US20060148745A1 - DNA-based aptamers for human cathepsin G - Google Patents

DNA-based aptamers for human cathepsin G Download PDF

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US20060148745A1
US20060148745A1 US11/236,197 US23619705A US2006148745A1 US 20060148745 A1 US20060148745 A1 US 20060148745A1 US 23619705 A US23619705 A US 23619705A US 2006148745 A1 US2006148745 A1 US 2006148745A1
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cathepsin
inhibiting
aptamers according
inhibiting aptamers
dna
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Manlio Palumbo
Barbara Gatto
Rodolfo Pescador
Roberto Porta
Laura Ferro
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Gentium SRL
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Publication of US20060148745A1 publication Critical patent/US20060148745A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--

Definitions

  • Cathepsin G is a serine protease commonly found in the azurophilic granules of neutrophils and monocytes. Together with elastase and proteinase 3 it belongs to the chymotrypsin family and cleaves extracellular matrix proteins such as elastin, collagen, fibronectin and laminin causing extensive lung tissue damage in the animal. Cathepsin G also plays a role in blood clotting; in fact, it is involved in an alternative pathway of leukocytes initiation of coagulation, and by activating coagulation factor X and factor V it can cleave and potentially modulate the thrombin receptor and it can activate platelets in vitro. It is also able to convert angiotensin I into angiotensin II with only minor cleavage occurring elsewhere in the molecule.
  • cathepsin G kills bacteria and fungi but this property is not related to its activity, in fact peptides derived from its cleavage showed direct antimicrobial properties. It can also degrade necrotic tissues and is therefore related to several inflammatory diseases like lung emphysema, bronchitis, cystic fibrosis and psoriasis.
  • the enzymatic activity of cathepsin G is regulated by two types of protein proteinase inhibitors: the so called “canonical” inhibitors and the serpins.
  • the former are relatively small proteins (29-190 amino acids) and are tight-binding reversible inhibitors; among them are Mucus proteinase inhibitor (MPI), eglin c and aprotinin.
  • serpins are larger proteins (400-450 residues) that form an irreversible complex with their cognate protein due to the formation of a non-hydrolysable acyl bond between the catalytic site of cathepsin G and their reactive site loop.
  • serpins 1-antichymotrypsin is the most important: inhibitors of this family are not selective because they are able to bind to and inhibit other chymotripsins. Moreover, their stability and distribution in vivo is affected by their peptidic nature.
  • EP-775745 discloses oligonucleotide cathepsin G-inhibiting aptamers having a chain length of about 40 nucleotides (and in any case lower than 55 nucleotides) and containing G-pairs repeating units which are useful in the treatment and prophylaxis of inflammatory occurrences and procoagulant conditions.
  • FIG. 1 represents a comparison of the Kd of the tested oligonucleotides.
  • FIG. 2 represents a summarization of the Log concentration-effect curves of GT and AC aptamers.
  • FIG. 3 represents a summarization of the Log concentration-effect curves of PolyT aptamers.
  • the present research is mainly directed to the identification of non-peptidic inhibitors of cathepsin G characterised by high levels of selectivity and which can be thus more efficaciously used in the treatment and prophylaxis of the above conditions and also in that of genetic diseases, degenerative diseases, DNA damages, neoplasia and/or skin diseases.
  • DNA molecules are able to assume a variety of tridimensional structures depending on their sequence. Some of these might be relevant for binding to the target.
  • SELEX Systematic evolution of ligands by exponential enrichment
  • Aptamer technology combines the capacity of generating huge structural diversity in random pools of oligonucleotides with the power of the polymerase chain reaction (PCR) to amplify selected sequences.
  • This technology involves the screening of large, random-sequence pool of oligonucleotides and is based on the fact that they assume a large number of tertiary structures, some of which may possess desirable binding or catalytic activity against target molecules.
  • the new cathepsin G-inhibiting aptamers of the present invention are single or double stranded linear DNA or polynucleotide sequences characterized by having a chain length of at least 60 nucleotides, preferably 70, and by being substantially not subjected to inter and/or intra molecular base pairing.
  • the DNA sequences may have a chain length of 70 ⁇ 120 nucleotides, preferably of 70 ⁇ 110 nucleotides, even more preferably of 80 ⁇ 100 nucleotides.
