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O D Zakharova

Publications and source records attributed to O D Zakharova.

At least 19 recordsLinked to original sources
PubMed2000

Inhibition of HIV-1 integrase-catalysed reaction by new DNA minor groove ligands: the oligo-1,3-thiazolecarboxamide derivatives.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the retrovirus, responsible for catalysing the insertion of the viral genome into the host cell chromosome. For this reason it provides an attractive target for antiviral drug design. We synthesized a series of novel thiazole (Tz)-containing oligopeptides (TCOs; oligo-1,3-thiazolecarboxamides), specifically interacting within the minor groove of DNA. The oligocarboxamide derivatives contained 1-4 Tz rings and different N- and C-terminal groups. The effect of these oligocarboxamides on the HIV-1 IN-catalysed reaction was investigated. Some of the compounds were able to inhibit the reaction. The inhibitory effect of the TCOs increased with the number of Tz units. The structure of various additional positively and/or negatively charged groups attached to the N- and C-termini of TCOs had a pronounced effect on their interaction with the DNA substrate complexed to IN. Modified TCOs having a better affinity for this complex should provide a rationale for the design of drugs targeting the integration step.

Amides↗
PubMed2000

Synthesis and evaluation of oligo-1,3-thiazolecarboxamide derivatives as HIV-1 reverse transcriptase inhibitors.

A set of oligo-1,3-thiazolecarboxamide derivatives able to interact with the minor groove of nucleic acids was synthesized. These oligopeptides contained different numbers of thiazole units presenting dimethylaminopropyl or EDTA moieties on the C-terminus, and aminohexanoyl or EDTA moieties on the N-terminus. The inhibition of such compounds on HIV-1 reverse transcriptase activity was evaluated using different model template primer duplexes: DNA x DNA, RNA x DNA, DNA x RNA and RNA x RNA. The biological properties of the thiazolecarboxamide derivatives were compared to those of distamycin, another minor groove binder which contains three pyrrole rings. Similar to distamycin, the thiazole containing oligopeptides were good inhibitors of the reverse transcription reaction in the presence of DNA x DNA. But in contrast to distamycin, the oligothiazolide derivatives were able to inhibit reverse transcription in the presence of RNA x DNA or DNA x RNA template primers. Both distamycin and oligothiazolecarboxamides had low affinity for RNA x RNA duplexes. The inhibition obtained with the newly synthesized thiazolecarboxamides showed that these compounds were more powerful and versatile inhibitors of the RT-dependent polymerization than the natural minor groove binder distamycin.

Anti-HIV Agents↗
PubMed1999

High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.

DNA Primers↗
PubMed1999

Interaction of oligonucleotides conjugated to substituted chromones and coumarins with HIV-1 reverse transcriptase.

Ten different pyranone-related substituents (chromones or coumarins) were covalently linked to the 5' end of various oligonucleotides (ODN). The interaction of these compounds with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was analyzed. A different behavior was found to depend on the structure of the oligonucleotide derivatives. Some compounds activated the enzyme at relatively low concentrations (0.1-0.5 microM), followed by an inhibition of the activity at higher concentrations (5-20 microM), whereas others behave just as inhibitors. Because the presence of some coumarin or chromone derivatives conjugated to ODNs enhanced the interaction with the reverse transcriptase, we analyzed the capacity of such ODN derivatives to be used as primers. The introduction of substituent I, a chromone derivative, the 2-[(3-(aminopropyl)amino]-8-isopropyl-5-methyl-4-oxo-4H-1-benzopyran-3-c arbaldehyde], and II, a coumarin derivative, the 1-(3-aminopropoxy)-2-ethyl-3H-naphto[2,1-b]pyran-3-one, into the 5' end of a noncomplementary ODN allowed these compounds to be used as primers. In the case of complementary primers, the presence of conjugated derivatives enhanced the affinity with Km values that were two to three orders of magnitude lower than that of a complementary primer of the same length. After addition of a ddT-unit to the 3'-terminal end of the ODN, some of these primers became very effective inhibitors of RT with Ki values in the nanomolar range.

Chromones↗
PubMed1998

Structural constraints in the HIV-1 reverse transcriptase-primer/template complex for the initiation of DNA synthesis from primer tRNALys3.

