SI24316A - Antagonists of Toll-like receptor 4 (TLR-4), detected by a virtual screening - Google Patents

Abstract

The invention provides a detection process and the use of compounds that bind to the TLR-4 protein interfere with the formation of the TLR4-MD-2 complex, thereby expressing antagonistic action. The latter means to prevent the operation of natural TLR-4 ligands such as LPS and signaling induced by this type of binding to said receptor. Said compounds have potential therapeutic use as drugs for the prevention of disease states associated with excessive TLR-4 activity, such as, for example, sepsis and other inflammatory diseases.

Description

Toll-like Receptor 4 (TLR-4) Antagonists Detected by Virtual Solving DESCRIPTION OF THE INVENTION

A. TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of pharmacy and relates to compounds that bind to the TLR4 protein and prevent the binding of molecules that activate elements of the innate immune response via the MD-2 / Toll-like receptor (TLR) -4 complex. The characteristic of the compounds is that they bind specifically to the TLR4 protein through several wave of polar interactions, thus preventing the formation of the TLR4-MD-2 complex. In this way, said compounds prevent immune activation in the context of TLR-4, which is one of the major mechanisms of immune-related diseases. This is especially true of systemic infections with gram-negative bacteria, leading to sepsis and other immune-related pathological conditions.

B. DISPLAY OF THE PROBLEMS SOLVED BY THE INVENTION

Innate immunity and toll-like receptors

The innate branch of the immune system is the first defensive species in protecting the body against pathogenic microorganisms. The major receptors that modulate the innate immunity response belong to a family that recognizes molecular patterns or. Pattem recognition receptors or PRRs. Despite the fact that innate immunity does not specifically act on a particular antigen, its advantage is in the rapid and potent immune response that occurs when PRR receptors recognize pathogen-associated molecular pattems or PAMPs. Toll-like receptors (TLRs) play a key role in this group and are one of the major PRRs. Toll-like receptors recognize many different PAMPs that originate from bacteria, viruses, fungi, as well as parasites (Kawai, Immunity, 2011).

Toll-like receptor 4 (TLR-4)

To date, more than 10 types of TLR receptors have been recognized in humans. Of these, the TLR-4 is definitely the best studied. It is also very interesting in clinical terms, since the mechanisms of signaling via TLR-4 have been linked to many immune system-related conditions. The TLR-4 receptor is located on the cell surface where it recognizes many PAMPs such as. lipopolysaccharide (LPS) (Akira, Celi, 2006). LPS recognition leads to heterodimerization of TLR-4 and myeloid differentiation factor-2, an MDR-2 protein, resulting in an intracellular signaling cascade, activating pathways associated with both Myd88 and TRIF (Kawai, Nat Immunol, 2006 ). The latter leads to the activation of the Nf-κΒ core factor and the activation of the IRF-3 transcription factor. The end result of this cascade is the transcription of many inflammatory genes and the activation of cells of innate immunity, which include important players such as macrophages and dendritic cells.

Clinical significance of TLR-4

The proper functioning of TLR-4 and the response to endotoxin binding (LPS) are crucial for protecting the body against gram-negative pathogens. The immune processes induced by the activation of TLR-4 (the release of inflammatory cytokines and the activation of immune cells) are crucial for defense against infections and the resulting damage. In any case, it can be incorrect or TLR-4 overexpression causes an overreaction of the immune system, which has been linked in many cases to many human conditions such as sepsis and neuropathic pain (Wittebole, Mediators Inflamm, 2010; Guo, Cellular and Molecular Life Sciences, 2007). For the latter, in particular, the incidence of sepsis in the world is a key reason for requiring the development of new treatment approaches. The number of sepsis-related deaths in America is prb. 250,000 a year and 200,000 a year in Europe. Mortality in people who develop septic shock is more than 50% (Angus, Crit Chare Med, 2001). These considerations make TLR-4 an ideal therapeutic target for novel immunomodulatory agents.

A technical problem

The most important object of the invention was the discovery of novel agents that would specifically bind to the TLR4 protein and act on it antagonistically.

C. TECHNICAL STATUS

The state of the art in the field of MD-2 / TLR4 synthesis inhibitors is described by the following examples:

Hedayat et al., Medicinal Research Reviews, 2012. 32: 294-325

Henessy et al., Nat Rev Drug Disc, 2010. 9: 293-307

O'Neill et al., Pharmacol Rev, 2009. 61: 177-197

Kondo et al., Trends Immunol, 2012. 33: 449-458

Connoly et al., Curr Op Pharmacol, 2012. 12: 510-518

Kang et al., Annu Rev Biochem, 2011. 80: 917-941

Makkouk et al., Immunopharmacol & Immunotoxicol, 2009. 31: 331-338

Bowen et al., Sci Signal, 2012. 5

Keogh et al., Trends Pharmacol Sci, 2011. 32: 435-442

Jin et al., Immunity, 2008. 29: 182-191

Horscroft et al., J Antimicrobial Chemotherapy, 2012. 67: 789-801

Chavez et al., J Med Chem, 2011. 54: 4659M669.

