WO2003095605A2 - A chimeric reverse transcriptase and methods for identifying telomerase inhibitors - Google Patents
A chimeric reverse transcriptase and methods for identifying telomerase inhibitors Download PDFInfo
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- WO2003095605A2 WO2003095605A2 PCT/EP2003/003874 EP0303874W WO03095605A2 WO 2003095605 A2 WO2003095605 A2 WO 2003095605A2 EP 0303874 W EP0303874 W EP 0303874W WO 03095605 A2 WO03095605 A2 WO 03095605A2
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- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
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Definitions
- the present invention is directed, in part, to nucleic acid molecules encoding a novel chimeric molecule based on HIV-1 Reverse Transcriptase (HIV-RT) and human telomerase reverse transcriptase (hTERT) catalytic site.
- HIV-RT HIV-1 Reverse Transcriptase
- hTERT human telomerase reverse transcriptase
- This novel protein has been named HIV-RT/hTERT and will be referred to herein with this term.
- the present invention is also directed to novel polypeptides, and assays for screening compounds which bind to HIV-RT/hTERT and/or inhibit the reverse transcriptase activity of hTERT.
- Cancer is one of the major causes of disease despite the great effort and investments in research and development during the last decades. In addition to that, most cancer patients still die due to metastatic disease. In the same time, despite the great increase in the knowledge and understanding of the regulatory mechanisms involved in the onset of malignancy, currently available treatments (including surgery, radiation and a variety of cytoreductive and hormone- based drugs, used alone or in combination) are still highly non specific and toxic to the patient, causing severe side effects including nausea and vomiting, hair loss, diarrhea, fatigue, ulcerations and the like. These evidences indicate the need for new and more effective anti-cancer therapies. Recently an understanding of the mechanisms by which normal cells reach the state of senescence, i.e. the loss of proliferative capacity that cells normally undergo in the cellular aging process, has begun to emerge and in this respect telomerase appears to have a central role .
- Telomerase is a ribonucleoprotein enzyme responsible in most eukaryotes for the complete replication of chromosome ends, or telomeres, that are tandemly repeated DNA sequences (in particular human telomeres are formed by 5 ' -TTAGGG repeats) . Telomerase synthesises one strand of the telomeric DNA using as a template a sequence contained within the RNA component of the enzyme necessary for the addition of the short sequence repeats (TTAGGG) to the chromosome 3' end (see Blackburn 1992, Annu. Rev. Biochem. , 61, 113-129).
- telomere activity cannot be detected and telomeres shorten with successive cell division: in fact, actively dividing normal cells have the potential to lose 50- 200 base pairs after each round of cell division, due to the discontinuous synthesis of DNA lagging strand, finally resulting in shortening of telomeres .
- telomere length constant Shay, J. W. and Bacchetti, S. Eur. J. Cancer, 1997, 33, 787-791.
- Such compounds are also expected to exhibit greater safety and to lack toxic effects in comparison with traditional chemotherapeutic anticancer agents, as they can be effective in providing treatments that discriminate between malignant and normal cells to a high degree, avoiding many of the deleterious side-effects present with most current chemotherapeutic regimes which rely on agents that kill dividing cells indiscriminately.
- the present invention is directed to, in part, isolated nucleic acid molecules comprising SEQ ID NOs. 1, 26, 27, 28 or 29, or a fragment thereof; a nucleotide sequence complementary to at least a portion of SEQ ID NOs. 1, 26, 27, 28 or 29; a nucleotide sequence homologous to SEQ ID NOs. 1, 26, 27, 28 or 29, or a fragment thereof; a nucleotide sequence that encodes a polypeptide comprising SEQ ID NOs. 2, 21, 22, 24 or 25, or a fragment thereof; or a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to SEQ ID NOs. 2, 21, 22, 24 or 25, or a fragment thereof.
- the present invention is also directed to recombinant expression vectors comprising any of the nucleic acid molecules described above.
- the present invention is also directed to methods of producing a polypeptide comprising SEQ ID NOs. 2, 21, 22, 24 or 25, or a homologue or fragment thereof, by introducing a recombinant expression vector comprising any of the nucleic acid molecules described above into a compatible host cell, growing the host cell under conditions suitable for expression of the polypeptide, and recovering the polypeptide from the host cell .
- the present invention is also directed to isolated polypeptides encoded by any of the nucleic acid molecules described above .
- the present invention is also directed to methods of identifying a compound which binds to HIV-RT/hTERT, by contacting HIV-RT/hTERT with a compound, and determining whether the compound binds HIV-RT/hTERT.
- the present invention is also directed to methods of identifying a compound which binds a nucleic acid molecule encoding HIV-RT/hTERT by contacting a nucleic acid molecule encoding HIV-RT/hTERT with a compound, and determining whether the compound binds the nucleic acid molecule.
- the present invention is also directed to methods of identifying a compound which inhibits the reverse transcriptase activity of hTERT by contacting HIV-RT/hTERT with a compound, and determining whether reverse transcriptase activity is inhibited.
- the present invention is also directed to compounds which inhibit the reverse transcripatse activity of hTERT identified by contacting HIV-RT/hTERT with the compound, and determining whether the compound inhibits reverse transcriptase activity.
- Figure 1 is a schematic representation of HIV-RT/hTERT chimera. HIV-1 RT sequence regions (1-63, 72-103, 121-178, 241-560) are drawn in black, whereas hTERT sequence regions (625-630, 706-722, 862-941) are drawn in white. The two lines below the scheme drawing indicate the length of the two subunits (p53: 1-430 and p67: 1-560) of the hetero-dimeric protein.
