structures of Dpo4 have revealed that this DNA lesion bypass polymerase can bind up to eight base pairs of double-stranded DNA which is entirely in B-type. Thus, the DNA binding cleft of Dpo4 is flexible and can accommodate both A- and B-type oligodeoxyribonucleotide duplexes as well as damaged DNA.

The Acyclic 2,4-Diaminopyrimidine Nucleoside Phosphonate Acts as a Purine Mimetic in HIV-1 Reverse Transcriptase DNA Polymerization

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

Potential Role of Group IIC-attCIntrons in Integron Cassette Formation

Integrons are natural expression vectors in which gene cassettes are integrated downstream of a promoter region by a site-specific recombinase. Gene cassettes usually consist of a single gene followed by a recombination site designated attC. A major unanswered question is how a gene becomes associated with an attC site. Here, we investigate the potential role of a specific lineage of group IIC introns, named group IIC-attC, in cassette formation. Group IIC-attC introns preferentially target attC while retaining the ability to target transcriptional terminators. We show using a PCR-based mobility assay with Escherichia coli that the S.ma.I2 intron from the genome of a clinical isolate of Serratia marcescens can target both attC site and putative terminator motifs of resistance genes. Quantitative results showed that S.ma.I2 is more efficient in targeting various attC sequences than three group IIC-attC introns (54 to 64% sequence identity) from the genomes of environmental isolates. We also show that purified group IIC-attC intron-encoded reverse transcriptases have both RNA-dependent and DNA-dependent DNA polymerase activities in vitro. These data permit us to suggest a new model for gene cassette formation, in which a group IIC-attC intron targets separately a transcriptional terminator adjoining a gene and an isolated attC, joins the gene and the attC by homologous recombination, and then splices and reverse transcribes a gene-attC RNA template, leading to the formation of a cassette.

Mechanism of Inhibition of HIV-1 Reverse Transcriptase by the Novel Broad-Range DNA Polymerase Inhibitor N-{2-[4-(Aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide,

Employing a novel strategy, we have virtually screened a large library of compounds to identify novel inhibitors of the reverse transcriptase (RT) of HIV-1. Fifty-six top scored compounds were tested in vitro, and two of them inhibited efficiently the DNA polymerase activity of RT. The most effective compound, N-{2-[4-(aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide (NAPETA), inhibited both RNA-dependent and DNA-dependent DNA polymerase activities, with apparent IC50 values of 1.2 and 2.1 microM, respectively. This inhibition was specific to the RT-associated polymerase activity and did not affect the RNase H activity. NAPETA also inhibited two drug-resistant HIV-1 RT mutants as well as HIV-2 RT and other DNA polymerases. Kinetic analysis of RT inhibition indicated that the DNA polymerase activity of HIV-1 RT was inhibited in a classic noncompetitive manner with respect to dTTP, demonstrating a Ki value of 1.2 microM. In contrast, the inhibition with respect to the RNA.DNA template was a mixed linear type with a Ki value of 0.12 microM and was not affected by the order in which the template.primer and inhibitor were added to the reaction mixture. Gel shift and surface plasmon resonance analyses confirmed that NAPETA interfered with the formation of the RT.DNA complex (that is crucial for the polymerization activity) by reducing the affinity of RT for DNA, accounting at least partially for the inhibition. It is likely that NAPETA inhibited RT via a mechanism that is different from that of the classic non-nucleoside RT inhibitors used for treating AIDS/HIV patients and, thus, may serve as a lead compound for the development of novel anti-HIV drugs.

Ribavirin and mycophenolic acid markedly potentiate the anti-hepatitis B virus activity of entecavir

MPA [the active metabolite of the immuno-suppressive agent CellCept] and ribavirin markedly potentiate the anti-HBV activity of the guanine-based nucleoside analogue entecavir (ETV) against both wild-type HBV and a lamivudine-resistant variant. Ribavirin (in its 5′-monophosphate form) and MPA are inhibitors of IMP-dehydrogenase and cause depletion of intracellular dGTP pools. The active triphosphorylated form of ETV may inhibit more efficiently the priming reaction, reverse transcription and DNA-dependent DNA polymerase activity of the HBV polymerase in the presence of reduced levels of dGTP. The potential for enhanced ETV activity is supported by the observation that exogenously added deoxyguanosine reversed the potentiating effect of ribavirin and MPA. Our observations may have important implications for those (liver) transplant recipients that receive MMF as part of their immunosuppressive regimen and who, because of a de novo or a persistent infection with HBV need antiviral therapy such as ETV. Further studies will need to be conducted to determine if combining ribavirin (a compound used for the treatment of HCV infections) with ETV could have an advantage for the treatment of HBV infections, in particular in patients co-infected with HCV.

Chlamydial DNA polymerase I can bypass lesions in vitro

We found that DNA polymerase I from Chlamydiophila pneumoniae AR39 (CpDNApolI) presents DNA-dependent DNA polymerase activity, but has no detectable 3′ exonuclease activity. CpDNApolI-dependent DNA synthesis was performed using DNA templates carrying different lesions. DNAs containing 2′-deoxyuridine (dU), 2′-deoxyinosine (dI) or 2′-deoxy-8-oxo-guanosine (8-oxo-dG) served as templates as effectively as unmodified DNAs for CpDNApolI. Furthermore, the CpDNApolI could bypass natural apurinic/apyrimidinic sites (AP sites), deoxyribose (dR), and synthetic AP site tetrahydrofuran (THF). CpDNApolI could incorporate any dNMPs opposite both of dR and THF with the preference to dAMP-residue. CpDNApolI preferentially extended primer with 3′-dAMP opposite dR during DNA synthesis, however all four primers with various 3′-end nucleosides (dA, dT, dC, and dG) opposite THF could be extended by CpDNApolI. Efficiently bypassing of AP sites by CpDNApolI was hypothetically attributed to lack of 3′ exonuclease activity.

