Source: https://patents.justia.com/patent/5846841
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US Patent for Motif Libraries Patent (Patent # 5,846,841 issued December 8, 1998) - Justia Patents Search
Justia Patents Involving An Insoluble Carrier For Immobilizing ImmunochemicalsUS Patent for Motif Libraries Patent (Patent # 5,846,841)
Nov 22, 1996 - Selectide Corporation
Latest Selectide Corporation Patents:
Topologically segregated, encoded solid phase libraries comprising linkers having an enzymatically susceptible bond
Topologically segregated, encoded solid phase libraries
Those skilled in the art have long recognized the immense effort required to synthesize individually thousands of chemical compounds to be tested for their pharmacologic activity. A potential alternative is to synthesize simultaneously a mixture of millions of compounds in solution which could then be screened in mass to detect the rare compound of potential utility. Methods to generate such mixtures have been described (Rutter, et al. U.S. Pat. No. 5,010,175; Huebner, et al., U.S. Pat. No. 5,182,366), however, the utility of such approaches is greatly limited by the unavoidable fact that as the complexity of a library increases the concentration of any particular species within it decreases. Thus, the task of identifying a compound of interest, present at less than 1 part in 10.sup.5, and of separating that compound from the other species of library has limited the utility of soluble libraries.
To overcome this impediment, previous workers have attempted to employ libraries containing larger ligands, but ones which are not inconveniently large because they do not contain all possible species or sequences. Such "incomplete" libraries are useful when employed in an iterative procedure: a first generation incomplete library is screened and several "low affinity" ligands are isolated and analyzed. Inspection of the sequences of these several ligands reveals some consensus sequence which is termed a primary motif. A second library is constructed in which the positions defined by the consensus sequence (i.e., the primary motif) and the remaining positions are randomized. Successive iterations can produce ligands having affinities more than 100 fold higher than the primary motif. For example, Lebl et al., 1994, in "INNOVATION AND PERSPECTIVES IN SOLID PHASE SYNTHESIS AND RELATED TECHNOLOGIES," Vol. 3, ed. by Epton, R. isolated a trisodecameric ligand of an anti-insulin monoclonal antibody having an affinity within a factor of 2 of that of the proband protein antigen. The isolation required the successive analysis of the products of four incomplete peptide libraries. By comparison, screening of a complete 13-mer random library, which would contain 20.sup.13 (8.times.10.sup.16) species is economically impractical.
The utility of such libraries requires that the binding of acceptor molecules be primarily due to the structure of the motif. Consider, for example, the case of a library of peptides having three nonmotif positions occupied by one of 20 amino acids. For a solid phase particle .about.80 .mu.m in diameter, or a fixed array library having locations of the same surface area, there will be .about.10.sup.10 molecules on the surface of each set, each molecule having the same motif. In addition there will be 10.sup.10 /20.sup.3 (8,000) (or .about.1.3.times.10.sup.5) copies of each of the individual species, each having some particular arrangement of residues at is the nonmotif position. Since most assays can detect as few as 1000 molecules on the surface, it was uncertain, prior to the present teaching whether the binding of an acceptor molecule to such a set would be due primarily to binding to the highly represented motif sequence or whether it would be due to binding to the less frequently represented, but potentially higher affinity ligands, formed by the different combinations of residues at the nonmotif positions. The operability of the present invention depends, in part, on the unpredictable observation that the binding of ligands due to the structure of the motif is not dominated by high affinity binding to other sequences in the set.
The invention further provides two particular embodiments of motif libraries. The first embodiment provides a library consisting, in approximately equal amounts, of all permutations of a predetermined number of motif and nonmotif positions. There are, for example, 20 different sequences of motif and nonmotif positions in a triplet, hexamer library. Thus a triplet, hexamer library composed of all the natural amino acids would contain 160,000 different motifs. By contrast a complete, natural amino acid, hexamer library contains 6.4.times.10.sup.7 peptide sequences. It would require several times that number of individual supports to ensure that all sequences were represented.
