Patent Application: US-20396902-A

Abstract:
the invention relates to the field of molecular recognition or detection of discontinuous or conformational binding sites or epitopes corresponding to a binding molecule , in particular in relation to protein - protein protein - nucleic acid , nucleic acid - nucleic acid or biomolecule - ligand interactions . the invention provides a synthetic molecular library allowing testing for , identification , characterisation or detection of a discontinuous binding site capable of inter - acting with a binding molecule , said library having been provided with a plurality of molecules , each molecule of said molecules comprising at least one first segment linked to a second segment , each segment having the capacity of being a potential single part of a discontinuous binding site .

Description:
a peptide with a n - terminal bromoacetamide group was synthesised at the surface of a solid support containing free amino groups . the peptide still contained the side - chain protecting groups of the amino acid residues . a second peptide containing a cysteine residue , which was deprotected and cleaved from another solid support was reacted with the bromoacetamide peptide on the first solid support . the formed construct was deprotected , but not cleaved from the support , and could be used in elisa . a polypropylene or polyethylene support , or of other suitable material , was grafted 15 with , for instance , polyacrylic acid . as an example : a polypropylene support in a 6 % acrylic acid solution in water , containing cuso 4 , was irradiated using gamma radiation at a dose of 12 kgy . the grafted solid support containing carboxylic acid groups was functionalised with amino groups via coupling of t - butyloxycarbonyl - hexamethylenediamine ( boc - hmda ) using dicyclohexylcarbodiimide ( d cc ) with n - hydroxybenzotriazole ( hobt ) and subsequent cleavage of the boc groups using trifluoroacetic acid . standard fmoc peptide synthesis was used to synthesise peptides on the amino functionalised solid support . after cleavage of the fmoc group of the last amino acid and washing , bromoacetic acid was coupled using dcc or dccihobt . if only dcc was used the peptide did contain a thiol reactive bromoacetamide group , however , if dcc / hobt was used to couple bromoacetic acid , the peptide essentially did not contain the bromo group , but another reactive group capable to react efficiently with thiol groups thus forming the same thioether link between the segments . coupling / ligation of a second peptide to a pep tide synthesised on a solid support : peptides were synthesised at polyethylene pins grafted with poly - hydromethylmethacrylate ( poly - hema ). this graft polymer was made by gamma irradiation of polyethylene pins in a 20 % hema solution in methanol / water 80 / 20 or 70 / 30 at a dose of 30 - 50 kgy . the functionalised support can be used for the synthesis of 1 μmol of peptide / cm 2 after coupling of β - alanine and an acid labile fmoc - 2 , 4 - dimethoxy - 4 ′-( carboxymethyloxy )- benzhydrylamine ( rink ) linker . the peptides were synthesised using standard fmoc chemistry and the peptide was deprotected and cleaved from the resin using trifluoroacetic acid with scavengers . the cleaved peptide containing a cysteine residue at a concentration of about 1 mg / ml was reacted with the solid support bound peptide described above in a water / sodium bicarbonate buffer at about ph 7 - 8 , thus forming a partially protected construct of two peptides covalently bound via a thioether and c - terminally bound to the solid support . the construct described above was deprotected following standard procedures using trifluoroacetic acid / scavenger combinations . the deprotected constructs on the solid support were extensively washed using disrupting buffers , containing sodium dodecylsulphate and p - mercaptoethanol , and ultrasonic cleaning and were used directly in elisa . subsequent cleaning in the disrupt buffers allows repeatingly testing against other antibodies in elisa . [ 0072 ] fig1 shows an example of the elisa results of screening a simple library of constructs , consisting of a dodecapeptide segment coupled via its c - terminally added cysteine residue to a n - terminally bromoacetylated second segment , scanning a protein sequence by steps of a single amino acid residue . the bromoacetamide peptide was covalently bound to a functionalised polypropylene / polyacrylic acid solid support in 3 μl wells as described above . the cysteine - containing sequences were synthesised on and cleaved from functionalised polyethylene pins as described above . as shown in fig1 high binding was observed in elisa for constructs around position 125 , which consists of the segments [ 125 - 136 ] and [ 139 - 150 ], linked via a the ether bond . a conventional linear pepscan of dodecapeptides or 15 - peptides did not show any binding in a reaction against the same monoclonal antibody . on a surface of a solid support peptides are synthesized with a bromoacetamide group at the n - terminus as described above . on this peptide functionalized support a second peptide segment containing a free thiol group was spotted using piezo drop - on - demand technology , using a microdosing apparatus and piezo autopipette ( auto drop - micropipette ad - k - 501 ) ( microdrop gesellschaft fur mikrodosier systeme gmbh . alternatively , spotting or gridding was done using miniature solenoid valves ( inkx 0502600a ; the ice company ) or hardened precision ground gridding pins ( genomic solutions , diameters 0 . 4 , 0 . 6 , 0 . 8 or 1 . 5 mm ). subsequent deprotection of the construct and extensive washing to remove uncoupled peptide gave dipeptide constructs at the spotted area . [ 0074 ] fig2 shows binding of the same antibody as was tested in fig1 with constructs consisting of two peptide segments , generated with different volumes of spotted peptides 2 to 8 , ranging from 1 μl - 0 . 25 nl ( x - axis ). within the square the whole surface was covered with peptide 1 , which was synthesised directly on this surface , only the spots contain constructs . the y - axis shows different constructs , consisting of peptide 1 with peptide 2 up to 8 . peptides 2 up to 8 are overlapping dodecapeptides , while peptide 1 is sequence [ 139 - 150 ] of the same protein as described in fig1 . fig2 shows that peptide constructs generated with peptide solution droplets in the nanoliter - range , bind enough antibody for detection , in this case using indirect fluorescence detection . spots generated with 0 . 25 nl - 50 nl are smaller than 1 mm 2 . thus , peptide construct density can be as large as 100 - 1000 spots per cm 2 . proteins and peptides can be screened using for instance antibodies , soluble receptors , which contain a fc - tail or a tag for detection , biotinylated molecules or fluorescent molecules . alternative building blocks could be , for instance , carbohydrates , non - natural amino acids , pna &# 39 ; s , dna &# 39 ; s , lipids , molecules containing peptide bond mimetics . in the examples $ is used as a symbol for the thioether link formed by reaction of the thiol group of a cysteine residue of one building block with a bromoacetamide at the n - terminus or at the side chain of a lysine residue from another building block . this symbol can also be used for other linking chemistries as described . the examples are divided into two types . type i is performed in the creditcard format minicards ( cf fig1 ). type ii is performed using gridding pins on a discontinuous porous matrix surface ( cf . fig2 ). for each example the type is indicated between brackets . ( type i ): ‘ standard ’ 24 - 30 - mer scan of linear sequence , containing two building blocks . in this example the consecutive sequences of the building blocks are both shifted one by one residue through the sequence of the protein to be tested as shown in fig3 a and exemplified in fig3 b and 3c for 30 - mers ( in fig1 the example is with 24 - mers ). the - c $- link between both building blocks replaces 0 - 2 or more amino acid residues of the native protein sequence . applications include replacement sets of peptides , in which amino acid residues are replaced systematically by other amino acid residues ( fig1 c ), deletion sets of peptides , in which amino acid residues are deleted systematically , and combination sets , in which peptides of different lenght ranging from 2 - 24 ( here building block 2 , 2 - 40 or more and building block 1 , 2 - 15 or more ) amino acid residues are used . this is a scan similar to example 1 described above , however , in this scan the cysteine is used to substitute the amino acid residues one by one in every position of the second building block as shown in fig4 a and exemplified in fig4 b and 4c . fig5 illustrates the reproducibility of coupling a 25 - mer that binds mab - 01 to all overlapping 15 - mers . in type - i , i . e . using the creditcard sized minicards only a few thousand peptides can be synthesized . in type - ii , i . e . using the gridding pins , many thousands of peptides ( in the order of 40 . 