CN105998013A - Application of IMB-NY compounds in preparation of antimycobacterial drugs - Google Patents

Application of IMB-NY compounds in preparation of antimycobacterial drugs Download PDF

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CN105998013A
CN105998013A CN201610343082.9A CN201610343082A CN105998013A CN 105998013 A CN105998013 A CN 105998013A CN 201610343082 A CN201610343082 A CN 201610343082A CN 105998013 A CN105998013 A CN 105998013A
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杨延辉
肖春玲
刘河涛
刘忆霜
蒙建州
鲁众阳
田静
杨志伟
杨玉荣
王浩
王大军
摆茹
梁锦屏
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Ningxia Medical University
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Abstract

The invention discloses application of IMB-NY compounds in preparation of antimycobacterial drugs. The IMB-NY compounds comprise IMB-NY1 and IMB-NY2. The invention further discloses a method for screening a lead compound containing an MTB (Mycobacterium Tuberculosis) IspD inhibitor and a ZYM-5052 liquid medium for expressing the MTB IspD inhibitor. The application disclosed by the invention has the advantages that enzyme inhibitors IMB-NY1 and IMB-NY2 are obtained by means of screening compounds from more than 70000 different sources through combined application of a high-throughout phenotype screening model and an MTB Rv3582c enzyme inhibitor screening model; the situation that the compound has specific antimycobacterial activity is confirmed by means of evaluating the antituberculous activity of the IMB-NY1 and the IMB-NY2.

Description

The application in preparation Killing Mycobacterium Tuberculosis medicine of the IMB-NY compounds
Technical field
The invention belongs to chemicals and drug screening field, specifically, relate to a kind of IMB-NY class Compound application in preparation Killing Mycobacterium Tuberculosis medicine.
Background technology
Tuberculosis (TB, Tuberculosis) is by mycobacterium tuberculosis (Mycobacterium Tuberculosis, MTB) infect the chronic infectious disease caused, remain at present and threaten mankind's life One of Infectious Diseases that life is healthy.The statistical result of up-to-date World Health Organization (WHO) shows, the current whole world there are about The population of 1/3rd suffers from tuberculosis, and wherein annual new cases is more than 8,800,000, and dead 1,400,000, Become and AIDS, malaria the three big infectious disease claimed, and China is that 22, whole world TB high burden is national One of, showing: first, mycobacterium tuberculosis infection person is more than 5.5 hundred million people, and patient numbers occupies the whole world the Two；Second, resistance problems is very serious, and multidrug resistance rate has been over the warning line that WHO specifies； 3rd, HIV sufferers growth rate is fast, may cause HIV and tuberculosis double fluid row；4th, China's stream Moving mouth is big, also certainly will expand the infection rate of this infectious disease.Especially at present fastbacteria and Tuberculosis, with the increase of acquired immune deficiency syndrome (AIDS) coinfection phenomenon, makes tuberculosis become the thorny problem of clinical treatment.Mesh Before, Antibiotic Resistance and the resistance levels of tubercule bacillus are continuously increased, but the present situation of research at present is, and nearly 60 The antituberculotics all not having brand-new mechanism of action and brand-new framing structure over Nian comes out, and bacillus calmette-guerin vaccine Protective rate is in developed country also less than 50%, and the protective rate in Chinese adult does not reaches far away flourishing state The level of family.Therefore, develop efficiently, the Newer Antibuberculotics of low toxicity solves threat lungy and is The main target of current numerous seminar.
Since penicillin occurs, research worker finds that the cell wall growth and breeding for antibacterial is to closing weight Want.Due to the complicated structure of cell wall, and be antibacterial be de novo synthesis, cause at cell wall The synthesis of each composition and assembling process there is numerous enzyme systems to participate in, and in wherein most enzymes is human body Institute is non-existent.Therefore, also exist many novel during Cell wall synthesis, and there is preferably choosing Select toxicity potential antibacterials target spot.MTB is also such.
Result of study in recent years shows: the 2-methyl of MTB-erythritol phosphoric acid (methylerythritol Phosphate, MEP) function of approach be synthesis isoprenoid precursor substance isovaleryl pyrophosphoric acid or Dimethylallyl pyrophosphoric acid.This approach in mammal and plant endochylema by diverse mevalonic acid (MVA) approach substitutes.In addition MEP approach synthesis isoprenoid and derivant at biological metabolism Most important with in Cell wall synthesis, therefore MEP approach has become the focus of medicine target research.This approach For MTB also it is critical that, the MEP approach of MTB comprises 8 enzymes altogether and participates in, wherein Rv3582c encodes the key enzyme ispD (2-C-methyl-D-erythritol-4-phosphate in this approach Cytidyltransferase, 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase) it is this approach In key gene, the expression product of gene can be catalyzed MEP and CTP (cytidine) synthesis CDP-ME (as shown in Figure 1).Still there is no the IspD inhibitor report at screening antituberculosis medicaments at present, The antituberculotics of brand-new mechanism of action is likely found by the IspD inhibitor of screening MTB.
