阅读说明：本技术 一种小分子化合物c34h22fn3o5s2在大肠杆菌性脑膜炎中的应用 (Small molecule compound C34H22FN3O5S2Application in E.coli meningitis ) 是由 陈誉华 徐晓倩 赵伟东 于 2019-11-06 设计创作，主要内容包括：本发明公开了小分子化合物C_(34)H_(22)FN_3O_5S_2在制备治疗大肠杆菌性脑膜炎药物中的用途,该化合物或其异构体、溶剂合物或前体,或它们的药学上可接受的盐,通过靶向竞争性结合细菌性脑膜炎毒力因子IbeA蛋白中与脑血管内皮上受体蛋白Caspr1的互作热点氨基酸残基,从而阻断二者结合,达到阻大肠杆菌穿过血脑屏障或阻断脑膜炎大肠杆菌侵袭神经元的目的。本发明从干预细菌-宿主的配体(IbeA)-受体(Caspr1)相互作用,封闭病原菌角度出发,采用靶向治疗新手段,将为新生儿细菌性脑膜炎的预防和治疗提供新的靶向药物。(The invention discloses a small molecule compound C 34 H 22 FN 3 O 5 S 2 The compound or an isomer, a solvate or a precursor thereof, or pharmaceutically acceptable salts thereof can be combined with hot spot amino acid residues of interaction of a brain vascular endothelial receptor protein Caspr1 in a bacterial meningitis virulence factor IbeA protein in a targeted competitive manner so as to block the combination of the two, thereby achieving the purpose of preventing the escherichia coli from passing through a blood brain barrier or blocking the meningitis escherichia coli from invading neurons. The invention starts from the interaction of ligand (IbeA) -receptor (Caspr1) for intervening bacteria-host and the closure of pathogenic bacteria, adopts a new targeted treatment method, and is characterized in thatThe prevention and treatment of neonatal bacterial meningitis provides new targeted drugs.)

1. Small molecule compound C₃₄H₂₂FN₃O₅S₂The application in preparing the medicine for treating the E.coli meningitis;

the small molecule compound C₃₄H₂₂FN₃O₅S₂The structural formula of (A) is shown as formula I:

。

2. the small molecule compound C of claim 1₃₄H₂₂FN₃O₅S₂The application of the compound shown in the formula I or isomers, solvates or precursors thereof or pharmaceutically acceptable salts of the compound and the isomers, solvates or precursors in preparation of the medicine for treating the meningitis of the escherichia coli is characterized in that the compound shown in the formula I or the isomers, solvates or precursors thereof is combined in a targeted and competitive manner with the interaction hotspot amino acid residue of a receptor protein Caspr1 on the inner skin of a cerebral blood vessel in bacterial meningitis virulence factor IbeA protein, so that the combination of the two is blocked, and the aim of preventing the escherichia coli from passing through the blood brain barrier or blocking the meningitis escherichia coli from invading neurons is fulfilled.

3. The small molecule compound C of claim 2₃₄H₂₂FN₃O₅S₂The application of the IbeA protein in preparing the medicine for treating the Escherichia coli meningitis is characterized in that the IbeA protein is used as pathogenic bacteria of neonatal meningitisE.coliVirulence factors of K1.

4. The small molecule compound C of claim 2₃₄H₂₂FN₃O₅S₂The application of the IbeA-Caspr1 in preparing the medicine for treating the E.coli meningitis is characterized in that the interaction hotspot amino acid residues of IbeA-Caspr1 are R189, W196, R331, V332, N333, L337, S202, G325, I328, F329, N330, D336, R340, H342 and R344 are derived from Caspr1, V255, L319, F322, G323, T325, V326, G259, L261, T265, E266, M324, R329 and S331 are derived from IbeA.

5. The small molecule compound C according to any one of claims 1 to 4₃₄H₂₂FN₃O₅S₂The application of the medicine for treating the meningitis of the escherichia coli is characterized in that the medicine for treating the meningitis of the escherichia coli is used for blocking pathogenic bacteria to block meningitis escherichia coli from passing through a blood brain barrier or block meningitis escherichia coli from invading neurons.

6. The small molecule compound C according to any one of claims 1 to 5₂₉H₂₈Cl₂N₆The application of O in preparing the medicine for treating the E.coli meningitis is characterized in that the medicine for treating the E.coli meningitis is a medicine for treating the central nervous system diseases which can easily pass through a blood brain barrier.

