OA16992A - Novel antibiotic preparation method and platform system based on same. - Google Patents
Novel antibiotic preparation method and platform system based on same. Download PDFInfo
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- OA16992A OA16992A OA1201400243 OA16992A OA 16992 A OA16992 A OA 16992A OA 1201400243 OA1201400243 OA 1201400243 OA 16992 A OA16992 A OA 16992A
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Abstract
Provided are a novel antibiotic preparation method and platform system based on the method, belonging to a novel drug development method. The method is based on a fixed structural formula: F-R, wherein F is an effect area, and R is an identification area. At the prior art level, the present invention can quickly develop a specific novel antibiotic for most pathogenic microorganisms or biological cells. Also provided is a platform for implementing the method, ensuring that the novel antibiotic is developed in an efficient streamlined process.
Description
Technical Field
This Invention relates to biological medicine developlng technology, especially to a novel antibiotic 5 préparation method and platform system based on same.
Background
Currently, the research and development of pharmaceutical industry, particularly antibiotic, is facing difficulties: 1. There are more and more drug-resistant pathogenic bacteria. Current antibiotics don't pose a threat to drug-resistant pathogenic bacteria. Mortality rate caused by these drug-resistant 10 pathogenic bacteria is increasing. 2. The speed of novel medidne developlng is far behind the occurring pace of drug-resistant pathogenic bacteria. It needs long time and costs much to screen antibiotics with traditional methods as well as obtain the achievement from the research and development. 3. The novel antibiotics developed by gene engineering or biotechnology are still easy to resuit in drug-resistance of pathogenic bacteria.
It was shown by statistics data of WHO that, In millions of people infected with drug-resistant staphylococcus aureus every year, around 30% of said people die finally—which is higher than the mortality rate of AIDS. In order to cure Infection of drug-resistant staphylococcus aureus, the cost of whoie worid exceeds 20 billion USD every year. After drug-resistant staphylococcus aureus occurred, vancomycin became the main rôle to cure staphylococcus aureus infection. However, 20 there has been vancomycin-resistant staphylococcus aureus occurred in hospital since 2002.
Although the spreading area of vancomycin-resistant staphylococcus aureus is small at présent, there is Utile medicine that can inhibit them effectively, so the mortality rate caused by infection of vancomycin-resistant staphylococcus aureus 1s very high. in the latest 10 years, another drug-resistant bacteria-multi-drug-resistant Gram-negative bacteria occurred, and they hâve 25 stronger drug-resistance. Almost none of antibiotics used in current clinical medidne can threaten said drug-resistant bacteria.
Currently most of antibiotics are produced by bacteria and fungi, or derived from naturel antibiotics i
by chemical modification. The traditional screening method for antibiotîcs comprises isolating microorganisms from soil samples, extracting sécrétions from medium for growing said microorganisms, detecting substance with antibacterial or sterilizing effects in said sécrétions and separating individual medicine ingrédients with potential médicinal value. The method was very 5 effective ln golden âge of developing antibiotics (1940-1950). But it cornes to the end now, as said screening method dépends on sécrétions of bacteria and fungi ln nature, namely the novei antibiotics are obtained by screening and modifying sécrétions of discovered microorganisms. Said traditional method for developing antibiotics requires long time unavoidably, and has uncertain outcome. Thus, in the past 50 years, pharmaceutical companles never stopped screening antibiotics, 10 but novei antibiotics screened are less and less. It's demonstrated by statistics data of Infectious
Diseases Sodety of America (IDSA) that, in recent new medicine applications of FDA in US, there are only 16 novei antibiotics applications, and there ls no one patent application about antibiotic against highly-drug-resistant Gram-negative bacteria. The famous popular science magazine Sdentific American’ had wamed continuously from 2009 to 2012 that, super bacteria are 15 threatening human life safety. CDC in US forecast that, the current used antibiotics would become
Invalid totally 10-20 years iater. The New York Times reports that, due to abusing antibiotics In agriculture, human would go back to the times without any available antibiotics 5 years Iater, and NDM-1 super bacterium mutant genes has lead to panic ln the worid. The problem of antibiotics and drug-resistant bacteria in stockbreeding is also a serious problem, and has influenced agricultural 20 product safety. The recent 40 years ls a vacuum period of antibiotics development, ln a word, the speed of developing and researchlng novei medidne is far behind the pace of drug-resistant bacteria mutation.
At présent, there are two methods for developing novei antibiotics: (1) modifying current antibiotics or synthesizing new type of antibiotics, (2) conducting gene modification on bacteria syntheslzing 25 antibiotics. However, both of said two methods hâve not overstepped such a keynote that:
antibacterial mechanism of said antibiotics ls not novei to bacteria. Therefore, without exception, the bacteria will generate drug-resistance to said ‘novei antibiotics soon.
ln order to reslst pathogenic microorganisms with strong variabîlity, strong viabiiity, strong pathogenlcity and various species, the problems urgent to be solved currently are as follows:
1. providing antibiotics with stronger antibacterial or sterilizing effects to pathogenic bacteria, especially to drug-resistant pathogenic bacteria;
providing methods for preparing said antibiotics In clause 1, preferably methods which can response to variability of pathogenic bacteria, i.e. developlng and researchlng methods of antibiotics with short development cycle, which can sensitively resïst new or variant pathogenic microorganisms;
3. said antibiotics developed by said method will not lead to drug-resistance of pathogenic microorganisms in a short time.
However, the above-mentioned problems are just conundrums which hâve not been solved by current several générations of antibiotics and traditional antibiotics developing methods..
Summary of invention in view of technical difficultés of obtaining novel antibiotics existing In above-mentioned fields, provided is a method and platform different from traditional strategies for developing antibiotics. Through said method and platform, novel antibiotics wîth spécifie récognition and killing capacity against any pathogenic microorganism, target cell or target tissue can be offered in a short term; Said methods can sensitively soive drug-resistance raised due to continuous mutation of pathogenic microorganism, i,e„ promptly provides corresponding novel antibiotics against newly-raised variant strains. And said novel antibiotics prepared by said method rarely lead to variance of pathogenic microorganism as well as drug-resistance, due to their novel sterilization mechanism. Technical solution of this Invention ls as follows:
A novel antibiotic préparation method, wherein steps are as follows:
(1) determining targets: said targets refer to prokaryotic cells, eukaryotîc cells, viruses or products thereof which said novel antibiotic wili react against directly;
(2) designing molecular structure of said novel antibiotic:
said molecular structure of said novel antibiotic is designed according to the following general formula:
( HR J
Wherein, R is récognition région, which specifically recognizes or combines said targets; F is effect région, which generates pharmaceutical effects to targets, and said pharmaceutical effects are effects of regulating, repairing, labeling, causing death and/or collapsing against said targets specifically;
establishing récognition région molecular structure library, establishing effect région molecular structure library, according to said general formula, designing recombinant molecular structure library on the basis of said récognition région molecular structure library and effect région molecular structure library said designing refers to the process of structural readjustment, structural recombination and/or structurai confirmation carried out on the basis of molecular structures of collected, selected or prepared substances used as effect région or récognition région;
(3) based on said recombinant molecular structure library, preparing and verifying recombinant so as to obtain candidate novel antibiotics;
(4) screening novel antibiotics which meet medicine requirement from candidate novel antibiotics. Said establishing récognition région molecular structure library refers to coilecting currently-known natural substances which specifically recognize and/or combine said targets by searching and analyzing; or/and artificlally preparing artificial substances which specifically recognize and/or combine said targets.
Said natural substances refer to natural bioactive molecuiar, or récognition région of bactériophage, which can be recognized by accepter of said targets;
said artificial substances refer to antibody mimetic, said antibody mimetic is designed according to amino add sequence of immunoglobulin which can spedfically recognize unique substance on said targets.
Said antibody mimetic is short peptide with a structure of VHCDR1-VHFR2-VLCDR3 from N-terminal to C-terminal, which is constituted by the régions of VMCDR1, VHFR2, VLCDR3 on Fab short arm of said immunoglobulin; or is mutamer of said short peptide; said mutamer refers to product obtained by artificial site-mutation to 5 amino acid residues of VHCDR1 and 9 amino acid residues of VLCDR3 on short peptide, which préserves récognition capability to unique substance on said targets.
Said immunoglobulin can be multiple. E.g., VHCDR1 of VHCDR1-VHFR2-VLCDR3 Is from one immunoglobulin, and VLCDR3 is from another immunoglobulin.
Said Immunoglobulin Is prepared by taking unique substance on target as immunogen to immunize animai.
Said Immunoglobulin Is prepared by taking common unique substances on multiple targets as Immunogens to Immunize animal.
Said Immunoglobulin Is prepared by taking multiple unique substances on target as Immunogens respectively to immunize animal.
When said target refers to virus, prokaryotic cell or eukaryotic cell with phospholipid biiayer membranes as the basic structure of its cell membrane or envelope, said pharmaceutical effect refers to causing death and/or collapsing;
said effect région refers to bioactive substance which can form ion channel or pore path on phospholipid biiayer membranes.
Said effect région refera to pseudomonas aeruglnosa barteriodn Pyosin.
Said effect région refera to collons E1, la, Ib, A, B or N; or domains of colidn molécules E1, la, Ib, A, B and/or N which can form Ion channels; or molécules obtained by allosterism from collcin molécules E1, la, Ib, A, B or N, or from domains of colidn molécules E1, la, Ib, A, B or N which can form ion channels, having function of forming Ion channels in said phospholipid biiayer membranes.
