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Biomedical subjects

G A Nevinsky

Publications and source records attributed to G A Nevinsky.

At least 37 records · Page 2Linked 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↗
PubMed2000

Natural catalytic antibodies (abzymes) in normalcy and pathology.

This review summarizes literature data on natural abzymes. Peculiar features of their functioning and substrate specificity are considered in comparison with traditional enzymes. Working hypotheses on the possible biological roles of natural abzymes in autoimmune processes and diseases accompanied by disorders of immune status are analyzed.

Antibodies, Catalytic↗
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

Human milk lactoferrin binds two DNA molecules with different affinities.

Evidence is presented that lactoferrin (LF), an Fe3+-binding glycoprotein, possesses two DNA-binding sites with different affinities for specific oligonucleotides (ODNs) (Kdl = 8 nM; Kd2 approximately 0.1 mM). The high affinity site became labeled after incubation with affinity probes for DNA-binding sites; like the antibacterial and polyanion-binding sites, this site was shown to be located in the N-terminal domain of LF. Interaction of heparin with the polyanion-binding site inhibits the binding of ODNs to both sites. These data suggest that the DNA-binding sites of LF coincide or overlap with the known polyanion and antimicrobial domains of the protein.

Anti-Infective Agents↗
PubMed1999

Structural requirements of double and single stranded DNA substrates and inhibitors, including a photoaffinity label, of Fpg protein from Escherichia coli.

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E. coli catalyzes excision of several damaged purine bases, including 8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine from DNA. In this study the interaction of E. coli Fpg with various specific and nonspecific oligodeoxynucleotides was analyzed. Fpg was shown to remove 8-oxoguanine efficiently, not only from double-stranded, but also from single-stranded oligodeoxynucleotides. The Michaelis constants (KM) of a range of single-stranded oligodeoxynucleotides (0.55-1.3 microM) were shown to be 12-170 times higher that those for corresponding double-stranded oligodeoxynucleotides (KM = 6-60 nM). Depending on the position of the 8-oxoguanine within the oligodeoxynucleotides, relative initial rates of conversion of single-stranded substrates were found to be lower than, comparable to, or higher than those for double-stranded oligodeoxynucleotides. The enzyme can interact effectively not only with specific, but also with nonspecific single-stranded and double-stranded oligodeoxynucleotides, which are competitive inhibitors of the enzyme towards substrate. Fpg became irreversibly labeled after UV-irradiation in the presence of photoreactive analogs of single-stranded and double-stranded oligodeoxynucleotides. Specific and nonspecific single-stranded and double-stranded oligodeoxynucleotides essentially completely prevented the covalent binding of Fpg by the photoreactive analog. All these data argue for similar interactions occurring in the DNA binding cleft of the enzyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude. Addition of the second complementary chain increases the affinity of the first single-stranded chain by a factor of approximately 10. It is concluded that Michaelis complex formation of Fpg with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which is found to lie in the kcat term for catalysis; the reaction rate being increased by 6-7 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides.

Base Sequence↗
PubMed1999

Human milk lactoferrin binds ATP and dissociates into monomers.

The physiological role of lactoferrin (LF) is still unclear, but it has been suggested to be responsible for primary defence against microbial infections. Many different unique functions have been attributed to LF, including DNA and RNA binding, and transport into the nucleus, where LF binds to specific DNA sequences and activates transcription. Here we present evidence that in addition to the above (and below) mentioned functions LF binds ATP with a stoichiometry of 1 mole of nucleotide per mole of the protein and a Kd = 0.3 mM. The ATP-binding site is localized in the C-terminal domain of LF, in contrast to the antibacterial and polyanion-binding sites, which are located in the N-terminal domain. Binding of ATP by LF leads to dissociation of its oligomeric forms and to a change of the protein's interaction with polysaccharides, DNA and proteins.

Adenosine Triphosphate↗
PubMed1999

Single-stranded oligodeoxyribonucleotides are substrates of Fpg protein from Escherichia coli.

The interaction of Escherichia coli Fpg protein, which catalyzes excision of several damaged purine bases including 8-oxoguanine (oxoG) from DNA with a set of single- (ss) and double-stranded (ds) 23-mer oligodeoxyribonucleotides (ODNs) containing 8-oxoguanine(s) at various positions, has been investigated. The affinities of different ss ODNs (KM = 0.55-1.3 microM) were shown to be 12-170 times less than those for corresponding ds ODNs (KM = 6-60 nM). Depending on the position of the oxoG within the ODNs, relative initial rates of conversion of ss substrates may be less than, comparable, or greater than those for ds ODNs. The enzyme can remove 5'-terminal oxoG from ODNs only if the 5'-end is phosphorylated. Fpg does not release oxoG residues from the ultimate and penultimate 3'-terminal positions. Duplexes containing two adjacent oxoG are poor substrates for the glycosylase.

DNA↗
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↗
PubMed1999

Possibilities of the method of step-by-step complication of ligand structure in studies of protein--nucleic acid interactions: mechanisms of functioning of some replication, repair, topoisomerization, and restriction enzymes.