7. A pharmaceutical composition for treating e.coli meningitis, comprising: a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier;

formula I:。

8. a pharmaceutical composition for treating e.coli meningitis, comprising: a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier; and other effective components for treating E.coli meningitis;

formula I:。

9. the small molecule compound C of claim 8₃₄H₂₂FN₃O₅S₂The application of the traditional Chinese medicine composition in preparing a medicine for treating the E.coli meningitis is characterized in that other effective components for treating the E.coli meningitis are broad-spectrum antibiotics.

10. The small molecule compound C according to any one of claims 7-9₃₄H₂₂FN₃O₅S₂In the preparation of medicine for curing colibacillosisUse in a medicament for treating meningitis, wherein said pharmaceutically acceptable carrier in said pharmaceutical composition is a pharmaceutically or comestibly acceptable solvent, suspending agent or excipient for delivering a compound of formula I, isomer, solvate, precursor, or pharmaceutically acceptable salt thereof, of the present invention to an animal or human; the carrier may be a liquid or a solid.

Technical Field

The invention relates to the technical field of medicaments, in particular to a new medicament target for blocking a pathogenic virulence factor IbeA of neonatal meningitis from passing through a blood brain barrier and attacking neurons, and application of the new medicament target in preparing new medicaments for the neonatal meningitis.

Neonatal bacterial meningitis is one of serious infectious diseases in pediatrics, and clinically, despite antibiotics and related supportive therapies against bacteria, 5% -40% of infants die due to bacterial drug resistance and difficulty in passing through barrier structures between blood and brain tissues of most antibiotics. Escherichia coli (e.coli) K1 strain is the most common gram-negative pathogen causing bacterial meningitis in newborns. The primary condition for causing e.coli meningitis is that bloodborne e.coli must cross the blood-brain barrier, which is mainly composed of Brain Microvascular Endothelial Cells (BMECs), where the key link is e.coli adhesion and invasion into BMECs. Recently, in the laboratory of the applicant, neuron contact chaperonin 1 (Caspr1) is identified in vivo as a receptor of Escherichia coli virulence factor IbeA on BMECs, and the IbeA is combined with a Laminin protein family domain consisting of Caspr1 extracellular segment 203-355aa through 229-343 amino acid fragment to realize the penetration of the blood brain barrier of bacteria. Based on the above research results, we first constructed the three-dimensional spatial structures and interaction sites of Caspr1 (203-. The presence of a stable small molecule binding pocket in IbeA (229-.

In view of the fact that the Caspr1 and IbeA protein purification cannot meet the technical requirements of structure analysis, the subject group of the applicant constructs three-dimensional space structures of Caspr1 (203) -355aa) and IbeA (229-343aa) by a computer-assisted homologous modeling method, predicts a binding mode and an interaction site between the Caspr1 (203) -355aa) and the IbeA (229-343aa), and determines stable binding of the Caspr1 (203) -355aa) and the IbeA (229-343aa) through MM/GBSA and RMSD. Based on the prediction model, the inventor finds that IbeA (229-.

CN108611411A discloses the use of Caspr1 membrane protein as a new drug for preparing neonatal meningitis drugs, this patent application discloses the use of Caspr1 membrane protein or IbeA-Caspr1 interacting domain as a new drug target for blocking meningitis escherichia coli from crossing the blood brain barrier and attacking neurons and as a new drug for preparing neonatal meningitis drugs, and discloses the preparation of oligopeptide drugs for blocking meningitis escherichia coli from crossing the blood brain barrier based on Caspr 1203-355 peptide fragment, but this technical scheme performs the knockout of Caspr1 gene on rats, thereby significantly reducing the incidence of neonatal mouse meningitis caused by e.coli K1, which is impractical in actual clinical and drug preparation, and the knockout of patient gene can cause a large number of problems that are difficult to verify subsequently.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a small molecule compound C₃₄H₂₂FN₃O₅S₂The application in preparing the medicine for treating the E.coli meningitis.

In order to achieve the purpose, the invention adopts the following technical scheme:

small molecule compound C₃₄H₂₂FN₃O₅S₂The application in preparing the medicine for treating the E.coli meningitis;

the small molecule compound C₃₄H₂₂FN₃O₅S₂The structural formula of (A) is shown as formula I:

furthermore, the compound shown in the formula I, or an isomer, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof is combined with hot spot amino acid residues of interaction between the bacterial meningitis virulence factor IbeA protein and the brain vascular endothelial receptor protein Caspr1 in a targeted and competitive manner, so that the combination of the two is blocked, and the purpose of preventing escherichia coli from passing through a blood brain barrier or blocking the meningitis escherichia coli from invading neurons is achieved.