Said recombinant is recombinant polypeptide, said preparing recombinant refers to that gene coding said recombinant polypeptide is transformed into biological expression system to express fusion protein, and candidate nove! antibiotic is obtained by separating and purifying fusion protein.
Said biological expression system refers to Escherlchia coli pET system engineering bacteria E.coli B834 (DE3).
Said pharmaceutical effect refers to labeltng, said effect région refers to label, said preparing recombinant refers to linkïng effect région and récognition région operably.
Novel antibiotic préparation platform System comprises 3 interoperable Systems: (1) goal proposing system, (2) designing system, (3) laboratory System;
said platform préparés novel antibiotic according to said method of any one of claims 1-14; said goal proposing System détermines development goal, and delivers task instruction to said designing System;
said designing system establishes récognition région molecular structure library as well as effect région molecular structure library, designs molecular structure library for said recombinant, and provides said molecular structure library to said laboratory System, said laboratory system offers experimental results to said designing System; said designing System reports the finally-selected candidate products as development results to marketing system;
said designing refers to the procedure of structural adjustment, structural recombination and/or structural confirmation based on molecular structure of the collected, selected or prepared substance used as effect région or récognition région.
Said laboratory system comprises at least one 31 party partner institution which undertakes said experiments.
By the method for preparing novel antibiotics of this invention, ifs available to préparé a batch of candidate novel antibiotics against most targets, and select novel antibiotics specifically against said targets with récognition région and effect région from candidates. In the method of this invention, said targets may be prokaryotic cells (e.g., staphylococcus aureus with drug-resistance in example 5, environmental pollutant-cyanobacteria in example 6, bacillus anthrads in example 3), eukaryotic cells (e.g., agricultural fungus against by antifungal polypeptide in example 4, EB virus-induced tumor cells in example 1), virus (e.g., unique envelope glycoprotein In EB virus In example 1), products of said prokaryotic or eukaryotic cells.
During préparation, in accordance with general formula
R , establishing récognition région molecular structure library as well as effect région molecular structure library, and designing molecular structure library for recombinants.
Because there exist substances with spedfic récognition for spedfic species of cells in nature, such as, pheromone, ligand of accepter on récognition cells, immunoglobulin produced In human or animal. Moreover, immunoglobulin against xenobiotic is produced naturally by immune system of , animais. On the basis of Immunoglobulin prepared through artifidally immunizing animais or existing naturally, it's basically available to obtain récognition substances against any said target, which makes said method of this invention has a wide applicability.
Additionally, there also exist substances in nature, which can form léthal change on spedfic cells. For example, both of colicln and pseudomonas aeruginosa bacteriodns can be conducted as effect région, but their disadvantage is that the species of target cells they react against are limited.
Taking advantages of above-mentioned two kinds of substances and adopting their strong points while overcomlng own deftdendes, as to novel antibiotics prepared by the method of this Invention, récognition région leads whole recombinant molecular to recognize target substances, and effect région complétés pharmaceutical effects.
The prindpie of the method of this invention Is preparing novel antibiotics based on a general formula, collecting and/or designing a batch of molecular structures of récognition région and effect région against target substance proposed to establish molecular structure library, then designing molecular structure of novel antibiotics according to established molecular structure library, and obtaining a batch of molecular structure libraries of recombinants; afterwards, preparing a batch of candidate novel antibiotics in line with molecular structural information of molecular structure libraries of recombinants, finaliy verifying said candidate novel antibiotics one by one to select novel 5 antibiotic which meets pharmaceutical standards. The advantages of the préparation method of this invention are as follows:
(1) The method of this invention only aims against prokaryotic cells, eukaryotic celis, virus or products thereof to préparé novel antibiotics, and these above-mentioned substances either hâve natural récognition substances or hâve unique surface substances or their own can be conducted as immunogen for immunizing animal to obtain immunoglobulin which recognize themselves, which ensures that, It's available to always obtain récognition région by préparation method of this invention, and effectively breaks a bottleneck existing in current antibiotic development, i.e., the reality that proteins as targets of drug effect (i.e., drug targets) hâve been exhausted gradually, and 15 the left targets are undruggable (i.e., do not react with drugs).
Moreover, there also exist substances as effect région in nature, but said substances hâve selectivity in connection with different pharmaceutical effects. For example, if the goal is to préparé a novel antibiotic to cause death of target cells, substances such as colidns are competent to achieve said goal; if the goal is to regulate, interfère or label, ifs suffiaent to select substance molécules with 20 such effects as effect région. On the basis of general formula the method of this invention premlsed on, it's ensured that novel antibiotics can be prepared against most target substances, i.e., the method of this invention has high success rate and wide applicability.
(2) The best advantage of the method of this invention is that, against one target substance, several 25 novel antibiotics can be prepared once time and they are well prepared to counter drug-resistance of pathogenic microorganism, because in this invention, novel antibiotics are prepared against some target substance based on a general formula; recombinants library is obtained by establishing effect région library and récognition région library respectively, and available recombinants with well effects are selected as novel antibiotics from recombinants library. This solves a problem existing In current new drug development that, the speed to occur drug-resistance in pathogenic microorganism Is high, and there is no other alternative antibiotic when drug-resistance occurs.
In the method of this Invention, when establishing récognition région library, spécifie monoclonai 5 antibodies are preferably selected as the designed récognition région, I.e., they are designed as the commonly-designed single-chain antibodies, small molecular antibodies In prior art or antibody mimetic with structure shown In Table 1 which was disclosed by inventer of this Invention previously. That is, molecular structure of the established récognition région may be antibody mimetic in Table 1 or point mutation products of said antibody mimetic; point mutation refers to short peptides obtained 10 by conducting artificial point mutation on amino acid residue of two récognition régions composing antibody mimetic, which hâve récognition capacity against said target.
This makes members of the established récognition région molecular structure library expanded unlîmitedly, so as to counter mutation of pathogen better, which amplifies the above second advantage of the method of this invention. For example, if target (pathogenic ceil) is not recognized 15 due to drug-resistance occurring on one récognition région, there can be many similar récognition région molecular structures as candidates.
Antibody mimetic has more advantages compared with antibody, and is easier to be obtained as well as operated artifidally. Owing to different directed targets, substances conducted as récognition 20 régions may be pheromone, phage récognition région or spécifie monoclonal antibodies which are collected from current database, but they are limited after ail. In bioactive substances found in plants, animais or microorganisms, the most effective bioactive substance to recognize single molécule Is antibody. in order to avoïd the disadvantage of nature antibody with huge size, people always design and préparé antibody mutamer with smaller size. However, these mutamers consist of hundreds of 25 amino acid residues, and still hâve huge size compared with récognition régions we seek. Fab short arm of each natural antibody has 6 antigen binding régions, and they as weil as their backbone form complicated spatial structure. Said spatial structure has function of recognizing and binding spécifie antigen, while in novel antibiotics developed by the platform of this invention, ifs sufficient that récognition région only has récognition function, and récognition région does not need to bind the corresponding antigen. According to Qiu et al ( Small antibody mimetic comprising two complementarity-determining régions and a framework région for tumor targeting, Nature B'totechnology 25(8):921-929(2007)) , Qiu et al selected 2 antigen binding réglons and one 5 backbone région on Fab short arm of natural antibody to form a short peptide antibody mimetic referring to structure of natural antibody; although its size was 50-300 times smaller than natural antibody, It préserves basic biological activity of natural antibody, I.e., it can recognize some antigen specifically. Because immune system In human or animais will respond to produce immunoglobulin specifically against said immunogen when im'tated by immunogen, this invention preferably 10 prépares monoclonal antibodies recognizing targets and provides récognition région based on the monoclonal antibodies and the antibody mimetic designing Idea disclosed in article of Qiu et al> 2007. Thus, aiming at any pathogenic microorganism cell or pending-treated cell, spécifie monodonal antibodies can be prepared theoretically, and suitable récognition région substances - antibody mimetic can be prepared correspondingly. This makes that the success rate to prépare novel 15 antibiotics against some targets by the method of this invention has qualitative leap comparing with traditional method for preparing antibiotics, and the time to obtain products is shortened a lot. Theoretically, the time to préparé a novel antibiotic even a batch of novel antibiotics against one target equals to the time to obtain spécifie monoclonal antibody and the time to prépare recombinant protein by bio-engineering method.
Said targets are multiple targets with common surface antigen, and said antibody mimetic is prepared by immunizing animal with said common surface antigen as antigen composition. Novel antibiotics prepared by taking said antibody mimetic as récognition région can recognize several said targets, i.e., they hâve effects of broad-spectrum antibiotics.
With regard to récognition région molecular structure library established by the method of this 25 invention, if spécifie substance on the selected target surface is common for various microorganism surfaces, the corresponding antibody mimetic becomes a broad-spectrum récognition région, that ls, only if its surface has similar substances, the microorganism cell can be recognized, and the prepared novel antibiotic against It ls broad-spectrum antibiotic. lf the selected spécifie substance is unique for one microorganism cell, the corresponding antibody mimetic becomes a narrow-spectrum récognition région, and only the microorganism cell with similar substance can be recognized.