Matsunaga et al., Mol Pharmacol 2011. 79: 34-41.

• · · • ·

E. DETAILED DESCRIPTION OF THE INVENTION WITH EXAMPLES AND DRAWINGS

The invention is described by the following figures / graphs:

Figure 1. TLR4 protein binding sites for compound 1 (A), compound 2 (B) and compound 3 (C) as predicted by LeadIT. The predicted polar interactions (A and B) are shown with a dark dashed line.

Figure 2. Inhibition by LPS of elicited TNF-α secretion from PBMCs with 100 μΜ concentration of the subject matter of the patent. The statistically significant difference between individual pairs (treated cells according to the LPS control) was determined by the odd Student's t test. (*** - p <0.001) Figure 3. Preliminary test of binding of compounds to immobilized TLR4. Each compound was used at two different concentrations (20 μΜ (black) and 200 μΜ (red)). Sensor diagrams for Compound 1 (A), Compound 2 (B) and Compound 3 (C) are shown. The binding of all three compounds at 20 μΜ concentration is visible. At higher concentrations, compounds 2 and 3 form aggregates due to their lower solubility.

The present invention provides compounds that bind to the TLR-4 protein and thus exhibit antagonistic activity. The latter means that TLR-4 natural ligands such as LPS are prevented and signaling triggered by such binding to said receptor. Specifically, the present invention discloses the discovery of novel TLR-4 receptor antagonists that have potential therapeutic utility as a means of preventing disease states associated with TLR-4 overexpression, such as e.g. sepsis and other inflammatory diseases. The novel compounds of the invention are represented by the formula and name 1UPAC:

o

NH;

compound 1

compound 3

Compound 1: 1- (4-fluorophenyl) -2- (5- (2-hydroxy-5-methoxybenzoyl) pyrimidin-2-yl) guanidine Compound 2: 6 - ((2- (benzo [diazothiazol-2-yl) ) pyrrolidin-1-yl) methyl) - N- (o-tolyl) -1,3,5-triazine-2,4-diamine

Compound 3: 2- (methyl ((4-oxo-6-phenyl-3,4-dihydrothieno [2,3-d] pyrimidin-2-yl) methyl) amino) ^r - (4morfolinofenil) acetamide

The present invention is based on intensive interdisciplinary studies of the development and quantification of new TLR-4 ligands and is illustrated by the following non-limiting examples:

Example 1:

The process of discovering new TLR4 antagonists

Previous approaches to solving the technical problem have not specifically targeted the inhibition of the interaction between TLR4 and MD-2, or have used compounds that bind to either the MD-2 domain or the intracellular domain on the TLR4 protein. Although the literature reported compounds that allegedly bind to the region of contact between TLR4 and MD-2, the compounds exhibit cytotoxicity on the HEK-293 cell line. From the crystal structure of the TLR4-MD-2 complex, it can be seen that the TLR4 surface in contact with the MD-2 protein is a long and narrow region with dimensions 40 * 20 A. The connection between the proteins occurs through a number of polar interactions, the most important of which are hydrogen. of arginine 106 on MD-2 protein with serine 183 and aspartic acid 209 on TLR4 protein. The resolved crystalline structure of the complex and the knowledge of key amino acids important for the interaction made it possible to structure-support the design of novel TLR4 receptor antagonists. The compounds defined in the present invention have been discovered by means of a structure-supported virtual library grid? ZINC containing 11.3 million drug-like compounds. We used LeadIT software to place each of the compounds in the virtual library in the TLR4-MD-2 contact area and evaluate their interaction. We obtained compounds from various manufacturers for which LeadIT envisaged strong interaction with the TLR4 protein. The predicted binding estimates for the compounds that are the subject of this patent are shown in Table 1. The binding site as predicted by the LealT program can be seen in Figure 1.

*

Example 2:

Biological evaluation of the activity of all 3 compounds in terms of LPS antagonism on TLR-4 was determined using a commercial HEK-Blue ™ hTLR-4 cell line from Invivogen. It is a HEK 293 cell line that is co-transfected with human proteins TLR-4, MD-2, CD14, and SEAP (secreted embryonic alkaline phosphatase) inducible phosphatase. The SEAP gene is bound to the binding sites for the transcription factor Nf-κΒ and AP-1. Activation of TLR-4 thus leads to the transcription and expression of SEAP, the rate of which in the cell culture supernatant can be determined by colorimetric methods (Quanti-Blue ™, Invivogen). The antagonism of the compounds was determined in this sense by pre-treating HEK-Blue ™ hTLR-4 cells with different concentrations of the compounds tested. After 2 hours of preincubation at 37 ° C, LPS was added to the cell cultures and cells were grown for a further 20 hours. The inhibition of LPS binding to TLR-4 was subsequently determined by quantification of SEAP in the culture supernatant after 20 h. The inhibition constants (ICs) of ₅₀ compounds were calculated based on their ability to inhibit the secretion of SEAP into cell culture substrates, which was shown to be a reduced absorbance value. We used the non-linear regression result analysis function to calculate it. All procedures in the test were performed according to the manufacturer's instructions (Invivogen). A commercially available TLR-4 inhibitor TAK-242 (Invivogen) was selected as a positive control of antagonism. The values (IC) ₅₀ for the compounds subject to this patent are shown in Table L