The invention encompasses polymers wherein the monomers are covalently connected by every sort of chemical bond which those skilled in the art will recognize can be synthesized on a solid phase support. The term polymer as used herein includes those compounds conventionally called heteropolymers, i.e., arbitrarily large molecules composed of varying monomers, wherein the monomers are linked by means of a repeating chemical bond or structure. The polymers of the invention of this type are composed of subunits or monomers that can include any bi-functional organic or herteronuclear molecule including, but not limited to amino acids, amino hydroxyls, amino isocyanates, diamines, hydroxycarboxylic acids, oxycarbonylcarboxylic acids, aminoaldehydyes, nitroamines and thioalkyls and haloalkyls having one of the previously noted functionalities. The monomers of the test ligand are linked by chemical bonds selected from the group consisting of amide, ester, urea, urethane, carbonate, amine, alkane, alkene, sulfide, and disulfide. A monomeric subunit can also be comprised of additional functionalities which are not used in the synthesis of the polymer, as for example the .epsilon.-amino group of lysine. The various chemistries which are within the scope of the present invention are described in co-pending, co-assigned U.S. patent applications Ser. No. 068,327, filed May 27, 1993, which is hereby incorporated by reference in its entirety.
A second type of screening assay is useful when the acceptor is present in a living cell and the practitioner desires to measure the effects of binding on the cell. The test ligands are connected to freely mixable solid phase supports by a selectively and controllably cleavable linker. The test ligands are released from the supports under conditions wherein the effects of ligands released by a particular individual support or a small group of supports can be identified. To this end the supports can be placed in a plaque or replica-plating assay or they can be divided into a multitude of portions, for example, in 8.times.12 microculture arrays. By observing the effects of the soluble ligand, the support that released the ligand of interest can be identified and isolated. The chemistries suitable to construct cleavable linkers are explained in detail in co-pending, co-assigned U.S. patent application Ser. No. 5 081,997, filed Jun. 23, 1993, which is hereby incorporated by reference in its entirety.
The support can be isolated by any conventional means known to those of ordinary skill in the art and the invention is not limited by the method of isolation. For example and not by way of limitation, it is possible to physically isolate a solid phase support/ligand combination that exhibits the strongest physico-chemical interaction with the specific acceptor molecule. In one embodiment based on physico-chemical interaction, a solution of a specific acceptor molecule added to a random peptide library which is equivalent to approximately 10.sup.5 to 10.sup.7 solid phase supports. The acceptor molecule is incubated with the resin for a time sufficient to allow coupling between the peptide and antibody, for example, one hour at 22.degree. C. Thereafter, the acceptor molecule coated bio-oligomer/solid phase support is isolated. More specific embodiments are set forth below. Although the following refers to libraries of peptides, it will be understood that motif libraries of having other chemistries may also be assayed.
(i) The acceptor is first labeled with a fluorescent moiety or "fluoresceinated" by techniques that are within the routine skill of those in this art. The antibody at a concentration of 1 ug/ml is then introduced to the library of peptides and, after gentle mixing at 22.degree. C. for one hour, the solid phase supports are washed, and the fluorescent antibody solid phase support/peptide combinations are identified and recovered with a fluorescence activated cell sorter. Alternatively, the fluorescent antibody solid phase support/peptide combinations are identified and physically picked up under a dissecting microscope with fluorescent attachment using a micromanipulator. The relative intensity of fluorescence is generally proportional to the affinity of the peptide-ligand to the monoclonal antibody in question.
(ii) The acceptor is first conjugated onto ferro-magnetic beads by techniques that are routine in the art. The conjugated antibody at a concentration of 1 ug/ml is then incubated with the library for one hour at 22.degree. C. The magnetic beads will form a rosette around the solid phase support/peptide of interest which can then be physically isolated with a strong magnet.
(iii) The acceptor is first conjugated is to an enzyme such as alkaline phosphatase by techniques that are routine in the art. This antibody-enzyme conjugate is then incubated with the random peptide library for 30 minutes to one hour at 22.degree. C. After washing, the whole library is poured into a petri dish which contains a substrate for alkaline phosphatase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitro-blue tetrazoleum (NBT). After incubating for several minutes, the antibody-solid phase support/peptide combination changes color (becomes blue) due to precipitation of the converted substrate on the solid phase support, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. The relative intensity of the color reaction is generally proportional to the affinity of the peptide for the monoclonal antibody in question.