Taking various surface substances on the selected targets as immunogens, generating corresponding monoclonal antibodies by taking said surface substances separately to immunize animal to obtain many kinds of antibody mimetic and mutamer thereof which can recognize said microorganism cell specifically, Is another approach to expand récognition région library. Ifs available to firstly select any one of these antibody mimetic as the first récognition région of novel antibiotic developed by the method of this invention. After being used practically for a period of time, If said microorganism has occurred drug-resistance against said récognition région (e.g., structure of the corresponding surface substance (antigen) Is modified to keep said récognition région from being recognized), ifs available to select another from these antibody mimetic as the second récognition région, the third récognition région, the fourth récognition région......of novel antibiotîc developed by the method of this invention, accordingly to offer various optional novel antibiotics, which effectively overcomes the difficulty of failing to immediately provide alternative drug for treatment after drug-resistance occurring against antibiotics in prior art. The method of this invention is capable to extend the effective application lifetime of one novei antibiotîc.
When said target refers to virus, prokaryotic cell or eukaryotlc cell with phospholipid bilayer membranes as the basic structure of its cell membrane or envelope, said pharmaceutical effect refers to causing death and/or collapslng, said effect région refers to bioactive substance which can form ion channel or pore path on phospholipid bilayer membranes, such as the currently known colidn and pseudomonas aeruginosa barteriodn Pyosin.
Said effect région refers to colidns E1, la, Ib, A, B or N; or refers to domains of colidn molécules E1, la, Ib. A, B and/or N which can form ion channels;
or refers to molécules obtained by allosterism from colidn molécules E1, la, Ib, A, B or N, or from domains of colidn molécules E1, la, Ib, A, B or N which can form ion channels, having function of forming ion channels In said phospholipid bilayer membranes.lt's illustrated according to description of background art that, main pathogenic microorganisms confronted by human currently n
and in future hâve a common characteristic that their cell membranes hâve a structure of phospholipld bilayer membranes, ln nature, there exist many bacteriocins which ktll bacteria by directly forming Ion channel through cell membranes of bacteria. The typical représentation Is a bacteriodn secreted by Escherichla coli-colicin, and Its fonction Is to kill Escherichia coli of the same species but different strains, rather than hurt other species of bacteria and host of Escherichia coli-human as well as animais. As a model sample of colidns which forms ion channel, after colidn la was found by Jacob ln 1952, transmembrane spatial structure of ion channel formed by colicin la In artificial lipid bilayer membranes ln the state of opening or dosing was finally demonstrated in 1996 (Qiu et al, Major transmembrane movement assodated with colicin la channel gating. J. Gen. Physiology, 107:313-328 (1996)), which laid a theoretical basis for deslgning and preparing novel antibiotics on molecular level. However, wild-type colicin only reacts on Escherichia coli ofthe same species but different strains, and It’s necessary to alter its targeting to make colidn attack other pathogenic bacteria. Therefore, colidn is an idéal candidate of effect région of novel antibiotic developed by the piatform of this Invention.
When said recombinant is recombinant polypeptide, above-mentioned llnking effect région and récognition région refers to that gene coding said recombinant polypeptide is transformed Into biologlcal expression system to express fusion protein, and novel antibiotic is obtained by separating and purifying fusion protein.
Said pharmaceutical effect refers to labeling, said effect région refers to label, said preparing recombinant refers to linking effect région and récognition région operatively. For example, the molécule of récognition région can be labelled with radioactive marker.
Conceming the method of this Invention, after récognition région molécule is confirmed against target, effect région molécule is selected, and sélection of effect région molécule is relative to the goal of establishing said antibiotic. For example, if the goal is to regulate against target, it’s availabie to select molécule with repalring fonction; if the goal is to cause target dead or iimit its growth and development, it's availabie to select biological polypeptide molécule which can form léthal Ion channel through target's cell membrane, like colidn; if the goal Is to label or image target, it's availabie to link label or imaging agent to récognition région to obtain novel antibiotic for labeling, and label Is bound to target through récognition région, in order to conduct continuous therapy or research against labeling cells.
In the method of this invention for developing novel antibiotics, the important work is to estabiish récognition région library, because most targets hâve phospholipid bilayer as the basic structure of their cell membrane or envelop. ln the method of this invention, colicin, pseudomonas aeruginosa barteriodn Pyosin are preferably selected as effect région. Colicin is an effective régulation motivation for survival compétition and maintaining biodiversity among Escherichia coli, and Escherichia coli varies continuously in order to avoid the sterilization from colicin; colicin has been tO existing for billions of years, and It's still playing a irreplaceable physiological rôle in alimentary canal of each individual multicellular organism. Since the attacking target of novel antibiotics prepared by the method of this invention is microorganism or cell of other species, and these microorganisms or cells hâve never been attacked by colicin, it will cost much time for these bacteria to produce immune proteins similar with bacteria; accordingly, it's forecasted that novel antibiotics obtained by 15 the method of this invention will hâve longer application lifetime than traditional antibiotics. By means of forming ion channel through target’s membrane, colicin has strong sterilization effect, which is appropriately hundreds of even tens of thousands of times as that of traditional antibiotics, like penicfllln, cynnematin, vancomycin, streptomycin, carbapenem, tigecycline, etc. Effect région selected preferably by the method of this invention will give much stronger sterilization effect to 20 novel antibiotics prepared by the method of this invention, compared with traditional antibiotics.
ln the case that both récognition région and effect région are recombinants of polypeptide molécule, the method of binding récognition région to effect région is preferably to synthesize the nucléotide sequence coding said recombinant, then to build recombinant expression vector loading said nucleotîde sequence, finally to transform said recombinant expression vector into biological 25 expression system, e.g., said recombinant is expressed in engineered strain of Escherichia coli and separated. Furthermore, engineered strain of Escherichia coli pET system, i.e., E.coli B834 (DE3) is selected preferably as biological expression system in the method of this invention, and the expression rate of said recombinant protein in this system is higher through vérification.
About novel antibiotics prepared by the method of this Invention, a batch of recombinants are galned In steps of obtaining recombinant, and the recombinants having récognition and pharmacdoglcal effects at least against targets, I.e., novel antiblotics are finally selected through conventional function vérification, I.e., vérification of récognition and pharmacologlcal effects of recombinants by 5 using their targets and non-targets. For some recombinants without effect on targets but with effects on non-targets, it’s available to confirm their effect spectrum by further vérification.
This invention also provides platform system of operating the above-mentioned method to develop novei antïbiotics, which makes operation of the method to develop novel antibiotics standardized and modeled to enhanœ the efficiency of developing novel antibiotic. Said platform comprises 3 iO coordînated System groups; (1) goal proposing system, (2) designlng System and (3) laboratory system;
Said goal proposing system détermines development goal and give task instruction to designîng system; the task of said goal proposing system Is confirming which targets should be aimed at for 15 developing novei antiblotics, what pharmacological effects the novel antibiotics carry out against sald targets; for example, through search, demands of medidne field, drug-reslstance status of antiblotics are analyzed, and new pathogens as well as information thereof and experimental data or outsourced contracting projects are collected as well as counted to confirm development goal. In this invention, said pharmaceutical effects are effects of regulating, repairing, marking, causing death 20 and/or collapsing. Sald goal proposing System may include core members of R&D team, marketing team; the fixed goal may be outsourced contracting projects, which désignâtes to develop novel antiblotics against some pathogenic microorganlsms;
Designîng system designs récognition région, effect région and molecular structure of said 25 recombinants and proposes the desired experimental task to laboratory system according to task
Instruction from goal proposing system and said general formula; it’s required for designîng system of novel antiblotics to establish récognition région library and effect région library in accordance with task instructions from goal proposing system. Based on instructions from goal proposing system, substances specifically recognizing said targets are collected, selected and prepared as récognition région; said preparing substances specifically recognizing said targets refers to preparing and screening to obtain monoclonal antibody specifically recognizing said targets through immunizing animais by using unique substances separated from said targets or said targets as immunogen;
Said laboratory system offers experimental results to novel antibiotic deslgning system, and novel antibiotic deslgning system reports candidate products of finally-selected development achievements to goal proposing system;
said designing refers to the procedure of structural adjustment, structural recombination and/or structural confirmation based on molecular structure of the collected, selected or prepared 10 substance.
In platform system of this invention, said laboratory system complétés experimental work according to instruction from novel antibiotic designing system, for example, preparing monoclonal antibody of candidates as récognition région, preparing polypeptide as effect région and preparing said recombinants as well as carrying out a sériés of vérification work. Basically, the work of said 15 laboratory system includes preparing monoclonal antibody against target, obtaining amino acid sequence of monodonal antibody by sequendng. synthesizing the gene of the designed antibody mïmetic, synthesizing the gene coding recombinant, preparing as well as purifying recombinant or preparing recombinant, verifying recombinant, generating experimental report and giving feedback to designing system.
In development platform of this invention, designing system Is the core of technology, and ail works in laboratory system can be completed by prior art, thus, Ifs available to build a own completed laboratory work team, and ifs also available to oniy arrange administrator for laboratory work team, and outsource laboratory work by laboratory system administrator to the partner Institutes who focus on experiment works of the corresponding stages. Through the above-mentioned process, 25 development work can be completed with high-quality efficlently; research and development resources are integrated cost-effedively. Development platform established by this invention equals to a R & D factory of novel antibiotics.