Evidence That HIV-1 Reverse Transcriptase Employs the DNA 3′ End-directed Primary/Secondary RNase H Cleavage Mechanism during Synthesis and Strand Transfer

We previously analyzed strand transfers catalyzed by human immunodeficiency virus, type 1 reverse transcriptase (RT) in a hairpin-containing RNA template system. In this system, RT produces a series of adjacent RNase H cuts before the hairpin base on the first, or donor template that clears a region of the donor, facilitating invasion by the second, or acceptor RNA. Here we analyze characteristics of the prominent cuts before the hairpin base and their role in strand transfers. Analysis of the template cleavage pattern during synthesis suggested that the RT performs DNA 3' end-directed primary and secondary cuts while paused at the hairpin base and that these cuts contribute to creation of the invasion site. RT catalyzed similar cleavages on a substrate representing a paused cDNA-template intermediate. DNA 3' end-directed secondary cuts, which require positioning of the polymerase active site downstream of the primer terminus, had previously not been specifically identified during synthesis. Our findings indicate that during synthesis DNA 3' end-directed primary and secondary cuts occur at pause sites. RT mutants with substitutions at the His(539) residue in the RNase H active site were defective in secondary cleavages. Analysis of the template cleavage pattern generated by the His(539) mutants during synthesis revealed inefficient cleavage at the invasion site, correlating with defects in strand transfer. Overall, results indicate RT can catalyze pause-associated DNA 3' end-directed primary and secondary cuts during synthesis and these cuts can contribute to strand transfer by creation of an invasion site.

Effects of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis on HIV-1 reverse transcriptase.

It has been recently demonstrated that HIV-1 reverse transcriptase is the target of two diterpenes, (6 R)-6-hydroxydichotoma-3,14-diene-1,17-dial (compound 1) and (6 R)-6-acetoxydichotoma-3,14-diene-1,17-dial (compound 2), that inhibit HIV-1 replication in vitro. In this work, the effects of both diterpenes on the kinetic properties of the recombinant HIV-1 reverse transcriptase (RT) enzyme were evaluated. RNA-dependent DNA-polymerase (RDDP) activity assays demonstrated that both diterpenes behave as non-competitive inhibitors with respect to dTTP and uncompetitive inhibitors with respect to poly(rA).oligo(dT) template primers. The K(i) values obtained for compounds 1 and 2 were 10 and 35 microM, respectively. Neither of these diterpenes affected the DNA-dependent DNA-polymerase (DDDP) activity of the HIV-1 RT. The RDDP activities of AMV-RT and MMLV-RT enzymes were also inhibited by compounds 1 and 2. In contrast to the HIV-1 enzyme, the DDDP activities of AMV-RT and MMLV-RT enzymes were significantly reduced by compound 1. Taken together, our results demonstrate that compound 1 is a more effective inhibitor of the viral reverse transcriptases from HIV-1, AMV and MMLV than compound 2. The kinetic behavior analyses of the HIV-1 RT demonstrate that both diterpenes have similar mechanisms of inhibition of RDDP activity.

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives β-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). β-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 μM, while the IC50 for Escherichia coli and human RNases H was 50 μM and 5.7 μM, respectively. In contrast, the related tropolone analog β-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 μM and 40 μM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both β-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 μM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between β-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

Mechanism of action and resistant profile of anti-HIV-1 coumarin derivatives

Dicamphanoyl khellactone (DCK) is a coumarin derivative that can potently inhibit HIV-1 replication. DCK does not inhibit RNA-dependent DNA synthesis. However, an HIV reverse transcriptase (RT) inhibitor-resistant strain, HIV-1/RTMDR1, is resistant to DCK. Thus, it is possible that HIV-1 RT is the target of DCK. To test this possibility, DCK-resistant viruses were selected in the presence of DCK. Our results indicate that a single amino acid mutation, E138K in HIV-1 RT, is sufficient to confer DCK resistance. Interestingly, a DCK derivative, 3′R,4′R-Di- O-(-)-camphanoyl-2-ethyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (DCP8), is effective against HIV-1/RTMDR1. However, the DCK-escape virus carrying the E138K mutation remains resistant to DCP8. Since DCK did not inhibit the RNA-dependent DNA polymerase activity of HIV-1 RT when using poly-rA or poly-rC as template, we evaluated the effect of DCK on the DNA-dependent DNA polymerase activity of HIV-1 RT. Our results indicate that DCK can inhibit the DNA-dependent DNA polymerase activity of HIV-1 RT. In conclusion, DCK is a unique HIV-1 RT inhibitor that inhibits the DNA-dependent DNA polymerase activity. In contrast, DCK did not significantly affect the RNA-dependent DNA polymerase activity when poly-rA or poly-rC was used as templates. An E138K mutation in the non-nucleoside RT inhibitors (NNRTIs) binding pocket of HIV-1 RT confers resistance to DCK and its chromone derivative, DCP8.