(iv) The acceptor is first conjugated to an enzyme such as horseradish peroxidase by techniques that are routine in the art. This antibody-enzyme conjugate is then incubated with the random peptide library for 30 minutes to one hour at 22.degree. C. After washing, the whole library is poured into a petri dish which contains a substrate for peroxidase, for example, 3,3',4,4'-diaminobenzidine (DAB); 3,3',5,5'-tetramethylbenzidine (TMB); or 4-chloro-1-napthol (4CN). After incubating for several minutes, the antibody-solid phase support/peptide combination changes color, and can be identified and isolated physically under a dissecting microscope with a micromanipulator. The relative intensity of the color reaction is generally proportional to the affinity of the peptide for the monoclonal antibody in question.
(v) The acceptor is first labeled with biotin or "biotinylated" by techniques that are routine in the art and is thereafter incubated with the random peptide library for 30 minutes to one hour at 22.degree. C. After washing, a streptavidin-alkaline phosphatase or streptavidin-horseradish peroxidase complex is added and incubated for 30 minutes. The support is then washed, and the color is developed as described above in (iii) with the enzyme method. The peptide/solid phase support of interest is physically isolated as above.
Alternatively, in another embodiment within the scope of the invention, it is possible to isolate a single solid phase support particle, such as a bead, with its coding peptide sequence attached and introduce the bead to a sequencer for peptide sequencing without previously cleaving the coding peptide from the bead. It is estimated that a single 100 .mu.m diameter resin bead with 0.5 mEq/gram of functionalizable sites contains approximately 100 pmole of peptide if one half of the sites are used to link coding peptides. For a similar degree of substitution with coding peptides, a single 250 .mu.m diameter PAM resin bead with 0.5 mEq/gram resin of functionalizable sites contains approximately 1500 pmole of coding peptide. With state of the art peptide sequencer, only 5-10 pmole is required for adequate sequencing. Therefore, for a standard PAM resin a single bead of 100 .mu.m in diameter can be loaded to contain more than an adequate amount of coding peptide for sequencing.
Reading a non-sequential code requires only determining whether a given signal is present or not. The baseline resolution of two peaks which differ by about 0.3 minutes in retention time can be achieved using the standard reversed phase HPLC analysis with gradient elution. Therefore, a 45 minute gradient can discrimate among 150 compounds. A coding molecule consisting of subunits selected from a group of 150 different coding moieties is equivalent to a 150 digit binary number. Hence, 2.sup.150 or about 10.sup.45 different species of test compound could be so encoded. Thus, non-sequential codes are easily adequate to encode both the sequence and the identity of the monomers of the test compounds of even the largest practical libraries.
Side Chain Derivatives of Diamino Acids
Suitable for a Non-Sequential Code
.alpha.,.beta.-
.alpha., .gamma.-
Diamino  Diamino
bulyric
No.   Acid     Acid     ornithine
1    *        *        *      *    acetyl
2    *        *        *      *    propionyl
3    *        *        *      *    butyryl
4    *        *        *      *    valeryl
5    *        *        *      *    caproyl
6    *        *        *      *    pivaloyl
7             *        *      *    c-hexyl
8             *        *      *    trichloroacetyl
10             *        *      *    phenylacetyl
11             *        *      *    2,2-diphenylacetyl
12             *        *      *    phenylbutyryl
13             *        *      *    1-naphtylacetyl
14             *        *      *    2-naphtylacetyl
15    *        *        *      *    1-adamantyl-
16             *        *      *    1-adamantylacetyl
17             *        *      *    tosylglycyl
18             *        *      *    dansylglycyl
19    *        *        *      *    beiizoyi
20    *        *        *      *    succinamyl
21             *        *      *    succinyl
22             *        *      *    glutaryl
23    *        *        *      *    isobutyryl
24             *        *      *    4-chlorobenzoyl
25             *        *      *    2,2-
diphenylpropionyl
26             *        *      *    N,N-dimethylglycyl
27    *        *        *      **   heptanoyl
28    *        *        *      *    octanoyl
29    *        *        *      *    3,3-di-ph-propionyl
30             *        *      *    N,N-dimethylamino
butytyl
31    *        *        *      *    3-ph-propionyl
32    *        *        *      *    4-bi-ph-carbonyl
33    *        *        *      *    4-bi-ph-acetyl
34    *        *        *      *    crotonyl
One embodiment to achieve the simultaneous cleavage of coding moieties provides that every coding moiety is an .alpha.-amino acid, attached as an N-terminal amino acid with its amino group free. The backbone of the coding structure is constructed from diamino carboxylic acids (Daa). The amino groups of these amino acids are acylated by the N-protected amino acids used for the coding. Acylation is performed using a mixture of the moieties defined as a code for the given monomer and its position in the test compound.
