RU2399612C2 - Method of detecting compounds reducing functional activity of human immunodeficiency virus protease and method of inhibiting dimerisation of hiv protease subunits - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of detecting a compound which is a noncompetitive inhibitor of human immunodeficiency virus (HIV) protease (SEQ ID No:1) involves detection using alanine scanning methods and molecular docking of the compound at least with one atom of an amino acid residue selected from a group consisting of Asn98, Phe99, Asp29, Asp30, Arg8, Gly49, Gly51 and Gly52 SEQ ID NO:1, at a distance of not more than 5 Å.

EFFECT: increased effectiveness of the compounds.

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Description

FIELD OF THE INVENTION

The invention relates to the field of medical pharmacology, and more particularly to the search for compounds that reduce the functional activity of the human immunodeficiency virus protease (HIV protease) by inhibiting the association of its subunits. The invention is focused on the development of new drugs.

BACKGROUND OF THE INVENTION

Therapy based on the use of HIV protease inhibitors prolongs the life of patients with acquired immunodeficiency syndrome (Sepkowitz, K. A. AIDS - the first 20 years. N. Engl. J. Med. 2001, 344, 1764-1772). HIV protease is an enzyme that breaks down a viral polyprotein into functionally active mature proteins necessary for the reproduction and assembly of HIV. Typically, competitive protease inhibitors are used that directly bind the active site of the enzyme. The effectiveness of the use of such inhibitors during treatment is rapidly decreasing due to point mutations in the viral genome, which inhibit the protease binding of the inhibitor to a greater extent than its enzymatic activity. As a result, the body is affected by the emerging strains of HIV that have become resistant to the inhibitor used.

An alternative is protease inhibition by blocking the formation of a functionally active form of the enzyme (Bannwarth L., Reboud-Ravanx M. An alternative strategy for inhibiting multidrug-resistant mutants of the dimeric HIV-1 protease by targeting the subunit interface. Biochem. Soc. Trans. 2007 Jun; 35 (Pt 3): 551-4), which is a dimer of homologous subunits. The main site of their contact is a beta structure, the cords of which are C- and N-terminal fragments of subunits. Peptides and peptidomimetics mimicking these fragments have been shown to interfere with subunit assembly by acting as non-competitive HIV protease inhibitors (Zutshi R., Brickner M., Chmielewski J. Inhibiting the assembly of protein-protein interfaces. Current Opinion in Chemical Biology, 1998 , 2 (1): 62-66 (5)). Inhibitors of this kind are preferable as drugs, since resistance mutations require combined mutations in obviously conservative terminal regions of the protease polypeptide chain (Archakov AI, Govorun VM, Dubanov AV, Ivanov YD, Veselovsky AV, Lewi P., Janssen P. Protein-protein interactions as a target for drugs in proteomics. Proteomics. 2003 Apr; 3 (4): 380-91).

Currently studied dimerization inhibitors are modified peptides or peptidomimetics, as well as substances of synthetic and natural origin (Camarasa MJ, Velázquez S., San-Félix A., Pérez-Pérez MJ, Gago F. Dimerization inhibitors of HIV-1 reverse transcriptase , protease and integrase: a single mode of inhibition for the three HIV enzymes? Antiviral Res. 2006 Sep; 71 (2-3): 260-7). From a structural point of view, these substances mimic extended sections of the beta structure.

Disclosure of the present invention

The invention relates to a method for searching for new chemical compounds - dimerization inhibitors (IDs) - capable of affecting the assembly of HIV protease subunits by competitive interaction with the contact area of the protease subunits and also affecting the conformation of subunits, reducing their affinity for each other.

