JP2005524409A - Methods for identifying chimeric reverse transcriptase and telomerase inhibitors - Google Patents

Abstract

【Task】
The present invention provides a nucleic acid molecule encoding a chimeric molecule HIV-RT / hTERT produced based on the catalytic sites of HIV-1 reverse transcriptase (HIV-RT) and human telomerase reverse transcriptase (hTERT). To do. Assays for screening HIV-RT / hTERT polypeptides and compounds that bind to HIV-RT / hTERT and / or compounds that inhibit the reverse transcriptase activity of hTERT are also disclosed.

Description

Field of Invention

  The present invention relates in part to nucleic acid molecules encoding novel chimeric molecules made based on HIV-1 reverse transcriptase (HIV-RT) and human telomerase reverse transcriptase (hTERT) catalytic sites. This new protein is named HIV-RT / hTERT and this terminology is used herein. The present invention also relates to novel polypeptides and assays that screen for compounds that bind to HVI-RT / hTERT and / or inhibit the reverse transcriptase activity of hTERT.

Background of the Invention

  Despite significant effort and investment in research and development over the last decade, cancer is one of the major etiologies. In addition, many patients still die because of metastatic disease. In addition, despite the significant increase in knowledge and understanding of the control mechanisms involved in malignant tumor development, currently available treatments (surgery, radiation, various cytoreductive agents used alone or in combination) And hormone-based drugs) are extremely nonspecific and harmful to patients, resulting in serious side effects such as nausea, vomiting, hair loss, diarrhea, fatigue, and ulcers. These evidences indicate the need for more effective new anticancer therapies.

  In recent years, understanding of the mechanism by which normal cells reach an aging state, that is, the loss of proliferation ability normally experienced by cells in the aging process of cells, has begun to be understood. In this respect, telomerase plays a central role. It is seen.

  Telomerase is used in many eukaryotic cells to fully replicate telomeres, which are chromosomal ends, ie, DNA sequences that repeat in tandem (especially human telomeres are formed by 5′-TTAGGG repeats). It is a required ribonucleoprotein enzyme. Telomerase synthesizes one strand of telomeric DNA using as a template a sequence contained within the RNA component of the enzyme required to add a short repeat (TTAGGG) to the 3 ′ end of the chromosome (Blackburn). 1992, Annu. Rev. Biochem., 61, 113-129). In most human somatic cells, telomerase activity cannot be detected, and telomeres become shorter as cell division continues. Indeed, actively dividing normal cells may lose 50-200 base pairs per cell division due to the discontinuous synthesis of the DNA lagging strand, and ultimately the telomere is short. Become. Recently, cumulative loss of telomeric DNA due to repeated cell division can act as a trigger for cell senescence and aging, and that telomerase control can have important biological implications. Has been proposed as a hypothesis by scientists (Harley 1991, Mutation Research, 256, 271-282). In fact, in the absence of telomerase, it is likely that telomere shortening ultimately causes cellular senescence by various mechanisms. This phenomenon, which is considered to be the cause of aging of cells, has been named “mitotic clock” (Holt et al. Nat. Biotechnol., 1996, 15, 1734-1741).

  In contrast, telomerase has been restored in immortalized cell lines and in over 85% human tumors, thus maintaining a constant telomere length (Shay, JW and Bacchetti, S .; Eur. J. Cancer, 1997, 33, 787-791). For this reason, cancer cells that have telomerase activity and have a malignant phenotype due to loss of cell cycle or growth control, or other genetic damage, do not lose telomeric DNA during cell division. Causes immortalization of cancer cells and death of the patient.

  In fact, it has been demonstrated that inhibiting telomerase shortens telomeres in tumors and leads to an aging phenotype (Feng et al Science, 1995, 269, 1236-1124). In addition, human TERT (Telomerase Reverse Transscriptase, the catalytic subunit of this enzyme) that does not have catalytic ability is expressed in tumor cells to inhibit telomerase activity, thereby shortening telomeres and stopping cell growth. It has recently been shown to be obtained (Hahn et al. Nature Med., 1999, 5, 1164-1170). In addition, peptide-nucleic acid and 2'-O-MeRNA oligomers complementary to the template region of the RNA component of the enzyme can cause telomerase activity inhibition, telomere shortening, and cell death in certain tumor cell lines. Have been reported (Herbert et al. PNAS, 1999, 96, 14276-14281; Shamass et al. Oncogene, 1999, 18, 6191-6200). These data strongly suggest that inhibiting telomerase activity is an innovative, selective and useful method for developing new anticancer agents.