000 ) can be synthesized simultaneously . in a complete matrix - scan the n - terminal sequence of , for instance , sequence [ 1 - 11 ] of a protein , is linked as a building block with each overlapping peptide sequence of a complete scan of the same protein as shown in fig6 a . next , sequence [ 2 - 12 ] is linked with the same set of overlapping sequences and so on . the link can be formed , for instance , by reaction of a cysteine at the c - terminus of the second building block with a bromoacetamide modified n - terminus of the first building block . this means that every combination of , for instance , undecapeptides from the protein sequence is being synthesised on a seperate , known , position of the solid support . this is the same scan as the complete matrix scan from example 2a , however , in this scan the cysteine of the second building block is located at its n - terminus , providing a reversed or tail - to - tail orientation of both building blocks in the construct as also shown in fig6 a . both example 2b and 2c are illustrated in fig6 b , 6c and 6 d . in this example a thiol fuction is introduced on an amino - functionalised solid support . this can be made by a direct reaction of the amino groups with , for instance , iminothiolane , or by coupling of fmoc - cys ( trt )- oh , followed by fmoc cleavage using piperidine , acetylation , and trityl deprotection using tfa / scavenger mixtures . this thiol - functionalised solid support can be reacted with , for instance , a bromoacetamide - peptide , containing a protected cysteine residue . after coupling of the first peptide , the cysteine can be deprotected , using , for instance , a tfa / scavenger mixture . the formed free thiol group can be used to couple a second bromoacetamide - peptide , again containing a protected cysteine . this procedure can be repeated to make multi - building block constructs . several types of scans , as described in the other examples , can be used in combination with this multi building , block scan . in fig7 a an example is shown for a three multi building block scan . an working example with two building block scan is illustrated in 7 b , 7 c and 7 d . in a matrix combi - scan , a matrix scan from two different proteins is tested against a labeled soluble protein . fig8 a shows two examples . in the first example ( fig8 a ) soluble protein 1 ( growth hormone , gh ) was tested against a combined matrix scan of protein 2 ( gh - receptor - 1 ) and protein - 3 ( gh - receptor - 2 ). in the second example a part of soluble protein 1 ( cd - 4 ) was tested against a combined matrix scan of protein 2 ( hiv ) and protein 3 ( chemokine - receptor ccr4 ). in fig8 b the usage of receptors and hormones is illustrated by using a biotin - labeled part of the protein human follicle stimulating hormone tested on all overlapping 30 - mers covering te human human follicle stimulating hormone receptor . examples 1 to 4 describe methods using peptide building blocks and screening with proteins . these constructs can also be screened against non - proteins . also non - peptide building blocks can be used . below , examples of whole proteins in combination with peptides ( example 5 ), or peptides / proteins in combination with non - peptide / proteins , or non - peptide / protein with non - peptide / protein ( example 6 , dna / rna / pna ) are shown . this example is similar to example 4 . the difference is that the building block 2 sequences , derived from one protein ( abcdefghijkc etc .) are replaced by a complete protein , which contains an added thiol group for coupling ( see fig9 a ). to illustrate that native proteins can be used to be coupled in this way the protein glucose oxidase was used as an example ( fig9 b ). this example is similar to that of examples 1 to 5 with the difference that one or more other non - peptide building blocks are used ( dna , rna or a peptide nucleic acid ( pna ) instead of a peptide building block ). the nucleotide building blocks or pna &# 39 ; s are modified with groups that enable linking of the building blocks as in examples 1 to 5 . screening is performed with labeled dna strands , peptides or proteins ( see fig1 ). as alternative labeled dna or pna strands