  • sequences according to the present invention may be single or double stranded, single stranded sequences are preferred.
  • sequences according to the present invention are also preferably characterized by having a molar content in guanine of about 25 ⁇ 50%, preferably 35 ⁇ 45% and/or by having a molar ratio AG/TC of about 1.0 ⁇ 2.0, preferably 1.2 ⁇ 1.8 (for the purposes of the present invention AG means the total number of A and G nucleotides of the sequence whereas TC means the total number of T and C nucleotides of the sequence).
  • the expression “substantially not subjected to inter and/or intra molecular base pairing” means that the DNA or polynucleotide sequences do not undergo inter and/or intra molecular base pairing to an extent higher than 20%, preferably than 10%, even more preferably than 5%, under both stringent and non stringent conditions. Such a result is the direct consequence of their structure, since the fact of:
  • the aptamers according to the present invention do selectively and efficaciously inhibit cathepsin G and, consequently, they can be used in the manufacture of a medicament for the treatment and prophylaxis of inflammatory occurrences, procoagulant conditions, genetic diseases, degenerative diseases, DNA damages, neoplasia and/or skin diseases, which represents therefore an object of the invention.
  • a further object of the invention is also represented by the pharmaceutical composition containing the cathepsin G-inhibiting aptamers of the invention together with customary eccipients and/or adjuvants.
  • Other objects of the invention may be represented by the cathepsin G-inhibiting aptamers selected from those reported in the sequence listing (i.e. from SEQ ID NO: 1 to SEQ ID NO: 18).
  • Cathepsin G was purchased from Europa Bioproducts or from Calbiochem. All oligonucleotides were obtained from Eurogentec Bel SA (Belgium) and purified by PAGE before use. Some oligonucleotides, already purified by PAGE were obtained from Gibco BRL Custom Primers. Taq polymerase was from Pharmacia Amersham Biotech while dNTPs were purchased as sodium salt from Boehringer Mannheim. T4-polynucleotide kinase, ligase and the restriction enzymes were from Gibco Life Technologies. Qiagen kits were used for plasmid miniprep purification, and sequencing was performed using T7 Sequenase (Pharmacia Amersham Biotech) and [gamma- 33 P]dATP (Nen Life Sciences).
  • the synthesised random pool is 96 base length, the central part of the molecule has a randomised region that is flanked by two constant regions for amplification, cloning and sequencing; its sequence is 5′-CGTACG GAATTC GCTAGC(N) 60 GGATCC GAGCTCCACGTG-3′.
  • the underlined sequences refer to restriction sites for EcoRI and BamHI enzymes respectively.
  • the pool was amplified by PCR using primer II-up, which sequence is 5′-CGTACGGAATTCGCTAGC-3′, and primer III-Down 5′Biot-CACGTCGAGCTCGGATCC-3′ which is biotinylated at the 5′ end in order to be bound to a streptavidin column to get ssDNA.
  • the starting random pool was radioactive labelled with 32 P, denaturated at high temperature and incubated with cathepsin G in Incubation buffer (buffer IB: 30 mM Tris HCl pH7.5, 150 mM NaCl, 5 mM KCl and 5 mM MgCl 2 ) which is close to the physiological conditions.
  • the incubation was conducted for 90 minutes in ice, then the sample was loaded in an affinity chromatography mini-column filled with Sepharose SP (Amersham Pharmacia Biotech), swollen and equilibrated in buffer IB.
  • the ssDNA/protein solution was incubated with the resin for 30 minutes at 4° C.
  • the unbound oligonucleotide molecules were washed away with buffer IB, while the remaining, more selective ones were eluted from the column a high ionic force elution buffer (buffer EB: 0.8 M NaCl e 50 mM Tris pH 7.8).
  • washing volumes were modified during the selection in order to increase the stringency as well as the DNA concentration which was twice the protein at the first cycle, but it was progressively reduced.
  • the fractions were counted and the yield of the Selex cycle was expressed as a percentage of the total radioactivity.
  • the flow through and the first two fractions of the EB wash were collected and amplified.
  • Polymerase chain reaction was done using Taq polymerase at a concentration of 0.3-0.5 u/50 ⁇ l in the buffer indicated by the producer. The number of cycles was adjusted after every different selection.
  • the DNA was subjected to a polishing reaction in order to get blunt ends: an aliquot of the normal PCR reaction was incubated with 2.5 u/ ⁇ l of Pfu Turbo polymerase (Stratagene) in the suggested buffer at 72° C. for 30 minutes.
  • Pfu Turbo polymerase (Stratagene)
  • E. coli competent cells SURE strain Stratagene
  • E. coli pulser Biorad
