CN107898784A - Application of the eltrombopag olamine monoethanolamine in Killing Mycobacterium Tuberculosis infection - Google Patents

Application of the eltrombopag olamine monoethanolamine in Killing Mycobacterium Tuberculosis infection Download PDF

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Publication number
CN107898784A
CN107898784A CN201711059498.9A CN201711059498A CN107898784A CN 107898784 A CN107898784 A CN 107898784A CN 201711059498 A CN201711059498 A CN 201711059498A CN 107898784 A CN107898784 A CN 107898784A
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mycobacterium tuberculosis
monoethanolamine
eltrombopag olamine
tuberculosis infection
compound
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CN201711059498.9A
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CN107898784B (en
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杨海涛
郑金姣
刘祥
李俊
张晓玲
王泽方
陈成
蔡岩
饶子和
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TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
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TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention provides one kind to be directed to mycobacterium tuberculosis (Mycobacterium tuberculosis, Mtb the compound of the leucine amino peptidase (LAP) in), the compound is eltrombopag olamine monoethanolamine, there is significant inhibitory activity to the leucine amino peptidase in mycobacterium tuberculosis, therefore compound provided by the invention can be used for preparing the micromolecular inhibitor for leucine amino peptidase in mycobacterium tuberculosis, be expected to become the potential drug of anti-Mtb infection.

