Application of a polyketone compound with a special cage structure

Abstract

The present invention relates to the technical field of microbial pharmaceutical chemistry, specifically to a method for preparing a Penicillium fungus ( Penicillium daleae The present invention relates to the medical use of a polyketide compound with a unique cage-like structure isolated from the fermentation product of a strain of the bacterium Mycobacterium spp. Using patch-clamp techniques to measure the action potential discharge frequency and spontaneous excitatory post-synaptic currents (sEPSCs) of hippocampal basket cells, the present invention confirmed that the compound has significant inhibitory activity on neuronal excitability and is non-toxic to N2a neurons at active concentrations. The compound can be used to prepare new drugs for treating diseases associated with neuronal hyperexcitability.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pharmaceutical chemistry, and in particular to the use of a cage-structured polyketide compound derived from Penicillium daleae in the preparation of a drug for inhibiting neuronal overexcitation. Background Art

[0002] Neurons are the fundamental structural and functional units of the nervous system, capable of receiving, emitting, and transmitting electrical signals. Synapses are specialized structures within neurons that connect with surrounding nerve cells to form neural networks, serving as the structural foundation for information transmission within organisms. Normally, signal transmission between two neurons is completed when neurotransmitters are released from the presynaptic membrane and bind to receptors on the postsynaptic membrane, generating either an excitatory postsynaptic current (EPSC) or an inhibitory postsynaptic current (IPSC). When the excitatory electrical signals transmitted by a neuron's synapses continuously change, the cell will compensate by regulating its response to maintain a relatively stable state. This process is known as homeostatic plasticity in the nervous system. When this homeostatic plasticity is disrupted, leading to neuronal overexcitation, various diseases such as epilepsy and stroke can occur.

[0003] Epilepsy is a chronic, recurrent syndrome of transient brain dysfunction, a neurological disorder characterized by recurrent seizures caused by highly synchronized neuronal discharges. The primary etiology of epilepsy is trauma and recurrent seizures. Brain damage following an epileptic seizure can alter the expression levels of various regulatory nervous system molecules within brain cells, leading to excessive neuronal proliferation and ectopic growth, further triggering neuronal hyperexcitation and leading to epileptic seizures.

[0004] Currently, the primary treatment for epilepsy is medication, and inhibiting neuronal hyperexcitation is the primary target of epilepsy drugs. Frontline medications used in clinical practice, such as carbamazepine, lamotrigine, and gabapentin, have numerous side effects and are prone to developing drug resistance. For example, carbamazepine can cause allergic rashes, while lamotrigine and gabapentin may cause dizziness, nausea, and drowsiness. Furthermore, most anti-epileptic drugs have side effects on liver, kidney function, and the blood system. Therefore, the development of new drugs that can inhibit neuronal hyperexcitation with fewer side effects holds great potential for market development.

[0005] The applicant has extracted a variety of new compounds from the fermentation products of five fungi including Xylaria longifolia (see: Wang Qingyuan, Research on Chemical Composition and Biological Activity of Five Fungi Including Xylaria longifolia [D], South-Central University for Nationalities, Issue 03, 2024), including the cage-structured polyketide compounds described in the present invention. It is of great significance to study their pharmacological activity. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a cage-structured polyketide compound isolated from the fermentation product of Penicillium daleae for use in the preparation of a drug having the activity of inhibiting neuronal hyperexcitability.

[0007] The cage-structured polyketone compound is a compound represented by Formula I:

[0008]

[0009] Furthermore, the drug having the activity of inhibiting neuronal hyperexcitability is a drug intended to inhibit neuronal hyperexcitability, such as a drug used to treat epilepsy, stroke, and Alzheimer's disease.

[0010] The cage-structured polyketide compound has the activity of inhibiting neuronal excitability, can be used as a lead compound for inhibiting neuronal overexcitation, and can be used for preparing drugs for inhibiting neuronal overexcitation.

[0011] The cage-structured polyketone compound of the present invention is soluble in dichloromethane, chloroform, acetone, methanol, pyridine, dimethyl sulfoxide and the like, but is insoluble in water.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] 1. The drugs currently used in clinical practice to inhibit neuronal hyperexcitability are mainly benzodiazepines The present invention provides polyketide compounds with a completely different skeleton type from currently used drugs for inhibiting neuronal hyperexcitability. These compounds can be further developed into drugs for inhibiting neuronal hyperexcitability, providing more options for the market.