Summary of the invention
In view of this, the present invention is directed to the problems referred to above, it is provided that a kind of IMB-NY compounds is in preparation Application in Killing Mycobacterium Tuberculosis medicine, use in conjunction high flux phenotypic screen model of the present invention and knot Core mycobacteria Rv3582c inhibitor sifting model, from the compound of more than 70000 separate sources Middle screening obtains enzyme inhibitor IMB-NY1 and IMB-NY2；By to IMB-NY1 and IMB-NY2 Anti-tubercular evaluation, confirm that this compound has clear and definite tuberculosis bar mycomycete activity.
In order to solve above-mentioned technical problem, the invention discloses a kind of IMB-NY compounds anti-in preparation Application in mycobacterium tuberculosis medicine, IMB-NY compounds includes IMB-NY1 and IMB-NY2, The chemical formula of described IMB-NY1 is as shown in (I):
The compound of described IMB-NY2 is as shown in (II):
The invention also discloses the screening technique of a kind of lead compound containing MTB IspD inhibitor, Comprise the following steps:
1) preparation of strain seed liquor；
2) primary dcreening operation of positive compound sample；
3) the multiple sieve of positive compound sample；
4) the checking positive compound MIC to CG, determines MTB IspD inhibitor.
Further, step 1) in strain seed liquor prepare particularly as follows: the best solid put down Streak inoculation CG in plate BHI culture medium, recovers and cultivates fresh CG, condition of culture 37 DEG C, falling Put cultivation 12h, then picking list colony inoculation in 20mL liquid B HI culture medium 37 DEG C, 200rpm Incubated overnight；At rear mensuration 600nm, the light under visible wavelength absorbs (OD600) is 4.0～5.5；Will Bacterium solution liquid B HI culture medium is diluted to OD600=3.0, is dispensed in the sterile centrifugation tube of 1.5mL, Every 1mL, 4 DEG C preserve 10 pipes.
Further, step 2) in positive compound sample primary dcreening operation particularly as follows: with 0.3% connect bacterium CG is accessed in precalculated culture medium and screens compound in compound library by amount, except blank Every hole outside comparison adds 198 μ L bacterium solution with 8 channel pipettor, by observing the growth feelings of this pattern bacterium Condition, screens positive；The compound of compound library is carried out 100 times of dilutions, to final concentration of 20 μ g/mL, under this concentration its result of perusal be muddiness be then the sample that CG can not be suppressed to grow, It is judged to primary dcreening operation negative sample；Under this concentration, its result of perusal is then to suppress this bacterium for clarification The sample of growth, it is determined that for primary dcreening operation positive.
Further, step 3) in positive compound sample multiple sieve particularly as follows:
(1) positive compound that is ready to obtain for primary dcreening operation, CG strain are (with strain kind in primary dcreening operation The preparation of sub-liquid), aseptic 96 orifice plates, 8 channel pipettor, V-groove and 8 aseptic passages move liquid Device rifle head；
(2) 96 orifice plates, 8 channel pipettor, V-groove are put in super-clean bench medium ultraviolet and irradiate 15min, Rear venting 3～5min；
(3) take primary dcreening operation positive compound 2 μ L to add in 96 orifice plates, take two parts of numberings 1,2, respectively Add 198 μ L with 0.3% the dilution bacterium solution of BHI and BHIS-I of the CG connecing bacterium amount in being ready for 96 orifice plates having primary dcreening operation positive compound in；
(4) 96 orifice plates are particularly as follows: 8 (A～H) row * 12 (1～12) row, and the last string in the right side stays Four holes are ethambutol positive drug control；The left side first row of every plate arranges 4 hole blanks and 4 Hole growth control, wherein, blank group is not added with bacterium, and growth control group is not added with medicine, at 96 orifice plates The final compound concentration planting remaining 84 hole samples is 20 μ g/mL, and reaction system cumulative volume is 200 μ L； In two kinds of methods of BHI with BHIS-I, way is identical；
(5) by all add excellent 96 orifice plates as in 37 DEG C of constant incubators cultivate 12～16h, The absorption at visible wavelength 600nm is measured by microplate reader；
The suppression that in BHI or BHIS-I culture medium, sample grows is calculated for CG according to formula (1) Rate；
WhereinRefer to CG bacterium normal growth matched group absorption value under porose 600nm average Value；Refer to culture medium blank group without CG bacteria growing absorption value under porose 600nm Meansigma methods；T affects after referring to add compound under the experimental test hole 600nm of CG bacteria growing Absorption value；
Suppression rate variance (Δ I) is for deduct pressing down in BHIS-I culture medium by the suppression ratio in BHI culture medium Rate processed, the Δ I of recycling positive drug determines positive criteria；
Δ I=IBHI-IBHIS-I
IBHI: sample or control compound are to CG suppression ratio in BHI culture medium；
IBHIS-I: sample or control compound are to CG suppression ratio in BHIS-I culture medium；
Pick out the sample of suppression ratio both greater than 70% in two kinds of culture medium to carry out splitting screening, and right BHI and BHIS-I culture medium suppress the rate variance the carrying out more than 20% split screening；To each chemical combination Thing screens, and specifically determines it is a certain compound role or certain two or more on earth Synergism；
Choose suppression ratio still greater than or equal to more than 70% multiple sieve positive compound sample, according to sample Numbering, the numbering of the lookup each compound corresponding to sample, and take out each single compound sample Product；Checking each compound is carried out same multiple sieve；Still have and press down more than or equal to more than 70% The compound made, carries out the checking of the paper disk method to CG；It is 100 that each scraps of paper add 50 μ L concentration The testing sample of μ g/mL, records antibacterial circle diameter；It is active ingredient by the Compound nomenclature of multiple sieve checking Thing, is lead compound, containing IMB-NY compounds.
