WO2004008151A2 - New combinatorial peptide libraries containing markers and methods for their preparation and utilization - Google Patents

New combinatorial peptide libraries containing markers and methods for their preparation and utilization Download PDF

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Publication number
WO2004008151A2
WO2004008151A2 PCT/HU2003/000056 HU0300056W WO2004008151A2 WO 2004008151 A2 WO2004008151 A2 WO 2004008151A2 HU 0300056 W HU0300056 W HU 0300056W WO 2004008151 A2 WO2004008151 A2 WO 2004008151A2
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WIPO (PCT)
Prior art keywords
groups
marker
protein
combinatorial
proteins
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PCT/HU2003/000056
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English (en)
French (fr)
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WO2004008151A3 (en
Inventor
Ferenc Darvas
László ÜRGE
György Dormán
Péter KRAJCSI
Dániel SZALAY
Mariann BÉRES
Ágota BUCSAI
Tamás Nagy
Ferenc KÁLMÁN
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Comgenex Rt.
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Priority to AU2003251072A priority Critical patent/AU2003251072A1/en
Publication of WO2004008151A2 publication Critical patent/WO2004008151A2/en
Publication of WO2004008151A3 publication Critical patent/WO2004008151A3/en

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention pertains to substantially new combinatorial protein marker compound libraries including methods for their preparation and utilization.
  • affinity chromatography small molecules are bound to cellulose or other polymers, and a protein mixture is streamed through this so-called affinity column.
  • the proteins leave the column separately and at different speeds depending on the binding strength to the small molecules.
  • affinity labeling involves a specific chemical or photochemical reaction occurring by means of a reactive marker group, resulting in the formation of a stable covalent bond between the protein and ligand.
  • the main advantage afforded by this method is that the adduct remains stable even after denafuration of the protein, so that the ligand and the reporter groups linked to it remain in a state of attachment to the protein (at the binding site).
  • reporter structural unit or group may be a fluorescent, radioactive, biotin, spin, or other unit used for the purposes of marking the target. (The name “reporter” refers to the fact that the group provides information regarding conformation, binding, etc.)
  • the ligand or substrate is often used up in non-specific reactions with the nucleophiles before binding.
  • photochemically activated groups photophores such as benzophenone or aromatic azides
  • photophores such as benzophenone or aromatic azides
  • groups are remotely controllable 'clean reagents' and that they have numerous other favorable properties, in contrast to groups that are simply chemically reactive. Thus, they are stable in the "dark" and in a biological non-covalently bound state.
  • a photochemical reaction resulting in a covalent bond in a well-defined time will occur only upon irradiation of light by the researcher.
  • the chemically and photochemically reactive marker groups such as the photophores
  • reporter groups were attached to the library as a marker unit, in a single, parallel, robotized step, through functional groups initially introduced at different diversity points of the structure.
  • the functional group may be positioned directly on the core structure, or preferably, as the terminal group of a side-chain what is called tether.
  • this invention involves combinatorial protein marker molecule libraries containing groups that may be chemically or photochemically activated, with or without reporter groups, attached at different diversity points or spatial directions via side chains around a molecular core.
  • the invention consists furthermore of combinatorial marker unit libraries having chemically or photochemically reactive marker groups, reporter groups, and various types of side-chains attached variably to a common molecular, preferably lysine- based, structural core.
  • the library described as part of this invention contains: benzophenonee and nitro-phenilazide groups as marker groups; biotin and fluorescent groups as reporter groups; saturated carbon chains as side chains; and polyethylene-glycol units.
  • the invention consists furthermore of a method for combinatorial chemical tethering, whereby a side chain is introduced in optimal position, ensuring structural diversity and protein binding effectiveness.
  • a marker unit is attached to a side chain with a terminal functional group, preferably an amino group.
  • the invention consists furthermore of the application of tethered combinatorial libraries toward the study of non-covalent interactions between affinity-based biopolymers, preferably proteins and small molecules, such as ligands, substrates and other compounds, either directly, or immobilized to a solid support, preferably using affinity chromatography, chemical microarrays, or microchips.
  • affinity-based biopolymers preferably proteins and small molecules, such as ligands, substrates and other compounds, either directly, or immobilized to a solid support, preferably using affinity chromatography, chemical microarrays, or microchips.
  • the tethered chemicals immobilized to the solid support described as part of this invention may be applied toward the study of interactions between macromolecules, preferably proteins and small molecules, such that they are used in established protocols or processes for using proteins, DNA, or any other type of reader chip, that have been developed for application to the study of molecules immobilized to microchips or microarrays .
  • the invention also consists of a method for robotized parallel derivatization, in which the side chain or, directly, the marker unit is linked to the intermediate of the molecular library.
  • the invention consists furthermore of the application of a high throughput biological test that enables parallel affinity labeling of a large number of samples obtained from different tissues including the detection and separation of covalently bound proteins. This method is also suitable for identifying protein markers specific to diseases and may also be used as a diagnostic method. Application of the covalent labeling method contributes to simplifying complex proteomics.
  • Our invention consists furthermore of the application of a high-throughput analytical method suitable for sequencing the covalently bound proteins using mass spectrometric methods, and for comparing them to known sequence databases.
  • a combinatorial library of simple marker units may also be formed that are attached en masse to appropriately prepared, small molecule libraries using parallel, robotized methods.