  • Plasmids were purified by alkaline lysis and their quality was every time tested by agarose gel electrophoresis.
  • the sequence of the aptamers was determined with the Sanger's method, labeling with [gamma- 33 P]dATP and employing two different primers EleA457: 5′-ACG-CCA-AGC-TTG-CAT-3′ (sense) and Ele S: 5′-GGG-TTT-TCC-CAG-TCA-CGA-3′ (antisense).
  • the affinity of the oligonucleotides was determined by affinity chromatography as performed in the selection. Different aliquots of each oligonucleotide were previously incubated with 15 ⁇ g of Cathepsin G in ice. The solution was then loaded in the min-chromatography column used for the selection and washed with 15 volumes of buffer IB. After one hour incubation, it was washed with six volumes of buffer EB. Fractions of the same volumes were collected and counted.
  • Cathepsin G from human neutrophils, dissolved in HBS EP buffer, pH 7.40 (Biacore) was immobilized on the surface of a CM 5 research grade sensor chip flow cell, according to the procedure suggested by Biacore and using the Biacore amine coupling kit.
  • a blank flow cell was prepared using all the above reagents but Cathepsin G.
  • the amount of Cathepsin G immobilized on the surface of the flow cell was 5178.91 ⁇ 129.63 RU.
  • Aptamers [Poly GT (chain length: 20, 30, 40, 60, 80 and 100) and Poly AC (chain length: 20, 40 and 80,] were dissolved in 30 mM Tris-HCl buffer, pH 7.50, 150 mM NaCl, 5 mM KCl, and 5 mM MgCl 2 and injected over the Cathepsin G surface or the blank surface. Three sets of experiments were run. The first at a concentration of 500 nM, for all the aptamers, the second at a concentration of 6595 ⁇ g/L, for all the aptamers, and the third one at concentrations ranging from 15.6 to 8000 nM, according to the aptamer being tested.
  • aptamers for cathepsin G starting from a DNA pool with a randomised region of 60 nucleotides flanked by two regions with conserved sequence for the PCR reaction and restriction sites for the following cloning step (see above).
  • the selected molecules were then efficiently removed from the column, together with the bound protein, using a high ionic strength buffer (buffer EB), and then counted by radioactivity.
  • buffer EB high ionic strength buffer
  • the first two fractions and the flow through were then collected, amplified by PCR and reduced to single stranded molecules in order to be used for the next cycle (see methods section for details).
  • the selected molecules were cloned into E. coli cells as described in the experimental selection and sequenced. We found 19 different sequences out of 50 clones. We used two sequence alignment programs, Clustal W and FastA-align, searching for a repeated consensus motif, but the molecule diversity was too high to yield a good alignment even within subsets of the sequenced molecules. Further analysis showed that GT motifs are clearly repeated in 14 sequences. Moreover, a closer look at these molecules showed that they are not prone to undergo either inter and intra molecular base pairing to an appreciable extent, nor do they form more complex tridimensional structures like G quartets. It seemed that the selection led to unstructured, linear and flexible molecules that can tightly bind to the positive protein because of a charge-charge interaction.
  • the selected CG51 showed a high affinity for cathepsin G (Kd 0.9 nM). Besides, its Kd was comparable with AC and GT oligonucleotides of the same length (Kd 0.8 nM and 1 nM respectively) and with cmpCG51 (Kd 0.6 nM) ( FIG. 1 ). These data indicate that our hypothesis about tight binding by unstructured and flexible molecules was correct.
  • Molecules longer than the 60mer like (AC) 60 and (GT) 40 which are respectively a 120mer and a 80mer, showed an affinity of 1.2 nM.
  • the shorter (GT) 20 and (GT) 10 that are shorter molecules have a Kd of 1.5 nM and 2 nM respectively, suggesting that the length of the selected oligonucleotides is important to grant efficient binding.
  • Aptamer THR that was selected against thrombin, was also included as a control in order to prove whether the oligonucleotide structure was important for cathepsin binding. This aptamer is known to form stable G quartets.
  • GT 80 has a relative potency of about 0.32, GT 60 of about 0.144, AC 80 of about 0.017, GT 40 of about 0.016, AC 40 of about 0.0047 and GT 30 of about 0.0020.
  • GT 20 and AC 20 were not evaluable because of their poor binding.
  • the aptamers can be divided into three families ( FIG. 2 ); first family: GT 100, GT 80 and GT 60; second family: AC 80, GT 40, AC 40 and GT 30; third family: AC 20 and GT 20.
  • a linear and flexible single stranded DNA chain with a length of at least 60, preferably more than 70-80, is more effective in binding cathepsin G than the chromosomal counterpart and also more effective than shorter DNA chains.

