Inhibitor of reproduction of human immunodeficiency virus

 

(57) Abstract:

The proposed use of alkylating derivatives phosphothioate analogues oligodeoxyribonucleotides as an inhibitor of reproduction of HIV. These compounds are characterized by the highest efficiency in the range of the tested derivatives phosphothioate analogues oligodeoxyribonucleotides. The invention is used to inhibit the reproduction of HIV type 1. 2 Il., table 1.

The invention relates to Virology, medicine, namely to the use of alkylating derivatives phosphothioate analogues of oligonucleotides General formula

< / BR>
where is the rest of thymine, cytosine, adenine or guanine; RCl - balance alkylating an amine, such as 4-(N-methyl-N-2-chloroethylamino)benzylmethylamine

< / BR>
n = 12-30, as an inhibitor of reproduction of the human immunodeficiency virus (HIV).

Phosphothioate analogues of oligonucleotides (FTAA) and their derivatives have found wide application as biologically active compounds [1-8]. In its properties, FTA differ from the natural oligonucleotides resistant to the action of cellular nucleases that contributes to their long-term preservation in the cellular environment and leads to an increase in biological Activ derivative FTAA, tested for anti-HIV activity, have a cholesterol derivative FTA [3-4]. However, such derivatives have a high enough activity, as blocking the reproduction of the virus in this case is by linking them with the components of viral particles, which is reversible.

In the event of exposure of the reactive derivatives of FTA can occur not only reversible binding of the reagent components of viral particles, but also irreversible modification of the components of the virus, providing various stages of its reproduction. As a consequence, the introduction of reactive groups in the composition of FTA can greatly improve the efficiency of inhibition of virus reproduction. However, due to the high nucleophilicity phosphothioate groups are not excluded self-modification mezhnukleotidnyh phosphothioate groups under the influence of the reactive groups introduced into the composition of FTO [9]. As a result, such modification may result in the loss of reactivity of the reagent. The manifestation of the increased effect of inhibiting the reproduction of the virus in this case is not obvious and may largely depend on nucleoplasty modifitsiruemoi group, introduced into the composition of FTAA.

Known reactive alkylating derivatives of FTA General formula (I) that in the presence of nucleophilic compounds in the environment can modify [9-11]. However, to date such derivative FTA on anti-HIV activity not tested.

The present invention is to increase the efficiency of inhibition of reproduction of HIV in the number of derivatives of FTO and expansion of the range of the derivative of FTA possessing anti-HIV activity.

The problem is solved by application of the described [10, 11] reactive alkylating derivatives of FTA General formula (I) as inhibitors of reproduction of the human immunodeficiency virus.

These derivative gain on the basis of a fully released phosphothioate oligonucleotides General formula (I) where RCl = -HE, by pre-activation of the 3'-terminal phosphate group in the presence of condensing vapors triphenylphosphine - piperidinomethyl according to previously described method [10, 11] .

Reactive RCl group in the compounds (I) is a residue of an aromatic nitrogen mustard, which is capable, through the formation of intermediate e is placed to assess anti-HIV effect was used cholesterol derived FTAA, because from literature data it is known that among the known derivatives of FTA cholesterol derivative FTAA are most anti-HIV activity [3-4].

For testing has been synthesized antisense 12-tier phosphothioate analogue of the oligonucleotide structure

GpsCpsApsTpsCpsApsApsGpsCpsApsGpscp (II) the complementary region (+)-chain DNA of HIV-1 (nucleotides 7516-7527) [13]. Based phosphothioate of the oligonucleotide (II) according to the method of [10] were obtained his cholesterol GpsCpsApsTpsCpsApsApsGpsCpsApsGpscpnh(CH2)2C(O)-Chol (III) and alkylating GpsCpsApsTpsCpsApsApsGpsCpsApsGpscp-RCl (IV) derivatives modified at the 3'-end and bearing in this position, the remainder of the molecule of cholesterol or reactive alkylating RCl group. Was the comparative evaluation of the effectiveness of inhibition of reproduction of HIV infection by these compounds (oligonucleotide derivative (III) and (IV)).

Inhibition of reproduction of HIV includes the cultivation of primary HIV-positive lymphoid cells MT-4 in the presence of oligonucleotide drugs (III) and (IV), the final concentration in the culture medium is 10.0 - 0.01 µm, for one passage within 4 days.