  • the library of the marker unit is actually the set of all possible combinations of various types of attaching side-chains, (photo)reactive groups, and different reporter groups.
  • this produces a lysine-based branched system, where the marker and reporter groups, as well as the qualify of the side-chains, can be varied.
  • the marker libraries of this invention are suitable for designing combinatorial affinity ligands. We have determined that the following points must be biochemically considered when designing the affinity ligands:
  • the binding of the affinity-based probe molecules should expectably occur at the same site as in the case of ligands not containing these modifications, and their biological activation should be in the same order of magnitude.
  • the formation of the covalent bond and its irreversible activation or inactivation should be proportional to the intensity of the signal transferred by the reporter group, in other words, besides the bound proteins, the nonspecific signals should be kept to a minimum.
  • the excitation wavelength of the light employed should not cause any damage to the protein; said wavelength should not be less than 320 nm, while the value of Gmax (the molar extinction coefficient) should be high.
  • the covalent adduct should be stable under in the condition of chemical and enzymatic protein fragmentation. This is inherent in the specific mechanism of the chemically or photochemically reactive groups applied.
  • the covalent modification of the protein should preferably be point or regio- selective to some degree. This property provides a single, modified or labeled protein fragment with the labeling of one or two neighboring amino acids. This makes easier the identification of the protein using S-based sequence identification or via comparison with sequence databases.
  • the tissue sample containing the target protein is incubated with the ligand for a short period of time.
  • the crosslink forms directly.
  • photoreactive groups if the procedure is carried out in the "dark," the photochemical reaction characteristic of the given group takes place when the non-covalent linkage formed is irradiated by a wavelength appropriate for the photosensitive group, and an irreversible bond forms between the receptor and the ligand. This bond can be detected by means of the reporter group in several ways:
  • the ligand binding protein/proteins can be distinguished from the polypeptide that does not show binding activity /affinity.
  • biological reporter groups are also suitable for separation: for example, a protein-ligand adduct containing a biotin unit can be easily separated on an avidin affinity column. The separated proteins may be broken down into smaller fragments using enzymatic or chemical methods and subsequently analyzed using mass spectrometry or classical sequencing analysis. If the protein fragment containing the binding region is known, then without labeling, the exact location of the binding site can be identified directly by means of its MS fingerprint without recourse to special reporter groups.
  • the affinity-based interaction labeling experiment using ligands made chemically or photochemically reactive by means of the present invention, provides information on the following levels:
  • the amino acid sequence within the binding region of the protein may be identified from several perspectives, with the result that the minimum active fragment of the binding region can be determined
  • MDR efflux pumps
  • the reactive marker group/units are attached expectably to biologically active chemicals. It is known that in one group of affinity-labeling analogues (so-called exo-type tethered ligands; for naming, see: Baker: Design of Active Site Directed Irreversible Enzyme inhibitors, 1967, John Wiley & Sons, Inc., New York), the light-sensitive group is linked to the ligand via a side chain. This method is advantageous in isolating the receptor. In such cases, when the pharmacophore is a part of the reactive group and mimics its structural units, the mapping of the binding region of the receptor (endo-type) becomes possible. When exo-type affinity probes are used, the reactive group must be introduced at a sterically flexible site, far away from the pharmacophor, in order not to change the conformation of the compounds.
  • the chemically reactive group, or photophore must not undergo degradation. For this reason, it is preferable that a stable precursor should be formed or built into the molecule to be converted into a photoreactive group during the last step of the reaction (the linear approach).
  • the reactive group or its precursor may be attached most simply directly to an available functional group of the natural ligand (endo-type), or we may form the photoreactive group by directly appending a simple group (semi-synthetic approach, such as when a substituted-benzophenonee is formed by directly benzoylating an aromatic ringbenzophenone).
  • Photoaffinity and other affinity-based bioconjugate techniques require careful planning and modeling if biological activity is to be sustained.
  • placement of the tether can be determined in 3 different ways, when analogue molecules are desired to be tested on a known protein:
  • the excited photophore has a greater chance of reaching a functional group if it attaches itself to a flexible alkyl side chain (also called a tether, see below).
  • the flexibility of the attaching side chain substantially effects the success of affinity labeling. Selection of this side chain depends both on the set goal and on the characteristics of the target protein.
  • the selection of a rigid or flexible linker generally means a compromise between two conflicting factors. More flexible and longer side chains increase the degree of freedom and offer a greater opportunity for covalent bonds to form, though they may result in a signal more distant from the binding region or in multiple points of attachment. If the "tether" is too short, the photoreactive group may embed itself deeply between the functional groups of the ligands, depending on ligand conformation, and may not be capable of effective signaling. Furthermore, intermolecular reactions may also occur. A rigid tether will probably label a single amino acid point-selectively, although its effectiveness is expected to be less, since the probability of forming the reactive protein parts is proportional to tether flexibility.
