Scientific Centre of Anti-Infectious DrugsScientific Centre of Anti-Infectious DrugsKazakhstanKazakhstan

Gulnara Yuldasheva, Aleksandr IlinGulnara Yuldasheva, Aleksandr Ilin

A Quantum-Chemical Model of the A Quantum-Chemical Model of the Inhibition HIV-1 intergrase action by Inhibition HIV-1 intergrase action by

Iodine Complex CompoundsIodine Complex Compounds

HIV integrase inhibitors have a high therapeutic effect for two reasons. First, integrase is one of the key participants in the virus replication cycle. Second, integrase has no cellular equivalent and, hence, the suppression of its activity should not disturb normal cellular metabolism processes.

Sen, S., Mathur, A.G., Gupta, R.M., Kapila, K., and Chopra, G.S. (2008) Recent Pat Antiinfect Drug Recent Pat Antiinfect Drug Discov., 3(3), 199-205.

In the paper the authors offer a comparison of the inhibiting activity of a series of antiretroviral agents towards HIV-1 integrase and the in vitro-isolated PIC. The results of the study show that inhibitors active towards the integrase enzyme may not be active towards the PIC. The capability to inactivate a PIC was exhibited only by three antiretroviral agents: quinalizarin, purpurin, and alizarin.

Farnet, C.M., Wang, B., Lipford, J.R., and Bushman, F.D. (1996) Differential Inhibition of HIV -1 Preintegration Complexes and Purified Integrase Protein by Small Molecules. Proc.Natl. Acad. Sci. USA, 93, 9742-9747.

We propose a model of inhibition of HIV-1 intergrase action where the active center of those drugs inhibits the integrase catalytic center inside the PIC.

Preintegration complexPreintegration complex

Well-known are quite a number of iodine-polymer complexes that possess a broad spectrum of antimicrobic and antiviral action including human immunodeficiency virus (HIV). Among them is Armenicum which is a drug to treat HIV infection.Patent Pub.No. WO/2001/078751 International Application No.: PCT/AM2000/000002, International Filling Date 24.11.2000.Recently a new anti-infective drug with anti-HIV action has been patented. At present the new drug (FS-1) is in the third clinical trial and for that reason has no name yet.Patent KZ (A) №15116, 15.12.2004, bull 12.Armenicum is composed of the LiI5--dextrin complex. The FS-1 contains polypeptides along with the LiI5 --dextrin complex. Gulnara A. Yuldasheva, Georgii M. Zhidomirov, and Aleksandr I. Ilin «Effect of Organic Ligands with Conjugated π-Bonds upon the Structure of Iodine--Dextrin Complexes.» Biotechnology and Apply Biochemistry 2012 Jan-Feb, 59(1), P.29-34.We managed to get the this electronic structure of I2 only by using the quantum-chemical calculations. However, using UV-IR – spectroscopy and the quantum-chemical method DFT-B3PW91/midi we researched the water-glycine – KI3 - LiCl – ethanol system as a model of the FS-1 active centre.[Gulnara A. Yuldasheva, Georgii M. Zhidomirov, Jerzy Leszczynski, Aleksandr I. Ilin «The effect of the amphoteric properties of amino acids in the zwitterionic form on the structure of iodine complex compounds in aqueous solutions containing halogenides of alkaline metals and amino acids» Journal of Molecular Structure 1033 (2013) 321–330.

ArmenicumArmenicum

Active center of FS-1Active center of FS-1

∆E=-16,60 ∆E=-14,26

∆E= -6,25 ∆E=-24,25 Energy of formation (∆E, kkal/mol) active complex (amine acid residues, ethanol,

molecular iodine LiCl). Red balls - oxygen atoms, blue - carbon, nitrogen – dark blue, violet-iodine, lithium-brown, chlorine - yellow.

Model of interaction of the active complex Armenicum and Model of interaction of the active complex Armenicum and FC-1 with nucleotides of HIV DNAFC-1 with nucleotides of HIV DNA

red balls - oxygen atoms, blue - carbon, nitrogen-dark blue, violet-iodine, yellow –chlorine, lithium-white.