[0014] 2. This invention is the first to reveal that the cage-structured polyketide compounds isolated from the fermentation products of Penicillium daleae have the activity of inhibiting neuronal excitability. In the future, the scientific community will focus on searching for lead compounds with the activity of inhibiting neuronal excitability from Penicillium daleae.

[0015] 3. The cage-structured polyketone compounds to be protected by the present invention can be obtained by microbial fermentation. The entire production process is chemical-free and environmentally friendly.

[0016] 4. The compounds of the present invention have the ability to significantly inhibit the action potential discharge frequency and spontaneous excitatory post-synaptic current (sEPSCs) frequency of basket cell neurons in the hippocampus of mice, and are non-toxic to nerve cells N2a, and are useful for preparing new drugs that inhibit neuronal hyperexcitability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : is the hydrogen spectrum (600 MHz, CDCl3) of the compound of formula I prepared in a specific embodiment.

[0018] Figure 2 : is the carbon spectrum (150 MHz, CDCl 3 ) of the compound of formula I prepared in a specific embodiment.

[0019] Figure 3 This is a graph showing the cytotoxicity of the compound of formula I at different concentrations in Example 1 to N2a cells.

[0020] Figure 4 This is a graph showing that the compound of formula I in Example 2 reduces the firing frequency of basket cells.

[0021] Figure 5 The frequency signal graph (AD left), cumulative probability graph (AD right) and statistical graph of the rate of change of sEPSC frequency before and after administration of different concentrations of the compound of Formula I in Example 2 are shown (ns, not significant; *P<0.05, statistically significant; **P<0.01, statistically significant; ***P<0.001, extremely statistically significant). DETAILED DESCRIPTION

[0022] In order to make the purpose and content of the invention of this application more clear, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0023] In the following examples, if no specific conditions are specified, all experiments were carried out according to conventional conditions or those recommended by the manufacturer. Reagents or instruments used, if no manufacturer is specified, are conventional commercially available products.

[0024] The animal experiments in the following examples have been approved by the Laboratory Animal and Biomedical Ethics Committee of South-Central University for Nationalities (No. 2019-SCUEC-AEC-021).

[0025] The Penicillium daleae strain used was isolated from soil derived from the rhizomes of the orchid "Monstera cloversa," which was obtained from Tiantangzhai Scenic Area in Yingshan County, Hubei Province, in August 2018. The strain is currently deposited with the Medicinal Fungi and Ethnomedicine Research Group, School of Pharmacy, South-Central University for Nationalities, under the registration number L3SO. The address is: 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province, China. The strain is available to the public from this unit.

[0026] The cage-like polyketide compound studied in the present invention is derived from the fermentation product of Penicillium daleae. Its structure is shown in Formula I:

[0027]

[0028] The cage-structured polyketone compound represented by formula I of the present invention is obtained as follows:

[0029] (1) Preparation of culture medium:

[0030] The rice culture medium formula is: 400g of rice and 400mL of water are added to a 3L conical flask, respectively, and the mixture is autoclaved at 121°C for 20 minutes and then cooled to room temperature (room temperature in this application refers to 25°C). In this embodiment, 4kg of rice culture medium is prepared according to the above formula ratio.

[0031] (2) Fermentation

[0032] 1 mL of bacterial culture solution (about 1×10 8 After the spores were inoculated into rice culture medium, they were cultured at room temperature in a dark environment for 60 days to complete the fermentation.

[0033] (3) Extraction

[0034] (a) After fermentation, the extracts were extracted using anhydrous methanol (10 L each time, three days each time, for a total of three times). After each soaking, the methanol extract was separated from the fermentation product using a centrifuge (Zhangjiagang Dingshi Machinery). The three extracts were combined and then the methanol extract was concentrated under reduced pressure using a rotary evaporator, a vacuum pump, and a low-temperature cooling circulation pump (i.e., a vacuum concentration device, the same below) to remove the methanol and obtain a concentrate.