Further, the IbeA protein is used as a virulence factor of pathogen E.coli K1 of neonatal meningitis.

Further, the interaction hotspot amino acid residues of the IbeA-Caspr1 are R189, W196, R331, V332, N333, L337, S202, G325, I328, F329, N330, D336, R340, H342 and R344 derived from Caspr1; v255, L319, F322, G323, T325, V326, G259, L261, T265, E266, M324, R329, S331 are derived from IbeA.

Furthermore, the medicine for treating the meningitis of the escherichia coli is used for blocking pathogenic bacteria to block meningitis escherichia coli from crossing a blood brain barrier or blocking meningitis escherichia coli from invading neurons.

Furthermore, the medicine for treating the E.coli meningitis is a medicine for treating central nervous system diseases which can easily pass through a blood brain barrier.

Furthermore, the medicine for treating the E.coli meningitis is a small molecular compound medicine.

A pharmaceutical composition for treating e.coli meningitis, comprising: a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier;

formula I:

a pharmaceutical composition for treating e.coli meningitis, comprising: a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier; and other effective components for treating E.coli meningitis.

Formula I:

furthermore, the other effective components for treating the E.coli meningitis are broad-spectrum antibiotics.

Further, in the pharmaceutical composition, the pharmaceutically acceptable carrier is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the compound of formula I, isomer, solvate, precursor, or pharmaceutically acceptable salt thereof of the present invention to animals or humans; the carrier may be a liquid or a solid.

The invention discloses an interaction site of a virulence factor IbeA of bacterium E.coli K1 and a receptor Caspr1 on human brain microvascular endothelial cells, so that a small molecule compound medicine for preventing and treating neonatal bacterial meningitis, which blocks the interaction, is screened and prepared on the basis of an IbeA-Caspr1 interaction domain.

In another aspect of the invention, a structural model of the interaction between Caspr1(203-355 amino acid peptide fragment) and IbeA (229-343 amino acid peptide fragment) was constructed for the first time, and the interaction amino acid hot spot residues were reported. Wherein the hotspot residues in IbeA (229-343 peptide segment) are mainly concentrated on alpha 1 and alpha 3 helices and form a small molecule binding pocket at the periphery, which is closely related to bacterial invasion.

In another aspect of the invention, IbeA (229-343 peptide fragment) is used as a target for the first time, and the obtained molecular model is virtually screened from a drug-like small molecular compound library to search for potential inhibitors from 213279 commercially available compounds. The first 50 small molecules with ordered binding energy were tested in vitro by Surface Plasmon Resonance (SPR) for their binding capacity titration to IbeA (229-343aa) purified protein, and 9 compounds showed direct binding capacity. Furthermore, in vitro toxicity tests and bacterial invasion experiments prove that the 4 molecules can obviously reduce or prevent the invasion of bacteria into human brain microvascular endothelial cells (hBMECs) within a low cytotoxicity range.

Compared with the prior art, the invention has the beneficial effects that:

(1) the prior art has no effective method aiming at preventing the occurrence of the neonatal bacterial meningitis, the neonatal meningitis is treated by adopting broad-spectrum antibiotics, and the defect that 5 to 40 percent of the sick children still die due to bacterial resistance and the fact that most of the antibiotics are difficult to pass through a barrier structure between blood and brain tissues; and the principle of antibiotic treatment is to kill bacteria, but at the same time is associated with a large amount of inflammatory reactions, such as hyperthermia, so that even if cured, 30% of the surviving children remain with permanent neurological sequelae, such as epilepsy, mental and movement disorders, etc. In this context, it is of great interest to develop targeted drugs that are effective in preventing bacterial meningitis in newborn infants.

(2) The invention provides a novel targeted medicine for preventing and treating the bacterial meningitis of the newborn by adopting a novel targeted treatment method from the viewpoints of intervening the interaction of a ligand (IbeA) -receptor (Caspr1) of a bacterium-host and blocking pathogenic bacteria.

Drawings

FIG. 1 is a diagram showing the interaction structure pattern of Caspr1 (203) -355 amino acid peptide fragment) and IbeA (229-343 amino acid peptide fragment) and hot spot amino acid residues, wherein A is the hot spot amino acid residue in the Caspr1 (203) -355aa) -IbeA (229-343aa) complex for maintaining stable structure, and the key site sequence and the amino acid name are marked with orange characters at the corresponding sites; b is a Caspr-IbeA interaction pattern diagram, and key amino acid sites are marked by black characters; c is a surface structure pattern diagram of an IbeA (229-343) amino acid peptide segment, and orange color shows a small molecule binding pocket related to bacterial invasion.