- Figure 2 is a 1% agarose gel electrophoresis demonstrating the reverse transcriptase activity of the chimera of the invention.
- the RT-PCR reaction products of the experiments described in Example 5.1 were subjected to electrophoresis on 1% agarose gel .
- the amplified 324 bp ⁇ - actin DNA band is visible in the positive control reaction (lane 1: AMV RT) as well as in the reactions using recombinant HIV-RT/hTERT chimera (lane 2) or recombinant HIV- 1 RT (lane 3), respectively.
- the negative control (lane 4) without reverse transcriptase did not yield any amplified DNA product .
- Figure 3 HIV-RT/hTERT chimera sequence alignment with hTERT and HIV-1 RT. Alignments of the three modified regions in HIV-RT/hTERT chimera with the corresponding sequences from hTERT and HIV-1 RT. # Residue numbers, of * HIV-RT/hTERT chimera with respect to residue 1 of HIV-1 RT.
- Telomerase Reverse Transcriptase: Homo sapiens: Hs; Mus musculus: Mm; Mesocricetus auratus : Ma,- Xenopus laevis: XI; Schizosaccharomyces pombe : Sp; Arabidopsis thaliana: At; Oryza sativa: Os; Euplotes aediculatus : Ea; Oxytricha trifallax: Ot; Paramecium caudatum: Pc; Cryptosporidium parvum: Cp; Tetrahymena thermophila: Tt .
- the term "activity" refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e. having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event.
- binding means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc.
- the physical interaction, the binding can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates .
- the term "compound” means any identifiable chemical or molecule, including, but not limited to, small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound can be natural or synthetic.
- the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.
- the term "contacting" means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention.
- the polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc.
- Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding HIV-RT/hTERT or fragment thereof.
- homologous nucleotide sequence or “homologous amino acid sequence,” or variations thereof, refers to sequences characterised by a homology, at the nucleotide level or amino acid level, of at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% to the entire SEQ ID NOs. 1, 26, 27, 28 or 29, or to at least a portion of SEQ ID NOs. 1, 26, 27, 28 or 29 which encodes a functional domain of the encoded polypeptide, or to SEQ ID NOs. 2, 21, 22, 24 or 25.
- Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI) , using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein by reference in its entirety) .
- Homologous amino acid sequences can include those amino acid sequences which encode conservative amino acid substitutions in SEQ ID NOs. 2, 21, 22, 24 or 25.
- Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
- Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
- hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
- an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
- substitution of amino acids whose hydrophilicity values are within +2 is preferred, those that are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
- isolated nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment .
- isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
- oligonucleotide refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR) . This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They can be chemically synthesized and can be used as probes .
- probe refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences . Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Shorter probes can be chemically synthesized. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA- like technologies.
- stringent hybridization conditions refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium.
- Tm thermal melting point
- stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at least about 60°C for longer probes, primers or oligonucleotides.
- Stringent conditions may also be achieved with the addition of destabilising agents, such as formamide .
- nucleic acid molecules comprising novel nucleotide sequences encoding the chimeric molecule HIV-RT/hTERT.
- the nucleic acid molecules are preferably either RNA or DNA, but can contain both RNA and DNA monomers or peptide nucleic acid monomers .
- a nucleic acid molecule can be single stranded or double stranded.
- Monomers of nucleic acid molecules can be linked via conventional phosphodiester bonds or modified bonds, such as, for example, phosphorothioate bonds and the like.
- the sugar moieties of the monomers can be modified by, for example, addition of 2' substitutions which help confer nuclease resistance and/or cellular uptake.
- a nucleic acid molecule comprises a fragment of SEQ ID NOs. 1, 26, 27, 28 or 29.
- the fragment comprises from about 10 to about 100 contiguous nucleotides, from about 101 to about 200 contiguous nucleotides, from about 201 to about 300 contiguous nucleotides, from about 301 to about 400 nucleotides, from about 401 to about 500 nucleotides, from about 501 to about 600 nucleotides, from about 601 to about 700 nucleotides, from about 701 to about 800 nucleotides, from about 801 to about 900 nucleotides, from about 901 to about 1000 nucleotides, from about 1001 to about 1100 nucleotides, from about 1101 to about 1200 nucleotides, from about 1201 to about 1300 nucleotides, from about 1301 to about 1400 nucleotides, from about 1401 to about 1500, from about 1501 to about 1600, from about 1601 to about 1700,
- oligonucleotides which hybridize under stringent hybridization conditions to at least a portion of SEQ ID NOs. 1, 26, 27, 28 or 29.
- Preferred oligonucleotides comprise at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesised and can be used as probes, primers, and as antisense agents.
- a nucleic acid molecule comprises a nucleotide sequence homologous to SEQ ID NOs. 1, 26, 27, 28 or 29.
- the nucleotide sequence is at least about 60% homologous, more preferably at least about 70% homologous, more preferably at least about 80% homologous, more preferably at least about 90% homologous, and most preferably at least about 95% homologous to the entire SEQ ID NOs. 1, 26, 27, 28 or 29.
- a nucleotide sequence homologous to SEQ ID NOs. 1, 26, 27, 28 or 29 also includes a fragment of the nucleotide sequence homologous to SEQ ID NOs. 1, 26, 27, 28 or 29 of the lengths described above .
- Homologous sequences include also variant sequences as defined above .
- the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NOs. 2, 21, 22, 24 or 25.
- the nucleic acid molecule preferably comprises SEQ ID NOs. 1, 26, 27, 28 or 29 containing codon substitutions which reflect the degeneracy of the genetic code.