Each system of said platform system of this invention performs its own function, coordinates to make the whole platform system work effidently, which makes available that it only costs around half a year to generate a target novel antibiotic. In fad, a batch of novel antibiotics against one target was produced during this half a year, and antibiotic préparation efficiency is far higher than traditional préparation. In addition, the work of each system is carried out pointedly, accordingly fond of corporation or research institution Is assigned with definite object, rather than Invested blindly in projects without practically applicable significance or market demand. In particular, in platform of this invention, some Works of system can be outsourced to a 3^ partner specialized in corresponding field. Consequently, not only can the high-effident intégration and utilization of R&D equipments and resources be reaiized, but also the R&D cost is lowered and the consumed time is shortened.
Workflow chart of said platform system of this invention is shown as Figure 5, and it avoids wasting and repeatlng in development work at a maximum extent.
In summary, the method and platform system of this invention provide a new method for preparing medidne, and the advantages superior to traditional antibiotic préparation methods are as follows:
(1) non-subjecting to limitation of traditional method for screening antibiotics: novel antibiotics prepared by the method of this invention hâve common structural constitution, I.e., consist of effect région and récognition région. In development projects against targets with phospholipid bilayer celi membranes, it’s available to seled the current colicin as effect région; under introduction of récognition région, colicin of novel antibiotics can form léthal ion channel through almost ail cell membranes wilh phospholipid bilayer cell membrane structure.
As there exists genus-unique or species-unlque or strain-unique surface substances on surfaces of cell membranes in most microorganisms, preparing monocional antibody specifically recognizing the target by using said surface substances or cells containing said surface substances as immunogen Is a very mature technology at présent, and after obtaining said monoclonal antibody, antibody mimetic will be obtained as récognition région in light of the idea of designing antibody mlmetic In previous inventions by Inventer. Thereby. the development method of this invention will not be subjected to limitation that proteins as targets of drug effect (i.e., drug targets) discovered at présent hâve been exhausted gradually, and the left targets are pharmacological significance (undruggable Le., do not react with drugs), and It is capable to develop novel antibiotics specifically against most 5 pathogenic microorganisms.
(2) capadty of sensitively countering drug-resistance of pathogenic mtcroorganism: because there Is not only one surface antigen substance in pathogenic microorganlsm, it's available te select various surface substances on the surface of target microorganlsm for immunizing animal to generate corresponding monoclonal antibody, accordingly many kinds of antibody mimetic and mutamer thereof which can recognlze said microorganlsm cell specifically are gained. As shown in Figure 3, it’s available to firstly select any of these antibody mimetics as the first récognition région of novel antibiotic developed by the method of this invention. After being used practically for a period of time, if said mlcroorganism has occurred drug-resistance against said récognition région (e.g., structure of 15 the corresponding surface substance (antigen) is modified to keep said récognition région from being recognized), It’s available te select another from these antibody mimetics as the second récognition région, the third récognition région, the fourth récognition région......of novel antibiotic developed by the method of this invention, accordingly to offer various optional novel antibiotics, which effectively overcomes the difficulty of failing to immediately provide alternative drug for 20 treatment after drug-resistance occurrlng against antibiotics in prior art. The method of this invention is capable to extend the effective application lifetime of one novel antibiotic.
(3) the récognition région substances provided In said platform or method of this Invention comprise, preferably comprise antibody mimetic, and mutamer by point mutation on short peptide of said antibody mimetic. Therefore, when mutation occurs on surface antigen of target pathogenic mlcroorganism, there has exlsted not only one candidate antibody mimetic waitïng for recognizing with said surface antigen, which makes the capadty of development method of this invention to counter drug-resistance further improved, as shown in Figure 4.
(4) It's difficult for target to occur drug-resistance. In the method of this invention, it’s préférable to select colicins as substances of effect région of novel antibiotics; bacteriddal mechanism of coiicin is different from that of most current antibiotics, and is not known well by pathogenic microorganisms; it will cost much time to occur drug-resistance in pathogenic microorganisms, which provides plenty of time to deveiop the next-generation antibiotics.
(5) By means of forming Ion channel through target’s membrane, coiicin has strong sterilization effect, which is appropriately hundreds of even tens of thousands of times as that of traditional antibiotics, like penicillîn, cynnematin, vancomycin, streptomycin, carbapenem, tigecycline, etc. As a preferred effect région of the method of this invention, It wiii give much stronger steriiizatîon effect to novel antibiotics prepared by the method of this invention, compared with traditional antibiotics.
(6) Different from traditional method of screening antibiotics with iong-time consuming, ifs oniy required to cost 4-6 months to produce a batch of novei antibiotics against various pathogenic microorganisms through the method of this invention. The procedure is thaï, animai (mouse) is immunized using different antigens; the corresponding monoclonai antibody will be screened by haemospasia after 2 to 3 weeks of antibody production period; the nucléotide sequences coding variable région and backbone of heavy chain and light chain on Fab segment of monoclonai antibody are obtained by using protein sequencing and gene translation technology; according to the obtained nucléotide sequences, gene coding récognition région - antibody mimetic is designed, and it Is bound to effect région -gene coding coiicin to construct the recombinant plasmid; the engineered bacteria are transfected by said recombinant plasmid to express a novel antibiotic by prolifération. Afterwards, specîfi city and sensitivity of antibiosis and cytocidal effect of the separated and purified novel antibiotic are verified. It's only required about haif a year to screen a batch of novel antibiotics by the whole procedure. Such high efficiency of construction and production wiii b ring the revolutîonary changes to traditional construction and production of antibiotics.
(7) The platform of this invention provides teamwork approach and resource utilization method to is efficiently develop novel antibiotics. Since the developed antibiotics hâve common structural characteristics, through sald platform of this invention, research and development resources are integrated effectively, and development work can be performed as flow-line production, which equals to a R & D factory of novel antibiotics.
In summary, method and platform system to develop novel antibiotics of this Invention can offer novel antibiotics with spécifie récognition région and effect région against most pathogenic microorganisms and targets. For most targets, the currently-known bacteriodns are competent as the rôle of effect région, so development cycle of a novel antibiotic dépends on the time to design molecular structure and the time to préparé as well as verify recombinants. Because récognition région dépends not only on the selected nature substances but mainiy dépends on artificiai-prepared antibody mimetic as récognition région, while obtaining monoclonal antibody through immunizing animais by using an immunogen and then getting amino add sequence of said monoclonal antibody ls a current mature technology, based on the current biotechnological level, the time to develop a novel antibiotic can be controlled In a short period baslcally. Platform for operating said development method of this Invention fully optimizes, utilizes and Intégrâtes human resources and technology resources, to ensure effident conduct of development process and make novel antibiotic development operating as flow-line production.
Description of figures
Figure 1 shows general formula of novel antibiotic developed by the method of this Invention: wherein F is effect région; R is récognition région.
Figure 2 shows structure of antibody mimetic.
Figure 3 shows strategy 1 to construct récognition région.
Figure 4 shows strategy 2 to construct récognition région.
Figure 5 shows platform work flow chart, wherein M represents goal proposing system; D represents designïng system; L represents laboratory system; double-headed arrow represents information exchange ways during préparation of novel antibiotics.
Figure 6 shows comparison of in vitro killing effect of novel antiblotics against EB virus-induced Burkitt’s lymphoma.
(A) Control group, (B) group treated by novel antiblotics group 1
Figure 7 shows comparison of survival curves about inhibition of novel antibiotics prepared by this invention, wild-type colicin and anti-staphylococcus aureus polypeptide (ZL 01128836.1) on methicillin-resistant staphylococcus aureus (ATCC BAA-42), vancomydn-resistant enterococcl (ATCC 700802), multi-drug résistant pseudomonas aeruginosa (clinical Isolated strain 13578 In West China Hospital);
Ordinate represents the minimum inhibltory concentration (nMol);
wherein A Is vancomydn-resistant enterococd, (1) anti-staphylococcus aureus polypeptide, MIC = 0.91 nMol, (2) wild-type colicin la, MIC - 0.91 nMol, (3) PMC-AM1, MIC = 0.23 nMol; B Is methidllin-resistant staphylococcus aureus, (1) anti-staphylococcus aureus polypeptide, MIC = 0.06 nMol, (2) wild-type colidn la, MIC = 0.23 nMol, (3) PMC-AM1, MIC = 0.06 nMol; C is multi-drug résistant pseudomonas aeruginosa, (1) anti-staphylococcus aureus polypeptide, MIC = 0.91 nMol, (2) wild-type colidn la, MIC = 0.91 nMol, (3) PMC-AM1, MIC = 0.23 nMol.
Figure 8A shows test results of inhibition of anti-cyanobacteria polypeptide against microcystis aeruginosa growing in liquid medium; the left flask is control, and the right flask is anti-cyanobacteria polypeptide of 35pg/ml.
Figure 8B shows test results of inhibition of anti-cyanobacteria polypeptide against anabaena growing in liquid medium; the left flask Is control, and the right flask Is anti-cyanobacteria polypeptide of 35pg/ml.
Figure 8C shows test results of inhibition of anti-cyanobacteria polypeptide against chiorella growing in liquid medium; the left flask is control, and the right flask is anti-cyanobacteria polypeptide of 35pg/ml.
Figure 8D shows test results of inhibition of anti-cyanobacteria polypeptide against scenedesmus growlng in liquid medium; the left flask is control, and the right flask ls anti-cyanobacteria polypeptide of 35pg/ml.
EMBODIMENTS
The method and platform of this Invention will be described by the following currently-completed development examples.
Example 1 préparation of novel antibiotics against virus-induced tumor (1) determining targets: to détermine léthal novel antibiotics against EB virus-induced tumor cells.