  Cancer cells express telomerase activity, whereas normal human somatic cells are biologically significant levels (ie, levels sufficient to maintain telomere length during many cell divisions) Therefore, compounds that inhibit telomerase activity can be used for the treatment of cancer. In particular, as demonstrated by a great variety of human tumor cell lines and tumors with telomerase activity, compounds that can inhibit telomerase activity are useful for treating many, if not most, malignant tumors. A very general method can be given. In addition, these compounds provide a treatment that differentiates malignant tumor cells from normal cells, avoiding many of the harmful side effects associated with many current chemotherapy regimens that use substances that indiscriminately kill dividing cells. Therefore, it is expected to have high safety and no toxic effect as compared with conventional chemotherapeutic anticancer agents.

  Unfortunately, to date, few such compounds have been identified. This is because, despite the vast amount of attempts to obtain recombinant telomerase preparations with enzymatic activity, this approach has resulted in a minimal amount of impure enzyme. Furthermore, biochemical assays using recombinant enzymes failed to efficiently identify potent telomerase inhibitors.

  Reverse transcriptase activity is also present in human immunodeficiency virus (HIV). Replication of the HVI genome is a series of enzymatic reactions involving two virally encoded enzymes, reverse transcriptase (“HIV-RT”) and integrase, and DNA and RNA polymerases encoded by the host cell. proceed. HIV-RT polymerizes deoxyribonucleotides by using viral RNA as a template, and also uses newly synthesized minus-strand DNA as a template to generate double-stranded DNA, which can also be used as a DNA polymerase. Works. Because of the vital role of HIV-RT that plays when the virus enters the host organism, therapeutic approaches have been based on attempts to inhibit HIV-RT.

  The above discussion clearly shows that there is a current need to identify compounds that act as telomerase inhibitors.

  The present invention solves this problem by providing a bioactive chimera of human telomerase reverse transcriptase (hTERT) catalytic subunit and HIV-1 reverse transcriptase, as described below. This novel chimeric molecule was obtained by replacing a functionally and structurally important domain of the enzyme pocket of HIV-RT with a homologous domain of hTERT. The construction of this functional chimera is based on the juxtaposition and comparison of the amino acid sequences of the catalytic sites of both enzymes and the consideration by the three-dimensional structure. Analysis of these data allows one skilled in the art to make predictions about the functionality of key regions (especially active sites) of these molecules. It is then possible to exchange the related domains of the two enzymes.

  This approach resulted in a functional chimeric enzyme that exhibited reverse transcriptase activity in conventional RT and RT PCR assays. Furthermore, this chimera is a product of E. coli. It can be expressed at high levels in E. coli and can be easily purified.

  Furthermore, obtaining structural information on the active site is essential for optimizing lead compounds based on the structure.

Summary of the Invention

The present invention relates in part to a sequence of SEQ ID NO: 1, 26, 27, 28 or 29 or a fragment thereof, a nucleotide sequence complementary to at least a portion of the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 A nucleotide sequence that is homologous to the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 or a fragment thereof, a nucleotide sequence encoding a polypeptide having the sequence of SEQ ID NO: 2, 21, 22, 24 or 25 or a fragment thereof; An isolated nucleic acid molecule having a nucleotide sequence encoding a polypeptide having an amino acid sequence homologous to the sequence of SEQ ID NO: 2, 21, 22, 24 or 25 or a fragment thereof. The present invention also relates to a recombinant expression vector having any of the above nucleic acid molecules.

  The present invention also relates to a host cell transformed with a recombinant expression vector having any of the above nucleic acid molecules.

  The present invention introduces a recombinant expression vector having any of the above nucleic acid molecules into a compatible host cell, propagates the host cell under conditions suitable for the expression of the polypeptide, and converts the polypeptide from the host cell. To a method for producing a polypeptide comprising the sequence of SEQ ID NO: 2, 21, 22, 24 or 25 or a homologue or fragment thereof.

  The present invention also relates to an isolated polypeptide encoded by any of the above nucleic acid molecules.

  The present invention also relates to a method for identifying a compound that binds to HIV-RT / hTERT by contacting HIV-RT / hTERT with the compound and determining whether the compound binds to HIV-RT / hTERT. Related.