FIG. 2 is a graph showing the affinity of Top50 small molecules to IbeA protein screened by SPR in vitro test SPECS database, wherein 9 small molecules show the characteristic of direct binding to IbeA (229-343 aa).

FIG. 3 is a diagram of toxicity of small molecules capable of directly binding to IbeA in human brain microvascular endothelial cells (hBMEC) detected by CCK-8 assay.

Fig. 4 is a graph of bacterial invasion experiments to verify the blocking effect of 5 low-toxicity small-molecule compounds on the invasion of escherichia coli e.coli K1 into human brain microvascular endothelial cells.

FIG. 5 is a schematic diagram of the docking structure of a small molecule compound and IbeA (229-343 amino acid peptide) for blocking E.coli K1 from invading human brain microvascular endothelial cells, wherein A is C₂₈H₂₆Cl₂N₈(AK-968/11283147) and IbeA (229-; b is C₂₉H₂₈Cl₂N₆The interaction pattern diagram of O (AE-641/30154051) and IbeA (229-; c is C₃₄H₂₂FN₃O₅S₂(AF-399/42017932) and IbeA (229-; d is C₂₉H₂₂N₂O₈(AN-652/14563011) and IbeA (229-.

Detailed Description

The inventor of the invention, through the intensive research, reveals a small molecule compound C for the first time₃₄H₂₂FN₃O₅S₂The application of the polypeptide in preparing the medicine for treating the meningitis of the escherichia coli can block the meningitis escherichia coli from passing through a blood brain barrier or blocking the meningitis escherichia coli from invading neurons by targeting and competitively combining meningitis virulence factor IbeA protein or IbeA-Caspr1 interaction hot spot amino acid residues. The present invention has been completed based on this finding.

In the present invention, a "pharmaceutically acceptable" component is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

In the present invention, the "pharmaceutically acceptable carrier" is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the compound of formula (I), isomer, solvate, precursor, or pharmaceutically acceptable salt thereof of the present invention to animals or humans. The carrier may be a liquid or a solid.

It will be understood by those skilled in the art that, once the structure of the compounds of the present invention is known, the compounds of the present invention can be obtained by a variety of methods well known in the art, using well known starting materials, such as chemical synthesis or extraction from organisms (e.g., animals or plants), which are encompassed by the present invention.

The invention discloses an interaction site of a virulence factor IbeA of bacterium E.coli K1 and a receptor Caspr1 on human brain microvascular endothelial cells, so that a small molecule compound medicine for preventing and treating neonatal bacterial meningitis, which blocks the interaction, is screened and prepared on the basis of an IbeA-Caspr1 interaction domain.

In another aspect of the invention, a structural model of the interaction between Caspr1(203-355 amino acid peptide fragment) and IbeA (229-343 amino acid peptide fragment) was constructed for the first time, and the interaction amino acid hot spot residues were reported. Wherein the hotspot residues in IbeA (229-343 peptide segment) are mainly concentrated on alpha 1 and alpha 3 helices and form a small molecule binding pocket at the periphery, which is closely related to bacterial invasion.

In another aspect of the invention, IbeA (229-343 peptide fragment) is used as a target for the first time, and the obtained molecular model is virtually screened from a drug-like small molecular compound library to search for potential inhibitors from 213279 commercially available compounds. The first 50 small molecules with ordered binding energy were tested in vitro by Surface Plasmon Resonance (SPR) for their binding capacity titration to IbeA (229-343aa) purified protein, and 9 compounds showed direct binding capacity. Further, in vitro toxicity tests and bacterial invasion experiments prove that 4 molecules can obviously reduce or prevent the invasion of bacteria into hBMECs in a low cytotoxicity range.

The dosage form of the pharmaceutical composition of the present invention may be various, as long as it is a dosage form that enables the active ingredient to efficiently reach the body of a mammal. Such as may be selected from: a solution, suspension, tablet, capsule, powder, granule, or syrup. Depending on the type of disease to be treated by the compounds of the present invention, one skilled in the art may select a dosage form that is convenient to use.

The effective administration dose of the compound of formula (I) as an active ingredient may vary depending on the mode of administration and the severity of the disease to be treated. Or in a sustained release form, which may be adjusted to provide the optimum therapeutic response. For example, divided doses may be administered several times daily, or the doses may be proportionally reduced, or used in combination with other drugs having an effect on the treatment of E.coli meningitis, as is urgently required for the treatment condition.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.