- SEQ ID NOs. 1, 26, 27, 28 or 29 containing codon substitutions which reflect the degeneracy of the genetic code.
- the present invention therefore, contemplates these other DNA and RNA molecules which, on expression, encode the polypeptide of SEQ ID NOs. 2, 21, 22, 24 or 25.
- DNA and RNA molecules other than those specifically disclosed herein characterised simply by a change in a codon for a particular amino acid are within the scope of the present invention.
- codons constitute triplet sequences of nucleotides in mRNA molecules and, as such, are characterised by the base uracil (U) in place of base thymidine (T) (which is present in DNA molecules) .
- U base uracil
- T base thymidine
- a nucleic acid molecule comprises a nucleotide sequence that encodes a fragment of a polypeptide encoding SEQ ID NOs. 2, 21, 22, 24 or 25.
- the fragment comprises from about 5 to about 20 contiguous amino acids, from about 21 to about 40 contiguous amino acids, from about 41 to about 60 contiguous amino acids, from about 61 to about 80 contiguous amino acids, from about 81 to about 100 contiguous amino acids, from about 101 to about 120 contiguous amino acids, from about 121 to about 140 contiguous amino acids, from about 141 to about 160 contiguous amino acids, from about 161 to about 180 contiguous amino acids, from about 181 to about 200 contiguous amino acids, from about 201 to about 220 contiguous amino acids, from about 221 to about 240 contiguous amino acids, from about 241 to about 260 contiguous amino acids, from about 261 to about 280 contiguous amino acids, from about 281 to about 300 contiguous amino acids, from about 301 to
- a nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to SEQ ID NOs. 2, 21, 22, 24 or 25.
- the nucleic acid molecule comprises a nucleotide sequence that encodes a fragment of the polypeptide comprising an amino acid sequence homologous to SEQ ' ID NOs. 2, 21, 22, 24 or 25.
- the fragment comprises from about 5 to about 20 contiguous amino acids, from about 21 to about 40 contiguous amino acids, from about 41 to about 60 contiguous amino acids, from about 61 to about 80 contiguous amino acids, from about 81 to about 100 contiguous amino acids, from about 101 to about 120 contiguous amino acids, from about 121 to about 140 contiguous amino acids, from about 141 to about 160 contiguous amino acids, from about 161 to about 180 contiguous amino acids, from about 181 to about 200 contiguous amino acids, from about 201 to about 220 contiguous amino acids, from about 221 to about 240 contiguous amino acids, from about 241 to about 260 contiguous amino acids, from about 261 to about 280 contiguous amino acids, from about 281 to about 300 contiguous amino acids, from about 301 to about 320 contiguous amino acids, from about 321 to about 340 contiguous amino acids, from about 341 to about 360 contiguous amino acids, from about 361 to about 380 contiguous amino acids, from
- a nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence of SEQ ID NOs. 2, 21, 22, 24 or 25 wherein one or more conservative, non- conservative, or both amino acid substitutions have been made.
- amino acid substitutions can be made at amino acid numbers 107, 110, 115, 177, 179, 181, 182, 193, 194, 196, 197, 200, 201, 203, 204, 212, 213, 214, 215, 216, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 244, 253 and 256 of SEQ ID NO: 2.
- a substitution can occur at one or more of these amino acid positions.
- vectors or recombinant expression vectors, comprising any of the nucleic acid molecules described above.
- Vectors are used herein either to amplify DNA or RNA encoding HIV-RT/hTERT and/or to express DNA which encodes HIV-RT/hTERT.
- Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination) .
- Preferred viral particles include, but are not limited to, adenoviruses, parvoviruses , herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses .
- Preferred expression vectors include, but are not limited to, pET-20b (Novagen) pGEX-6p2 (Amersham Biosciences) and pKK-223-3 (Amersham-Pharmacia) .
- Other expression vectors include, but are not limited to, pcDNA3 (Invitrogen) , pSVL (Pharmacia Biotech) , pSPORT vectors, pGEM vectors (Promega), pPROEXvectors (LTI, Bethesda, MD) , Bluescript vectors (Stratagene) , pQE vectors (Qiagen) , pSE420 (Invitrogen) , and pYES2 (Invitrogen) and the range of Gateway expression plasmids (LifeTechnologies) .
- Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding HIV-RT/hTERT is operably linked to suitable control sequences capable of effecting the expression of the HIV-RT/hTERT in a suitable host.
- DNA regions are operably linked when they are functionally related to each other.
- a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
- Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants .
- the need for control sequences into the expression vector will vary depending upon the host selected and the transformation method chosen.
- control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, and sequences which control the termination of transcription and translation.
- Preferred vectors preferably contain a promoter which is recognised by the host organism.
- the promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral.
- suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973) , which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed.,
- Additional promoters include, but are not limited to, mouse mammary tumor virus promoter, long terminal repeat of human immunodeficiency virus promoter, Maloney virus promoter, cytomegalovirus immediate early promoter, Epstein Barr virus promoter, Rous Sarcoma virus promoter, human actin promoter, human myosin promoter, human hemoglobin promoter, human muscle creatine promoter, and human metalothionein promoter.
- Additional regulatory sequences can also be included in preferred vectors .
- Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene ell of bacteriophage lambda. The Shine-Dalgarno sequence cam be directly followed by the DNA encoding HIV-RT/hTERT and result in the expression of the mature HIV-RT/hTERT protein.
- suitable expression vectors can include an appropriate marker which allows the screening of the transformed host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al . , supra.
- An origin of replication can also be provided either by construction of the vector to include an exogenous origin or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter can be sufficient.
- one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and HIV-RT/hTERT DNA.