  The present invention 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 to the nucleic acid molecule. It also relates to a method for identifying a compound to be identified.

  The present invention also relates to a method for identifying a compound that inhibits the reverse transcriptase activity of hTERT by contacting HIV-RT / hTERT with the compound and determining whether the reverse transcriptase activity is inhibited.

  The present invention also relates to compounds that inhibit the reverse transcriptase activity of hTERT identified by contacting HIV-RT / hTERT with the compound and determining whether the compound inhibits reverse transcriptase activity.

  These and other aspects of the invention are described in further detail below.

Detailed Description of the Preferred Embodiment

  The invention includes, inter alia, isolated and purified polynucleotides encoding HIV-RT / hTERT or portions thereof, vectors containing these polynucleotides, host cells transformed with these vectors, HIV-RT / Method for producing hTERT, method using the above polynucleotide and vector, isolated and purified HIV-RT / hTERT, compound that binds to HIV-RT / hTERT and / or compound that inhibits the reverse transcriptase activity of hTERT A method for screening is provided.

  Various definitions are given throughout this specification. Many terms have the meaning commonly used by those skilled in the art for the word. Terms specifically defined below or elsewhere in this specification generally have the meaning given in the context of the present invention as commonly understood by one of ordinary skill in the art.

  As used herein, the term “activity” implies binding (including both direct and indirect) that affects the response (ie, has a measurable effect that occurs in response to some exposure or stimulus) or Various measurable indicators to reveal, such as the affinity of a compound that directly binds a polypeptide or polynucleotide of the invention, or upstream or downstream proteins or other similarities that have occurred, for example, after some stimulus or phenomenon Means the measured value of the amount of function.

  As used herein, the term “binding” refers to a physical or chemical interaction that occurs between two proteins or compounds or associated proteins or compounds or combinations thereof.

  Bonds include ionic, non-ionic, hydrogen bonds, van der Waals, hydrophobic interactions, and the like. Physical interaction, binding can be either direct or indirect, and indirect interaction occurs through or by the effect of another protein or compound. By direct binding is meant an interaction that does not occur through or due to the effect of another protein or compound, but without other substantial chemical intermediates.

  As used herein, the term “compound” means any identifiable chemical or molecule, including but not limited to small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids. Rather, such compounds can be natural or synthetic.

  The term “complementary” as used herein means that a Watson-Crick base pair is formed between nucleotide units of a nucleic acid molecule.

  As used herein, the term “contact” means that a compound is in direct physical proximity, either directly or indirectly, to a polypeptide or polynucleotide of the invention. The polypeptide or polynucleotide can be in any number of buffers, salts, solutions, and the like. For example, the compound may be placed in a beaker, a microtiter plate, a cell culture flask, or a microarray (such as a gene chip) containing a nucleic acid molecule or a polypeptide encoding HIV-RT / hTERT or a fragment thereof. Included in contact.

  As used herein, the term “homologous nucleotide sequence” or “homologous amino acid sequence”, or modified examples thereof, refer to the entire sequence of SEQ ID NO: 1, 26, 27, 28, or 29, or the coding Against at least part of the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 encoding the functional domain of the polypeptide to be processed, or against the sequence of SEQ ID NO: 2, 21, 22, 24, Having at least about 60% homology at the nucleotide or amino acid level, 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%. Means the characteristic sequence. Percent homology is determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version) using the algorithm of Smith and Waterman (Adv. Appl. MAT., 1981, 2, 482-489, which is incorporated by reference in its entirety). 8 for Unix, Genetics Computer Group, University Research Park, Madison WI) with default settings. Homologous amino acid sequences can include amino acid sequences that encode conservative amino acid substitutions in the sequence of SEQ ID NO: 2, 21, 22, 24, or 25. Conservative substitutions are well known in the art, eg, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glutamine to asparagine; 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; valine to isoleucine or leucine It is included.

  In giving such changes, the hydropathic index of amino acids can be examined. It is generally understood in the art that the hydropathic amino acid index is important in conferring interacting biological functions on proteins (Kyte and Doolittle, 1982). The relative hydropathic properties of the amino acids contribute to the secondary structure of the resulting protein, and then with other molecules of the protein (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.) It is supposed to define the interaction.

  Each amino acid is given a hydropathic index based on their hydrophobicity and charge properties (Kyte and Doolittle, 1982). The hydropathic index of each amino acid is as follows. Isoleucine (+4.5); valine (+4.2); leucine (+3.8); fanylalanine (+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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); Arginine (-4.5).