[0035] (b) The concentrate was diluted with water to 2 L and then extracted with ethyl acetate in a 10 L separatory funnel in a volume ratio of 1:1. After standing and stratification, the upper ethyl acetate layer was taken and the lower aqueous layer was further extracted with ethyl acetate / water in a volume ratio of 1:1. After repeated extraction three times, the ethyl acetate layers were combined and concentrated under reduced pressure to remove ethyl acetate to obtain 80.5 g of crude extract.

[0036] (c) Add an appropriate amount of methanol to the crude extract as a solvent to dissolve it. Transfer the extract to a suitable evaporating dish and add 100 g of normal-phase silica gel (80-100 mesh, Qingdao Ocean Chemical Co., Ltd.) to adsorb the sample. Evaporate the solvent in a water bath (constant temperature 50°C) and grind into a powder. Add 300 g of fresh normal-phase silica gel and the powder to a glass column. Elute with a gradient of petroleum ether and acetone (in this order: 2 L of pure petroleum ether, 2 L of a 20:1 volume ratio, 2 L of a 15:1 volume ratio, 2 L of a 10:1 volume ratio, 2 L of a 5:1 volume ratio, 2 L of a 2:1 volume ratio, and 1 L of pure acetone). Bottle each 200 mL volume. Concentrate under reduced pressure until the solvent is dry. Dissolve the solution in a small amount of methanol and transfer the solution to a 10 mL vial with a rubber-tipped pipette. All vial samples were sampled with a capillary and spotted on a silica gel plate G254 (Qingdao Ocean Chemical Co., Ltd.), and developed in a 200 mL developing cylinder with 5 mL of a mixed solvent of petroleum ether and acetone (volume ratio of 3:2). After the solvent reached the top, it was taken out and dried. The fluorescence of the compound was observed under an ultraviolet lamp (254 nm). Vanillin-concentrated sulfuric acid-ethanol solution was evenly sprayed on the silica gel plate and heated on an electric furnace for color development. The same or similar components were combined, and the elution component at a volume ratio of petroleum ether to acetone of 5:1 was collected and recorded as component E.

[0037] (d) Component E was dissolved in a small amount of methanol, filtered through cotton and added to a Sephadex column (LH-20). The eluent was pure methanol. One tube was connected for every 10 mL. TLC was used for detection (the developing solvent was a mixed solvent of petroleum ether and acetone in a volume ratio of 3:2). The fluorescent spot under 254 nm ultraviolet light was used as the main reference. Vanillin-concentrated sulfuric acid-ethanol solution was used for color development. f Fractions with spots of ≈0.5-0.6 were combined and evaporated to dryness under reduced pressure, designated as Fraction E1. Fraction E1 was dissolved in a small amount of chromatographic-grade methanol, filtered through a 0.22 μm filter into a 1.5 mL liquid phase vial, and separated and purified on a preparative HPLC (Agilent 1260, USA) to obtain the compound of Formula I. Conditions: Zorbax SB-C 18 Column (particle size 5 μm, size 9.4×150 mm, flow rate 4 mL·min -1 ), the mobile phase was acetonitrile and water (v / v, 35:65 to 50:50, 30 min), the detector was DAD (photodiode array detector), the detection wavelengths were 205, 220, 254, 280 and 365 nm, and the peak at a retention time of 27 minutes was the compound of formula I.

[0038] Structure identification:

[0039] Purify the obtained compound: The solvent was concentrated and evaporated to dryness under reduced pressure, dissolved in 0.45 mL of deuterated chloroform, and then transferred to a nuclear magnetic resonance tube. The hydrogen spectrum and carbon spectrum ( Figure 1 , Figure 2 ) and two-dimensional spectra, HR-ESI-MS confirmed that its molecular formula is C 31 H 40 O 10 (m / z 573.26941[M+H] + , the calculated value is C 31 H 41 O 10 ,573.26997) has a total of 12 degrees of unsaturation, and the final confirmed structural formula is shown in Formula I, which belongs to a cage-structured polyketone compound.