Further, step 4) the middle checking positive compound MIC to CG, determine MTB IspD Inhibitor particularly as follows:
(1) 96 orifice plates, V-groove, pipettor etc. are irradiated under uviol lamp 30min；
(2) seed liquor is inoculated in fresh BHI/BHIS-I fluid medium with the inoculum concentration of 0.3%；
(3) with row's type micro pipettor, fresh bacterium solution is joined in 96 orifice plates, the first horizontally-arranged 198 μ L, 2～8 horizontally-arranged every hole 100 μ L；
(4) add the antibacterials 2 μ L mixing that concentration is 10 μ g/mL in the 1st every hole of row, make mixing The final concentration of 0.05 μ g/mL of liquid Chinese medicine；
(5) draw 100 μ L liquid from the 1st row and join mixing the 2nd row, make mixed liquor Chinese medicine Final concentration of 0.025 μ g/mL；
(6) 2～12 rows press the 2 times of dilutions successively of step (5) method antibacterials concentration, and the 12nd row is dense Degree is 0.1 μ g/mL；
(7) 96 orifice plates are placed in 37 DEG C of constant incubators cultivation 16h；
(8) observed result: be designated as minimum inhibitory concentration with the complete downtrod concentration of bacterial growth；From Compound library obtains the influential lead compound of cell wall totally 147 that pattern substitutes bacterium CG.
The invention also discloses a kind of ZYM-5052 liquid culture expressed for MTB IspD inhibitor Base, the culture medium of the ZYM-5052 of every liter containing reagent is: the tryptone of 1%, the ferment of 0.5% Female extract, the Na of 25mM2HPO4, the KH of 25mM2PO4, the NH of 50mM4Cl、5mM Na2SO4, the glycerol of 0.5%, the glucose of 0.05%, the alpha-lactose of 0.2%, the MgSO of 2mM4。
Compared with prior art, the present invention can obtain and include techniques below effect:
1) abduction delivering of IspD albumen have employed self-induction culture medium (ZYM-5052 fluid medium), Improve the amount of soluble protein IspD.
2) combine Bacterial phenotype screening model and target specificity screening, improve positive rate.
3) newfound compound IMB-NY1 and IMB-NY2 and the structure of existing antituberculotics It is different from.
Certainly, the arbitrary product implementing the present invention it is not absolutely required to reach all the above skill simultaneously Art effect.
Accompanying drawing explanation
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes of the present invention Point, the schematic description and description of the present invention is used for explaining the present invention, is not intended that the present invention's Improper restriction.In the accompanying drawings:
Fig. 1 is the catalytic reaction figure of Rv3582c in background of invention
Fig. 2 is that in the embodiment of the present invention 1, the sample of phenotypic screen arranges figure；
Fig. 3 is the method flow diagram measuring MIC in the embodiment of the present invention 2；
Fig. 4 is absorption value and the standard curve fit figure of PPi concentration in the embodiment of the present invention 3.
Detailed description of the invention
Embodiments of the present invention are described in detail, thereby to the present invention below in conjunction with drawings and Examples How application technology means solve technical problem and reach the process that realizes of technology effect and can fully understand And implement according to this.
Embodiment 1: the screening of the lead compound of potential Killing Mycobacterium Tuberculosis in vitro cell wall
By applying the high-throughout replacement bacterium corynebacterium glutamicum targeting Mycobacterium cell wall (CG) phenotypic screen model, screens the lead compound of potential Killing Mycobacterium Tuberculosis in vitro cell wall, Comprise the following steps:
1, the preparation of strain seed liquor: at the best solid plate BHI culture medium (BD of 3.7% The brain-heart infusion medium of company adds the agar of 1.5%, article No.: 211065) upper streak inoculation CG, Recovering and cultivate fresh CG, condition of culture 37 DEG C, being inverted and cultivate 12h, then picking list bacterium colony connects Kind to 20mL liquid B HI culture medium (brain-heart infusion medium of the BD company of 3.7%, article No.: 211065) in 37 DEG C, 200rpm incubated overnight.At rear mensuration 600nm, the light under visible wavelength is inhaled Receive (OD600) it is about 4.0～5.5.Bacterium solution liquid B HI culture medium is diluted to OD600=3.0, point Installing in the sterile centrifugation tube of 1.5mL, every 1mL, 4 DEG C preserve 10 pipes.Postmenstruation, test proved 1 week can be used.