  An amino acid can be replaced by another amino acid with a similar hydropathic index or score, and still have a protein with similar biological activity, i.e., a biofunctionally equivalent protein This is known in the art. When giving such changes, amino acid substitutions with a hydropathic index within ± 2 are preferred, amino acids with a hydropathic index within ± 1 are more preferred, and amino acids with a hydropathic index within ± 0.5 are more preferred. Should.

  It is also understood in the art that similar amino acid substitutions can be made effectively based on hydrophilicity. US Pat. No. 4,554,101 (incorporated by reference) states that the maximum local average hydrophilicity of a protein governed by the hydrophilicity of adjacent amino acids correlates with the biological properties of the protein. ing. As detailed in US Pat. No. 4,554,101, amino acid residues have been assigned the following hydrophilicity values: Arginine (+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 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); fanylalanine (-2.5); Tryptophan (-3.4).

  It is understood that one amino acid can be replaced with another amino acid having a similar hydrophilicity value to obtain a biologically equivalent and immunologically equivalent protein. When giving such a change, it is preferable to substitute an amino acid having a hydrophilicity value of ± 2 or less, particularly preferably a hydrophilicity value of ± 1 or less, more preferably a hydrophilicity value of ± 0.5 or less. Amino acids should be substituted.

  The term “homologous nucleotide sequence” or “homologous amino acid sequence” also includes variants of the nucleotide and amino acid sequences reported herein. In particular, “variant” means a nucleotide sequence that encodes a chimeric enzyme of the invention or a fragment thereof having substantially the same biological properties as a polypeptide encoded by the nucleotide sequence described herein. To do. Typically, a variant of the chimeric molecule described herein consists of a telomerase reverse transcriptase (TERT) from a non-human organism and a reverse transcriptase from an organism other than HIV-1. Indeed, since there is a high degree of sequence homology between the active sites of TERT from various organisms, for example, between the reverse transcriptase domains of the HIV family, those skilled in the art will be described herein. The present teachings could be used to create a variety of chimeric molecules based on the same concept and based on the present invention. In particular, the use of the reverse transcriptase domain of HIV-2 is to be considered within the scope of the present invention since it is included in the definition of variants provided herein.

  As used herein, the term “isolated” nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment. Examples of isolated nucleic acid molecules include recombinant DNA molecules contained in vectors, recombinant DNA molecules retained in heterologous host cells, partially or substantially purified nucleic acid molecules, and synthesis Examples include, but are not limited to, DNA or RNA molecules.

  As used herein, the term “inhibit” means a reduction in the amount, quality, or effect of an activity or protein.

  As used herein, the term “oligonucleotide” refers to a linked series of nucleotide residues having a sufficient number of bases for use in polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and amplifies the presence of identical, similar, or complementary DNA or RNA in a cell or tissue. Used to confirm, clarify. Oligonucleotides have at least about 10 nucleotides and a maximum of about 50 nucleotides, preferably about 15 to 30 nucleotides, and constitute part of a DNA sequence. Oligonucleotides can be chemically synthesized and used as probes.

  As used herein, the term “probe” means a nucleic acid sequence that can vary in length, depending on the application (preferably at least about 10 and up to about 6,000 nucleotides). Probes are used for the detection of identical, similar or complementary nucleic acid sequences. Longer probes are usually obtained from natural or recombinant sources, are more specific than oligomers, and are much slower to hybridize. Shorter probes can be synthesized chemically. Probes can be single-stranded or double-stranded and are carefully designed to exhibit specificity with techniques such as PCR, hybridization, membrane-based techniques, or ELISA.

  As used herein, the term “stringent hybridization conditions” or “stringent conditions” refers to the hybridization of one probe, primer, or oligonucleotide to its target sequence but not to other sequences. Refers to a condition that does not.

Stringent conditions will vary with the sequence and will vary with different ambient conditions. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ° C. lower than the thermal melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Tm is a temperature (under a predetermined ionic strength, pH, and nucleic acid concentration) at which 50% of probes complementary to the target sequence hybridize to the target sequence in an equilibrium state. Since the target sequence is generally present in excess, at Tm, 50% of the probe is occupied in equilibrium. Stringent conditions are pH 7.0 to 8.3, sodium ion with a salt concentration of less than about 1.0M, typically about 0.01 to 1.0M sodium ion (or other salt). For short probes, primers, or oligonucleotides (eg, 10-50 nucleotides), the temperature is typically at least about 30 ° C., and for longer probes, primers, or oligonucleotides, conditions are typically at least about 60 ° C. Will. Stringent conditions can also be created with the addition of destabilizing substances such as formamide.