(2) designing molecular structure of novel antibiotics:
the following designing work was performed according to general formula
, wherein F ls effect région; R is récognition région.
Establishlng récognition région molecular structure library: monoclonal antibodies specifically-recognlzlng EB virus - anti-EB virus envelope glycoproteln antibodies gp320, i.e., monoclonal antibodies secreted by ATCC HB-168 hybridoma cells and amino acid sequences Information thereof which had existed in prior art were found by searching in database.
Based on said monoclonal antibody, inventors designed a sériés of antibody mimetic structures as shown in Table 1, and obtained a sériés of mutamers through random point mutation on the first 5 and the last 9 amino acids of antibody mimetics with structures listed in Table 1,
Table 1 the designed antibody mimetic structures
VlCDR1 -VhFR2-VhCDR3
VlCDR2-VhFR2-VhCDR3
Establishing effect région molecular structure library: because the préparation goal was léthal novel antibiotics against EB-virus induced tumor cells, colicin could form léthal Ion channel through cell membrane of Escherichia coli of the same species but different strains by Itself to cause death of Escherichia coli of the same species but different strains, and it was a competent candidate 5 substance for effect région. Thus, colidns la, Ib, A, B and N or mutant sequence were selected as substances of effect région library and offered to laboratory System.
Prelïminarily obtaining the designed molecular structure library of recombinants: from amino terminal to carboxyl terminal: colidn or mutamers thereof + 28 peptides mimetic recognizing EB virus envelop glycoprotein, and molecular structures of some recombinants are shown in Table 2:
| No. | Récognition région molécule | effect région molécule | Recombinant molécule (amino terminal - carboxyl terminal) |
| 1 | VhCDR1 -VhFR2-VhCDR3 | la | la-VHCDR1-VHFR2-VHCDR3 |
| 2 | VlCDR1 -VHFR2-VLCDR3 | la | la-VLCDR1 -VHFR2-V(CDR3 |
| 3 | VhCDR1-VhFR2-V|CDR3 | la | la-VHCDR1-VHFR2-VLCDR3 |
| 4 | VhCDR2-VhFR2-VlCDR3 | la | la-VHCDR2-VHFR2-VLCDR3 |
| 5 | VlCDR1 -VhFR2-VhCDR3 | la | la-VLCDR1 -VHFR2’VHCDR3 |
| 6 | VlCDR2-VhFR2-VhCDR3 | la | la-VLCDR2-VHFR2-VHCDR3 |
| 7 | VhCDR1-VhFR2-VhCDR3 | mla (Seq ID No.1) | mla-VHCDR1 -VHFR2-VHCDR3 |
| 8 | VlCDR1 -VhFR2-VlCDR3 | mla | mla-VLCDR1-VHFR2-VLCDR3 |
| 9 | VhCDR1-VhFR2-VlCDR3 | mla | mla-VHCDR1 -VHFR2-VLCDR3 |
| 10 | VhCDR2-VhFR2-VlCDR3 | mla | mla-VHCDR2-VHFR2-VLCDR3 |
| 11 | VlCDR1 -VhFR2-VhCDR3 | mla | mla-VLCDR1 -VHFR2-VHCDR3 |
| 12 | VLCDR2-VHFR2-VHCDR3 | mla | mla-VLCDR2-VHFR2-VHCDR3 |
Note* monoclonal antibodies secreted by ATCC HB-168 hybridoma cells, VLCDR1, VLCDR2, VhCDR1, VhCDR2 VhFR2, VlCDR3, VhCDR3, colidn la and amino acid sequence thereof as well as nucléotide sequence thereof are known, accordingly amino acid sequence and nucléotide sequence of recombinants can be deduced, and they will take too much space. Thereby, such sequence information will not be listed in this description.
(3) Laboratory system: recombinant library was obtained by binding the provided effect région and récognition région; gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; a batch of recombinant polypeptides were obtained by transforming said recombinant expression vector into engineered bacteria.
Anti-target vérification experiment was conducted on the obtained recombinants (vérification method and experimental design were the same as recorded in ZL200410081446.8). Recombinant 3 and 9 in Table 2 had the best killing effect against EB-virus induced tumor cells, and their resuits of in vitro killing experiment on EB-virus induced Burkitt's lymphoma are shown In Figure 6; other 9 kinds of recombinants had different killing effects against EB virus4nduced tumor, which are weaker than recombinant 3 and 9; ali recombinants had no toxic and side effects on normal cells. The experimental process of vérification ls the same as recorded in example 2-5 of ZL200410081446.8. Recombinants prepared by taklng the mutants of antibody mimetics in recombinants 3 and 9 as récognition région were verified that, in Table 3, recombinants with Seq ID No.2-6 as récognition région has basically équivalent killing effects against EB virus induced tumor cells as that of recombinant 3 or 9.
Table 3 amino acid sequences of 28 anti-EB virus induced tumor peptide mimetic
VhCDR1-VhFR2-VlCDR3 and mutamers thereof
| NO. | VHCDR1-VHFR2-VLCDR3 and point mutants thereof |
| Seq ID No.2 | SFGMHWVRQAPEKGLEWVAGQGYSYPYT |
| Seq ID No.3 | SYGMHWVRQAPEKGLEWVAGQGYSYPYT |
| Seq ID No.4 | SFGMHWVRQAPEKGLEWVAQQWSSNPYT |
| Seq ID No.5 | SFGMHWVRQAPEKGLEWVALQGTHQPYT |
| Seq ID No.6 | SFGMHWVRQAPEKGLEWVAQQLHFYPHT |
| Seq ID No.7 | RQGMHWVRQAPEKGLEWVAGQGYSYPYT |
It took less than 6 months for this préparation, and a batch of candidate novei antibiotics with spécifie killing effect against targets were obtained successfully.
Experimental methods and materials adopted to obtain each recombinant in this example were exactly the same as recorded In Patent No. ZL200410081446.8, except for the inserted gene sequences when constructing vectors, so they are not repeated here.,
Example 2 préparation of novei antibiotics against dlplococcus Intracellularis (1) determining targets: diplococcus intracellularis.
(2) designïng molecular structure of novei antibiotics:
the following designing work was performed according to general formula
, wherein F ls effect région; R is récognition région.
Establishing récognition région molecular structure library:
porin is one outer membrane protein which is common in gram-positive bacteria, like staphylococcus, streptococcus, enterococcus, gram-negative bacteria, like escherichia coli, klebsiella pneumonia, pseudomonas aeruginosa, bowman acinetobacter, enterobacter cloacae, bacillus breslaviensis, serratia marcescens, aeromonas, vibrio, myxococcus, and mycobacterium tuberculosis; it is an idéal antigen protein, and PorA is one kind of porin.
Monoclonal antibody speclfically-recognizing porin PorA which had existed in prior art was found by searching in database; PUBMED ID of its heavy chain peptide ls 2MPA_H, and PUBMED ID of its light chain peptide ls 2MPA_L. Based on said monoclonal antibody, inventera deslgned a sériés of antibody mimetic molecular structures as shown ln Table 1 of example 1.
Establîshing effect région molecular structure library: because the préparation goal was léthal novel antibiotics against diplococcus intracellularis, col ici n was a competent candidate substance for effect région. Thus, colicins la, Ib, A, B and N were selected as substances of effect région library, and colicins la, Ib, A, B and N, ion channel domain molécules thereof and mutant molécules thereof constitute effect région molecular structure library.
The prelimïnarily deslgned molecular structure of recombinant library was: from amino terminal to I0 carboxyl terminal: colicin or ion channel domain thereof or mutamers thereof + anti-PorA antibody mimetic and molecular structures of some recombïnants are shown in Table 4:
| NO. | Récognition région molécule | effect région molécule | Recombinant molécule (amino terminal to carboxyl terminal) |
| 1 | VhCDR1-VhFR2-VhCDR3 | la | la-VHCDR1 -VhFR2-VhCDR3 |
| 2 | VlCDR1 -VhFR2-VlCDR3 | la | la-VLCDR1 -VhFR2-VlCDR3 |
| 3 | VHCDR1-VHFR2-VLCDR3 | la | la-VHCDR1-VHFR2-VLCDR3 |
| 4 | VhCDR2-VhFR2-VlCDR3 | la | la-VHCDR2-VHFR2-VLCDR3 |
| 5 | VlCDR1 -VhFR2-VhCDR3 | la | la-VLCDR1 -VHFR2-VHCDR3 |
| 6 | VlCDR2-VhFR2-VhCDR3 | la | la-VLCDR2-VHFR2-VHCDR3 |
Note* monocional antibodies spedfically-recognizing porin PorA, and PUBMED ID of Its heavy chain peptide ls 2MPA_H; PUBMED ID of its light chain peptide ls 2MPA_L, which are all-known. Thus,
VlCDR1, VlCDR2, VhCDR1, VhCDR2 VhFR2, VlCDR3, VhCDR3 are known, accordingly amino acid sequence and nudeotide sequence of recombinant molécules can be deduced exactly.
Thereby, such sequence Information will not be listed ln this description.
(3) Laboratory system: gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector, a batch of recombinant polypeptides were obtained by transforming said recombinant expression vector into engineered bacteria.