2, the application of primary dcreening operation model
Preliminary screening based on 96 well plate method: CG is accessed precalculated with the bacterium amount that connects of 0.3% Culture medium (derives from Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences's screening real to compound library Test room and build the compound sample storehouse in the cut-off end of the year in 2014 preserved) in compound screen, remove Every hole outside blank adds 198 μ L bacterium solution with 8 channel pipettor, by observing the growth of this pattern bacterium Situation, screens positive.The compound of compound library is carried out 100 times of dilutions, to final concentration of 20 μ g/mL, under this concentration its result of perusal be muddiness be then the sample that CG can not be suppressed to grow, It is judged to primary dcreening operation negative sample；Under this concentration, its result of perusal is then to suppress this bacterium for clarification The sample of growth, it is determined that for primary dcreening operation positive；Shown in comprising the following steps that:
(1) it is ready to the compound for screening, gone out the 8 channel pipettor rifle heads of bacterium.
(2) 96 orifice plates, 8 channel pipettor, V-groove are put in super-clean bench medium ultraviolet and irradiate 15min, Rear venting 3～5min.
(3) taking compound 2 μ L in 96 orifice plates, plate one_to_one corresponding, in order to avoid obscuring, prepares above-mentioned Take 198 μ L in 96 orifice plates having compound with the 0.3% CG bacterium solution connecing bacterium amount.
(4) left side first row is left comparison, and front four holes only add the bacterium solution of 198 μ L CG and 2 μ L are aseptic Water only adds 198 μ L BHI pure culture bases and 2 μ L water as negative control as positive control, rear four holes.
(5) excellent 96 orifice plates are added as cultivation 12～16h in 37 DEG C of constant incubators, use by all Microplate reader measures the absorption value at visible wavelength 600nm；
The suppression ratio that sample grows in BHI culture medium is calculated for CG according to equation below.
WhereinRefer to CG bacterium normal growth matched group absorption value under porose 600nm average Value；Refer to culture medium blank group without CG bacteria growing absorption value under porose 600nm Meansigma methods；T affects after referring to add compound under the experimental test hole 600nm of CG bacteria growing Absorption value.
The suppression ratio hole more than 70% is as primary dcreening operation positive compound sample.
3, multiple sieve verifies positive compounds based on 96 well plate method primary dcreening operations
The positive compound being previously obtained is carried out multiple sieve, applies BHI (the brain heart of the BD company of 3.7% Infusion medium, article No.: 211065) and BHIS-I (in BHI culture medium, with the addition of 10.92% Sorbitol, the sodium chloride of 1% and the magnesium sulfate of 0.05%) two kinds of culture medium carry out multiple sieve；Concrete steps are such as Under:
(1) positive compound that is ready to obtain for primary dcreening operation, CG strain are (with strain kind in primary dcreening operation The preparation of sub-liquid), aseptic 96 orifice plates, 8 channel pipettor, V-groove and 8 aseptic passages move liquid Device rifle head.
(2) 96 orifice plates, 8 channel pipettor, V-groove are put in super-clean bench medium ultraviolet and irradiate 15min, Rear venting 3～5min.
(3) take primary dcreening operation positive compound 2 μ L to add in 96 orifice plates, take two parts of numberings 1,2, respectively Add 198 μ L with 0.3% the dilution bacterium solution of BHI and BHIS-I of the CG connecing bacterium amount in being ready for 96 orifice plates having primary dcreening operation positive compound in.
(4) as in figure 2 it is shown, 96 orifice plates are particularly as follows: 8 (A～H) row * 12 (1～12) row, right The last string in face stays four holes to be ethambutol (EMB) positive drug control.In the left side first row of every plate Arranging 4 hole blanks and 4 hole growth control, wherein, blank group is not added with bacterium, growth control group Being not added with medicine, in 96 orifice plates, the final compound concentration of remaining 84 hole samples is 20 μ g/mL, reaction System cumulative volume is 200 μ L.In two kinds of methods of BHI with BHIS-I, way is identical.
(5) by all add excellent 96 orifice plates as in 37 DEG C of constant incubators cultivate 12～16h, The absorption at visible wavelength 600nm is measured by microplate reader；
The suppression that in BHI or BHIS-I culture medium, sample grows is calculated for CG according to formula (1) Rate.
Suppression rate variance (Δ I) is for deduct pressing down in BHIS-I culture medium by the suppression ratio in BHI culture medium Rate processed, the Δ I of recycling positive drug determines positive criteria.
Δ I=IBHI-IBHIS-I
IBHI: sample or control compound are to CG suppression ratio in BHI culture medium；
IBHIS-I: sample or control compound are to CG suppression ratio in BHIS-I culture medium.