  One form of the invention relates to nucleic acid molecules having a novel nucleotide sequence encoding the chimeric molecule HIV-RT / hTERT. The nucleic acid molecule is preferably either RNA or DNA, but can also contain both RNA and DNA monomers or peptide nucleic acid monomers. Nucleic acid molecules can be single-stranded or double-stranded. Monomeric nucleic acid molecules can be linked via, for example, conventional phosphodiester bonds such as phosphorothioate bonds or modified bonds. Furthermore, the sugar moiety of the monomer can be modified, for example, by the addition of 2 'substitutions that serve to confer nuclease resistance and / or cellular uptake.

  In a preferred embodiment of the invention, the nucleic acid molecule has the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 encoding the HIV-RT / hTERT chimeric protein.

  Alternatively, the nucleic acid molecule has a fragment of the sequence of SEQ ID NO: 1, 26, 27, 28 or 29. Preferably, 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 About 501 to about 600 nucleotides, about 601 to about 700 nucleotides, about 701 to about 800 nucleotides, about 801 to about 900 nucleotides, about 901 to about 1000 nucleotides, about 1001 to about 1100 nucleotides, about 1101 to about 1200 nucleotides, About 1201 to about 1300 nucleotides, about 1301 to about 1400 nucleotides, about 1401 to about 1500, about 1501 to about 1600, about 1601 to about 1700, about 1701 Stone about 1728, and any combination thereof. The fragment can be located within any portion of the sequence of SEQ ID NO: 1, 26, 27, 28 or 29.

  In another preferred embodiment of the invention, the nucleic acid molecule has a nucleotide sequence that is complementary to at least a portion of the sequence of SEQ ID NO: 1, 26, 27, 28, or 29. Preferably, the nucleic acid molecule has a nucleotide sequence complementary to the entire sequence set forth in SEQ ID NO: 1, 26, 27, 28 or 29. Alternatively, the nucleic acid molecule has a nucleotide sequence that is complementary to a portion of the sequence of SEQ ID NO: 1, 26, 27, 28, or 29 (ie, complementary to any of the above fragments). A nucleotide sequence complementary to at least a portion of the sequence of SEQ ID NO: 1, 26, 27, 28, or 29 includes, for example, at least a portion of SEQ ID NO: 1, 26, 27, 28, or 29 under stringent hybridization conditions. Oligonucleotides that hybridize to are included. Preferred oligonucleotides have at least about 10 nucleotides and a maximum of about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and can be used as probes, primers, and antisense agents.

  In another preferred embodiment of the invention, the nucleic acid molecule has a nucleotide sequence that is homologous to the sequence of SEQ ID NO: 1, 26, 27, 28 or 29. Preferably, the nucleotide sequence is at least about 60% homologous, more preferably at least about 70% homologous, more preferably at least about 80%, relative to the entire sequence of SEQ ID NO: 1, 26, 27, 28 or 29. Homology, more preferably at least about 90% homology, most preferably about 95% homology. Further, the nucleotide sequence homologous to the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 is a fragment of the nucleotide sequence having the above-mentioned length and homologous to the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 Is also included. Homologous sequences include variant sequences as described above.

  In another preferred embodiment of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having the sequence of SEQ ID NO: 2, 21, 22, 24 or 25. Said nucleic acid molecule preferably comprises the sequence of SEQ ID NO: 1, 26, 27, 28 or 29 containing codon substitutions reflecting the degeneracy of the genetic code. As is well known in the art, due to the degeneracy of the genetic code, other DNA and RNA capable of encoding the same polypeptide as the polypeptide encoded by SEQ ID NO: 1, 26, 27, 28 or 29 There are many. The present invention thus also envisions such other DNA and RNA molecules that, when expressed, encode the polypeptide of SEQ ID NO: 2, 21, 22, 24 or 25. DNA and RNA molecules other than those specifically disclosed herein, which are simply characterized by changes in the codons of certain amino acids, are also within the scope of the present invention.