Example 3:

For further biological evaluation of the antagonistic activity of the compounds, we used a cytokine assay assay in the peripheral blood mononuclear cell (PBMC) cultures of the leukocyte cultures. PBMC cells were isolated from the blood of voluntary donors at the RS Institute for Transfusion Medicine, in accordance with the rules of the Institute. Cells were then preincubated with compounds 1, 2, and 3 at ΙΟΟμΜ for 2 h and then exposed to LPS for 48 h. A commercially available TLR-4 signaling inhibitor, compound TAK-242 (Invivogen), was used to positively control antagonism. After 2 days, cell supernatants were removed and analyzed for the presence of inflammatory cytokine tumor necrosis factor (TNF) -a (Figure 2). Cytokine levels were determined by ELISA (Biolegend) method. The percent inhibition of TNF-α secretion for the compounds subject to this patent are shown in Table 1.

Table 1. Biological results and assessment of LeadIT binding.

Compound IC50 (μΜ) ± SEM % inhibition of secretion TNF-α at concentration 100μΜ LeadIT (kcal / mol) rating 1 16.6 ± 1.2 87.4 -31.70 2 64.1 ± 1.4 80.8 -30.83 3 167.5 ± 1.4 93.3 -25.42

Example 4:

Compounds with the properties described above bind specifically to the TLR4 protein. The binding experiment was performed using surface plasmon resonance (SPR). We used a Biacore T100 instrument (GE Healthcare) equipped with a Series S Sensor chip CM5 (GE Healthcare). HBS-EP buffer (10 mM Hepes, 150 mM NaCl, 0.005% P20, pH 7.4) was used to immobilize the protein. TLR4 was covalently bound to the chip via amine groups pj by the chip manufacturer. The surface of the CM5 chip (flow cells 1 and 2) was activated with a 12-minute pulse of a mixture of 0.4 M l-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 0.1 M Nhydroxysuccinimide according to the manufacturer's instructions. Recombinant human TLR4 (R&D Systems) was dissolved in 10 mM acetate buffer (pH 5.0), to a final concentration of 200 pg / ml and injected twice after 600 s via another flow cell. Both cells were blocked with a 7 minute pulse of 1.0 M ethanolamine. The final binding level after deactivation of both cells was approximately 8000 RU. Screening was performed at 25 ° C using PBS (10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , 137 mM NaCl, 2 mM KC1, pH 7.4) and 2% DMSO (Merck) added as carrier buffer. All 3 active compounds from cellular assays were tested at 2 different concentrations, at 20 μΜ and 200 μΜ. Each compound was injected for 1 minute at a flow rate of 30 μΐ / min and dissociation was monitored for 30 seconds. The surface was not regenerated, but after each injection, the buffer flow was allowed to stabilize for 30 seconds. We compared the responses of the compounds with the buffer response and found that all 3 compounds showed affinity for the TLR4 protein (Fig. 3). Compound 1 was also soluble at a concentration of 200 μΜ, while compounds 2 and 3 formed aggregates at a higher concentration.

METHOD OF INDUSTRIAL OR OTHER USE OF THE INVENTION

The compounds which are the subject of this patent are used for the preparation of drugs that act antagonistically to the TLR4 receptor. Medicaments having a pharmacologically effective content of the compounds of this patent are used to treat diseases characterized by an overactive immune response via the TLR4 receptor, such as sepsis and neuropathic pain.

Claims (5)

1. Compounds designated by the structural formula, compound 3 and the name IUPAC: Compound 1: 1- (4-fluorophenyl) -2- (5- (2-hydroxy-5-methoxybenzoyl) pyrimidin-2-yl) guanidine Compound 2: 6 - ((2- (benzo [d] thiazol-2- yl) pyrrolidin-1-yl) methyl) -A ₂ - (o-tolyl) -1,3,5-triazine-2,4-diamine Compound 3: 2- (methyl ((4-oxo-6-phenyl-3,4-dihydrothieno [2,3-d] pyrimidin-2yl) methyl) amino) -A- (4-morpholinophenyl) acetamide, with antagonistic action to the TLR4 receptor. Compounds of the structure and name of IUPAC according to claim 1, characterized in that they are used as therapeutically effective substances. Use of compounds of the structure and name of IUPAC according to claim 1 for the preparation of drugs that act antagonistically to the TLR4 receptor. Use of compounds of the structure and name of IUPAC according to claim 1 for the preparation of medicaments for use in the treatment of diseases characterized by excessive immune responses via the TLR4 receptor, such as sepsis and neuropathic pain. Pharmaceutical preparations characterized in that they contain a therapeutically effective amount of compounds of the structure and name of IUPAC according to claim 1 and pharmaceutically acceptable excipients.