Step    Action          Reagent and Solvent
1       1 g Peptide Resin
10 mL DMF
2       3 fold-excess amino
3       3 equivalent    DIC
4       3 equivalent    HOBt
5       Couple for 120 min
6       Wash (3 .times. 8 mL)
7       Ninhydrin test
8       Deprotection (10 min)
8 mL 50% Piperidine/DMF
9       Wash (6 .times. 8 mL)
10      Repeat from step 2
Sequencing by Edman degradation was performed on an ABI 4778 protein sequencer (Applied Biosystems, Foster City, Calif.) and Porton PI 3010 instrument (Porton Instruments, Tarzana, Calif.). Both analytical and preparative HPLC were carried out on a Waters 625 LC system with a Waters 490E Programmable Multiwavelength Detector using Vydac Peptide and Protein C18 analytical (0.46.times.250 mm, 5=E6 m, 1 mL/min) and preparative (10.times.250 mm, 10=E6 m, 3 mL/min) columns, respectively. UV/VIS absorption spectra were recorded on a Hewlett Packard HP 8452A Diode-Array spectrophotometer using a 1-cm quartz cuvette. Amino acid analyses were carried out on a D-500 system (Durrum Corp., Palo Alto, Calif.) system.
Depending on the application, additional non-cleavable linkers such as Fmoc-aminocaproic acid, Fmoc-aminobutyric acid, and/or Fmoc-.beta.-alanine, can first be added onto the resin prior to the amino acid randomization steps.
In the split synthesis randomization steps of the library synthesis, the resins were first divided into 19 aliquots contained in 19 polypropylene vials. Nineteen Fmoc protected natural amino acids (all but cysteine) were then added separately into each of the resin aliquots. Minimal amount of dimethylformamide (DMF) was used. The amino acids were added in 3 fold excess and coupling was initiated by adding 3 fold excess of BOP and HOBt. In some experiments DIC and HOBt were used instead. A trace amount of bromophenol blue was added into the reaction mixture. The vials were capped tightly and rocked gently for approximately 120 minutes at room temperature or until all beads turned from blue to colorless 14. Completion of the coupling was then confirmed by a ninhydrin test. For the aliquots in which coupling reactions were incomplete, the beads were allowed to settle and the supernatant gently removed. Fresh Fmoc-amino acid was then added to that vial followed by BOP and HOBt and the reaction allowed to proceed for another hour. In general, most randomization cycles required only one coupling and only on rare occasions was double coupling needed. The resins were then mixed in a siliconized cylindrical glass vessel fitted with a frit at the bottom. Dried N2 was bubbled through for mixing of the resin. After washing (8.times.) with DMF, 50% piperidine (in DMF) was added. After 10 minutes of bubbling with N2, the piperidine was removed and the resins washed 10 times with DMF. The amount of released fulvene-piperidine adduct was quantitated by UV spectrometry (302 nm). Stable level of substitution determined in this way throughout the library synthesis served as one of the quality control measures. The resins were then divided into aliquots again for another cycle of randomization or amino acid mixture coupling. After all steps were completed, the Fmoc group was removed with 20% piperidine (v/v) in DMF and the side chain protecting groups were removed with a mixture of trifluoroacetic acid-phenol-anisole-ethanedithiole (94:2:2:2; v/w/v/v) or reagent K (TFA-phenol-water-thiophenol-ethanedithiole, 82.5:5:5:5:2.5; v/w/v/w/v) (31). The resin was then washed thoroughly in DMF, neutralized with 10% DIEA (in DMF), thoroughly washed again, and stored in DMF at 4.degree. C.