Anti-target vérification experiment of the obtained recombinants was carried out. Vérification experiment was conducted on the killing effects of the obtained recombinants against multi-drug résistant pseudomonas aeruginosa, vancomycin-resistant enterococci, methicillin-resistant staphylococcus aureus, bowman acinetobacter, klebsiella pneumoniae and mycobacterium tuberculosis (vérification method and experimental design were the same as recorded ln ZL200910092128.4). Recombinant 3 in Table 4 had the best kiliing effect against said pathogenic bacteria: comparison of survival curves about inhibition of novel antibiotics prepared by this invention on methiciliin-resistant staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococci (ATCC 700802), muiti-drug résistant pseudomonas aeruginosa (clinical Isolated strain 13578 In West China Hospital) Is shown In Figure 7; other 5 kinds of recombinants had different killing effects against said drug-resistant bacteria, which are weaker than recombinant 3; ali recombinants had no toxlc and side effects on normal ceils. The experimental process of vérification is the same as recorded in example 2-6 of ZL200910092128.4.
Recombinants prepared by taking the mutants of antibody mimetics ln recombinant 3 as récognition région and taking colidn la as effect région were verified that, in Table 5, recombinants with Seq ID No.9-13 as récognition région has baslcally équivalent killing effects against the above-mentioned pathogenic bacteria as that of recomblnant 3.
Table 5 amino acid sequences of anti-diplococcus Intracellularis antibody mimetic
VHCDR1-VHFR2-VLCDR3 and mutamers thereof
| No. | VhCDR1-VhFR2-VlCDR3 and point mutants thereof |
| Seq ID No.8 | SYWLHWIKQRPGQGLWIGSQSTHVPRT |
| Seq ID No.9 | SYGMHWIKQRPGQGLWIGSQSTHVPRT |
| Seq ID No.10 | SYWIEWIKQRPGQGLWIGSQSTHVPRT |
| SeqlDNo.11 | NYWMHWIKQRPGQGLWIGSQSTHVPRT |
| Seq ID No. 12 | SYWLHWIKQRPGQGLWIGMQNIGLPWT |
| Seq IDNo.13 | SYWLHWIKQRPGQGLWIGQQFTSSPYT |
It took less than 6 months for this préparation, and a batch of candidate novel antibiotics with broad-spectrum antibacterial effect were obtained successfully.
Experimental methods and materials adopted to obtain each recombinant in this example were 5 exactly the same as recorded in Patent No. ZL200910092128.4, except for the Inserted gene sequences when constructing vectors, so they are not repeated here.
Example 3 préparation of novel antibiotics against badllus anthracis (1) Goal proposing system determined anthrax toxin or badllus anthracis as targets; léthal infection dîseases caused by anthrax toxin or badllus anthracis hâve been posing a huge threat against human health; in terrorist attacks, anthrax toxin is also the most homble pathogen or toxin as weapon.
The goal of this préparation is to provide a novel antibiotic to destroy the toxidty of badllus anthrads or anthrax toxin, i.e., to inhibit or interfère anthrax toxin PA antigen from forming active PA heptamer.
(2) Designing novel antibiotics:
The following designing work was performed according to general formula
, wherein F is effect région; R is récognition région.
The general characteristic of anthrax toxin is that, anthrax toxin is a binary toxin with high harmfulness to organisms, and consists of protein antigen PA, necrosîn and edema factor (LF/EF);
protein antigen PA is a transport structure and can recognize target cells, and it transports necrosin and edema factor (LF/EF) into target cells. It’s illustrated by animal experiments that, a combination of protein antigen and necrosin can immediately lead to cell death, while no réaction will be caused as applying said two components separately. The novel antibiotic was designed preliminarily that récognition région of said novel antibiotic can recognize anthrax PA antigen, and effect région of said novel antibiotic can inhibit or Interfère anthrax toxin PA antigen from forming active PA heptamer. Establishing récognition région molecular structure library: anti-bacillus anthracis protein antigen léthal factor complex antibody (NCBI CAL58671) generated in cynomolgus and antî-badlius anthrads protein antibody (NCBIABF69350) generated in house mouse were found by searching database, and they are competent to spedficaliy recognize protein antigen PA of anthrax toxin. According to amino add information of said antibodies, a sériés of antibody mimetic structures and mutants thereof with a structure of VHCDR1-VHFR2-VLCDR3 which can recognize wild-type anthrax toxin were designed to build récognition région molecular structure library, and provided to laboratory system.
Establishing effect région molecular structure library: because préparation goal is to inhibit anthrax toxin PA antigen from forming PA heptamer, In accordance with infection mechanism of anthrax toxin, in this experiment, some mutant anthrax toxin PA antigens (see Seq ID No.10 recorded In ZL200810045212.6) obtained by artificial mutation on anthrax toxin PA antigen were conducted as member of effect région molecular structure library, and PA lost récognition capadty to corresponding receptor on target cells; said mutant anthrax toxin PA antigen and wild-type anthrax toxin PA antigen constituted heterozygous PA heptamer, which lost transmembrane activity completely or partially, accordingly interfered with infection abîlity of anthrax toxin.
(3) Laboratory system: recombinant library was obtained by binding the provided effect région and récognition région; gene coding said recombinant was inserted Into expression vector to obtain recombinant expression vector; a batch of recombinant polypeptides were obtained by transforming said recombinant expression vector into engineered bacteria.
A batch of recombinants with amino acid sequence listed in Table 6 as récognition région and mutant anthrax toxin PA antigens (see Seq ID No.10 recorded in ZL200810045212.6) as effect région were obtained through vérification, and they could protect mice infected by bacillus anthrads. Vérification experiment and results thereof were similar to the effects of pCHCA-PA1 recorded in ZL200810045212.6.
Table 6 amino add sequences of antibody mimetics and mutamers thereof recognizing wild-type anthrax toxin PA
| NO. | VhCDR1-VhFR2-VlCDR3 and its point mutants |
| Seq ID No.14 | STALHWRQAPGKGLEWVPRYDEFPYT |
| Seq iD No. 15 | SFGMHWRQAPGKGLEWVPRYDEFPYT |
| Seq ID No.16 | NYWMHWRQAPGKGLEWVPRYDEFPYT |
| Seq ID No.17 | STALHWRQAPGKGLEWVFQGSHVPFT |
| Seq IDNo.18 | STALHWRQAPGKGLEWVYCHQWSMYT |
| Seq ID No. 19 | STALHWRQAPGKGLEWVQQWSSNPYT |
| Seq ID No.20 | STALHWRQAPGKGLEWVQQFTSSPYT |
It took less than 6 months for this préparation, and a batch of novei antibiotics which hâve protection 10 effects against bacillus anthrads infection were obtained successfuily.
Experimental methods (e.g., vector construction, transformation, vérification experiment, etc.) and materials adopted to obtain each recombinant In this example were exactly the same as examples recorded in Patent No.ZL200810045212.6, except for the inserted gene of novel antibiotics, so they are not repeated here.
Example 4 préparation of novel antibiotics against fungi (1) Goal proposing system determined candida albicans as targets, and the goal was determined to préparé novel antibiotics killing agriculture) fungus - candida albicans.
(2) Designing novel antibiotics:
The following designing work was performed according to general formula
R , wherein F Is effect région; R is récognition région.
Establishing récognition région molecular structure library: the great progress in fungus basic research had been achieved in recent years; amino acid sequence (Seq ID No.21) of candida albicans pheromone consists of 14 amino acid residues. it can move around freely in bioioglcal media, and has bioioglcal activity of automatically searching the corresponding receptor on cell membranes of the same species of fungi cells. Thus, based on such automatically searching activity, it's available to utilize fungus pheromone as récognition région to induce effect région like bacterlal exotoxin such as collcin to kill these fungi by forming ion channel through cell membranes, and a batch of novel blological biocides were constructed accordingly.
Therefore, candida albicans pheromone represented by Seq ID No.21 was selected as récognition région.
Establishing effect région molecular structure library: because the préparation goal was léthal novel antibiotics against target of agricultural fungus-candida albicans, colicln was a competent candidate substance for effect région owing to its characteristics. Thus, colidns la, lb, A, B and N, and ion channel domains thereof were selected as members of effect région molecular structure library, and were provided to laboratory system.
The preliminarily designed molecular structure of novel antibiotic was: from amino terminal to carboxyl terminal: colicin or ion channel domain thereof or mutamers thereof + candida albicans pheromone.
(3) Laboratory system: recombinant library was obtained by binding the effect région and récognition région; gene coding said recombinant was inserted into expression vector to obtaln recombinant expression vector; recombinant polypeptides were expressed by transforming said recombinant expression vector Into engineered bacteria, which realized opérable binding between effect région and récognition région.
The obtained recombinants were verified that, they ail hâve protection effects on rice (Oryza sativa) infected by fungi like pyricularia oryzae, aspergilius flavus, and their protection effect on rice blast 5 infection Is thousands of times hlgher than that of current agriculture) antifungal.
Expérimente and data (e.g., vector construction, transformation) related to obtaining each recombinant ln this préparation was recorded ln examples of Patent No. ZL200710050926.1 or based on the record in prior art, person skilled ln the art can obtain the experimental methods required for the préparation of this Invention by a limited number of experiments, so they are not to repeated here.
It took 6 months for this préparation, and cost only 1.5-2 years including field experiments, whose efficiency and success rate are far hlgher than that of current préparation for a new drug.