can also be tested against peptide construct described in examples 1 to 5 . the binding binding between peptide and pna is illustrated in fig1 b and 10c , 10 d . in addition to scanning interaction regions of proteins and non - proteins ( dna / rna ) in elisa , chip or or blot format it is also possible to use to - c $- coupling in in vitro bio - assays . firstly , it is possible to use soluble constructs as explained in example - 3 as potential ( ant ) agonists for membrane bound receptors . secondly , it is possible to use membrane - transporting proteins such as transportan or penetratin to get any of the above mentioned combinations of peptides or peptides with pna or peptides with ( small ) proteins into the cell . in fig1 it is illustrated that it is possible to couple two peptides in solution . in this example peptides similar to these shown in for example fig1 a , 1b , 2 b and 3 b . an intracellular protein , like a kinase , can be scanned using overlapping peptides on a solid support , containing a c - terminal cleavable linker . the peptides were synthesised with a n - terminal bromoacetamide group . next , a membrane penetrating transportan peptide , containing a label and a thiol group was coupled with the sequences . these constructs were selectively cleaved from the solid support and tested in a bioassay . labels that can be used are , for instance , biotine or fluorescent labels ( fig1 ). 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or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal peptide derived from hfsh with an additional c - or n - terminal glu lys glu glu ala arg phe cys ile ser ile asn thr thr trp ala ile ser glu leu his pro ile cys asn lys ser ile leu arg cys val leu his pro ile cys asn lys ser ile leu arg gln glu val cys met ile cys asn lys ser ile leu arg gln glu val asp tyr met cys thr lys ser ile leu arg gln glu val asp tyr met thr gln thr cys gln leu arg gln glu val asp tyr met thr gln thr arg gly gln cys ser glu val asp tyr met thr gln thr arg gly gln arg ser ser cys glu tyr met thr gln thr arg gly gln arg ser ser leu ala glu cys glu gln thr arg gly gln arg ser ser leu ala glu asp asn glu cys tyr gly gln arg ser ser leu ala glu asp asn glu ser ser tyr cys gly ser ser leu ala glu asp asn glu ser ser tyr ser arg gly cys met ala glu asp asn glu ser ser tyr ser arg gly phe asp met cys thr asn glu ser ser tyr ser arg gly phe asp met thr tyr thr cys asp ser tyr ser arg gly phe asp met thr tyr thr glu phe asp cys leu val thr cys ser pro lys pro asp ala phe asn pro cys glu ser pro lys pro asp ala phe asn pro cys glu asp ile met pro asp ala phe asn pro cys glu asp ile met gly tyr asn asp leu cys asn glu val val asp val thr cys ser pro lys cys ala phe asn pro cys glu asp ile met gly tyr asn ile leu arg cys glu asp ile met gly tyr asn ile leu arg val leu ile thr cys ser pro lys pro asp ala phe asn pro cys glu asp cys gly pro lys pro asp ala phe asn pro cys glu asp ile met gly cys ile asp ala phe asn pro cys glu asp ile met gly tyr asn ile cys val asn pro cys glu asp ile met gly tyr asn ile leu arg val cys trp glu asp ile met gly tyr asn ile leu arg val leu ile trp cys ser met gly tyr asn ile leu arg val leu ile trp phe ile ser cys ala asn ile leu arg val leu ile trp phe ile ser ile leu ala cys gly arg val leu ile trp phe ile ser ile leu ala ile thr gly cys ile ile trp phe ile ser ile leu ala ile thr gly asn ile ile cys val ile leu thr thr ser gln tyr lys leu thr val pro arg phe tyr ala asx pro val glu ser asx asp arg arg phe xaa xaa ile arg ile his thr gly gln lys pro phe gln asx arg ile his thr gly gln lys pro phe gln asx arg xaa xaa ile his thr gly gln lys pro phe gln asx arg ile his thr gly gln lys pro phe gln asx arg ile asx xaa xaa thr gly gln lys pro phe gln asx arg ile asx met ile asx met arg asn phe ser arg ser asp his leu xaa xaa asx met arg asn phe ser arg ser asp his leu thr his ile arg thr his thr gly glu lys pro phe ala xaa xaa ile arg thr his thr gly glu lys pro phe ala asx arg thr his thr gly glu lys pro phe ala asx asp xaa xaa thr his thr gly glu lys pro phe ala asx asp ile his thr gly glu lys pro phe ala asx asp ile asx xaa xaa thr gly glu lys pro phe ala asx asp ile asx gly gly glu lys pro phe ala asx asp ile asx gly arg xaa xaa glu lys pro phe ala asx asp ile asx gly arg lys xaa asp ile asx gly arg lys phe ala arg ser asp glu ile asx gly arg lys phe ala arg ser asp glu arg xaa cys cys pro val glu ser asx asp arg arg phe ser arg ser his ile