Ratios of Fmoc amino acids affording
their equimolar incorporation onto
TentaGel by DIC/HOBt procedure
Molar Amino Acid Ratio
A              Ala    0.37
D              Asp    0.32
E              Glu    0.45
F              Phe    0.49
G              Gly    0.65
H              His    1.30
I              Ile    3.10
K              Lys    1.05
L              Leu    0.52
M              Met    0.42
N              Asn    0.69
P              Pro    0.70
Q              Gln    0.91
R              Arg    1.36
S              Ser    1.84
T              Thr    3.63
v              Val    1.50
W              Trp    0.95
Y              Tyr    0.41
To verify the quality of the library, several randomly chosen beads were submitted for sequencing and the average amount of the peptide per bead was determined. This value was confirmed by quantitative amino acid analysis of a random sample from the library (1 mg). Amino acid analysis, as well as sequence analysis of pooled sample of beads (.about.50 beads) must confirm at the random distribution of all amino acids.
Polyoxyethylene modified polystyrene (TentaGel S-NH2, Rapp Polymere Tubingen, Germany, 90 mm particle size, 0.29 mmol/g, 10 g) was swollen in DMF and Fmoc-Gly, Fmoc-.beta.-Ala, Fmoc-Gly and Fmoc-.beta.-Ala were coupled to it by diisopropylcarbodiimide/N-hydroxybenzotriazole (DIC/HOBt) procedure. Fmoc group was removed and resin was separated into two parts. One part ("1R1") was divided into 19 vessels and 19 Fmoc protected amino acids were coupled by DIC/HOBt procedure with bromophenol blue (BB) monitoring. The second part ("1M1") of the resin was acylated by the mixture defined in Table II using the same procedure. Part 1R1 was divided into two parts--"2R2" (1/3) and "2M2" (2/3), rather than 2/5 and 3/5 as specified above. Part 2R2 was divided into 19 aliquots and the coupling of Fmoc amino acids was performed. Part 2M2 was acylated by the mixture of amino acids. Part 1M1 was similarly divided into two parts--2M1 (1/3) and 2R1 (2/3), instead of the preferred mode of 2/5 and 3/5, and randomization was performed on part 2R1 and mixture of amino acids was coupled to part 2M1. No pooling was done until after the third coupling. The procedure of dividing into M and R parts continued. After the third step of the synthesis parts of the mixtures to follow identical paths were recombined. After all library aliquots were recombined, the library was deprotected and analyzed.
Set of building blocks (and their coding)
used in the diastereoisomeric library of libraries
Coded   added
Amino acid    by      when   M.w. Abre.  R.t.c
Fmoc-L-Alanine               311.3
A      14.07
G       mix    311.3
Fmoc-Aminoisobutyric acid
A       pre    325.3
Aib    18.6
Fmoc-3-(3-pyridyl)-L-
A       mix    389.6
3-Pal  14.53
Fmoc-3-(3-pyridyl)-D-
f       mix    389.6
Fmoc-beta-thienyl-L-         393.4
Tha    25.9
Fmoc-beta-thienyl-D-
G       mix    393.4
Fmoc-cyclohexyl-L-           393.5
Cha    38.88
Fmoc-cyclohexyl-D-
G       mix    393.5
Fmoc-aminobutyric acid       325.3
Abu    18.77
Fmoc-N-b-Boc-L-
Dab     mix    426.5
Dap    26.28
Diaminopropionic
Fmoc-N-g-Boc-L-
Dap     mix    441.5
Dab    26.9
diaminobutyric
Fmoc-L-Arginine(Pmc)         662.8
R      20.62
Fmoc-D-Arginine(Pmc)
G       pre    662.8
Fmoc-L-Asparagine(Trt)       596.7
N      6.77
G       pre    596.7
Fmoc-L-Aspartate(OBut)       411.5
D      6.33
Fmoc-D-Aspartate(OBut)
G       mix    411.5