Pick out the sample of suppression ratio both greater than 70% in two kinds of culture medium to carry out splitting screening, and right BHI and BHIS-I culture medium suppress the rate variance the carrying out more than 20% split screening.Because of above-mentioned for sieving The compound of choosing is all five in one, so carrying out screening each compound, specifically determines The end is a certain compound role or certain two or more synergism.
Choose suppression ratio still greater than or equal to more than 70% multiple sieve positive compound sample, according to sample Numbering, the numbering of the lookup each compound corresponding to sample, and take out each single compound sample Product.Checking each compound is carried out same multiple sieve.Still have and press down more than or equal to more than 70% The compound made, carries out the checking of the paper disk method to CG.It is 100 that each scraps of paper add 50 μ L concentration The testing sample of μ g/mL, records antibacterial circle diameter.It is active ingredient by the Compound nomenclature of multiple sieve checking Thing, is lead compound, the IMB-NY compounds containing the present invention.
4, the reactive compound MIC to CG is measured
Measure further the reactive compound MIC to CG, by the CG seed liquor of preservation with 0.3% connect The amount of kind is seeded in BHI and BHIS-I culture medium respectively.On 96 orifice plates, every hole final volume is 200 μ L. After cultivating 12h in 37 DEG C, microplate reader is used to read the absorption value at visible wavelength 600nm, result Middle cell density numerical value is considered as minimal inhibitory concentration less than 0.02OD；96 well plate method measure MIC value tool Body step is following (as shown in Figure 3):
(1) 96 orifice plates, V-groove, pipettor etc. are irradiated under uviol lamp 30min；
(2) seed liquor is inoculated in fresh BHI/BHIS-I fluid medium with the inoculum concentration of 0.3%；
(3) with row's type micro pipettor, fresh bacterium solution is joined in 96 orifice plates, the first horizontally-arranged 198 μ L, 2～8 horizontally-arranged every hole 100 μ L；
(4) add the antibacterials 2 μ L mixing that concentration is 10 μ g/mL in the 1st every hole of row, make mixing The final concentration of 0.05 μ g/mL of liquid Chinese medicine；
(5) draw 100 μ L liquid from the 1st row and join mixing the 2nd row, make mixed liquor Chinese medicine Final concentration of 0.025 μ g/mL；
(6) 2～12 rows press step (5) method antibacterials concentration 2 times of dilutions, the 12nd row's concentration successively It is 0.1 μ g/mL；
(7) 96 orifice plates are placed in 37 DEG C of constant incubators cultivation 16h；
(8) observed result: remember with the concentration of bacterial growth the most suppressed (consistent with blank) For minimum inhibitory concentration.
From compound library, obtain the cell wall to pattern replacement bacterium CG by above-mentioned screening study and have shadow The lead compound totally 147 rung.
The expression of embodiment 2:MTB IspD, purification and applied research
By applying high-throughout MTB IspD inhibitor screening model, screening is with MTB IspD as target The Newer Antibuberculotics of point.
The construction method of the cloning vehicle of IspD albumen sees patent (patent name: IspD inhibitor screening Model and the new application of IspD inhibitor oradol, patent publication No. CN 102277411A, invention Day: 2010-06-10)；
PET28a that application build is successful (+)-Rv3582c plasmid (pET28a (+)-Rv3582c plasmid adopts Build by conventional technique means) thermal shock conversion Escherichia coli BL21 (DE3) plysS competence After, coat the LB flat board containing kanamycin (Kan), 37 DEG C, incubated overnight obtains efficient table Reach MTB IspD albumen pET28a (+)-Rv3582c::BL21 (DE3) plysS bacterial strain carries out the table of albumen Reach, purification and applied research.Concrete grammar is:
1) by can the escherichia coli of high efficient expression tubercule bacillus IspD albumen PET28a (+)-Rv3582c::BL21 (DE3) plysS streak inoculation in the LB containing 50 μ g/ml Kan put down Plate, 37 DEG C, incubated overnight.
2) picking list colony inoculation is in the LB fluid medium containing 50 μ g/ml Kan, 200r.p.m., 37 DEG C, incubated overnight；
Overnight culture is inoculated in the fresh ZYM-5052 liquid containing 200 μ g/ml Kan by 1:1000 Body culture medium (in the culture medium of the ZYM-5052 of every liter containing reagent is: the tryptone of 1%, 0.5% Yeast extract, the Na of 25mM2HPO4, the KH of 25mM2PO4, the NH of 50mM4Cl、 The Na of 5mM2SO4, the glycerol of 0.5%, the glucose of 0.05%, the alpha-lactose of 0.2%, 2mM MgSO4), 210r.p.m., cultivates 5h to thalline OD by 37 DEG C600≈0.3。
3) temperature is adjusted to 16 DEG C, 210r.p.m., cultivates 19h, the expression of induction IspD albumen.