To identify new IDs, a method was used based on a computational technique for selecting compounds according to a computer model of the protease structure. Using an approach known as “alanine screening,” amino acid residues were identified that determined the strength of the interaction between the protease subunits. It was found that substitution of the amino acid residue listed in Table 1 (hereinafter referred to as contact points) for the alanine residue in the protease structure leads to a sharp decrease in the interaction energy of subunits. For each contact point, a molecular docking method was used to screen a database of low molecular weight compounds and a series of designed peptidomimetics to identify candidates with high binding energies with the subunit. Using known IDs, a threshold value was established for the function of evaluating the effectiveness of the interaction of the compound with the contact point. As a result, compounds were selected that were characterized by binding energies above the established threshold, as well as those that were characterized by high affinity for the surface areas of the HIV protease molecule. The latter was evaluated by the molecular dynamics method, during which the effect of the binding of the compound to the surface elements of the protein molecule on the conformational properties of the contact areas was recorded. Table 1 shows the numbers of amino acid residues in the protease subunit, which are crucial for the molecular mechanism of action of ID. Thus, the applied computational technique made it possible to select structures that selectively bind to HIV protease subunit interaction sites and affect the spatial conformation of the subunit. The IDs selected by this method are shown in Table 3.

Table 1 The list of amino acid residues of HIV protease subunits involved in contact with ID, specified in the form of coordinates of spatial rectangular cells in the coordinate system according to Appendix 1. Mechanism of action Zone Amino Acid Residue Linking to subunit contact sites one ASN98 RNE99 2 ASP29 ASP30 Binding to the surface of subunits one ARG8 2 GLY49 GLY51 GLY52

To confirm the mechanism of action, the selected compounds were selectively tested according to a measuring scheme based on high-precision registration of intermolecular interactions on an optical biosensor of the Biacore-3000 model. HIV protease molecules in dimeric form were covalently immobilized in the measuring and control cuvette of the device. Then, in the control cuvette, the HIV protease dimer subunits irreversibly bind to each other by additional covalent bonds, while in the measuring cuvette, dimers were dissociated to monomers by washing with a stream of buffer mixture.

The indicated experimental scheme made it possible to record the interaction of the inhibitor with individual subunits (monomers) of the protease located in the measuring channel, minus the interaction with the complex of two subunits (dimers) in the control channel. It was found that the inhibitors found affect the characteristics of the interaction of subunits and do not affect the characteristics of the whole (assembled) protease dimer.

To check the effect of the IDs found on the enzymatic activity of the protease, a standard technique was used, which is based on spectrophotometric measurement of the process of enzymatic hydrolysis of the test substrate. The measuring cell contained the volume of a buffer solution containing a protease into which ID was added in various concentrations and the absorbance at a wavelength of 300 nm was measured for 20 min. Based on the data obtained, the values of the inhibition constant were calculated. The values obtained for 4 out of 15 selectively tested compounds varied from 1.5 to 0.01 μM, which is comparable to the effectiveness of currently known HIV-1 protease dimerization inhibitors.

Thus, the present invention also relates to a method for inhibiting the dimerization of HIV protease subunits, comprising contacting at least one HIV protease inhibitor compound identified in accordance with the above method with an HIV protease containing medium. The effectiveness of inhibiting the dimerization of HIV protease subunits by these compounds is clearly confirmed by the experimental data presented below.

Brief Description of the Drawings

Figure 1 shows, as an example, kinetic records of changes in absorbance at 300 nm over time in the presence of ID (preparation No. 10).

Figure 2 shows the dependence of the activity of HIV in the presence of different concentrations of ID (drug No. 10). From the dependence of the HIVp activity in the presence of various ID concentrations, IC ₅₀ values were calculated.

An example implementation of the present invention

Example 1. The selection of compounds (a) and testing using a virtual HIV protease (b)

(but)

1. Materials

1.1. HIV protease structure

The coordinates obtained by the X-ray diffraction method deposited in Protein Data Bank under the code 1A30 were used as the coordinates of the initial HIV-1 protease structure. Date of deposit: 27/12/1998. The enzyme code for the EU: 3.4.23.16. Resolution PCM 2.0 angstroms.