Description

Application of the eltrombopag olamine monoethanolamine in Killing Mycobacterium Tuberculosis infection
Technical field
The present invention relates to the technical field of pharmacy, and specifically eltrombopag olamine monoethanolamine is in Killing Mycobacterium Tuberculosis infection Application.
Background technology
Mycobacterium tuberculosis (Mycobacterium tuberculosis, Mtb) is cause tuberculosis (TB) pathogenic thin Bacterium, mainly by respiratory infectious, can cause the symptom such as cough, pectoralgia, spitting of blood, expiratory dyspnea.Healthy People infects tulase It might not fall ill, just fall ill only when immunity of organisms declines.The World Health Organization (WHO) statistics shows that the whole world is every Tuberculosis 800~10,000,000 occur for year, there are about 3,000,000 people every year and die of tuberculosis, are the most single biographies of the number of causing death Catch an illness.WHO announces " the global tuberculosis state of emergency " within 1993, it is believed that tuberculosis has become the important public health in the whole world and asked Topic.
Tuberculosis Pseudomonas mycobacteria, was found in 1882 by German microbiologist Robert Koch.Under the microscope, Tulase is elongated slightly bent or straight bacillus.Mycobacterium tuberculosis is obligate aerobes, and growth is very slow, in solid medium On, the tulase generation time is 18-20h, and incubation time needs 8 days so that up to 8 weeks, bacterium colony was in coarse on most of culture medium Type.Mtb has wax cell membrane, all has extremely strong resistance to dry environment and strong acid and strong base, will not be by a variety ofization Learn disinfectant infiltration.Tulase actually includes human-like, ox type, mouse type and African type, is mycobacterium tuberculosis complex, wherein Human-like, ox type and African type are pathogenic bacteria.
Tuberculosis is a global health problem, can cause the death of more than 100 ten thousand people, in recent years, tying every year The control aspect of core disease, due to the abuse and its drug combination of antibiotic, causes the appearance of multiple drug-resistant bacteria strain, especially Mtb Coinfection with HIV has been found to be lethal.In recent years, from TB national immigrant, HIV carriers and society occurred frequently The incidence of TB is also in rising trend in edge crowd (such as drug addicts and prisoner), these present situations all make us control tuberculosis Mycobacteria is faced with formidable challenges.So our urgent needs find the new target spot in mycobacterium tuberculosis, and then The medicine of new treating tuberculosis branch is developed for these target spots.The protease of mycobacterium tuberculosis coding has kind more than 100, this Play an important role in the growth cycle of the mycobacterium tuberculosis of a little protease, however, the research for protease is but very It is few.In the translation process of albumen, aminopeptidase is the protease of the selective N- ends for removing new synthetic protein and peptide, is Many pathogen microorganisms are maintained to grow essential protease.Leucine aminopeptidase (LAP) is oligomeric as cytosol Metal amino peptase, is the prototypical member of peptide family M17, internal survival of the leucine aminopeptidase for mycobacteria and is caused a disease Property is all indispensable.Therefore, leucine amino peptidase also becomes a crucial against mycobacterium tuberculosis drug targets, institute Have with the relevant medicament research and development infected for leucine amino peptidase progress inhibitor screening mycobacterium tuberculosis very big Meaning.
Eltrombopag olamine monoethanolamine, English name are Eltrombopag, are the compounds of double hydrazone classes, it is thrombopoietin The non-peptide agonists of acceptor (TpoR), are mainly used for treating the patient of long-term decrease of platelet.But so far, eltrombopag olamine second Application of the hydramine in Killing Mycobacterium Tuberculosis infection has no report.
The content of the invention
The problem of in correlation technique, the present invention provide eltrombopag olamine monoethanolamine in Killing Mycobacterium Tuberculosis infection Using.
Present invention also offers the inhibitor for leucine amino peptidase in mycobacterium tuberculosis.
Eltrombopag olamine monoethanolamine CAS involved in the present invention is 496775-62-3, is bought in Selleck.cn companies. On molecular level, by setting up negative control, find eltrombopag olamine monoethanolamine to the leucine amino peptidase in mycobacterium tuberculosis There is good inhibitory activity, compound is measured to Mycobacterium Smegmatis' by resazurin Microdilution plate method afterwards Minimal inhibitory concentration, find eltrombopag olamine monoethanolamine for the high mycobacterium smegmatis of mycobacterium tuberculosis tetraploid rice (Mycobacterium Smegmatis) has very strong inhibitory action, therefore the compound is expected to as suppression tuberculosis branch The potential drug of bacillus infection.
The present invention provides a kind of leucine amino peptidase (MtLAP) infection being used to prevent or treat in mycobacterium tuberculosis Medicine, its active ingredient are eltrombopag olamine monoethanolamine, which contains eltrombopag olamine monoethanolamine and one kind or more comprising above-mentioned Kind pharmaceutically acceptable carrier.It is the diluent of the carrier including pharmaceutical field routine, excipient, filler, adhesive, wet Moisten agent, disintegrant, sorbefacient, surfactant, absorption carrier, lubricant, synergist.The medicine can be made into injection, Tablet, pill, capsule, the form of suspending agent or emulsion use.Its method of administration can be oral, percutaneous, vein or intramuscular injection.
The present invention has the advantages and positive effects of：
The inhibitor of the leucine amino peptidase (MtLAP) being directed in mycobacterium tuberculosis of the present invention, this inhibitor are Eltrombopag olamine monoethanolamine.Eltrombopag olamine monoethanolamine has the leucine aminopeptidase activity in mycobacterium tuberculosis significant suppress Effect.
Brief description of the drawings
Fig. 1 is inhibitory action schematic diagram of the eltrombopag olamine monoethanolamine to the leucine amino peptidase in mycobacterium tuberculosis
Fig. 2 is IC of the eltrombopag olamine monoethanolamine to the leucine amino peptidase in mycobacterium tuberculosis50Measure schematic diagram
Embodiment：
In order to which the present invention is better described, the embodiment of the present invention will be described below.
1. the expression and purification of leucine amino peptidase (MtLAP) in mycobacterium tuberculosis
According to document (The Activity of a Hexameric M17Metallo-Aminopeptidase IsAssociated With Survival ofMycobacterium tuberculosis[J].Frontiors in Microbiology,2017,27(3):00504.)
(1) by the bacterium of the pET28a carrier Transformed E scherichia coli BL21 (DE3) containing coding MtLAP genes Strain, and with LB plating mediums (kanamycins containing 50mg/L) screening positive clone.
(2) the picking positive colony on tablet, be transferred to after 37 DEG C of overnight incubations 0.8L LB culture mediums (card containing 50mg/L that Mycin), when its light absorption value (that is, OD600) at 600nm wavelength reaches 0.6, add 0.1mM IPTG (isopropylthios Galactoside, Isopropyl β-D-Thiogalactoside) when 20 DEG C of cultures 16 are small.