Fmoc-L-Aspartate(OAllyl)     395.4       24.02
Fmoc-N-methyl-L-             459.5       36.03
Aspartate(OBzl)
Fmoc-L-Cysteine(Acm)         414.5
C(Acm) 12.75
Fmoc-L-Glutamate(OBut)       425.5
E      10.87
Fmoc-D-Glutamate(OBut)
G       mix    425.5
Fmoc-L-Glutamate(OAllyl)     409.4       26.85
Fmoc-L-Glutamine(Trt)        611.8
N      8.42
Fmoc-D-Glutamine
G       pre    368.4
Fmoc-Glycine                 297.3
G      9.62
Fmoc-MeGly(Sarcosine)        311.3
Sar    13.82
Fmoc-L-Histidine(Trt)        619.7
H      14.75
G       pre    619.7
Nle     pre    353.4
I      28.68
G,Nle   pre    353.4
Fmoc-allo-Isoleucine
V       mix    354.3
aIle   28.6
Fmoc-N-methyl-L-             367.4
MeIle  35.3
Fmoc-L-Leucine               353.4
L      29.55
G       mix    353.4
Fmoc-N-methyl-L-
A       pre    367.4
MeLeu  35.15
Fmoc-L-Lysine(Boc)           468.6
K      29.12
G       pre    468.6
Fmoc-L-Lysine(Alloc)         452.5       26.45
Fmoc-L-Methionine            371.5
M      23.23
G       mix    371.5
Fmoc-L-Norleucine
A       mix    353.4
Nle    30.11
Fmoc-L-Ornithine(Boc)        454.5
Orn    27.53
Fmoc-D-Ornithine(Boc)
G       pre    454.5
Fmoc-L-Phenylalanine         387.4
F      28
G       mix    387.4
Fmoc-p-amino(Boc)-L-         502.6
Aph    30.26
Fmoc-p-nitro-L-
A       mix    432.4
Nph    28.19
Fmoc-p-fluoro-L-
F       mix    405.4
Phe(F) 29.26
Fmoc-N-methyl-L-             401.4
MePhe  32.77
Fmoc-L-Proline               337.4
P      21.93
G       mix    337.4
Fmoc-L-Serine(But)           383.4
S      8.03
Fmoc-D-Serine(But)
G       pre    383.4
Fmoc-L-Threonine(But)        397.5
T      9.18
Fmoc-D-Threonine(But)
G       pre    397.5
Fmoc-L-Tryptophan            426.5
W      27.13
G       pre    426.5
Fmoc-L-Tyrosine(But)         459.5
Y      18.5
Fmoc-D-Tyrosine(But)
G       mix    459.5
Fmoc-3-nitro-L-Tyrosine      448.4
Ty(NO2)
Fmoc-L-Valine                339.4
V      23.62
Fmoc-D-Valine G       pre    339.4
Fmoc-L-Norvaline             339.4
Nva    24.76
Fmoc-L-tert-leucine          353.5
Tle    28.56
Fmoc-D-tert-leucine
G       pre    353.5
Fmoc-cyclohexyl-Glycine      379.5
Chg    34.1
Fmoc-p-hydroxy-L-
A       pre    390.4
Hgl    20.85
Fmoc-(1-napthyl)-L-          437.5
1-Nal  35.75
Fmoc-(2-napthyl)-L-          437.5
2-Nal  35.05
Fmoc-L-Tic                   399.4
Tic    36.4
Fmoc-D-Tic    G       pre    399.4
Fmoc-L-7-OH-Tic
Tic     mix    415.4
Tic(OH)
Fmoc-S-benzyl-D-             461.6
pen    40.2
Fmoc-L-Citrulline
A       pre    397.6
Cit    9.06
Fmoc-D-Citrulline
G, A    pre    397.6
Fmoc-L-3-(4-thiazolyl)       395.5
Ala(Th)
R.t. = retention time
We have prepared both a hexamer, triplet defined parameter library and a triplet, random trimer interval library by randomized synthesis with 19 natural amino acids (Cys omitted). The library was screened in three model systems -- binding to anti-.beta.-endorphin monoclonal antibody, to streptavidin, and to thrombin. Colorized beads were selected, destained and incubated with the same concentration of the acceptor in the presence of the known specific competitor (YGGFL, LHPQF, fPRPG) to prove the specificity of the binding. Beads which did not stain in this second experiment were regarded as specific binders and were washed and stained again in the absence of the competitor. Numbers of stained, is competed and restained beads are given in Table IV is a comparison of the numbers obtained in screening conventional one bead one structure library.