Structure Composition:

1. The symmetric dimer of the following amino acid composition (99 amino acid residues in each chain)

PRO GLN ILE Thr LEU TRP Lys ARG PRO LEU Val Thr ILE Lys ILE GLY GLY GLN LEU Lys GLU ALA LEU LEU ASP Thr GLY ALA ASP ASP Thr Val ILE GLU GLU MET SER LEU PRO GLY ARG TRP Lys PRO Lys MET ILE GLY GLY ILE GLY GLY RNE ILE Lys Val ARG GLN Tyr ASP GLN ILE ILE ILE GLU ILE Cys GLY His Lys ALA ILE GLY Thr Val LEU Val GLY PRO Thr PRO Val ASN ILE ILE GLY ARG ASN LEU LEU Thr GLN ILE GLY Cys Thr LEU ASN RNE

2. Tripeptide competitive inhibitor

GLU ASP LEU

3.26 water molecules.

1.2. Databases of low molecular weight compounds

1. The NCI Diversity Set database, which includes information on 1990 chemical compounds selected from the NCI database (140,000 compounds available at the US National Cancer Institute), with the exception of structurally similar compounds.

2. NCI database, including information on more than 250,000 chemical compounds.

2. Methods

2.1. Structure optimization

From the original structure were removed: inhibitor molecules, water molecules. The coordinates of the hydrogen atoms were restored using the Reduce program (Word, JM; Lovell, SC; LaBean, T.N .; Taylor, HC; Zalis, ME; Presley, BK; Richardson, JS; Richardson, DCJ Mol. Biol. 1999, 285 , 1711-1733). The structure of the dimer was subjected to the simulation of molecular dynamics in vacuum. The CHARMM program was used (Brooks, BR; Bruccoleri, R .; Olafson, B .; States, D .; Swaninathan, S .; Karplus, MJ Comp. Chem. 1983, 4, 187-217), the selected molecular-mechanical force field Merck (Halgren, TAJ Comp. Chem. 1996, 17, 490-519). The system balancing time is 50 ns in increments of 1 fs.

2.2. Alanine Screening

Simulation of the alanine scanning procedure was also performed using the CHARMM software using the molecular dynamics procedure. The proper simulation time after balancing is 10 ps (1 fs step) with the recording of intermediate states every 10 steps. For each intermediate state, a sequential virtual replacement of each amino acid residue of the protease subunit with alanine was carried out, during each of them the changes in the internal energy of the complex of subunits (in kcal / mol) were estimated using the formula

,

,

where E _int is the energy of interaction between subunits, E _AB is the energy of the dimer E _A , E _B is the energy of each of the chains.

2.3. Molecular docking

Structures of low molecular weight compounds were selected from testing databases using the molecular docking method using the Autodock software (Morris, GM; Goodsell, DS; Halliday, RS ;; Huey, R .; Hart, WE; Belew, RK; Olson, AJJ Comp. Chem . 1998, 19, 1639-1662). As a seat in the docking procedure, we used the zones on the surface of one of the dimer subunits determined as a result of the alanine scan. The second subunit was deleted, the conformation of the working subunit corresponded to that obtained after the structure optimization procedure. The locking procedure was carried out in two stages. The first stage included locking all the molecules of the NCI Diversity Set database no 10 attempts for each. The search procedure using the genetic algorithm was used, default search parameters. The second stage is the refinement of the position of the molecules selected in the first step. The same algorithm and set of parameters were used, however, the number of attempts was increased to 50 and the entry point to the pseudo-random number generator was changed. For each variant of the complex obtained as a result of the last procedure, the binding energy of the compounds to the HIV protease subunit, independent of the Autodock evaluation function, was estimated using the DS program. For testing, compounds with binding energies below 6 kcal / mol were proposed.

2.4. Additional search for similar structures

For molecules selected based on the results of locking, a search for structurally similar compounds was carried out using the complete NCI database. For similar compounds found, the molecular docking procedure was repeated completely and compounds with binding energies below 6 kcal / mol were selected.