X-Ray structure analysis is one of the most informative methods for investigation of enzymes. However, it does not provide quantitative estimation of the relative efficiency of formation of contacts revealed by this method, and when interpreting the data this does not allow taking into account the relative contribution of some specific and nonspecific interactions to the total affinity of nucleic acids (NA) to enzymes. This often results in unjustified overestimation of the role of specific enzyme--NA contacts in affinity and specificity of enzyme action. In recent years we have developed new approaches to analysis of the mechanisms of protein--nucleic acid interactions allowing quantitative estimation of the relative contribution of virtually every nucleotide unit (including individual structural elements) to the total affinity of enzymes to long DNA and RNA molecules. It is shown that the interaction between enzymes and NA on the molecular level can be successfully analyzed by the methods of synthesis and analysis, that is, step-by-step simplification or complication of the structure of a long NA-ligand. This approach allows the demonstration that complex formation including formation of contacts between enzymes and specific NA units can provide neither high affinity of the enzymes to NA nor the specificity of their action. Using a number of sequence-independent replication and repair enzymes specifically recognizing a modified unit in DNA and also some sequence-dependent topoisomerization and restriction enzymes as examples, it was shown that virtually all nucleotide units within the DNA binding cleft interact with the enzyme, and high affinity mainly (up to 5-7 of 7-10 orders of magnitude) is provided by many weak additive interactions between these enzymes and various structural elements of the individual NA nucleotide units. At the same time, the relative contribution of specific interactions to the total affinity of NA is rather small and does not exceed 1-2 orders of magnitude. Specificity of enzyme action is provided by the stages of the enzyme-dependent NA adaptation to the optimal conformation and directly of catalysis: kcat increases by 3-7 orders of magnitude when changing from nonspecific to specific NA. In the present work we summarized our experience in studies of enzymes by the method of step-by-step complication of the ligand structure and performed a detailed analysis of the features of this approach and its possibilities for the study of protein--nucleic acid interactions on the molecular level.

Binding Sites↗
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

Catalytic DNA- and RNA-hydrolyzing antibodies from milk of healthy human mothers.

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab')2 and Fab fragments. Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of catalytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infections until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.

Adult↗
PubMed1998

Secretory immunoglobulin A from human milk catalyzes milk protein phosphorylation.

This article presents evidence that protein kinase activity is an intrinsic property of secretory immunoglobulin A (sIgA) from milk of healthy human mothers. Polyclonal sIgA was purified by sequential chromatography on protein A-Sepharose, DEAE-cellulose, and gel filtration on Toyopearl HW-55 and Sepharose 4B columns. Its purity was established by one- and two-dimensional SDS-PAGE. The protein kinase activity was inhibited by specific antibodies (Abs) against sIgA, and was stable to acidic and alkaline conditions. Catalytic sIgA showed optimal reaction conditions (pH and MgCl2 concentration) and substrate specificity different from those of known protein kinases; i.e., sIgA phosphorylated the serine residues of various milk proteins in the presence of different gamma-[32P]nucleoside- and deoxynucleoside-5'-triphosphates. The homogeneous Fab fragment of sIgA also showed kinase activity. An ATP-binding activity of fractions of sIgA was demonstrated by affinity chromatography on ATP-Sepharose and by covalent binding of an affinity analog of ATP; this activity was mediated by the L chain of sIgA. The authors believe these observations are the first example of the catalytic activity of IgA Abs and of natural catalytic Abs with synthetic activity. In addition, the findings suggest the likelihood that catalytic Abs are generated by the immune system of healthy mothers.

Adult↗
PubMed1998

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

The interaction of human DNA topoisomerase I (topo I) with specific sequence oligodeoxynucleotides (ODNs) of different length and structure has been investigated. All the ODNs used were shown to be effective enzyme inhibitors and to inhibit the topo I catalyzed relaxation of scDNA in a competitive manner. Among two DNA regions (A and B) required for topo I-mediated DNA cleavage, the former was found to display the higher affinity for the enzyme. The enzyme's affinity for ODNs corresponding to the scissile strand (five and nine nucleotide units in length) is about 2-4 orders of magnitude higher than that for non-specific ODNs of the same length. Topo I can efficiently recognize even extremely short specific ODNs containing only two or three bases (AGA and pAG, Ki = 15 and 60 microM, respectively): the sequence AAGA (Ki = 10 microM) is essential for tight DNA binding to topo I. The affinities of ODNs corresponding to the non-scissile strand are significantly lower. The ligand's affinity increases with its length. Additionally, about a ten-fold enhancement of specific sequence affinity occurs due to stable duplex formation during enzyme preincubation with ligands before addition of scDNA. We believe the possibility of using the short specific oligonucleotides and its derivatives as topoisomerase I-targeting drugs could not be excluded.

Base Sequence↗
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↗
PubMed1998

Role of DNA definite structural elements in interaction with repair enzyme uracil-DNA glycosylase.

Interaction of different oligodeoxyribonucleotides (oligos), their analogs and oligonucleopeptide with uracil-DNA glycosylase (UDG) from human placenta was investigated. It is shown that there is no considerable contribution of heterocyclic bases of DNA to UDG-substrate binding but the UDG interaction with some DNA phosphate groups is necessary for enzyme-substrate recognition. However the phosphate group adjacent to single dU from the 3'-end in oligo is not involved into the electrostatic contact with UDG. It is found that UDG has the high affinity to its reaction product. An oligonucleotide containing a single 2'-deoxy-2'-aminouridine is a non-hydrolyzable substrate analog for UDG.

DNA↗