- a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Patent No. 4,399,216).
- Nucleotide sequences encoding HIV-RT/hTERT can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al . , supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al . , Mol. Cell. Biol., 1983, 3, 280, Cosman et al . , Mol.
- Another aspect of the present invention is directed to transformed host cells having an expression vector comprising any of the nucleic acid molecules described above .
- Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell .
- Introduction of the vector into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods.
- Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes . If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus. In a preferred embodiment of the invention, the prokaryotic host is E. coli.
- eukaryotic cells are cells of higher eukaryotes .
- Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.
- Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells) , African green monkey kidney cells (COS cells) , human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973) , which is incorporated herein by reference in its entirety) .
- yeast host can be employed as a host cell.
- Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluveromyces .
- Preferred yeast hosts are S. cerevisiae and P. pastoris.
- Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS) , a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
- ARS autonomously replication sequence
- Shuttle vectors for replication in both yeast and E. coli are also included herein.
- the polypeptide comprises an amino acid sequence homologous to SEQ ID NOs. 2, 21, 22, 24 or 25 or a fragment thereof as described above . It is to be understood that the present invention includes proteins homologous to, and having essentially the same biological properties as, the polypeptide encoded by the nucleotide sequences described herein, i.e., a variant.
- the variant forms can be characterised by, for example, amino acid insertion (s) , deletion (s) or substitution (s) .
- substitutions or “deletions” are changes to or within an amino acid sequence.
- the variation allowed in a particular amino acid sequence can be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the HIV-RT/hTERT nucleic acid sequence using recombinant DNA techniques .
- Alterations of naturally occurring amino acid sequence can be accomplished by any of a number of known techniques. For example, mutations can be introduced into the polynucleotide encoding a polypeptide at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis, which is described by Walder et al . , Gene, 1986, 42, 133, Bauer et al . , Gene, 1985, 37, 73, Craik, BioTechniques, January 1985, pp.12-19, Smith et al . , Genetic Engineering: Principles and Methods, Plenum Press (1981), and U.S.
- a HIV-RT/hTERT variant of the present invention will exhibit substantially the same biological activity of a naturally occurring HIV-RT/hTERT polypeptide.
- exhibit substantially the same biological activity of a naturally occurring HIV-RT/hTERT polypeptide is meant that HIV-RT/hTERT variants within the scope of the invention can comprise conservatively substituted sequences, meaning that one or more amino acid residues of a HIV-RT/hTERT polypeptide are replaced by different residues that do not alter the secondary and/or tertiary structure of the HIV-RT/hTERT polypeptide.
- Such substitutions can include the replacement of an amino acid by a residue having similar physicochemical properties, as described above.
- Other HIV-RT/hTERT variants which might retain substantially the same biological activities of HIV-RT/hTERT are those where amino acid substitutions have been made in areas outside functional regions of the chimera.
- Other preferred variants of the chimeric molecules described herein consists of a Telomerase Reverse Transcriptase (TERT) from an organism other than human and of a reverse transcriptase from an organism other than HIV-1.
- the chimeric molecule hTERT / HIV-2 reverse transcriptase constitutes a specific example of such variants.
- a suitable binding partner includes glutathione-S- transferase (GST) domain, which is easily purified to homogeneity by affinity chromatography on a column of immobilized glutathione.
- GST glutathione-S- transferase
- the protein can then be used directly, with the GST domain still attached; alternatively, the GST fusion protein is constructed with a protease cleavage site between the GST domain and the protein, so that digestion with a protease such as thrombin or blood coagulation Factor X a will remove the GST domain altogether.
- melatin leader in sf9 insect cells 6-His tag, thioredoxin tag,, hemaglutinin tag, IgG binding domains of protein A, and OmpA signal sequence tag.
- the binding partner which recognizes and binds to the peptide can be any molecule or compound including
- polypeptide can be expressed in E. coli without signal sequence.
- polypeptide is
- the present inventors succeeded in expressing HIV-RT/hTERT in E. coli as insoluble inclusion body at very high levels (100 mg/liter) as will be illustrated in the Examples section.
- the chimeric molecule thus obtained can be purified using protein purification techniques well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to peptide and non- peptide fractions. Having separated the protein from other proteins, the protein of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) .
- Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
- a particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
- purified will refer to an enzyme preparation • that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a preparation in which the enzyme forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
- High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks . This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
- Gel chromatography, or molecular sieve chromatography is a special type of partition chromatography that is based on molecular size.
- gel chromatography The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
- Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
- the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
- the present inventors set up an efficient purification protocol based on affinity chromatography followed by gel filtration specifically described in the experimental section.
- Such purification protocol yields dimeric chimeric enzyme.
- the purified enzyme preparation is subjected to refolding by dilution and the renatured chimera is dialyzed and concentrated.
- the chimeric enzyme can present other post- translational modifications, such as glycosylation, acylation, sialylation.
- HIV-RT/hTERT expressed in yeast, insect or mammalian expression systems can then be similar to or significantly different from a HIV-RT/hTERT polypeptide in molecular weight and glycosylation pattern.
- expression of HIV-RT/hTERT in bacterial expression systems will provide non-glycosylated HIV-RT/hTERT.
- One aspect of the present invention provides a method of identifying compounds which bind to either HIV-RT/hTERT or nucleic acid molecules encoding HIV-RT/hTERT, comprising contacting HIV-RT/hTERT, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds HIV-RT/hTERT, or a nucleic acid molecule encoding the same. Binding to the chimera protein can be determined by isothermal titration calorimetry (ITC) .
- ITC isothermal titration calorimetry
- the heat of binding released upon compound binding to protein can be directly measured by compensation of heat changes in the temperature controlled reaction vessel .