(3) height crushes bacterium after collecting cell with 5000rpm centrifugations 10min；Broken bacterium solution is received after centrifuging 30min with 10000rpm Collect supernatant.
(4) supernatant is added to the Ni- of broken bacterium buffer (20mM Tris-HCl, 0.5M NaCl, pH 7.9) pre-equilibration In NTA affinity columns, destination protein is fully combined with Ni, destination protein is fully enriched with.
(5) uncombined foreign protein, Coomassie brilliant blue G250 detection stream are washed off with the broken bacterium buffer containing 30mM imidazoles Go out liquid it is constant blue when, illustrate that major part foreign protein is rinsed totally.MtLAP is eluted with the broken bacterium buffer of the imidazoles containing 300mM, so Liquid is changed with the concentration tube concentration of 30kD afterwards, is purified with anion-exchange chromatography to obtain the purpose egg with electric charge homogeneity In vain.
The determination of activity of 2.MtLAP
Substrate is used as using Lec-AMC (be purchased from Shanghai gill biochemistry Co., Ltd) of the purity more than 95%；Fluorescence intensity The instrument of measure isThe wavelength point of the multi-functional micropore board detector of M1000Pro all-wave lengths, exciting light and transmitting light Wei not 360nm and 460nm.
Albumen buffer composition is 20mM Tris-HCl, 250mM NaCl, and 50% glycerine, pH=7.5, is matched somebody with somebody with buffer solution MtLAP (final concentration 80nM) is put, adds the compound (final concentration of 20 μM) for being dissolved in DMSO (dimethyl sulfoxide (DMSO)), room temperature is placed 5min, is rapidly added fluorogenic substrate Lec-AMC, and concentration of substrate is 20 μM.The record first order fluorescence reading per 30s, measures altogether 4500s.654rpm shakes 10s, detects fluorescent value.Negative control is not added with alternative sample, other experiment condition all sames.
Using the time as X-axis, fluorescent value can obtain enzyme activity kinetic curve for Y-axis.The phase of the enzyme reaction recorded by microplate reader Related parameter, according to fluorescence intensity and reaction time, using the enzymatic reaction of 540s before GraphPad Prism5 software analysis Speed.Set V0For the initial velocity of the enzymatic reaction of without inhibitor, ViFor the initial velocity of the enzymatic reaction of inhibiting.According to Enzyme's reaction speeding, calculates residual activity Ra (Residual Activity, Ra) (V of each compoundi/V0) and suppress Rate Ir (Inhibition Rate, Ir) (1-Vi/V0)。
For residual activity<30% compound carries out secondary screening, excludes the possibility that operation error causes false positive.For surplus Remaining activity<30% compound, design fluorescent quenching experiment.Consider residual activity percentage and the fluorescent quenching of compound Rate it may determine that compound to the inhibition of MtLAP.Because the system is mainly screened by fluorescence intensity, when this Body has the compound of fluorescence compound either similar with AMC all can produce interference to system.In addition, itself, which contains, is quenched base Dough compound also may can be quenched the fluorescence of system and cause false positive, in order to exclude false positive results, using first by MtLAP with Fluorogenic substrate reaction a period of time, the system fluorescence of allowing reach maximum Q1, again addition and the chemical combination of experimental group equivalent in system Thing, and the fluorescent value size of detection architecture is Q2.Fluorescent quenching rate Qr ((Qr are calculated according to formula in both fluorescent values =Q1-Q2)/Q2*100%).Work as fluorescence
When rate is quenched higher than 20%, it is false positive, as a result needs to exclude；When fluorescent quenching rate is less than 20%, for positive knot Fruit.
3. compound eltrombopag olamine monoethanolamine IC50Measure
In measure IC50When, we configure albumen MtLAP, the final concentration of 80nM needed for experiment first, then with 95% DMSO configures substrate Lec-AMC, makes its final concentration of 20 μM.We are first according to rough 10 inhibitor of setting of primary dcreening operation result Concentration (obtains) generally by gradient dilution, the concentration of eltrombopag olamine monoethanolamine be respectively 200uM, 100uM, 50uM, 25 μM, 12.5μM、6.25μM、3.125μM、1.5625μM、0.78125μM、0μM.It is essentially the same with operation before afterwards, first by egg It is white to add in ELISA Plate in microplate reader with inhibitor after 37 DEG C are incubated 5min, be rapidly added 2 μ L substrates, the record time with it is glimmering Light change curve.We obtain protease fluorescence reaction first rate by 6.0 softwares of Graphpad prism, and are fitted chemical combination The amount effect relation curve of thing concentration and residual activity, draws IC50Value.
4. resazurin Microdilution plate method measures minimal inhibitory concentration of the compound to Mycobacterium Smegmatis
(1) bacterium solution culture
According to 1 in super-clean bench:The glycerol stock of the MC2155 of 200 ratio inoculation 50ul is pressed in the centrifuge tube of 50ml 7H9 culture mediums, 37 DEG C of 220rpm shaken cultivations, to OD are added according to ratio600=1.2, (may be reused in three weeks).Add With 1 in super-clean bench:100 ratios transfer seed liquor into 5mL7H9 fluid nutrient mediums, 37 DEG C of 220rpm shaken cultivations, to OD600 =0.5 (logarithmic phase).Bacterium solution is taken to be adjusted to OD600=0.15, and dilute 100 times of uses.
(2) dilution of positive compound rifampin
Using rifampin as positive control in experiment, gradient dilution is followed successively by, 64ug/ml, 32ug/ml, 16ug/ml, 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml, 0.03125ug/ml, minimal inhibitory concentration of the 6th dilution gradient of general warranty for the medicine to wild-type strain.
(3) dilution of medicine eltrombopag olamine monoethanolamine
Drug dilution is carried out by 2 times of dilution gradients, concentration is 512ug/ml, 256ug/ml, 128ug/ml, 64ug/ successively Ml, 32ug/ml, 16ug/ml, 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml.
(4) 40 μ l 7H9 (containing Tween-80 and ADS) fluid nutrient mediums are added into 96 orifice plates, each detected bacterial strain is set 3 medicines are parallel, and 2 μ l comparable sodium medicines are added per a line, then add the 40 μ l of bacterium solution diluted, gently shake mixing.Put It is placed in 37 DEG C of incubators and is incubated 48h.
(5) 0.02% (w/v) resazurin of 8 μ l filtration sterilizations is added in super-clean bench, continues to be incubated 4h, amplifies in being inverted Bacterial growth situation is observed on mirror, pinkiness is the positive；It is then feminine gender in blueness.
The present invention relates to the technical field of pharmacy, and specifically eltrombopag olamine monoethanolamine is in Killing Mycobacterium Tuberculosis infection Application, eltrombopag olamine monoethanolamine Ir in leucine amino peptidase in suppressing mycobacterium tuberculosis>More than 95%, it is anti-preparing There is very big application potential in terms of the micromolecular inhibitor of leucine amino peptidase in mycobacterium tuberculosis, be expected to become treating tuberculosis branch The potential drug of bacillus infection.
Method used above, is method commonly used in the art unless otherwise specified.
It these are only the embodiment of the present invention, be not intended to limit the invention, it is all in the spiritual and former of the present invention Within then, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention.