System (a) AGA triplet

AGA 2.SampleName

Н2ОDescription

200 300 400 500 600 700

0,00,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

nm

A

256,56nm; 0,61A

199,43nm 0,94676A

R10.Sample

Name

Н2О

Description

200 300 400 500 600 700

0,00,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

nm

A

223,08nm; 0,59A

460,99nm; 0,01A355,15nm; 0,01A

256,43nm 0,14933A

System (c) AGA triplet+FS-1

System (b) FS-1

UV UV –– spectrum of system (a-c) spectrum of system (a-c)

Table 1. Theoretical wavelengths (λ, nm) of electronic transitions in iodine complex with adenosine and lithium halogenides (TD-DFT/B3PW91/midi)

λ theor.

206,51 1A→1A (π(I2)→π (nucleot.))1A→1A (π (nucleot.)→π (nucleot.))

210,20 1A→1A (π(I2)→π (nucleot.))1A→1A ((Cl—Li--I)→ π(I2))

218,63 1A→1A (π (nucleot.)→ π(I2))1A→1A ((N—I-I)→ π(I2))

226,57 1A→1A (π (nucleot.)→ π(I2))

234,55 1A→1A (π(I2)→ π(I2))1A→1A (π (nucleot.)→ π(I2))

244,34 1A→1A (π (nucleot.)→π (nucleot.))

Table 2. Theoretical wavelengths (λ, nm) of electronic transitions in iodine complex with guanosine and lithium halogenides (TD-DFT/B3PW91/midi)

λ theor.

215,09 1A→1A (π(I2)→ π(I2))

233,71 1A→1A (π(I2)→π (nucleot.))

244,34 1A→1A (π(I2)→ π(I2))

Mechanism of Inhibition of the catalytic center of the Mechanism of Inhibition of the catalytic center of the HIV-1 IntegraseHIV-1 Integrase

The structure of the catalytic domain of the HIV-1 integrase is determined by the X-ray structural analysis. According to the data obtained, the catalytic domain of the enzyme in the crystal forms a spherical dimer with each monomer forming a semi-sphere. The three amino acid residues - Asp64, Asp116 and Glu152 closely located in the tertiary structure of the catalytic domain – form its active catalytic center with the active centers of each integrase monomer located on the opposite sides of the dimer sphere 35Å apart.

The X-ray structural analysis data clearly show one Mg2+ ion coordinated by Asp64 и Asp116 and two water molecules.

Dyda, F., Hickman, A.B., Jenkins, T.M., Engelman, A., Craigie, R., and Davies D.R. (1994) Crystal Structure of Catalytic Domain of HIV-1 Integrase Similarity to Other Polynucleotidyl Transferases. Science, 266, 1981-1986.

Goldgur, Y., Dyda, F., Hickman, A.B., Jenkins, T.M., Craigie, R., and Davies, D.R. (1998) Three New Structures of the Core Domain of HIV-1 Integrase: An Active Site that Binds Magnesium. Proc.Natl.Acid.Sci USA, 95(16), 9150-9154.

Based on data regarding the action mechanism of nucleotidyltransferases, referred to the same family as the HIV-1 integrase, it was suggested in article that two ions may take part in the act of catalysis, but, due to a higher conformational mobility of the catalytic enzyme, they are not coordinately bound prior to being bound with the virus DNA.

With the use of the molecular dynamics method it was shown that the interaction with the virus DNA involves the integration of two Mg2+ ions into a stable bi-nuclear structure where the amino acid residue Glu152 simultaneously coordinates the two Mg2+ ions, while Asp64 and Asp116 interact only with one of the Mg2+ ions.

Chen, X., Tsiang, M., Yu, F., Hung, M., Jones, G.S., Zeynalzadegan, A., Qi, X., Jin, H., Kim, C.U., Swaminathan, S., and Chen J.M. (2008) Modeling, Analysis, and Validation of a Novel HIV Integrase Structure Provide Insights into the Binding Modes of Potent Integrase Inhibitors. J.Mol.Biol., 380, 504-519.