- This method represents a useful tool to assess the binding specificity and selectivity of test compounds to HIV-RT/hTERT chimera and it provides important indications for screening telomerase inhibitors .
- the compounds to be screened include, but are not limited to, molecules of extracellular, intracellular, biologic or chemical origin.
- the HIV-RT/hTERT polypeptide or polynucleotide employed in such a test can either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly.
- One skilled in the art can, for example, measure the formation of complexes between HIV-RT/hTERT and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between HIV-RT/hTERT and its substrate caused by the compound being tested.
- Another aspect of the present invention is directed to methods of identifying compounds which inhibits the reverse transcriptase activity of HIV-RT/hTERT comprising contacting HIV-RT/hTERT with a compound, and determining whether the compound inhibits the reverse transcriptase activity of HIV- RT/hTERT.
- the activity in the presence of the test compound is compared to the activity in the absence of the test compound. Where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.
- the present invention is particularly useful for ⁇ screening compounds by using HIV-RT/hTERT in any of a variety of drug screening techniques .
- the compounds to be screened (which can include compounds which are suspected to modulate HIV-RT/hTERT activity) include, but are not limited to, molecules of extracellular, intracellular, biologic or chemical origin.
- the HIV-RT/hTERT chimeric enzyme employed in such a test can be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.
- One skilled in the art can, for example, measure the formation of complexes between HIV- RT/hTERT and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between HIV-RT/hTERT and its substrate caused by the compound being tested.
- the activity of HIV-RT/hTERT polypeptide of the invention can be determined by conventional measurement of RT activity.
- the assay is based on the detection of incorporation of radioactivity in RNA/DNA hybrids, which can be precipitated with trichloro acetic acid (TCA) .
- TCA trichloro acetic acid
- Beta- emitting nucleotide scintillation liquids are used for detecting radioactivity.
- modified nucleotides instead of radio-labeled bases, modified nucleotides are used containing antigenic epitopes or structures (e.g. 5-bromo-deoxy-uridine triphosphate or dioxygenine-labeled dUTP) having a high affinity for defined ligands .
- antigenic epitopes or structures e.g. 5-bromo-deoxy-uridine triphosphate or dioxygenine-labeled dUTP
- the presence of these epitopes or structures in the RNA/DNA hybrid is revealed by antibodies or ligands conjugated with ELISA enzymes. The amount of bound ELISA enzymes is then determined with a secondary enzymatic test .
- Another method for showing RT activity is to use a series of specific probes for detecting newly synthesized cDNA.
- This enzymatic reaction makes use of a heteropolymer RNA molecule with a 20-bases oligonucleotide primer which is complementary to the RNA sequences close to the 5' end. During the RT reaction a complete cDNA strand is produced.
- cDNA is hybridized with two different oligonucleotide probes, the capture and the detection probe, respectively.
- the capture probe is used for binding cDNA to wells in a microtiter plate.
- the detection probe is conjugated to horseradish peroxydase, resulting in a colorimetric reaction.
- a first one is aimed at ascertaining the presence or absence of reverse transcriptase activity (i.e. a qualitative assay), whereas the second one is aimed at measuring the reverse transcriptase activity of the chimeric molecule of the invention (i.e. a quantitative assay) .
- the TitanTM RT-PCR System offers sensitivity and reproducibility in one tube RT-PCR.
- the TitanTM system exploits the thermal stability of AMV reverse transcriptase for the cDNA synthesis step to reduce secondary structure.
- AMV also provides better processivity and fuller length transcripts when compared to RNase H negative M-MuLV reverse transcriptase mutants .
- the Titan system uses the enhanced fidelity and ' high yield of the Expand High Fidelity enzyme blend.
- Access RT-PCR System is designed for the reverse transcription (RT) and polymerase chain reaction (PCR) amplification of a specific target RNA from either total RNA or mRNA.
- the Retrosys ® is a non-radioactive 96-well microtiter plate reverse transcriptase assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5 ' -triphosphate (BrdUTP) as dNTP.
- the whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase- conjugated anti-BrdU antibody and colorometric reading.
- the assay can be used with various types of cell-culture material. The results are discussed in the Example section.
- Chemiluminescent is another non-radioactive 96-well microtitre plate reverse transcriptase assay, based on the reverse transcriptase mediated synthesis of DNA, using a poly (A) template and oligo (T) primer.
- the reaction mixture contains Digoxigenin- and biotin-labeled TTP as well as unlabeled TTP, all of which are incorporated into the same newly synthesized DNA molecule.
- the incorporated biotin serves to immobilize the DNA onto streptavidin-coated microplates and the incorporated digoxigenin are then specifically detected with Anti- Digoxigenin-antibodies conjugated with a peroxidase.
- inhibitor any reagent, drug or chemical which is able to inhibit a reverse transcriptase activity in vitro or in vivo.
- Such inhibitors can be readily identified using standard screening protocols in which the chimeric molecule of the invention is placed in contact with a potential inhibitor, and the level of reverse transcriptase activity is measured in the presence or absence of the inhibitor, or in the presence of varying amounts of inhibitor. In this way, not only can useful inhibitors be identified, but the optimal level of such an inhibitor can be determined in vitro for further testing in vivo.
- an organic compound When an organic compound is identified, it is used as a "lead” compound.
- the design of mimetics to known pharmaceutically active compounds is a well known approach in the development of pharmaceuticals based on such "lead” compounds.
- Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.
- structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.
- the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such, as in tablets.
- a composition or pharmaceutical composition can be prepared thereof for oral intake, such, as in tablets.