Comparison of the screening of model
targets with classical pentapeptide
library and with library of libraries
Sample                 Restaine
Target Library  Size     Stained
Anti-.beta.-
5 (19)*  10.sup.6 >1000  160/-  35/35
endo                            300
6 (3F(19))
16     16/16  16/16
endo   **
Avidin 5 (19)   10.sup.6 >1000         43/43
Avidin 6 (3F(19))
10.sup.6 65     62/65  62/62
Thromb 5 (19D)  10.sup.6 380    126/380
Thromb 6 (3F(19)
30     28/30  13/28
*A complete pantamer library having one species per support, 19 amino
acids used in each split synthesis.
**A defined parameter triplet, hexamer library, 19 amino acids used in
each coupling.
The identified beads are sequenced by Edman degradation. Positions containing defined amino acids are clearly marked by a single amino acid signal equal to the amount of total content of peptide on the bead (.about.50-150 pmoles). Positions containing a mixture of amino acids yield all amino acids used for the randomization in 20 times lower amount (2-7 pmoles).
Results of the screening of variable length library with anti-.beta.-endorphin and streptavidin are given in Table VA.
Results of the screening of variable
interval motif library (tri to
pentadecapeptides)
A. Anti-.beta.-endorphin
X G A F X X X
X G G F
B. Streptavidin
X X W X X X H P X X X
X X N X P X F X X X
X W X X X P Q X X X
Anti-.beta.-endorphin binding was observed on beads containing XGAF or XGGF sequence. Surprisingly, an N-terminal tyrosine was not observed. The reason for this finding might be statistical -- the tested sample of the library was not large enough to completely represent all motifs.
Results obtained with the defined prameter motif libraries are given in the Tables VI-VIII. It is obvious that antibody screening (Table IV) confirmed earlier findings Lam et al., 1991, NATURE 354:82; Lam et al., 1993 BIOORG MED. CHEM. LETL. 31:419 of YG(G/A)(F/W) as the very strong binding motif for the anti-.beta.-endorphin antibody.
Sequences binding to anti-.beta.-endorphin
antibodies (sequences are listed in the
order of staining intensity)
1st experiment      2nd experiment
Y G x F x x highest Y G A x x x highest
Y ? x F x x         Y x G F x x
Y x G F x x         Y G x W x x
x G A F x x         Y G x F x x
Y G x F x x         Y G x F x x
Y G A F x x         x G G F x x
Y G x F x x         Y G G x x x
Y x A F x x         x G G F x x
Y ? A F x x         Y x G F x x lowest
x ? G F x x
x G A F x x
x G A F x ? lowest
Sequences found positive and specific for
binding to streptavidin (sequences are
listed in the order of staining intensity)
? = position undeterminable.
x x x H P Q highest x x x H P M highest
x x x H P Q         x x x H P Q
x x x H P Q         x ? x ? H P
x x x H P Q         x x x H P -
x x x H P Q         x x x ? P Q
x x H P Q x         H P Q x x x
W x x x P ?         x x H P M x
x x x H P M         x R x H P x
x W x H P x         w x x x P Q
W ? x H P x         x x W H P x
W x x H P x         x H P Q x x
H P Q x x x         x x P Q F x
H P x F x x lowest  x H x x F ?
F x x ? P Q
w x x x P M
However, alternative motifs (W/F).sub.-- -- -- P(Q/M) and HP.sub.-- F were identified, as well as a motif in which tryptophan is either directly attached or separated by up to three amino acid residues from histidyl-proline sequence. The longer motifs identified in this library could not be observed in pentapeptide libraries and they were apparently overlooked in the longer libraries.
Sequences identified as positive and
specific in the thrombin screening
(sequences are listed in the order of
staining intensity)
I R x W x x highest
I x F x Y x
I F x W x x
I x F W x x
I R W x x x
L R x W x x
L R Y x x x
I x F R x x
I R Y x x x lowest
Binding to these analogs was tested both on the solid surface and in solution. Alanine substitution in position 1 and 4 of anti-.beta.-endorphin ligand and in all positions of thrombin ligand dramatically decreased binding. We can conclude that in these cases that more than three residues are necessary for the binding. Thus, only fraction of the peptide mixture containing the proper motif accounts for all the observable binding. These results will illustrate the superiority of the use of mixtures of large numbers of species of amino acids rather than one or two supposed indifferent amino acids at the nonmotif positions.