Nucleoprotein complex formed by nucleotide of virus DNA, Nucleoprotein complex formed by nucleotide of virus DNA, active center of FS-1, active catalytic center.active center of FS-1, active catalytic center.

Energy of formation (∆E, kkal/mol) of nucleoprotein complex. Red balls - oxygen atoms, blue - carbon, nitrogen – dark blue, violet-iodine, lithium-brown, chlorine – yellow,

magnesium-green.

I, ∆E = -22,3 II, ∆E = -19,7

IV, ∆E = -20,3III, ∆E = -28,3

Experimental Study of FS-1 Substance in Relation to HIV-1 VirusExperimental Study of FS-1 Substance in Relation to HIV-1 VirusA study of the antiviral effect of FS-1 substance in relation to HIV-1 virus was

conducted. The study was conducted using a line of human lymphoblastoid cells (MT-2). Line H9 / HTLV-IIIB of human cells chronically infected with HIV-1LAI was used a source of HIV-1. Anti-HIV effect of FS-1 was determined based on the level of protein p24 and reverse transcriptase activity.

Table 3 shows the results of a study of viral activity of the HIV-1 stock solution in MT-2 cell culture.

Table 3. Results of Infectivity in terms of Cytopathic Effect, Level of p24 Antigen and RT-Activity of HIV-1 in MT-2 Cells.

Dilution of Stock Solution MTT

A450

P24

A450

RT

A405

Volume of

Liquid

% Living Cells

100 0,506 2,907 1,300 0,05ml 0,506/1,157=44

10-1 0,918 2,699 1,724 0,05ml 0,918/1,157=79

10-2 0,908 2,771 1,085 0,05ml 0,908/1,157=78

10-3 1,012 0,746 1,010 0,05ml 1.012/1,157=87

10-4 1.000 0,423 0,954 0,05ml 1,000/1.157=86

Experiment DesignExperiment Design:

200 l (80,000-100,000) of uninfected cells МТ-2 were placed into 96-well plates. 24 hours later they were infected with HIV-1 (Stock 13) and were put into an oven for 1 hour at 37 °С. Then the supernatant was removed and the test substance at various concentrations was introduced at the rate of 6 repetitions per each concentration. A number of uninfected cells were used as negative controls, while a number of virus-infected cells not treated with the test substance were used as positive controls. Percentage of survived cells was calculated after the computation of the average value of optical density of the experiment.

The results of the experiment have shown that FS-1 has a clear cut anti-HIV activity in MT-2 cell culture.

The experiment was conducted at The National Centre for Infectious Diseases of the Republic of Bulgaria under the guidance of Professor R. Argirova

Table 4 Anti-HIV Action of FS-1 as shown by the Level of Reverse Transcriptase Activity

At 1:4,000 dilution the activity of reverse transcriptase was 92% inhibited after 7 days

Experiment Date

FS-Dilution

1:4,00 1:8,000

(MNC)

1:16,000(1/2MNC)

1:8,000(1/4MNC)

% of Killed Cells

18.12.07 3 35 50 -

21.0108 7 39 55 90

25.01.08 10 45 51 83

% inhibition of HIV Infection in Terms of RT Activity

18.12.07 - - - -

21.0108 81 60 50 8

25.01.08 92 65 54

ConclusionsConclusions

The computation results show that drug FS-1 may be referred to compounds inhibiting the catalytic center of the HIV-1 integrase.

Our research have shown that active center of FS-1 prevents the formation of PIC and inhibits the HIV-1 integrase inside the nucleoprotein complex where the binuclear I2LiOCOMg complex interacts both with the virus DNA and the active center of the catalytic domain of the HIV-1 integrase.

The results of the experiment have shown that FS-1 has a clear cut anti-HIV activity in MT-2 cell culture.