The topography and functional implications of the complex formed in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNALys3 were studied in this work. On the basis of previous results showing the high affinity both of the native primer, tRNALys3, as well as that of mismatched short oligonucleotide primers for HIV-1 RT, we synthesized chimeric primers containing tRNALys3 linked to U and T residues of different lengths. We found that the affinity of the oligonucleotide primers for HIV-1 RT is dramatically increased when linked to primer tRNA. Our results also show that in the tRNA.RT complex, before annealing tRNALys3 to the retroviral RNA genome, the 3'-terminal nucleotide of tRNALys3 is positioned at a distance of one nucleotide unit away from the template in the active polymerization site of the enzyme.

Binding Sites↗
PubMed1998

HIV-1 reverse transcriptase is capable of elongating derivatives of sequence specific noncomplementary oligodeoxynucleotides.

We have carried out a comparison of KM and Vmax values for various primers in the polymerization reaction catalyzed by the HIV-1 RT. The affinity of RT for complementary d(pT)6 containing two different 5'-end pyranone derivatives was 2-3 orders of magnitude higher (KM = 3-15 nM) than that of d(pT)6 (KM = 12.6 mM). Oligodeoxynucleotides (ODNs) noncomplementary to poly(A) template were not elongated by RT. However, derivatives of d(CAGGTG) containing the 5'-terminal chromone and coumarin related groups were efficient primers showing KM (30-300 nM) and Vmax (75-93%) values comparable with that for d(pT)10 (800 nM; 100%). The [d(CAGGTG)]ddT ODN derivatives were effective inhibitors of RT. The primer function of derivatives of noncomplementary ODNs appears to be due to the additional interactions of their 5'-terminal groups with the enzyme tRNA-binding site.

Binding Sites↗
PubMed1998

Interaction of tRNA-derivatives and oligonucleotide primers with AZT-resistant mutants of HIV-1 reverse transcriptase.

While the molecular basis of HIV-1 AZT resistance has been widely studied, a biochemical explanation of this process is not well known. No significant changes in the binding affinity of reverse transcriptase (RT) mutants for AZT-triphosphate has been found. Here we analyzed the interaction of wild type and AZT-resistant mutant forms of HIV-1 RT with different primers. Site-directed mutagenesis was used to introduce point mutations on the retroviral enzyme. Primers were either synthetic oligonucleotides or tRNA(Lys3) derivatives containing d(pT)n or r(pU)n at the 3' end. In all cases, determination of kinetic parameters was done in the presence or absence of compounds known to modify protein conformation, such as dimethyl sulfoxide (DMSO), urea, and Triton X-100. Although we found similar K(m) values for all RTs, there was generally an increase in the affinity when enzymes were tested in the presence of DMSO, urea, and Triton X-100. Then, we analyzed the nucleation and elongation steps of the polymerization process. The efficiency of formation of the first base pair was determined by measuring K(m1), the affinity between RT and the 3' terminal nucleotide of the primer. An important difference was found: in the presence of DMSO, urea, and Triton X-100, the K(m1) values for mutated enzymes were higher than those of wild type RTs. Thus, the presence of compounds able to change protein conformation led to a marked destabilization of the interaction of mutated RTs with the 3' terminal nucleotide of the primer. From these results, it can be hypothesized that resistance to AZT is not due to the direct influence of mutations on RT, but rather to conformational changes of the mutated RT in complex with the template-primer altering the ability of the enzyme to select or reject an incoming dNTP.

DNA Primers↗
PubMed1996

The algorithm of estimation of the Km values for primers in DNA synthesis catalyzed by human DNA polymerase alpha.

DNA synthesis with various deoxyribo homo- and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN)n, primers (n = 1-10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template-primer structure in solution was analyzed and the objective laws of the changes of the KM and Vmax values were revealed.

Algorithms↗
PubMed1996

Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.

Animals↗
PubMed1995

High-affinity interaction of human immunodeficiency virus type-1 reverse transcriptase with partially complementary primers.

The comparison of Km and Vmax values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and Vmax, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1-1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT)4(dCdG)k(dT)4 showed higher affinity for the enzyme than (dT)4 or (dT)8 primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNA(3Lys), the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus, besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme.

DNA, Complementary↗
PubMed1995

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

DNA↗
PubMed1995

Interaction of primer tRNA(Lys3) with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation.

In the interaction between HIV-1 RT and tRNA(Lys3) each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo- and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed.

Anticodon↗
PubMed1995

Highly selective affinity labeling of DNA polymerase alpha-primase from human placenta by reactive analogs of ATP.