About the inhibition of reproduction, is also to increase cell viability, determined by the method of exclusion Trifanova blue, on the 4th day of cultivation compared to control.

The obtained experimental data are shown in table and graphic illustrations in Fig. 1 and 2, which show that the alkylating phosphothioate derivative (IV), the fragment which carries the reaction RCl-alkylating group on the 3'-end, at a concentration of 1 μm is practically 100% protects cells from death, whereas the protective effect of cholesterol derivative (III) of the same oligonucleotide GpsCpsApsTpsCpsApsApsGpsCpsApsGpscp in the same concentration does not exceed 40%. At this dose, the drug concentration required to protect cells from death (PD50and for inhibition of virus reproduction (ID50up to 50%, for alkylating and cholesterol derivative (IV) and (III) are 0.06 and 1.97 μm and 0.14 and 3.4 μm, respectively. I.e., alkylating derivative (IV) by these parameters (PD50(12ps-Chol)/PD50(12ps-RCl) = 1.97/0.06= 32.8; ID50(12ps-Chol)/ID50(R-RCl) = 3.4/0.14 = 24.3; ) more than 20 times more effective than cholesterol derivative (III).

Thus, the first set of ability alkylating derivatives of FTA to inhibit the reproduction of VIR is this drug significantly higher than the efficiency of cholesterol drug possessing the largest among the known derivatives of FTO anti-HIV activity. The effect was already in the chain length 12 nucleotide units. On the basis of the General laws of action of such drugs are effect in the range of 13-30 should be at least not less than the chain length equal to 12 [3, 4].

Below are specific examples in detail reveal the essence of the invention.

Example 1. Evaluation of the cytotoxicity of the compounds.

The cytotoxicity of the studied drugs was assessed by culture of human T lymphocytes person (line MT-4). The compounds are dissolved in culture medium RPMI-1640 without supplements and appropriate dilutions contribute to the wells of 96-well plates (three for each dilution) for the screening of cells. The seeding concentration is 0,5106cells/ml Cells were cultured in 96-well tablets for cell culture company "Costar" (USA) on growth nutrient medium RPMI-1640 containing 10% serum fetal cow, 0,06% L-glutamine, 100 µg/ml gentamicin at 37oC and 5% CO2within 4 days. After incubation count the proportion of viable cells in the cell Goryaeva after dyeing Trifanova blue. When evaluating citotoxicity 15 microns.

Example 2. Study of the effect of alkylating and cholesterol derived phosphothioate analogue of the oligonucleotide on the reproduction of HIV-1 in cell culture.

A suspension of HIV-1-infected with a multiplicity of infection of 0.2-0.5 cells MT-4 after 1 h incubation at 37oC (adsorption of the virus) was diluted to the seeding concentration 5105cells/ml growth nutrient medium RPMI-1640 containing 10% serum fetal cow, 0,06% L-glutamine, 100 µg/ml gentamicin, and make automatic pipette into wells of 96-well culture tablet 200 ál in each well. Then in the control wells (at least three) add 20 µl of nutrient medium RPMI-1640 without supplements, and in the remaining wells contribute 20 µl of the preparation of derivatives of oligonucleotides in a nutrient medium RPMI-1640 without supplements with a concentration of 100-0.1 μm to its respective final concentrations in the culture medium 10 - 0.01 µm (three wells for each concentration). In a few empty holes (at least three) contribute to the oversight of 200 μl of the suspension of uninfected cells MT-4 with a concentration of 5105cells/ml Tablets close and incubated in a humid chamber CO2incubator with 5% carbon dioxide at a temperature (36-37)oC for 4 days.

In the end, and control the level of virusprotection by ELISA. Preparation of samples for enzyme-linked immunosorbent assay (ELISA) carry out, inactivating HIV-1 by adding to the sample of tween-80 to a final concentration of 0.1%, followed by incubation at a temperature of (60.2)oC for at least 24 hours. Thus prepared samples analyzed, as described in [14]. Anti-HIV activity of drugs judged by the decrease in the accumulation of virousspecificakih protein p24, and, on the basis of the degree of protection of infected cells from death due to virus infection, calculating the percentage of viable cells after staining Trifanova blue in the camera Goryaeva.