3. The results obtained

3.1. Alanine Screening

The results of the alanine scan simulation procedure are presented in table 2.

table 2 Alanine scan results. Replaceable Amino Acid Number Standard Deviation (S) The energy of interaction of subunits, kcal / mol (E _int ) D 49 -310.28 -90,09469 -220,184 51 -310.28 -175,80623 -134,472 52 -310.28 -265.89658 -44.3816 29th -310.28 -279.93672 -30.3415 8 -310.28 -285,99873 -24.2795 25 -310.28 -307,00792 -3,2703

Replaceable Amino Acid Number Standard Deviation (S) The energy of interaction of subunits, kcal / mol (E _int ) D thirty -310.28 -308,0185 -2,25972 99 -310.28 -309,44921 -0.82901 60 -310.28 -311,43276 1,154543 98 -310.28 -311,66917 1,390953 35 -310.28 -312,21023 1,932013 fifty -310.28 -312.45246 2,174243 5 -310.28 -313,47315 3.194933 34 -310.28 -313,49734 3,219123

From the results of alanine screening, those were selected in which the difference between the standard deviation and the energy of the subunits (D = SE _int ) is negative. Table 2 shows the first 15 lines obtained after sorting by parameter D. Of the selected amino acid residues, only those that are on the surface of the protein were selected. Below are the numbers and names of amino acid residues, which according to the results of the alanine screening determine the contacts of the ID with the HIV protease subunit.

1) Asn98, Phe99. Cartesian coordinates of the rectangular locking region relative to the attached file (see Appendix I):

- the minimum values along the axes X, Y, Z - 15.290, 30.630, -4.210;

- the maximum values along the X, Y, Z axes are 37.790, 53.130, 18.290.

2) Asp29, Asp30 (interaction with region 3 on the opposite subunit).

3) Arg8 (interaction with region 2 on the opposite subunit).

4) Gly49, Gly51, Gly52.

3.2. Molecular docking

According to the results of the first locking step, 300 compounds with the best binding energy were selected.

According to the results of the second locking and independent of Autodock evaluation of the energy of interaction of the ligands with the HIV protease subunit (threshold value of -6 kcal / mol), 20 compounds were selected.

3.3. Additional search for similar structures

According to the similarity search results, the complete cycle of locking and energy selection (threshold value of -7 kcal / mol), 14 compounds were selected (see table 3).

Table 3 Compounds selected by search results by similarity, docking and energy characteristics. N Substance name DS rating *, kcal / mol one 7-chloro-5,6-dimethylbenzo [s] acridine -7.9 2 9-chloro-5,6-dimethylbenzo [s] acridine -7.77 3 5,5'-bis (2-methyl-1H-indol-3-yl) -3,3'-biisoxazole -7.68 four 10-chloro-5,6-dimethylbenzo [s] acridine -7.55 5 2-chloro-N- (2,5-dimethylphenyl) -7-methyl-7H-purin-6-amine -7.51 6 2,3,6,8-tetrachlorophenanthridine -7.43 7 2-chloro-4-methylbenzo [h] quinoline -7.32 8 2-chloro-4- (2-phenylvinyl) quinoline -7.29 9 1,3,6,8-tetrachlorophenanthridine -7.2 10 2- (2- (2,4-dichlorophenyl) vinyl) quinoline -7.12 eleven 4-chloro-2-phenylquinoline -7.04 12 N- (4-chlorobenzylidene) -N- (9H-fluoren-2-yl) amine -7.02 13 3,6,8-trichlorophenanthridine -7.02 fourteen 2-chlorobenzo [h] quinoline -7.01 * DS - free binding energy between the ligand and the HIV protease subunit

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Measurement of the enzymatic activity of HIV protease in the presence of inhibitors.

1. Materials

1.1. HIV-1 protease preparation

A recombinant HIV-1 protease from Bachem AG (Switzerland) (preparation N-9040) was used in the work.

1.2. Chromogenic substrate for HIV protease

To analyze the enzymatic activity of HIV protease, a special chromogenic substrate VII was used: H-Lys-Ala-Arg-Val-Tyr-p-nitro-Phe-Glu-Ala-Nle-NH ₂ manufactured by Bachem AG (Switzerland) (drug H- 1286).

1.3. HIV protease dimerization control inhibitor (ID)

For positive control, a well-known peptide ID (Scar inhibitor) (palmitoyl-TVSYEL) was used in the work.