[0040] The cage-structured polyketone compound represented by formula I of the present invention is soluble in solvents such as dichloromethane, chloroform, acetone, methanol, pyridine, and dimethyl sulfoxide, but is insoluble in water.

[0041] Example 1

[0042] The CCK-8 method was used to detect the cytotoxicity of cage-structured polyketide compounds on neuronal cells N2a (Neuro-2a, purchased from Wuhan Shanen Biotechnology Co., Ltd.).

[0043] Principle of the test: After CCK-8 solution (the active ingredient is WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt) is added to the cell suspension, WST-8 is oxidized and reduced by intracellular dehydrogenases in the presence of electron carriers to generate an orange-yellow formazan dye. This formazan dye can be dissolved in tissue culture medium, and the amount of formazan generated is proportional to the number of viable cells.

[0044] N2a cells were seeded into Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% fetal bovine serum and 1% penicillin / streptomycin and activated and cultured at 37°C and 5% CO2. To maintain the good condition of the cells, the medium was changed periodically and the cells were passaged at a density of 80% using 0.25% trypsin. The cultured N2a cell suspension was plated at 7×10 3 Cells / well were seeded in a 96-well plate, and the compound of formula I prepared by DMSO gradient dilution method was added to make the final concentrations 0.5, 1, 10, and 20 μM, respectively. DMEM was added to make the final volume of each well 200 μL. A negative control group (i.e., the following 0 加药(A group (an equal amount of DMSO was added and DMEM medium was supplemented to a final volume of 200 μL per well) was grown at 37°C and 5% CO for 24 hours. Then, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for another hour in the dark. Finally, the absorbance of each well was measured at 450 nm using a 96-well microplate reader (Thermo Fisher Scientific, USA), and cell viability was calculated.

[0045] Cell viability calculation:

[0046] Cell viability*(%)=[A (加药) -A (空白) ] / [A (0加药) -A (空白) ]×100%;

[0047] A (加药) : absorbance of wells with culture medium, cells, CCK-8 solution, and drug solution;

[0048] A (空白) : absorbance of wells with culture medium and CCK-8 solution but no cells;

[0049] A (0加药) : absorbance of the wells with culture medium, cells, and CCK-8 solution but no drug solution;

[0050] *Cell viability: cell proliferation activity or cytotoxic activity, in this example Figure 3 The cell viability is expressed in the middle.

[0051] The test results are as follows Figure 3 As shown, the compound of formula I showed no cytotoxicity to the neural cell N2a at a concentration of 500 nM-20 μM.

[0052] Example 2

[0053] The patch clamp technique was used to detect the inhibitory activity of the cage-structured polyketide compounds on the action potential discharge frequency and spontaneous excitatory post-synaptic current (sEPSCs) frequency of male rat hippocampal basket cell neurons.

[0054] Experimental principle: The patch clamp technique uses a glass microelectrode to contact the cell membrane, connecting them with an impedance of more than a gigaohm. The small area of the cell membrane (membrane patch) connected at the opening of the microelectrode tip is electrically separated from its surroundings. On this basis, the potential is fixed, and the ionic current (pA level) of the ion channel on this membrane patch is detected and recorded.

[0055] The patch clamp technique used in this example was performed as follows: All male mice were anesthetized with 2% isoflurane and rapidly decapitated. Preliminary trimmed brain tissue was immersed in an oxygen-saturated slicing solution (185 mM sucrose, 2.5 mM KCl, 2 mM MgSO₄, 2 mM CaCl₂, 26 mM NaHCO₃, 1.2 mM NaH₂PO₄, 25 mM glucose, pH 7.4) and ice-water. Coronal sections were cut using an oscillating microtome (Leica VT1200S) to a thickness of 300 μm. After dissection, brain slices were placed in an incubator containing oxygen-saturated ACSF (artificial cerebrospinal fluid): 124 mM NaCl, 1.25 mM NaH2PO4, 2.5 mM KCl, 1.3 mM MgCl2, 0.5 mM CaCl2, 26 mM NaHCO3, 10 mM glucose, pH 7.3-7.4. The slices were incubated at 32-34°C for half an hour. The temperature was controlled by a TC-324C dual automatic temperature controller (Warner Instruments, Inc., USA) and continuously bubbled with 5% CO2 and 95% O2. Following incubation, the slices were transferred to 25°C for the experiment and data were recorded.