4) 10000 × g is centrifuged 10min and collects thalline；Lysis buffer suspended bacteria somatic cell, ice bath, Using pressure breaking system to be crushed by somatic cells, at 4 DEG C, 14000 × g is centrifuged 1h and collects supernatant； UsePrime system, under conditions of pH 8.0, His-trap affinity column (GE on supernatant Company, 17-5247-01), carry out gradient elution with buffer, collect eluent；The sample collected adds Entering 15mL ultra-filtration centrifuge tube (10kDa, MilliPore company), at 4 DEG C, 5000g is centrifuged 15min, To sample size less than 2.5mL；Use PD-10 desalting column and desalination buffer by the sample desalination after ultrafiltration. To-80 DEG C of preservations after protein quantification.
Embodiment 3: enzyme activity assay
Reaction system: the 10 μ l Ion reagent (MgCl containing final concentration 10mM2, 20mM NaF and 1mM DTT)；The IspD of the purification that the embodiment 2 of 1 μ g obtains；CTP and MEP (its final concentration It is respectively 500 μMs and 250 μMs).Enzyme reaction buffer solution is 50mM Tris-HCl (pH 8.0), Reaction system cumulative volume is 100 μ l.If adding the IspD of heat inactivation as comparison.Set addition 8 simultaneously Variable concentrations (0 μM, 25 μMs, 50 μMs, 75 μMs, 100 μMs, 150 μMs, 200 μMs, 250 μM) 100 μ l PPi, determination of activity is drawn the standard curve of absorption value and PPi concentration, passes through Excel simulates associated straight lines formula (seeing Fig. 4)；Thus the reaction process of enzyme is calculated according to formula.
Y=3.589x+16.418
R2=0.9997
Determination of activity: at 37 DEG C, above-mentioned system is hatched 40min, adds 10 μ l color Reagent A (1M Beta-mercaptoethanol) and 40 μ l color Reagent B (2.5% ammonium molybdate is dissolved to the H of 2.5M2SO4After), 37 DEG C are continued to hatch 10min, and microplate reader detects the absorption value of reaction system under 590nm wavelength.
Result: by the absorption value reference standard curve of sample, calculate the concentration of the PPi that reaction generates. By the concentration of the PPi of generation divided by react completely may the concentration of PPi of generation, as reaction process.
Substitute into formula: the concentration of the PPi of the concentration of the PPi of reaction process=generation/react completely generation * 100%；
When reaction process is more than 40%, and the activity of enzyme is considered as i.e. normal.
The screening of embodiment 4:IspD inhibitor
Principle, reaction system and the activity determination method of screening method therefor (target specificity screening) is same Embodiment 3 is specifically screened packet and is shown in Table 1.
WhereinRefer to the most normal enzymatic reaction system absorption value meansigma methods as negative control group； Refer to the absorption value meansigma methods using the IspD inactivated as positive controls；T tries after referring to add compound The absorption value of test prospect hole.
In this screening system, come out owing to there is no the IspD inhibitor of MTB, with the IspD of inactivation As positive controls, the absorption value that it is given is minimum, and enzyme inhibition rate is maximum；With the most normal enzyme Rush reaction system is negative control, and the absorption value that this group is given is maximum, and suppression ratio is minimum.By calculating Go out the enzyme inhibition rate of sample to be sieved, be considered as i.e. positive findings when suppression ratio is more than 20%.
The selection result: in 147 compounds obtained from embodiment 1, screening obtains 2 positive chemical combination Thing, positive rate is 1.4%, and wherein, the chemical formula of IMB-NY1 is as shown in (I):
Wherein the IMB-NY1 of 20 μ g/ml is 51% to the suppression ratio of IspD.
The compound of IMB-NY2 is as shown in (II):
Embodiment 5: the anti-MTB evaluating MTB IspD inhibitor (IMB-NY1 and IMB-NY2) lives Property
MTB IspD inhibitor is in vitro to sensitive and drug resistance tubercule bacillus inhibitory activity assay method such as Under:
1) anti-tubercular measures and carries out in 96 aseptic well culture plates, and the final volume in each hole is 100 μL。
2) instrument connection is separately added into containing 5 × 105Mycobacterium tuberculosis H37Rv (the ATCC of cfu 25618) bacterial strain (960) sensitive to isoniazid and rifampicin that, be clinically separated, to isoniazid and profit (rear three strain bacterium are for the MDR bacterial strain (330) of the flat drug resistance of good fortune and the XDR bacterial strain (926) of extensive drug resistance Clinical laboratory of Nanjing Chest Hospital clinical samples is through changing Luo Fa and mycobacteria trace quick medicine-sensitive directly perceived Test multiple authentication bacterial strain, the numeral in bracket is strain number) culture fluid, be simultaneously introduced difference The positive control drug isoniazid (INH, Sigma company) of concentration and rifampicin (RFP, Sigma company) (selection of drug level is: isoniazid and rifampicin are being tested with in MTB (H37Rv) bacterial strain Time proceed by doubling dilution to 62.5ng/ml from final concentration of 32 μ g/ml, when using clinical isolates When strain is tested, the final concentration of medicine starts doubling dilution to 0.125 μ g/ml from 128 μ g/ml) or The testing compound of the variable concentrations of DMSO preparation, testing sample passes through two times in test determination system Dilution method obtains final concentration from 128.0 initial μ g/ml to 0.0625 μ g/ml；
3) surrounding of culture plate is isopyknic aseptic 7H9 culture medium, arranges 3 growths positive right simultaneously (isopyknic according to hole (isopyknic DMSO solvent without sample) and 3 growth negative control holes 7H9 culture medium without any tubercule bacillus)；
4), after covering 96 orifice plates, surrounding sealed membrane seals, and is placed in 37 DEG C of incubators and hatches；
5) cultivate to the 7th day microscope amplification 40 times and observe Growth positive control wells and negative growth pair According to hole, when observing that both have significant difference, the state of each test hole bacterial growth is observed, And judge and record suppression or drug resistance result；
6) within the 14th day, repeat observation and once confirm record result.