Example 5 préparation of novel antlblotlcs against drug-reslstant staphylococcus aureus (1) goal proposing system determined staphylococcus aureus as targets: since antibiotics like penicillin was applied in 1944, bacteria, espedally pathogenic bacteria threatenlng human life, like staphylococcus aureus, streptococcus pneumonia, pseudomonas aeruginosa, mycobacterium tuberculosis, etc., hâve generated drug-resistance, and human are urgent to develop novel antibiotics against drug-resistant bacteria.
(2) Deslgning novel antibiotics:
The following designing work was performed according to general formula
R , wherein F Is effect région; R Is récognition région.
Establishing récognition région molecular structure library: many cells secrete signal transduction polypeptides to the outside of cells; these polypeptides can automatically search for the corresponding receptors on cell membranes of the same spedes of bacteria, and bind to said receptors to transport information Into said bacteria; staphyiococcus sécrétés signal transduction polypeptides to the outside of cells; these polypeptides can automatically search for the corresponding receptors on cell membranes of the same spedes of bacteria, and bind to said 5 receptors to transport information into said bacteria. These signal transduction polypeptides consist of several to more than 10 amino adds, and are idéal récognition régions against staphyiococcus. Pheromone sequences as Seq ID No.22-26 were collected as the members of récognition région library through searching.
Providing effect région: because the préparation goal was léthal novel antibiotics against target of drug-resistant staphyiococcus aureus - candida albicans, colidn was a competent candidate substance for effect région owing to its characteristics. Thus, colidns la, Ib, A, B and N, and Ion channel domains thereof were selected as effect réglons, and were provided to laboratory system.
The preliminarily designed molecular structure of novel antibiotic was: from amino terminal to 15 carboxyl terminal:
colicin or ion channel domain thereof or mutamers thereof + pheromone, and pheromone + colidn or ion channel domain thereof or mutamers thereof.
(3) Laboratory system: recombinant library was obtained by binding the effect région and récognition région; synthetic gene coding said recombinant was inserted Into expression vector to 20 obtain recombinant expression vector; recombinant polypeptides were expressed by transformlng said recombinant expression vector into engineered baderia, which realized opérable binding between effect région and récognition région.
A batch of recombinants were obtained, such as recombinant polypeptide expressed by recombinant plasmids pBHC-SA1, pBHC-SA2, pBHC-SA3 pBHC-SA4, pBHC-SE and pBHC-PA (as 25 recorded in Patent No. ZL200910157564.5). They ail hâve significant killing effects against methidllin-resistant staphyiococcus aureus, penicillin-resistant staphyiococcus aureus, vancomydn-resistant enterococd, pseudomonas aeruginosa and multi-drug résistant pseudomonas aeruglnosa.
Experiments and data (e.g., vector construction, transformation) related to obtaining each recombinant in this préparation was recorded in examples of Patent No. ZL200910157564.5 or based on the record In prior art, person skilled in the art can obtain the experimental methods required for the préparation of this invention by a limited number of experiments, so they are not repeated here.
it took 5 months to préparé a batch of novel antiblotics against drug-resistant bacteria, whose efficiency and success rate are far higher than that of current préparation for a new drug.
Example 6 préparation of novel antiblotics against cyanobacteria (1) Goal proposing system determined cyanobacteria as targets: cyanobacteria prolifération caused by water eutrophication, water pollution are the severest harm threatening water environment in the worid, and they resuit in huge économie loss to human as well as cause unrepaired harm to earth's biosphère. As the accelerated industrialization and urbanization caused by Chinese économie development, ecological environment pollution and degeneration aggravate graduaily. and water environment ecological control has been major problem we must face and soive. The current antimlcroblal drugs almost hâve little effects against cyanobacteria; cyanobacteria Is prokaryotic ceil belonging to cyanobacteria phylum of bacteria kingdom, and only chemicals of heavy metals can control cyanobacteria at présent, e.g., cupric sulfate. However, in practical application, owing to limited effects, it’s required to use chemical with overdose repeatedly, and other bénéficiai algae, aquatic plants and aquatlc organisms are destroyed when killing cyanobacteria, which results in irréversible permanent damage to environment.
The purpose is preparing novel antiblotics killing or inhibiting cyanobacteria.
(2) Designîng novel antiblotics:
The following designîng work was performed according to general formula
, wherein F is effect région; R Is récognition région.
w
Establishing récognition région molecular structure library: a batch of hybridoma cells secretlng anti-cyanobacteria monoclonal antibodies were obtained by immunizing mice with cyanobacteria as antigen, and the deposit No. of one strain of said hybridoma cells is CGMCC No.4783.
Based on amino acid sequences of said monoclonal antibodies obtained by sequencing, a batch of antibody mimetlcs were designed, which are shown in Table 1, and mutamers of antibody mlmetics were obtained through random point mutation on the first 5 and the iast 9 amino acids of antibody mlmetics; récognition région molecular structure library was built by taking saïd antibody mimetic molécules and mutamers thereof as members. Wherein, the amino add sequence of antibody mimetic with a structure of VhCDR1-VhFR2-VlCDR3 of monodonal antibody secreted by 10 hybridoma cells(CGMCC No.4783) is shown as Seq ID No.27.
Establishing effect région molecular structure library: because the préparation goal was novel antibiotics kîliing or inhibiting cyanobacteria, colidn could form iethal ion channel through œil membrane of Escherichia coli of the same species but different strains by itself to cause death of Escherichia coli of the same spedes but different strains, and It was a competent candidate 15 substance for effect région. Thus, colidns la, Ib, A, B and N or mutant sequences thereof were selected as members of effect région molecular structure library.
Preliminarily obtaining the designed molecular structure of novel antibiotics: from amino terminal to carboxyl terminal: colicin or mutamers thereof + anti-cyanobacteria antibody mimetic/mutamers:
(3) Laboratory system: molecular structure of recombinants was obtained by binding the effect 20 région and récognition région; gene coding said recombinant was inserted into expression vector to obtain recombinant expression vector; the recombinants were expressed by transforming said recombinant expression vector Into engineered bacteria and Isolated.
It’s revealed by vérification on the Inhibition effect of obtained recombinants against cyanobacteria 25 (the design and operation of vérification experiments was the same as recorded in examples 3-5 of ZL201110155221.2) that, said recombinants had significant inhibition effects on mlcrocystis aeruginosa, anabaena but no inhibition on other benefidal algae like chlorella, diatom and scenedesmus. Some experimental results are shown as Figure 8A-8D. The molecular structures of some selected recombinants are listed In Table 7.
Table 7 amino acid sequences of antibody mimetics and mutamers against surface antigens of mlcrocystis aeruglnosa
| NO. | VHCDR1-VHFR2-VLCDR3 and point mutants thereof |
| Seq ID No.27 | SYWMQWVKQRPGQGLEWIGQQYWSTPPWT |
| Seq ID No.28 | SYGMHWVKQRPGQGLEWIGQQYWSTPPWT |
| Seq ID No.29 | DHYMHWVKQRPGQGLEWIGQQYWSTPPWT |
| Seq ID No.30 | SYWIEWVKQRPGQGLEWIGQQYWSTPPWT |
| Seq ID No.31 | SYWMQWVKQRPGQGLEWIGQQQFTSSPWT |
| Seq ID No.32 | SYWMQWVKQRPGQGLEWIGQQQSREYPYT |
| Seq ID No.33 | SYWMQWVKQRPGQGLEWIGQLQGTHQPYT |
It took less than 11 months for this préparation, and a batch of verified novel antibiotics which had kiliing effects against targets were obtained successfully.
Experimental methods and materials adopted to obtain recombinants in this préparation were exactly the same as recorded in Patent No.ZL201110155221.2, except for the Inserted gene of novel antibiotics In vector construction, so they are not repeated here.
Example 7. Experiments of screening suitable blologlcal expression Systems for the methods of this Invention.
Recombinant plasmids were obtained during novel antibiotics préparation recorded In examples 5*6: pBHC-SA1, pBHC-SA2, pBHC-SA3, pBHC-SA4, pBHC-SE, pBHC-PAand pBHC-PorA1.
Stepl. Transfomnlng competent cells
4040μΙ various englneered bacteria of pET system like BL-21 (DE3), B834(DE3), Nova Blue(DE3) and 618 (Novagen) were transfected by 100ng recombinant mutant plasmids respectiveiy; Ice incubate for 5 minutes, thermal shock at 42Ό for 30 seconds, Ice bathïng for 2 minutes, addîng 160ul SOC medium, 220 rpm, incubated at 37Ό by shaklng for 1 hour, and then spread on plate (LB medium with 1% agar and 50pg/ml ampicillin) to incubate at 37Ό ovemlght. Monoclonal colony was selected for prolifération to get strain, and the strain was preserved at low température.
Step2. Strain recovery
1. Strain recovery
Said preserved strain was unfrozen at 4Ό; 1.5ml strain was added in 10ml LB medium (containing AMP 50pg/ml), 220rpm, and incubated at 37Ό for 5-8 hours.
2. Inoculation of monoclonal strain
The recovered strain culturing liquid was diluted at 104 or 105 times; 10μΙ diluted strain culturing liquid was placed onto the prepared LB solid medium (AMP 50pg/ml) plate and spread. The plates were placed in moist box for Incubation at 37Ό for 10-12 hours till round single colonies were raised on the surface of plate.
Step3. Sélection and prolifération of strain (1) Regular round single colony with smooth edge was selected by the sterilized toothpick or Inoculation loop from the Incubated plate, and placed Into 1.5ml LB medium for culture by shaking at 220rpm and 37Ό for 5-8 hours.