Claims (5)

1. application of the eltrombopag olamine monoethanolamine in treatment mycobacterium tuberculosis infection.
2. eltrombopag olamine monoethanolamine is the micromolecular inhibitor of leucine amino peptidase in mycobacterium tuberculosis.
3. application according to claim 1 or 2, wherein, the structural formula of eltrombopag olamine monoethanolamine is：
4. a kind of medicine for treating mycobacterium tuberculosis infection, it is characterised in that it includes the Ai Qu described in claims 1 or 2 Ripple pa monoethanolamine and one or more pharmaceutically acceptable carriers；Diluent of the carrier including pharmaceutical field routine, Excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, surfactant, absorption carrier, lubricant, synergy Agent.
5. the medicine of the treatment mycobacterium tuberculosis infection according to claims 4, it is characterized in that it is containing the medicine Injection, tablet, pill, capsule, suspending agent or the emulsion of thing.
CN201711059498.9A 2017-11-01 2017-11-01 Application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection Active CN107898784B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111150725A (en) * 2019-10-31 2020-05-15 天津国际生物医药联合研究院 Potential application of closantel sodium in resisting mycobacterium infection

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WO2012121957A1 (en) * 2011-03-08 2012-09-13 Glaxosmithkline Llc Combination
CN102697745A (en) * 2007-05-03 2012-10-03 葛兰素史密斯克莱有限责任公司 Novel pharmaceutical composition
US20160184268A1 (en) * 2013-09-02 2016-06-30 Bandi Parthasaradhi Reddy Compositions of eltrombopag

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102697745A (en) * 2007-05-03 2012-10-03 葛兰素史密斯克莱有限责任公司 Novel pharmaceutical composition
WO2012121957A1 (en) * 2011-03-08 2012-09-13 Glaxosmithkline Llc Combination
US20160184268A1 (en) * 2013-09-02 2016-06-30 Bandi Parthasaradhi Reddy Compositions of eltrombopag

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111150725A (en) * 2019-10-31 2020-05-15 天津国际生物医药联合研究院 Potential application of closantel sodium in resisting mycobacterium infection

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