Result shows: compound IMB-NY1 is minimum to mycobacterium tuberculosis reference culture H37Rv's Mlc (MIC) is 4 μ g/ml；To the sensitive strain (960) being clinically separated, MDR bacterial strain (330) Having similar inhibitory activity with XDR bacterial strain (926), MIC is 2～4 μ g/ml.Compound IMB-NY2 is 8 to the minimum inhibitory concentration (MIC) of mycobacterium tuberculosis reference culture H37Rv μg/ml；To the sensitive strain (960) being clinically separated, MDR bacterial strain (330) and XDR bacterial strain (926) Having similar inhibitory activity, MIC is 8～16 μ g/ml (being shown in Table 1).
Table 1 compound MIC (μ g/ml) to all kinds of MTB
Note: for reaction final concentration in bracket.
Described above illustrate and describes some preferred embodiments of invention, but as previously mentioned, it should be understood that Invention is not limited to form disclosed herein, is not to be taken as the eliminating to other embodiments, and can For other combinations various, amendment and environment, and can pass through in invention contemplated scope described herein Above-mentioned teaching or the technology of association area or knowledge are modified.And the change that those skilled in the art are carried out and Change the spirit and scope without departing from invention, the most all should be in the protection domain of invention claims.

Claims (7)

  1. The application in preparation Killing Mycobacterium Tuberculosis medicine of the 1.IMB-NY compounds, it is characterised in that Described IMB-NY compounds includes IMB-NY1 and IMB-NY2, the chemistry of described IMB-NY1 Formula is as shown in (I):
    The compound of described IMB-NY2 is as shown in (II):
  2. 2. the screening technique of the lead compound containing MTB IspD inhibitor, it is characterised in that Comprise the following steps:
    1) preparation of strain seed liquor；
    2) primary dcreening operation of positive compound sample；
    3) the multiple sieve of positive compound sample；
    4) the checking positive compound MIC to CG, determines MTB IspD inhibitor.
  3. The screening side of the lead compound containing MTB IspD inhibitor the most according to claim 2 Method, it is characterised in that described step 1) in the preparing particularly as follows: the best of strain seed liquor Streak inoculation CG in solid plate BHI culture medium, recovers and cultivates fresh CG, condition of culture 37 DEG C, It is inverted and cultivates 12h, then picking list colony inoculation in 20mL liquid B HI culture medium 37 DEG C, 200 Rpm incubated overnight；At rear mensuration 600nm, the light under visible wavelength absorbs (OD600) is 4.0～5.5； Bacterium solution liquid B HI culture medium is diluted to OD600=3.0, is dispensed into the sterile centrifugation tube of 1.5mL In, every 1mL, 4 DEG C preserve 10 pipes.
  4. The screening side of the lead compound containing MTB IspD inhibitor the most according to claim 3 Method, it is characterised in that described step 2) in the primary dcreening operation of positive compound sample particularly as follows: with 0.3% The bacterium amount that connects CG is accessed in precalculated culture medium compound in compound library is screened, Every hole in addition to blank adds 198 μ L bacterium solution with 8 channel pipettor, by observing this pattern bacterium Growing state, screens positive；The compound of compound library is carried out 100 times of dilutions, to final concentration Be 20 μ g/mL, under this concentration its result of perusal be muddiness be then the sample that CG can not be suppressed to grow Product, it is determined that for primary dcreening operation negative sample；Under this concentration, its result of perusal is then to suppress for clarification The sample of this bacteria growing, it is determined that for primary dcreening operation positive.