1.4. Samples of low molecular weight compounds

For experimental verification of the action of potential dimerization inhibitors on the enzymatic activity of HIV protease, 3 samples of compounds taken during in vitro screening using OB were taken.

1.5. Other reagents and preparations

Other reagents and preparations used in the work were of analytical purity.

2. Methods.

2.1. Kinetic spectral measurements

To register the kinetics of the enzymatic reaction, a Libra S32PC spectrophotometer (Biochrom, England) in the “Kinetics” mode was used. The measurement was carried out at 25 ° C to reduce the absorption at 300 nm (peak absorption of the substrate VII).

2.2. Analysis of the enzymatic activity of HIV protease

To measure the enzymatic activity of HIV protease, a chromogenic substrate VII was used, which has a maximum absorption in UV at 300 nm.

HIV protease preparation (100 ng) was incubated for 35 min at 25 ° C in 240 μl of buffer (1 M NaCl, 1 mm EDTA, 0.1% CHAPS, 8% DMSO, 0.1 M sodium acetate, pH 5.0) in the presence (experience) or absence (control) of different concentrations of potential ID. The final HIV protease concentration was approximately 18 nM. The mixture was then transferred to a quartz semi-microcuvette and the reaction was started by adding 10 μl of substrate VII solution to a final concentration of 25 μM.

The mixture was stirred rapidly and HIV protease activity was recorded at 25 ° C to reduce absorbance at 300 nm for 35 minutes. HIV protease activity (μmol substrate / (min · mg enzyme)) was calculated by the formula

where ΔA ₃₀₀ is the change in optical density,

V is the total volume of the mixture (l),

E is the coefficient of molar extinction of the substrate (M ^-1 cm ^-1 ),

m is the mass of the protease (mg),

t is the measurement time (min),

l is the thickness of the measuring cell (cm).

The inhibitory effect of potential ID and KID was expressed by the value of IC ₅₀ (inhibitor concentration, inhibiting 50% of the enzyme activity).

3. Results.

Figure 1 shows, as an example, kinetic records of the change in absorbance at 300 nm over time in the presence of an ID. You can see that ID inhibits the enzymatic reaction. From the dependences of HIV protease activity in the presence of various ID concentrations (as an example, see FIG. 2), IC ₅₀ values were calculated. The results are shown in table 4.

Table 4 IC ₅₀ values for 3 potential IDs and KIDs. Drug number four 10 fourteen Kid IC ₅₀ (μm) 3.4 1,2 8.5 1,1

Thus, all three potential IDs selected in an in vitro experiment using the OB test system showed sufficient inhibitory activity comparable to the control peptide ID (Scar inhibitor).

Claims (4)

1. A method for detecting a compound that is a non-competitive inhibitor of the human immunodeficiency virus (HIV) protease (SEQ ID NO: 1), comprising detecting by alanine skinning and molecular docking methods a compound of at least one atom of which contacts at least one atom an amino acid residue selected from the group consisting of Asn98, Phe99, Asp29, Asp30, Arg8, Gly49, Gly51 and Gly52 SEQ ID NO: 1, at a distance of not more than 5 Å. 2. The method according to claim 1, whereby the alanine screening is carried out by means of the CHARMM program using the molecular dynamics procedure. 3. The method according to claim 1, according to which the proper simulation time after balancing is 10 ps (1 fs step) with the recording of intermediate states every 10 steps, and for each intermediate state a sequential virtual replacement of each amino acid residue of the protease subunit with alanine is performed , during each of which an assessment is made of changes in the internal energy of the complex of subunits (in kcal / mol) according to the formula:

,
wherein E _int - the energy of interaction between the subunits, E _AB - dimer energy E _A, E _B - the energy of each of the chains. 4. The method according to claim 1, according to which in the docking procedure, zones on the surface of one of the dimer subunits determined by alanine scanning are used as a seat, namely: Asn98, Phe99, Asp29, Asp30, Arg8, Gly49, Gly51 and Gly52 SEQ ID NO: 1.

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