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US11/236,197 2003-06-30 2005-09-27 DNA-based aptamers for human cathepsin G Abandoned US20060148745A1 (en)

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US12/015,663 US7838506B2 (en) 2003-06-30 2008-01-17 DNA-based aptamers for human cathepsin G

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EP03425428A EP1493810A1 (fr) 2003-06-30 2003-06-30 Aptamères pour la cathepsine G basés sur de l'ADN
EP03425428.4 2003-06-30
PCT/EP2004/006599 WO2005003347A2 (fr) 2003-06-30 2004-06-18 Aptameres a base d'adn pour la cathepsine g humaine

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US20150111306A1 (en) * 2013-10-21 2015-04-23 National Tsing Hua University HEMOGLOBIN A1c-SPECIFIC APTAMER, HEMOGLOBIN-SPECIFIC APTAMER, AND APPLICATIONS THEREOF

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US10744179B2 (en) 2014-09-18 2020-08-18 The Provost, Fellows, Foundation Scholars, & the Other Members of Board, of The College of the Holy Method for treatment of inflammatory skin disorders with inhibitors of IL-36 proteolytic processing
RU2683868C1 (ru) * 2018-11-30 2019-04-02 Акционерное общество "Молочный комбинат "Ставропольский" Способ производства молочного сахара

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US6316190B1 (en) * 1996-05-20 2001-11-13 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Oligonucleotides which specifically bind retroviral nucleocapsid proteins

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ES2110444T3 (es) 1990-06-11 1998-02-16 Nexstar Pharmaceuticals Inc Ligandos de acidos nucleicos.
WO1992014843A1 (fr) * 1991-02-21 1992-09-03 Gilead Sciences, Inc. Aptamere specifique de biomolecules et procede de production
DE19543750C2 (de) * 1995-11-24 1997-10-23 Crinos Industria Farmaco Cathepsin G inhibierende Aptamere
US6399302B1 (en) * 1998-08-21 2002-06-04 University Of Virginia Patent Foundation Signal generating oligonucleotide-based biosensor
AUPQ051099A0 (en) * 1999-05-24 1999-06-17 Tachas, George Dr Novel products and processes in treatment and/or prophylaxis

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Publication number Priority date Publication date Assignee Title
US6316190B1 (en) * 1996-05-20 2001-11-13 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Oligonucleotides which specifically bind retroviral nucleocapsid proteins

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150111306A1 (en) * 2013-10-21 2015-04-23 National Tsing Hua University HEMOGLOBIN A1c-SPECIFIC APTAMER, HEMOGLOBIN-SPECIFIC APTAMER, AND APPLICATIONS THEREOF
US9086406B2 (en) * 2013-10-21 2015-07-21 National Tsing Hua University Hemoglobin A1c-specific aptamer, hemoglobin-specific aptamer, and applications thereof

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CA2528686A1 (fr) 2005-01-13
CN1816625A (zh) 2006-08-09
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ZA200510093B (en) 2008-04-30
US20080176814A1 (en) 2008-07-24
MXPA05013800A (es) 2006-06-27
NO20060429L (no) 2006-01-26
EP2236609A3 (fr) 2011-07-20
EP1493810A1 (fr) 2005-01-05
RU2006102521A (ru) 2006-06-27
EP1618196A2 (fr) 2006-01-25
IL172407A0 (en) 2006-04-10
RU2360000C2 (ru) 2009-06-27
WO2005003347A3 (fr) 2005-09-15
EP2236609A2 (fr) 2010-10-06
KR20060035624A (ko) 2006-04-26
US7838506B2 (en) 2010-11-23
AU2004254014A1 (en) 2005-01-13
WO2005003347A2 (fr) 2005-01-13
JP2011078414A (ja) 2011-04-21
RS20050955A (en) 2008-06-05
AU2004254014B2 (en) 2010-06-10

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