Due to the number of building blocks and stereochemical diversity this library could not be screened completely. A complete library would contain 7.59.times.10.sup.7 motifs. However, screening of a sample provided important information about the motif required for the binding. An example of the results from the screening of this library with streptavidin is given in the Table IX. The known ligand HPQ was not found, but clearly its analogs were identified. The probability of finding HPQ sequence was 1:5,000,000 but only 1 million of beads were screened.
binding to streptavidin in the Library of
Libraries constructed from 78 building
blocks and mixtures of D and L amino acids
staining intensity). Lower case x denotes
mixture of D amino acids, upper case X
denotes mixture of L amino acids.
His - Tic -x-Lys(Alloc)-X - X
X - His-(Me)Phe -X-Lys-X
X - X - His-(Me)Phe - Lys(Alloc) - X
X - X - x - Tic - His-(Me)Phe
x - x - X - Tyr(3-NO2)-Pro - Tha
1. A library consisting of a multiplicity of sets, each said set comprising a multiplicity of species of test ligands, in which:
a) each species of test ligand of the library comprises a linker and a multiplicity of monomers, wherein:
i) said monomers are selected from a multiplicity of species of monomers; and
ii) said linker connects the monomers of a species of test ligand to a solid phase support particle or to an identifiable location of a solid support;
b) each monomer is disposed in one of a predetermined number of motif positions or in one of a predetermined or variable number of nonmotif positions, wherein:
i) the motif and non-motif positions have an ordered sequence; and
ii) there are a plurality of motif positions in each set of the library;
c) all species of test ligands of a particular set have:
i) a constant number of nonmotif positions and an identical, single ordered sequence of motif and nonmotif positions;
ii) an identical, single species of monomer, selected from a plurality of species of monomer, at each of the predetermined number of motif positions; and
iii) one of a plurality of species of monomers at each of the constant number of nonmotif positions;
d) all species attached to a particular solid phase particle or to a particular identifiable location of a solid support are encompassed by a single set; and
e) the library is a complete collection of sets in which all possible particular ordered
(i) sequences of the motif positions and nonmotif positions or
(ii) sequences of motif positions and intervals of nonmotif positions are represented.
2. The library of claim 1 in which the number of nonmotif positions is predetermined.
5182366 January 26, 1993 Huebner et al.
5194392 March 16, 1993 Geysen
5565325 October 15, 1996 Blake
86/00991 February 1986 WOX
Hirschmann et al., J. Am. Chem. Soc., vol. 114, pp 9699-9701 (1992) "The First Design and Synthesis of a Steroidal Peptidomimetic. The Potential Value of Peptidomimetics in Elucidating the Bioactive Conformation of Peptide Ligands". Brenner at al., Proc. Natl. Acad. Sci., vol. 89 (1992), pp5381-5383 "Encoded Combinatorial Chemistry". Furka, et al. 1991, Int. J. Protein Res. 37:487-493. Houghten et al., 1991, Nature, 354:82-86. Lam et al., 1991, Nature, 354:82-84. Lam et al., 1993, Bioorg Med. Chem. Lett. 31:419. Vagner et al., 1993, "Novel Methodology for Differentiation of " Surface and Interior Areas of Polyoxyethylene-Polystyrene (POE-PS) Supports: Application to Library Screening Procedures in Innovation and Perspectives in Solid Phase Synthesis and Related Technologies ed. by Epton, R. Chen et al., "Biased Combinatorial Libraries: Novel Ligands for the SH3 Domain of Phosphatidylinositol 3-Kinase", J. Am. Chem. Soc., vol. 115, pp 12591-12592 (1993).
Patent number: 5846841
Assignee: Selectide Corporation (Tucson, AZ)
Inventors: Nikolai Sepetov (Oro Valley, AZ), Victor Krchnak (Oro Valley, AZ), Michal Lebl (Oro Valley, AZ)
Application Number: 8/754,878
Current U.S. Class: Involving An Insoluble Carrier For Immobilizing Immunochemicals (436/518); 435/71; Biospecific Ligand Binding Assay (436/501); Synthesis Of Peptides (530/333); Polymer Supported Synthesis, E.g., Solid Phase Synthesis, Merrifield Synthesis, Etc. (530/334)