- the compositions, or pharmaceutical compositions, comprising the compounds identified by the screening methods described herein can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal .
- the nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A (ed.), 1980, which is incorporated herein by reference in its entirety.
- the dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
- For treating human or animals between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
- chimeric enzymes containing these or any other TERT active site sequences in combination with sequences from a reverse transcriptase, particularly HIV RT following the method claimed in this patent and exemplified for the HIV-RT/hTERT chimera with the aim of identifying telomerase inhibitors is a minor variation of the method claimed and therefore covered by the claims stated in this patent .
- an alternative chimeric constructs encompassed by the present invention is a chimeric molecule based on HIV-2 reverse transcriptase and human telomerase reverse transcripatese (hTERT) catalytic site.
- HIV-1 RT amino acid residues 1 to 560, SEQ ID NO. 6
- hTERT amino acid residues 1 to 1132, SEQ ID NO. 4
- HIV-1 RT nucleotide coding sequence 2550 - 4229, SEQ ID NO. 5.
- hTERT nucleotide coding sequence 56 - 3454, SEQ ID NO. 3 .
- VDDFLLVTPHLTHAKTFLRTLVRGVPEYGCWNLRKTWNFPVEDEALGGTAFVQMPAHGLF PWCGLLLDTRTLE SEQ ID NO. 7, restriction sites underlined
- a mixed HIV-RT / hTERT coding sequence was synthesized and amplified by standard PCR methods and introduced between the Clal and Ncol sites .
- This coding sequence corresponds to a dipeptide Isoleucin-Aspartate preceding amino acids 626 to 630 from hTERT followed by amino acids 72 to 103 from HIV-1 RT followed by a Glycine and amino acids 707 to 721 from hTERT followed by a Tryptophan with the following sequence: IJ PDGLRKLVDFRELNI-iTQDFWEVQLGIPHPAGLKKNGYFVKvDVTGAYDTIPW (SEQ ID NO. 9; hTERT sequence bold, restriction sites underlined) .
- the chimeric construct thus obtained (encoding the polypeptide sequence of SEQ ID NO. 10, construct 1 in the Table 1 below), was amplified by standard PCR methods and cloned into pET-20b vector (Novagen) using Ndel and HindiII restriction sites, thus obtaining construct 2, SEQ ID NO. 11.
- the new N-terminus contained a hexa-histidine- tag followed by residue 1 of HIV-1 RT.
- the C-terminus contains seven residues (from residue 593 to residue 599 of SEQ ID NO. 11) due to translated restriction sites (i.e.
- the following table 1 represents a first set of constructs of HIV-RT/hTERT chimera constructed (residue numbering according to HIV-1 RT and HIV-1 RT residues bold) :
- the additional hTERT coding sequence was introduced via two synthetic oligonucleotides each containing the coding sequence for 8 amino acids and a Sacl restriction site. Using this set of primers in a standard PCR reaction allowed the amplification of the entire vector except the coding sequence for the 16 modified amino acids. The amplified PCR product was purified using the PCR purification kit (Qiagen) and subsequently digested with Sad restriction enzyme and religated to obtain the modified chimeric construct. The modified chimera thus obtained (SEQ ID NO: 21) is represented as construct no. 12 in the following Table 3.
- the specific reverse transcriptase activity of the modified chimera was 10-30 fold- higher than the original chimera as determined using the Reverse Transcriptase Assay, chemiluminescent (Roche) .
- the original as well as the modified chimera protein were expressed as C-terminal fusions to Glutathion-S-Transferase (GST) using the commercial pGEX-6p2 vector (Amersham Biosciences) .
- GST-fusion system has been demonstrated to increase the amount of soluble protein for proteins that, when expressed alone, are predominantly insoluble. Since the original as well as the modified chimera are insoluble proteins when expressed in E. coli, the GST-fusion system was explored as a potential improvement that could allow the expression of soluble chimera in E. coli.
- the entire coding sequence of the original as well as the modified chimera was inserted into a slightly modified pGEX- 6p2 vector that contained a unique Ndel restriction site 3 base pairs (coding for Leucine) downstream of the BamHI restriction site that marks the first restriction site of the multiple cloning site of pGEX-6p2.
- the two chimeric pET-20b constructs were digested using Ndel and Notl restriction enzymes simultaneously.
- the resulting DNA fragment representing the coding sequence for the chimeric protein was separated using 1% agaraose gel electrophoresis and subsequently excised from the gel and purified using Gel Extraction Kit (Qiagen) .
- the modified pGEX-6p2 vector was digested using the same restriction enzyme combination (i.e. Ndel, Notl) and purified in an identical manner to that described above for the chimeric coding sequence insert .
- the two GST-fusion constructs obtained in this way (constructs nos. 13 and 14 in the following table 3; SEQ ID NOS: 22 and 23, respectively) still retained the amino acid sequence GPLHMKHHHHHHHGP (restriction sites underlined: 2 x Apal , 1 x Ndel; proline residue 1.from chimera shown in bold) after Prescission protease cleavage at the N-terminus of the chimeric enzyme (residues 227 to 241 of SEQ ID No: 22 and 23) .
- both the original and the modified chimeric enzymes are partially soluble and can be purified using Glutathione-Sepharose resin (Amersham- Biosciences) .
- the soluble GST- fused chimeric enzymes have a 10 -fold higher specific activity than the refolded enzymes expressed without GST-fusion .
- cysteine 931 was considered to potentially have an important role for enzyme activity and inhibitor specificity due to its vicinity to the active site. Therefore, the original cysteine residue was reintroduced into the modified GST-chimera protein.