  5. The screening side of the lead compound containing MTB IspD inhibitor the most according to claim 4 Method, it is characterised in that described step 3) in positive compound sample multiple sieve particularly as follows:
    (1) positive compound that is ready to obtain for primary dcreening operation, CG strain are (with strain kind in primary dcreening operation The preparation of sub-liquid), aseptic 96 orifice plates, 8 channel pipettor, V-groove and 8 aseptic passages move liquid Device rifle head；
    (2) 96 orifice plates, 8 channel pipettor, V-groove are put in super-clean bench medium ultraviolet and irradiate 15min, Rear venting 3～5min；
    (3) take primary dcreening operation positive compound 2 μ L to add in 96 orifice plates, take two parts of numberings 1,2, respectively Add 198 μ L with 0.3% the dilution bacterium solution of BHI and BHIS-I of the CG connecing bacterium amount in being ready for 96 orifice plates having primary dcreening operation positive compound in；
    (4) 96 orifice plates are particularly as follows: 8 (A～H) row * 12 (1～12) row, and the last string in the right side stays Four holes are ethambutol positive drug control；The left side first row of every plate arranges 4 hole blanks and 4 Hole growth control, wherein, blank group is not added with bacterium, and growth control group is not added with medicine, at 96 orifice plates The final compound concentration planting remaining 84 hole samples is 20 μ g/mL, and reaction system cumulative volume is 200 μ L； In two kinds of methods of BHI with BHIS-I, way is identical；
    (5) by all add excellent 96 orifice plates as in 37 DEG C of constant incubators cultivate 12～16h, The absorption at visible wavelength 600nm is measured by microplate reader；
    The suppression that in BHI or BHIS-I culture medium, sample grows is calculated for CG according to formula (1) Rate；
    WhereinRefer to CG bacterium normal growth matched group absorption value under porose 600nm average Value；Refer to culture medium blank group without CG bacteria growing absorption value under porose 600nm Meansigma methods；T affects after referring to add compound under the experimental test hole 600nm of CG bacteria growing Absorption value；
    Suppression rate variance (Δ I) is for deduct pressing down in BHIS-I culture medium by the suppression ratio in BHI culture medium Rate processed, the Δ I of recycling positive drug determines positive criteria；
    Δ I=IBHI-IBHIS-I
    IBHI: sample or control compound are to CG suppression ratio in BHI culture medium；
    IBHIS-I: sample or control compound are to CG suppression ratio in BHIS-I culture medium；
    Pick out the sample of suppression ratio both greater than 70% in two kinds of culture medium to carry out splitting screening, and right BHI and BHIS-I culture medium suppress the rate variance the carrying out more than 20% split screening；To each chemical combination Thing screens, and specifically determines it is a certain compound role or certain two or more on earth Synergism；
    Choose suppression ratio still greater than or equal to more than 70% multiple sieve positive compound sample, according to sample Numbering, the numbering of the lookup each compound corresponding to sample, and take out each single compound sample Product；Checking each compound is carried out same multiple sieve；Still have and press down more than or equal to more than 70% The compound made, carries out the checking of the paper disk method to CG；It is 100 that each scraps of paper add 50 μ L concentration The testing sample of μ g/mL, records antibacterial circle diameter；It is active ingredient by the Compound nomenclature of multiple sieve checking Thing, is lead compound, containing IMB-NY compounds.
  6. The screening side of the lead compound containing MTB IspD inhibitor the most according to claim 5 Method, it is characterised in that described step 4) the middle checking positive compound MIC to CG, determine MTB IspD inhibitor particularly as follows:
    (1) 96 orifice plates, V-groove, pipettor etc. are irradiated under uviol lamp 30min；
    (2) seed liquor is inoculated in fresh BHI/BHIS-I fluid medium with the inoculum concentration of 0.3%；
    (3) with row's type micro pipettor, fresh bacterium solution is joined in 96 orifice plates, the first horizontally-arranged 198 μ L, 2～8 horizontally-arranged every hole 100 μ L；
    (4) add the antibacterials 2 μ L mixing that concentration is 10 μ g/mL in the 1st every hole of row, make mixing The final concentration of 0.05 μ g/mL of liquid Chinese medicine；
    (5) draw 100 μ L liquid from the 1st row and join mixing the 2nd row, make mixed liquor Chinese medicine Final concentration of 0.025 μ g/mL；
    (6) 2～12 rows press the 2 times of dilutions successively of step (5) method antibacterials concentration, and the 12nd row is dense Degree is 0.1 μ g/mL；
    (7) 96 orifice plates are placed in 37 DEG C of constant incubators cultivation 16h；
    (8) observed result: be designated as minimum inhibitory concentration with the complete downtrod concentration of bacterial growth；From Compound library obtains the influential lead compound of cell wall that pattern substitutes bacterium CG.
  7. 7. the ZYM-5052 fluid medium expressed for MTB IspD inhibitor, its feature exists In, the culture medium of the ZYM-5052 of every liter containing reagent is: the tryptone of 1%, the ferment of 0.5% Female extract, the Na of 25mM2HPO4, the KH of 25mM2PO4, the NH of 50mM4Cl、5mM Na2SO4, the glycerol of 0.5%, the glucose of 0.05%, the alpha-lactose of 0.2%, the MgSO of 2mM4。
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