[0056] First, under an infrared differential interference microscope (Olympus, Japan), a suitable neuron with good activity was identified. The microelectrode (3-8 MΩ) was filled with electrode solution (122 mM potassium gluconate, 5 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 1 mM EDTA, 10 mM sodium phosphocreatine, 4 mM Na2ATP, 0.4 mM Na3GTP, pH 7.3). In bath mode, the micromanipulator was used to manipulate the microelectrode close to the neuron. When the resistance increased by 0.3-0.5 MΩ, the positive pressure was removed and an appropriate negative pressure was applied to allow the cell membrane to adhere to the opening of the electrode tip. When the seal resistance exceeded 1 GΩ, the negative pressure was removed, forming a high-resistance seal. The cell fast capacitance was compensated as needed, and the appropriate negative pressure was continued until the cell membrane at the seal was broken. The microelectrode was then switched to patch mode to observe whether the resistance remained at or above 1 GΩ. The whole cell mode was then switched to perform subsequent electrophysiological recordings.

[0057] (1) Depolarize the neuron by stimulating it with a 10pA gradient from 10 to 300pA for 1000ms, and then stimulate the membrane patch with a current of 90-110pA. Monitor and record the changes in action potential frequency before administration (final concentration 5μM, prepared by dissolving the mother solution of Formula I in DMSO and adding it to the electrode solution and diluting it, and perfusing the drug for 15 minutes), and after washing the drug with the electrode solution. The experimental results were analyzed by two-way ANOVA. P < 0.05 was considered statistically significant (the experimental results are shown in Figure 2). Figure 4 shown).

[0058] (2) 10 μM bicuculline (dissolved in DMSO) was added to ACSF to block GABA receptors. The signal was filtered at 2 kHz and sampled at 20 kHz through an Axon 700B amplifier and an Axon 440A analog / digital interface (Mercury Molecular Instruments, Inc., USA). Neurons were depolarized by stimulating with a 10pA gradient from 10 to 300pA for 1000ms. Spontaneous excitatory postsynaptic currents were recorded in the gap-free mode. All neurons were maintained at -70 mV in the voltage clamp mode. The membrane patches were divided into 0.5 μM, 1 μM, 10 μM, and 20 μM drug concentration groups (all obtained by dissolving the compound of formula I in DMSO) and a control group (DMSO group). The spontaneous excitatory postsynaptic currents were recorded before drug addition. Then, the drug was added and the spontaneous excitatory postsynaptic currents were recorded after 3-5 minutes. The experimental data were statistically analyzed for differences between the groups using one-way analysis of variance. P < 0.05 was considered statistically significant (the experimental results are shown in Figure 2). Figure 5 shown).

[0059] The test results are as follows Figure 4 As shown, the compound of formula I at a concentration of 5 μM significantly reduced the firing frequency of blue cells under 90-110 pA current stimulation. Figure 5 As shown, the compound of formula I can significantly reduce the frequency of spontaneous excitatory postsynaptic current (sEPSC) of blue cells at the used concentrations (0.5 μM, 1 μM, 10 μM, 20 μM). At a concentration of 20 μM, the frequency is reduced by nearly half, and the frequency of sEPSC is concentration-dependent with the dosage of the compound of formula I.

[0060] In summary, the cage-structured polyketide compounds isolated from the fermentation extract of Penicillium daleae provided by the present invention have significant activity in inhibiting neuronal excitability and can be used as candidate molecules of lead compounds with inhibitory activity for the preparation of drugs for inhibiting excessive neuronal excitability.

[0061] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of protection claimed in the present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

Claims

1. Use of a special cage-structured polyketide compound in the preparation of a drug capable of inhibiting neuronal hyperexcitability, characterized in that: The special cage-structured polyketone compound is a compound having the following chemical structural formula I:

2. The use according to claim 1, characterized in that The compound of formula I is used in combination with or without other active ingredients when preparing drugs for inhibiting neuronal hyperexcitability.

3. The use according to claim 2, characterized in that The special cage-structured polyketone compound is the only active ingredient.

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