- This single amino acid substitution was carried out by replacement of a Ncol/Xhol restriction fragment from the modified GST-chimera construct no. 14 with a Ncol/Xhol restriction fragment from an intermediate construct based on construct no. 2, which still contained the later two cysteines but had the first two cysteines mutated to serine.
- This intermediate construct was chosen because the Ncol/Xhol restriction fragment of construct no. 2 contains two coding sequences for cysteine (amino acid 896 and 931) while only the latter one was to be mutated.
- the modified GST-chimera cysteine mutant (construct no. 16 in table 4; SEQ ID NO: 25) displayed no difference with respect to expression and purification compared to construct no. 14 on which it was based. Also its enzymatic activity was comparable to the modified GST-chimera expressed with construct no . 14.
- the pET-20 and pGEX-6p2 constructs were used to transform E.coli BL21(DE3) cell line (Novagen) , while the pKK-233 constructs were used to transform E.coli DH5c- cell line (Life Technologies) .
- Freshly transformed bacteria were grown in 2xYT (Life Technologies) broth supplemented with 100 ⁇ g/ml carbenicillin at 37°C to an OD600 of 0.8, induced with 1 mM isopropylthiogalactopyranoside (IPTG) for 2 to 4 hours, harvested by centrifugation at 7000 rpm for 15 minutes after which the pellets were stored at -20°C until use.
- Example 3 Purification of chimera constructs 3.1 Purification of insoluble HIV-RT/hTERT chimera
- the washed white pellet containing the chimera protein was finally homogenized in 10 ml denaturing buffer A (6 M guanidinum hydrochloride in phosphate buffered saline (PBS) pH 7.8) using an electric homogenizer (PBI).
- the homogenized suspension was stirred overnight at room temperature using a magnetic stirrer. After extraction of the protein from the pellet, the suspension was centrifuged at 20.000 rpm for 30 minutes at 4°C. The clear supernatant was loaded on a 1.5 x 10 cm column containing 3 ml of ProBond resin (Invitrogen) previously washed with 10 ml Milli-Q water and equilibrated with 10 ml denaturing buffer A.
- ProBond resin Invitrogen
- DTT was added to the elution fraction from a 1 M stock solution to reach a final concentration of 10 mM DTT.
- the eluted protein was analyzed with 12.5% SDS-PAGE using the
- PhastSystem (Amersham-Pharmacia) .
- the protein was concentrated using Ultrafree ® -15 30K concentrators (Millipore) to a concentration of 3 to 7 mg/ml .
- SteriflipTM (Millipore)
- the protein solution was loaded on a HiLoad ® 26/60 Superdex ® 200 gel filtration column (Amersham- Pharmacia) using an AKTA-Explorer (Amersham-Pharmacia) .
- the GST-fused protein was expressed partially soluble and therefore the supernatent was subsequently loaded on a gravity column containing 5 ml Glutathion-SepharoseTM 4B resin (Amersham- Biosciences) . After loading the lysate the column was washed twice with 10 ml of 50 mM Tris-HCl pH 8.0, 5 mM DTT.
- the protein was eluted with 15ml 50 mM Tris-HCl pH 8.0, 5 mM DTT, 10 mM Glutathione.
- the elution protein was concentrated using 30 kDa cutoff Ultrafree concentrators (Millipore) to reach a final volume of 1 ml .
- the purified protein solution was loaded on a SuperoseTM 12 gel filtration column (Amersham-Pharmacia) using an AKTA-Explorer (Amersham- harmacia) .
- the protein was dialyzed against the refolding buffer or Milli-Q water overnight.
- the protein was reconstituted from the two differently sized subunits p67 and p53 (chimera) or p66 and p51 (HIV-1 RT) , respectively, equimolar amounts of the two independently expressed and purified protein samples were mixed and stirred for 30 minutes prior to refolding.
- the protein was concentrated using Ultrafree ® -15 3OK concentrators (Millipore) .
- Refolded and dialyzed protein samples at various concentrations were used in five different commercial reverse transcriptase assay formats described here below.
- Example 5.1 TitanTM One Tube RT-PCR kit (Roche).
- the TitanTM One Tube RT-PCR kit uses a two step RT-PCR technique where an initial reverse transcriptase reaction is followed by a PCR cycling protocol in the same reaction tube. In the first step a reverse transcriptase generates a complementary DNA product based on a RNA template . During the subsequent PCR cycling step the complementary DNA product is amplified in order to visualize the amplified DNA product on agarose gel containing ethidium bromide .
- the TitanTM One Tube RT-PCR kit contains an enzyme mixture containing AMV reverse transcriptase and ExpandTM High Fidelity System DNA polymerase.
- HIV-RT/hTERT chimera activity was qualitatively assessed using the positive control components provided in the kit (human control RNA: K562 total RNA with MS2 carrier RNA, human /3-actin upstream and downstream primer) .
- HIV- RT/hTERT chimera (construct 2) or HIV-1 RT (construct 8) protein in combination with ExpandTM High Fidelity System DNA polymerase was used in place of the TitanTM RT-PCR enzyme mix (Roche) to all reactions except for the control reactions.
- ExpandTM High Fidelity System DNA polymerase was used without any reverse transcriptase. Otherwise, the preparations of the reactions and the thermocycling protocol were carried out according to the manufacturers specifications .
- RT-PCR reaction products were subjected to electrophoresis on 1% agarose gel (see Figure 2) .
- the amplified 324 bp /3-actin DNA band was visible in the positive control reaction (AMV RT) as well as in the reactions using recombinant HIV-RT/hTERT chimera or recombinant HIV-1 RT, respectively.