  • the flexibility and length of the "tether" depends mainly on the set goals. For identifying a new, unknown receptor protein (e.g. in the case of natural materials), a longer, more flexible side chain is more beneficial. In other instances, when the main goal is to spatially map the receptor, a rigid side chain of known length may provide the desired information. In the latter case, it is practical to test the photoreactive group on various positions of the bioactive ligand, so that the regio-specificity and effectiveness of the formation of the covalent bond is studied from various "points of view".
  • the attaching photoreactive group may be guided towards various protein regions.
  • the hydrophobic milieu or hydrophobicity of the flexibly binding side chain the attaching photoreactive group may be guided towards various protein regions.
  • the hydrophobic milieu or hydrophobicity of the flexibly binding side chain it is preferable to choose the hydrophobic milieu or hydrophobicity of the flexibly binding side chain.
  • the main advantage of the solution proposed by this invention is that the tethered combinatorial library can be produced with high yield. Another important factor is that under robotized conditions, the functionalization and "tethering" of known and effective synthesis of the ligand or library can be carried out using minimal modification.
  • marker groups using a combinatorial approach (marker library). Accordingly, for unknown proteins or small molecule families where we do not have ample amounts of information for the above, we apply a combinatorial approach. This results in placing the tether at various points around the molecular core, in those positions that are actually diversity elements of the molecule.
  • the drugability of chemicals can be determined, and the protein target and attached biological ligands or substrates can be identified in a single step.
  • the combinatorial chemical libraries associated with this invention typically contain 3 or 4 diversity points, which generally point in different spatial directions.
  • the reactive marker groups can be placed in an appropriate, smaller, representative group of the library at variable points in such a way that they effect the expected biological activity of the unmodified chemicals only minimally.
  • the high- capacity, parallel derivatization of the combinatorial libraries for producing derivatives resulting in covalent bonds may be used as a method for fast protein profiling that allows for the detection and separation of the marked proteins, as well as their structure-based, sequential or functional characterization and classification.
  • This method is also suitable for identifying protein markers specific to diseases and can be used as a diagnostic method.
  • Use of the covalent labeling method contributes to simplifying complex proteomics.
  • This method may also be developed without reporter groups when previously identified, preferably recombinant proteins are used, through the application of mass spectrometry (MS), by seeking out MS photophore fingerprints.
  • MS mass spectrometry
  • groups that may be activated (photo)chemicalfy are necessary to be used, as these groups provide characteristic MS fragment patterns.
  • Step 1 Cbz-Boc-lyzine coupling using 5-amino-valeric acid ethyl ester hydrochloride
  • step No. 1 4.9 g initial material (the result of step No. 1) was dissolved in a 1:1 mixture of ethanol-ethyl acetate (100-ml solvent-mixture). First, ammonium formate (4.0 molar equivalent) then 10% palladium on charcoal (490 mg; 10% by weight) was added to the above solution. The mixture was heated up and stirred efficiently at reflux temperature for about 4 hours. After the initial compound had disappeared, the mixture was filtered through a short celite-pad. The catalyst filtered off was washed 2- 3 times with dichloromethane. All the filtrates were combined and evaporated under reduced pressure until dry. The residue was re-dissolved in dichloromethane and washed once with distilled wafer.
  • ammonium formate 4.0 molar equivalent
  • 10% palladium on charcoal 490 mg; 10% by weight
  • the initial compound (4 g (6.9 mmol) was dissolved in a solution of trifluoroacetic acid in dichloromethane (60 ml, 30%) and stirred at room temperature until the initial compound had disappeared.
  • Biotin (1.1 g, 0.8 equivalent calculated for the initial compound) was dissolved in hot N,N-dimethylformam ⁇ de (30 ml) at 80°C, then CDI was added (0.8 equivalent calculated for the initial compound). The reaction mixture was kept at 80°C for ten minutes until the bubbling had stopped, then the heating was terminated and the mixture was stirred for another two hours. A solution consisting of the product of the previous step mixed with 30 ml of dimethylformamide was added to the above prepared reaction mixture at room temperature and stirred for another 20 hours. Work-up: the solvent was removed in vacuo, and the residue suspended in dichloromethane and filtered off.
  • a biotin substituted compound (product of step 6a, 0.2 mmol) was dissolved in N,N-dimethylformamide (2-3 ml) at 80°C and 1 equivalent of CDI was added. The mixture was stirred for 1 hour, during such time the mixture cooled down to room temperature. Then 1 equivalent of a primary or secondary amine was added and the mixture was stirred at room temperature for 20 hours. The solvent was removed in vacuo, and the residue dissolved in chloroform and extracted first with solution of citric acid (1% in water), then with a solution of Na 2 C ⁇ 3 (5% in water), then with distilled water. The organic phase was dried and evaporated. Further purification was performed using preparative HPLC.
  • a benzophenone or dansyl substituted compound (the product of step 6b, 0.2 mmol) was dissolved in dichloroefhane (2-3 ml), and 1 equivalent of CDI was added. The mixture was stirred for 1 hour at room temperature. Then 1 equivalent of primary or secondary amine was added and the mixture was stirred at room temperature for 20 hours. The reaction mixture was extracted with a solution of citric acid (1% in water), then with a solution of Na 2 CO3 (5% in water), and finally with distilled water. The organic phase was dried and evaporated. Further purification was performed using preparative HPLC.
  • Boc-protected ⁇ mino acid amides originating from the previous step were dissolved in 2-ml of dichloromethane.
  • the flask was cooled in an ice water bath and 2-ml of trifluoroacetic acid was added to the cold solution dropwise.
  • the progress of the reaction was monitored by TLC using dichloroethane-ethanol in a 5:1 mixture as an eluent. Iodine vapour was used to render spots visible.
  • the reaction mixture was evaporated.
  • the residue was diluted with water and extracted with diethylether in order to remove the traces of the initial material.
  • the aqueous phase was basif ⁇ ed with a 20% aqueous Na2C ⁇ 3 solution.
  • the product was extracted with dichloromethane.
  • the organic phase was dried over MgSO 4 , filtered, and then the filtrate was evaporated.