The obtained experimental data are given in the table. Based on these data, construct a dose-dependent curve and determine the concentration of compounds by 50% reduces the accumulation virousspecificakih protein p24 (ID50). In addition, based on calculated according to [13] the degree of protection of infected cells from death due to virus infection on the 4th day of cultivation in the presence of drugs in different concentrations build dose-dependent curve and determine the concentration of compounds at 50% protect cells from death (PD50). Dose-dependent curves and quantitative characteristics Engibarov is th effect one of the high degree of suppression of the reproduction of HIV in cell culture cholesterolosis and alkylating derivatives (III) and (IV), expressed as quantitative indicators of inhibition: ID50= 3.4 and 0.14 μm (to reduce the accumulation of p24) and PD50= 1.97 and 0.06 μm (to protect infected cells from death), respectively.

Thus, for example, tested the specific compounds shown that alkylating derivatives of FTO have the ability to inhibit the reproduction of human immunodeficiency virus type 1 in cell culture, and the efficacy of such drugs can significantly (more than 20 times) to exceed the performance of known cholesterolemic derivative FTAA.

It should be noted that according to preliminary estimates the number one dose of the preparations required to fully inhibit the reproduction of HIV in the body and acting in the course of 4 days, will be 16 mg, and the cost of such doses on average, about 15-75 $. The number and cost of the same dose of cholesterol drug FTA required to fully inhibit the reproduction of HIV, should be higher no less than 20 times. This indicates a promising offer inhibitory:

1. Agrawal, S., Goodchild j, Civeira M., Sarin, P. S., P. C. Zamecnik Phosphoroamidate, phosphorothioate and methilphosphonate analogs of oligodeoxynucleotide: inhibitors of human immunodeficiency virus. // Nucleosides &Nucleotides, 1988, v. 8, p. 819-823.

2. Agrawal, S., Goodchild j, Civeira M. P., Thornton A. H., P. S. Sarin and Zamecnik, P. C. Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus // Proc. Natl. Acad. Sci. USA, 1988. v. 85. p. 7079-7083.

3. Stein C. A., Pal, R., De Vico A. L., G. Hoke, S. Mumbauer, Kinstler O., Sarngadharan, M. G. , R. L. Letsinger Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro // Biochemistry, 1991, v. 30, N 9, p. 2439-2444.

4. R. L. Letsinger, Znang G., Sun, D. K., Ikeuchi T., P. S. Sarin Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhhibitors of replicatioon of human immunodeficiency virus in cell culture // Proc. Natl. Acad. Sci. USA, 1989, v. 86, p. 6553-6556.

5. Temsamani J., Kubert M., Tang J., Padmapria A., Agraval S. Cellular uptake of oligodeoxynucleotide phosphorothioates and their analogs // Antisense research and development, 1994, v. 4, N 1, p. 35-42.

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9. Amirkhanov, N. In., Zarytova C. F. Reactive derivative phosphothioate analogues oligonu nitrogen mustard. Bioorgan. chemistry. - 1997, T. 23, N 7, S. 569-575.

10. RF patent N 94010679, 20.11.1996.

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12. Gimautdinova O. I., Karpov,,, Lomakina Ie, Selaqui E. L., Cemanova L. N., Grineva N. And. Alkylation of DNA complementary complexes with 4-(N-2-chloroethyl-N-methylamino)benzyl - 5'-phosphamide oligonucleotides. Bioorgan. chemistry. - 1980, so 6, No. 1, S. 70 - 80.

13. Svinarchik P. P. , Konevetz D. A., Pliasunova O. A., Pokrovsky, A. G., V. Vlassov V. Inhibition of HIV proliferation in MT-4 cells by antisense oligonucleotide conjugated to lipophilic groups. //Biochimie, 1993, v. 75, p. 49-54.

14. Plyasunov O. A., Egorychev I. N., Fedyk N. In. and other Studies of anti-HIV activity of glycyrrhizin acid. Questions Virology. - 1992, N 5-6, S. 235-238.

The use of alkylating derivatives phosphothioate analogues oligodeoxyribonucleotides General formula (I)

< / BR>
where B is the residue of thymine, cytosine, adenine or guanine;

RCL - balance alkylating an amine, such as 4-(N-methyl-N-2-creatinine)benzylmethylamine

< / BR>
n = 12 - 30;

as ing the

 

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