(2) 1.5ml LB medium was added into 100ml LB medium for culture by shaking at 220rpm and 37Ό for 5-8 hours.
(3) 1*’ grade amplification culture: 100ml strain culturing liquid obtained from the last step was added Into 700ml improved FB-M9 complex medium for culture by shaking at 220rpm and 37Ό for 5-8 hours.
(4) 21** grade amplification culture: 700ml strain culturing liquid obtained from the last step was added into 6x700ml improved FB-M9 complex medium for culture by shaking at 220rpm and 37Ό for 5-8 hours.
(5) 3rt grade amplification culture: 6x700ml strain culturing liquid obtained from the last step was added into 20L Improved FB-M9 complex medium for culture In fermenter with shaking speed at
220rpm and maximum oxygen content, at 37*C for 3-5 hours.
(6) Engineered bacteria fermentation and Induction of protein expression: 20L strain culturing liquid obtained from the last step was added Into 200L Improved FB-M9 complex medium for culture and protein expression In fermenter, with shaking speed at 220rpm and maximum oxygen content, at 30Ό for 2-4 hours; then at 42*C for 0.5 hours; finally at 37Ό for 1-2 hours. Note: IPTG with final concentration of 0.5mM was added when reaching 42*C.
Step4. Strain collection by centrifugation
Strain culturing liquid was centrifuged at 6000g, 4*C for 20mln. The precipitate was collected after centrifugation, and resuspended In 50mM borate buffer (pH9.0). Note: 2mM PMSF (benzyl sulfuryt fluoride serine proteases inhibitor) was added Into borate buffer, and the operation after thalli resuspending must be conducted at 4Ό.
Step5. Thalli fragmentation
After thalli were suspended In pH9.0 borate buffer totally, thalli were fragmentated by high pressure homogenizer at 500-600bar; fragmentation was repeated for 7 times, and there was 3-5 minutes interval between two fragmentations.
Step6. Précipitation of thalli DNA
Fragmentated strain culturing liquid was centrifuged at 55000g, 4*C for 40min. The supematant was isolated, added with streptomycin sulfate (16 bottles of streptomydn sulfate with 1 million units were added Into every 200ml liquid), and stirred on magnetic stirrer for 1 h.
Step7. Dialysis
Strain culturing liquid obtained by the last step was centrifuged at 55000g, 4Ό for 20min. The supematant was isolated, placed into dialysis bag, placed In borate buffer for dialysis for 8-12 hours, and the dialysate was changed once every 4 hours.
Step8. Antibacterial engineered polypeptide medicine obtained by protein purification
Strain culturing liquid after dialysis was centrifuged at 55000g, 4Ό for 20min. The supematant was placed into beaker to conduct protein purification by ion exchange method. The supematant was loaded in CM ion exchange column, and protein concentration was detected to count protein content per unit volume; the ratio of loading volume and CM Ion gel particles was fixed according to operation manual. After rinslng thoroughly, novel antibacterial englneered polypeptide was obtained through elution by 50mM borate buffer containing 0.2M NaCI.
It’s described by results shown in Table 8 that, the expression rate of PMC-SA In E.coli B834 (DE3) was the highest.
Table 8 comparison of expression rates In different strains (the average yield per unit=total production of extracted PMC-SA1Ahe volume of strain culturing liquid)
| Engineered strains | TG1 | BL-21 | 618 | NavaBlue | B834 |
| the average yield per unit (mg/L) | 0.8 | 10 | 5.8 | 8.1 | 24.4 |
The same operation and comparison were conducted on other 6 klnds of recombinant mutant plasmlds, and the results ail show the same tendency as shown in Table, that ls, compared with other englneered bacteria, the expression rates of 7 klnds of recombinant mutant plasmlds In E.coll B834(DE3) are ali the hlghest.
On the basis of this screening experiment, It's préférable but not limited to select E.coli B834(DE3) as expression system In the method of this invention, in order to efficiently express and préparé to obtain novel antibiotics.
In summary, novel antibiotic préparation method and platform system of this invention are capable of providing novel antibiotics with récognition région and effect région specifically against most pathogenlc microorganisms and targets. For most targets, the currentiy-known coltdn is competent to playlng the rôle of effect région, so the cycle time of preparing a novel antibiotic dépends on the time to design molecular structure, the time to provide récognition région molecular Information, as well as the time to préparé and verify recombinants. Because récognition région dépends not only on the selected nature substances but mainiy dépends on artifidal-prepared antibody mimetic as récognition région, in view of the current blotechnological level, the time to préparé a novel antibiotic can be basically controlled In a very short period. Platform for operating said préparation method of this invention fully optimizes, utilizes and Intégrâtes human resources and technology resources, to ensure efficient conduct of development process and make novel antibiotic development operating as flow-line production.
Claims (11)
1 « substance on said targets;
said immunoglobulin is prepared by taking multiple unique substances on target as immunogen respectively to immunizing animal.
(1) determining targets: said targets refer to prokaryotic cells, eukaryotic cells, viruses or products thereof which said novel antibiotic will react against directiy;
1. A novel antibiotic préparation method, wherein steps are as follows:
2. The method according to claim 1, said antibody mimetic is peptide with a structure of
(2) designing molecular structure of said novel antibiotic:
(3) based on said recombinant molecular structure library, preparing and verifying recombinant so 20 as to obtain candidate novel antibiotics;
4. The method according to any one of claims 1-3, wherein said target refers to virus, prokaryotic cell or eukaryotic cell with phospholipid bilayer membranes as the basic structure of its ce!) membrane or envelope; said pharmaceutical effect refers to causing death and/or collapsing;
(4) screening novel antibiotics which meet requirements as medicine from candidate novel antibiotics;
said establishing récognition région molecular structure library refers to artificially preparing artificial substances which specifically recognize and/or combine said targets as well as obtaining *
25 molecular structure thereof;
said artificial substances refer to antibody mimetic; said antibody mimetic is designed according to amino acid sequence of immunoglobulin which can specifically recognize unique
5. The method according to claim 4, wherein said effect région refers to barteriocin Pyosin of pseudomonas aeruginosa.
^5 said effect région refers to bioactive substance which can form ion channel or pore path in phospholipid bilayer membranes.
5 VhCDR1-VhFR2-VlCDR3 from N-terminal to C-terminal, which is constituted by the régions of VhCDR), VhFR2, VlCDR3 on Fab short arm of said immunoglobulin; or is mutamer of said short peptide; said mutamer refers to product obtained by artificial site-mutation to 5 amino acid residues of VhCDRI or 9 amino acid residues of VlCDR3 on short peptide, which holds récognition capability to unique substance on said targets.
5 said molecular structure of said novel antibiotic is designed according to the following general formula:
(τ X* ) wherein, R is récognition région, which specifically recognizes or combines said targets; F is effect région, which generates pharmaceutical effects to targets, and saîd pharmaceutical effects are effects of regulating, repairing, labeling, causing death and/or collapsing against said taigets specifically, establishing récognition région molecular structure library;
establishîng effect région molecular structure library, · according to said general formula, designing recombinant molecular structure library on the basis of said récognition région molecular structure library and effect région molecular structure 15 library, said designing refers to the process of structural readjustment, structural recombination and/or structrual confirmation carried out on the basis of molecular structures of collected, selected or prepared substances used as effect région or récognition région;
6. The method according to claim 4, wherein said effect région refers to colicins El, la, Ib, A, B or
2Q N; or domains of colicin molécules El, la, Ib, A, B and/or N which can form ion channels; or molécules obtained by allosterism finom colicin molécules El, la, Ib, A, B, N, domains of colicin molécules El, la, Ib, A, B or N which can form ion channels, having function of forming ion channels in said phospholipid bilayer membranes.
7. The method according to claim 4, wherein said recombinant is recombinant polypeptide, said je preparing recombinant refers to that gene coding said recombinant polypeptide is transformed into / ι * biological expression system to express fusion protein, and novel antibiotic is obtained by separating and purifying fusion protein.
8. The method according to claim 7, wherein said expression system refers to E.coli B8344DE3).
9. The method according to claim 1, wherein said pharmaceutical effect refers to labeling, said effect g région refers to label, said preparing recombinant refers to linking effect région and récognition région operably.
10. A platform system for novel antibiotic préparation, comprises 3 interoperable Systems: (1) goal proposing System, (2) designing system, (3) laboratory system;
said platform préparés novel antibiotic according to said method of any one of ciaims 1-9; said 10 goal proposing system détermines goal of research and development, and delivers task instruction to said designing system;
said designing system establishes récognition région molecular structure library, effect région molecular structure library as well as designs molecular structure library for said recombinant in line with task instruction offered by said goal proposing system and said general formula, and provides
15 said molecular structure library to said laboratory system;
said laboratory system offers experimental results to said designing system; said designing system reports the finally-selected candidate products as development results to marketing system;
said designing refers to the procedure of structural adjustment, structural recombination and/or structural confirmation based on molecular structure of the collected, selected or prepared substance 20 as effect région or récognition région.
10 3· The method according to any one of claims 1 or 2, wherein said immunoglobulin is prepared by taking common unique substances on multiple targets as immunogens to through immunize anima).
11. The platform according to claim 10, wherein said laboratory system comprises at least one 3 party partner institution which contracts to do said experiments.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110405775.3 | 2011-12-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA16992A true OA16992A (en) | 2016-02-26 |
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