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PCT/HU2003/000056 2002-07-10 2003-07-09 New combinatorial peptide libraries containing markers and methods for their preparation and utilization WO2004008151A2 (en)

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HUP0202212 2002-07-10
HU0202212A HUP0202212A2 (hu) 2002-07-10 2002-07-10 Új kombinatorikus fehérjemarker molekulakönyvtárak, eljárás azok előállítására és alkalmazására

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060051879A9 (en) * 2003-01-16 2006-03-09 Hubert Koster Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024517A2 (en) * 1992-05-21 1993-12-09 Furka Arpad Peptide sub-library kits
WO1994008051A1 (en) * 1992-10-01 1994-04-14 The Trustees Of Columbia University In The City Of New York Complex combinatorial chemical libraries encoded with tags
WO2000001802A2 (en) * 1998-07-01 2000-01-13 Cancerforskningsfonden Af 1989 (Fonden Til Fremme Af Eksperimentel Cancerforskning) Peptide antagonists of the human urokinase receptor and method for selecting them
WO2003080653A1 (en) * 2002-03-26 2003-10-02 Lars Baltzer Novel polypeptide scaffolds and use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024517A2 (en) * 1992-05-21 1993-12-09 Furka Arpad Peptide sub-library kits
WO1994008051A1 (en) * 1992-10-01 1994-04-14 The Trustees Of Columbia University In The City Of New York Complex combinatorial chemical libraries encoded with tags
WO2000001802A2 (en) * 1998-07-01 2000-01-13 Cancerforskningsfonden Af 1989 (Fonden Til Fremme Af Eksperimentel Cancerforskning) Peptide antagonists of the human urokinase receptor and method for selecting them
WO2003080653A1 (en) * 2002-03-26 2003-10-02 Lars Baltzer Novel polypeptide scaffolds and use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABSTRACTS OF PAPER. AT THE 223TH NATIONAL MEETING.OF THE AMERICAN CHEMICAL SOCIETY, vol. 223, no. 1-2, April 2002 (2002-04), page MEDI 245, XP008030884 USAMERICAN CHEMICAL SOCIETY, WASHINGTON, DC *
JOURNAL OF ORGANIC CHEMISTRY., vol. 67, no. 9, February 2002 (2002-02), pages 3120-3123, XP002966571 USAMERICAN CHEMICAL SOCIETY. EASTON. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060051879A9 (en) * 2003-01-16 2006-03-09 Hubert Koster Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US20100248264A1 (en) * 2003-01-16 2010-09-30 Koster Hubert Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US9034798B2 (en) * 2003-01-16 2015-05-19 Caprotec Bioanalytics Gmbh Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions

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AU2003251072A1 (en) 2004-02-02
HU0202212D0 (hu) 2002-09-28
HUP0202212A2 (hu) 2004-04-28
AU2003251072A8 (en) 2004-02-02
WO2004008151A3 (en) 2004-07-29

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