- the negative control without reverse transcriptase did not yield any amplified DNA product.
- the Access RT-PCR kit uses the two step RT-PCR technique where an initial reverse transcriptase reaction is followed by a PCR cycling protocol in the same reaction tube.
- a reverse transcriptase generates a complementary DNA product based on a RNA template .
- the complementary DNA product is amplified in order to visualize the amplified DNA product on agarose gel containing ethidium bromide.
- the Access RT-PCR kit contains an enzyme mixture containing AMV reverse transcriptase and Tfl DNA polymerase .
- the RT-PCR reaction products were subjected to electrophoresis on 1% agarose gel.
- the amplified 323 bp kanamycin resistance gene DNA band was visible in the positive control reaction (AMV RT) as well as in the reactions using recombinant HIV-RT/hTERT chimera or recombinant HIV-1 RT, respectively.
- the negative control without reverse transcriptase did not yield any amplified DNA product .
- This colorimetric ELISA type assay allows the quantification of the reverse transcriptase mediated DNA synthesis starting from an oligo (dT) 22 primer and an immobilized poly (A) template by incorporating Bromo- deoxyuridine-triphosphate (BrdUTP) that is subsequently detected with a specific antibody (Henric et al . , [1996] Biotechnol . Appl. Biochem. 23, 95-105).
- the BrdU specific antibody is conjugated to alkaline phosphatase.
- As a substrate for this phosphatase serves p-nitrophenylphosphate which gives rise to an absorbance increase upon cleavage.
- HIV-RT/hTERT chimera construct 2
- HIV-1 RT construct 8
- This chemiluminescent reverse transcriptase assay is based on the reverse transcriptase mediated synthesis of DNA, using a poly (A) template and oligo (T) ⁇ S primer.
- the HIV-RT/hTERT chimera is functionally active as a reverse transcriptase enzyme.
- This type of assay enables the quantification of the enzymatic activity of recombinant HIV- RT/hTERT chimera. Furthermore, it is possible to determine inhibition concentrations (IC 50 ) for potential telomerase inhibitors using this assay type when HIV-RT/hTERT chimera is employed as the active enzyme.
- the reaction mixture contains ratio optimized 3 H-TTP and unlabeled TTP.
- both thymidine species are incorporated by the reverse transcriptase into the same newly synthesized DNA molecule based on a biotinylated oligo (dT) primer.
- the incorporated radioactivity in combination with SPA beads coated with streptavidin is quantified using a microplate reader. This radioactive SPA- type assay allows the quantification of synthesized DNA as a measure for RT activity, HIV-1 RT of known activity serves as an internal standard.
- Example 6 Binding experiments using microcalorimetry A series of compounds, consisting of a nucleoside, a nucleoside analogue, a reverse transcriptase inhibitor and an analogue of a telomerase inhibitor were tested for binding to HIV-RT/hTERT chimera and HIV-1 RT using isothermal titration calorimetry (ITC) . All binding experiments were conducted using HIV-RT/hTERT chimera (construct 3) or HIV-1 RT (construct 8) at 10 °C.
- ITC isothermal titration calorimetry
- TTP (SIGMA) served as a representative nucleoside
- the nucleoside analogue AZT (SIGMA) was used as a known HIV-1 RT inhibitor
- NRTI non-nucleoside reverse transcriptase inhibitor
- R645176 (Aldrich) is a commercially available homologue of the potent telomerase inhibitor BIBR- 1532 (see Damm, EMBO Journal, 2001,20,6958-6968).
- AZT all compounds were tested for binding both HIV-RT/hTERT chimera and HIV-1 RT, respectively. AZT binding was measured only for HIV-RT/hTERT chimera.
- the nucleoside TTP and nucleoside analogue AZT bind HIV- RT/hTERT chimera and HIV-1 RT with similar affinity. This expected behavior is indicating that the active sites of both proteins share a common three-dimensional structure.
- the specific HIV-1 reverse transcriptase inhibitor Nevirapine shows a much higher (1000-fold) affinity for HIV-1 RT as compared to HIV-RT/hTERT chimera.
- Specific HIV-1 RT inhibitors exhibit strongly reduced selectivity for HIV- RT/hTERT chimera as compared to HIV-1 RT because the amino acid composition of the active site of HIV-RT/hTERT chimera is significantly different from HIV-1 RT.
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WO1990010230A1 (en) * | 1989-02-23 | 1990-09-07 | University Of Ottawa | Polypeptide having immunological activity for use as diagnostic reagent and/or vaccine |
WO2001007661A2 (en) * | 1999-07-23 | 2001-02-01 | Gen-Probe Incorporated | Polynucleotide amplification method |
WO2001046408A2 (en) * | 1999-12-23 | 2001-06-28 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Molecular clones with mutated hiv gag/pol, siv gag and siv env genes |
-
2003
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EP0185444A2 (en) * | 1984-10-10 | 1986-06-25 | Centocor, Inc. | Cloning and expression of HTLV-III DNA |
WO1990010230A1 (en) * | 1989-02-23 | 1990-09-07 | University Of Ottawa | Polypeptide having immunological activity for use as diagnostic reagent and/or vaccine |
WO2001007661A2 (en) * | 1999-07-23 | 2001-02-01 | Gen-Probe Incorporated | Polynucleotide amplification method |
WO2001046408A2 (en) * | 1999-12-23 | 2001-06-28 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Molecular clones with mutated hiv gag/pol, siv gag and siv env genes |
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AU2003229666A1 (en) | 2003-11-11 |
AU2003229666A8 (en) | 2003-11-11 |
EP1504090A2 (en) | 2005-02-09 |
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