Highly selective affinity labeling of a DNA-polymerase alpha-primase complex from human placenta by o-formylphenyl esters of ATP, ADP and AMP was performed in a two-step procedure in which a substrate analog attached to the active center was elongated by radioactive ATP. If the covalent attachment is performed in the presence of poly(dT) template, the ATP esters modify selectively the delta subunit of the complex. If poly(dT) is added after the covalent binding of the reagent, both delta and gamma subunits become labeled. With the o-formylphenyl ester of AMP the delta-subunit is modified. The ADP ester modifies both the delta and gamma subunit in the presence and absence of template. It is shown that formylphenyl ester of ATP is not the substrate in the reaction of elongation catalyzed by primase. The data obtained suggest the binding site of initiating substrate to be located in the region of contact of the two subunits of primase. The role of the template in the formation of the active site is discussed.

Adenosine Triphosphate↗
PubMed1993

[Study of the interaction of DNA primase from calf thymus and human placenta with oligonucleotides matrices of various length and structure].

Human placenta DNA-primase as a component of the DNA-polymerase alpha-primase multienzyme complex was examined with a view of establishing the dependence of Km values in the reaction of oligoriboadenylate synthesis from ATP on the length of a poly(dT) template. The pKm values increased linearly up to ten monomeric units of the oligo(dT)n template. These data favour oligo(dT)10 as an optimal template covered by the active site of this enzyme. The DNA-primase catalyzed processively the synthesis at each polymerization cycle of a unique length primer (7-10 nucleotides) as follows from the analysis of the primer length and its distribution with time. It is suggested that the 10 mer DNA-RNA duplex of the template and the primer is a critical size for dissociation of primase and further elongation of the primer by DNA-polymerase in the presence of dNTP.

Animals↗
PubMed1992

Recognition nucleotides for human phenylalanyl-tRNA synthetase.

The specificity of the interaction between tRNAPhe and phenylalanyl-tRNA synthetase isolated from human placenta was investigated. Using yeast tRNAPhe transcripts with different point mutations it was shown that all the five recognition points for the yeast phenylalanyl-tRNA synthetase (G20, G34, A35, A36 and A73) are also important for the reaction catalyzed by the human enzyme. A set of mutations in nucleotides involved in tertiary interactions of tRNAPhe revealed that mutations which maintained the proper folding of the molecule had almost no influence on the efficiency of aminoacylation. The most striking difference between the yeast and human phenylalanyl-tRNA synthetases involved a mutation in the lower two base pairs of the anticodon stem. This mutation did not affect aminoacylation with the yeast enzyme, but greatly reduced activity with human phenylalanyl-tRNA synthetase.

Anticodon↗
PubMed1990

[Phenylalanyl-tRNA-synthase from human placenta: isolation and characteristics].

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from human placenta was isolated and purified using fractionation with polyethyleneglycol and chromatography on hydroxylapatite, heparin-Sepharose and mono-S. The enzyme purified 14800-fold with a 8% yield had a specific activity of 260 U./mg. The molecular mass of the native enzyme as determined by gel filtration was 270 +/- 13 kDa. The molecular masses of the enzyme subunits according to SDS-PAGE data were 74 +/- 4 kDa (alpha-subunit) and 63 +/- 3 (beta-subunit). The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction were 6.6 X 10(-8) M, 8.3 X 10(-5) M and 5.8 X 10(-6) M, respectively.

Chromatography, Ion Exchange↗
PubMed1987

[Effect of diadenosine oligophosphates (Ap4A and Ap3A) and their phosphonate analogs on catalytic properties of phenylalanyl-tRNA synthetase from E. coli].

The influence of P1,P3-bis(5'-adenosyl)triphosphate (Ap3A), P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and its analogues, containing a residue of methylenediphosphonic acid in various positions of the oligophosphate chain, on the reactions catalysed by phenylalanyl-tRNA synthetase from E. coli MRE-600 has been studied. The compounds do not affect significantly the rate of ATP-[32P]PPi-exchange nor maintain this reaction in the absence of ATP. The diadenosineoligophosphates are shown to be noncompetitive inhibitors of ATP in the tRNA aminoacylation by phenylalanine (for Ap4A Ki = 1,45.10(-3) M). The phosphonate analogues of Ap4A inhibit the synthesis of Ap3A depending on their structure. The conclusion is thus drawn that the E. coli MRE-600 phenylalanyl-tRNA synthetase does not interact property with Ap4A and its phosphonate analogues.

Adenine Nucleotides↗