  Once the sequence of amino acid residues encoded by the HIV-RT / hTERT nucleic acid sequence is known, knowledge of all triplet codons for each amino acid residue is used to describe all such encoding RNA and DNA sequences. It becomes possible to do. DNA and RNA molecules other than those specifically disclosed herein, which are simply characterized by changes in the codons of certain amino acids, are also within the scope of the present invention.

本分野において周知であるように、コドンとは、ｍＲＮＡ分子中のヌクレオチドのトリプレット配列であり、このため、（ＤＮＡ分子中に存在する）塩基チミジン（Ｔ）が塩基ウラシル（Ｕ）に置き換えられていることを特徴とする。ポリヌクレオチド内の同一のアミノ酸残基に対するコドンに単純な変化が生じても、コードされるポリペプチドの配列又は構造は変化しないものと思われる。 A table of amino acids and their typical abbreviations, symbols, and codons is set forth in Table 1 below.

As is well known in the art, a codon is a triplet sequence of nucleotides in an mRNA molecule so that the base thymidine (T) (present in the DNA molecule) is replaced with the base uracil (U). It is characterized by being. A simple change in codons for the same amino acid residue in a polynucleotide will not change the sequence or structure of the encoded polypeptide.

  Alternatively, the nucleic acid molecule has a nucleotide sequence that encodes a fragment of a polypeptide that encodes the sequence of SEQ ID NO: 2, 21, 22, 24, or 25. Preferably, the fragment comprises about 5 to about 20 contiguous amino acids, about 21 to about 40 contiguous amino acids, about 41 to about 60 contiguous amino acids, about 61 to 80 contiguous amino acids, about 81 to about 100 contiguous amino acids, about 101 to about 120 contiguous amino acids, about 121 to about 140 contiguous amino acids, about 141 to about 160 contiguous amino acids, about 161 to about 180 contiguous amino acids, about 181 to about 200 contiguous amino acids, about 201 to about 220 contiguous amino acids, about 221 to about 240 contiguous amino acids, about 241 to about 260 contiguous amino acids, about 261 to about 280 contiguous amino acids, about 281 to about 300 consecutive amino acids, about 301 to about 320 consecutive amino acids Acid, about 321 to about 340 consecutive amino acids, about 341 to about 360 consecutive amino acids, about 361 to about 380 consecutive amino acids, about 381 to about 400 consecutive amino acids, about 401 to about 420 consecutive Amino acids, about 421 to about 440 consecutive amino acids, about 441 to about 460 consecutive amino acids, about 461 to about 480 consecutive amino acids, about 481 to about 500 consecutive amino acids, about 501 to about 520 consecutive Amino acids, about 521 to about 540 consecutive amino acids, about 541 to about 560 consecutive amino acids, about 561 to about 576 consecutive amino acids, and any combination thereof. The fragment can be located within any portion of the sequence of SEQ ID NO: 2, 21, 22, 24 or 25.

  In another preferred embodiment of the invention, the nucleic acid molecule has a nucleotide sequence encoding a polypeptide having an amino acid sequence homologous to SEQ ID NO: 2, 21, 22, 24 or 25. Alternatively, the nucleic acid molecule has a nucleotide sequence encoding a fragment of a polypeptide having an amino acid sequence homologous to the sequence of SEQ ID NO: 2, 21, 22, 24 or 25. Preferably, the fragment comprises from about 5 to about 20 consecutive amino acids, from about 21 to 40 consecutive amino acids, from about 41 to about 60 consecutive amino acids, from about 61 to 80 consecutive amino acids, from about 81 to about 100 About 101 to about 120 amino acids, about 121 to about 140 consecutive amino acids, about 141 to about 160 consecutive amino acids, about 161 to about 180 consecutive amino acids, about 181 to about 200 consecutive About 201 to about 220 consecutive amino acids, about 221 to about 240 consecutive amino acids, about 241 to about 260 consecutive amino acids, about 261 to about 280 consecutive amino acids, about 281 to about 300 consecutive About 301 to about 320 contiguous amino acids, about 3 1 to about 340 consecutive amino acids, about 341 to about 360 consecutive amino acids, about 361 to about 380 consecutive amino acids, about 381 to about 400 consecutive amino acids, about 401 to about 420 consecutive amino acids, about 421 to about 440 consecutive amino acids, about 441 to about 460 consecutive amino acids, about 461 to about 480 consecutive amino acids, about 481 to about 500 consecutive amino acids, about 501 to about 520 consecutive amino acids, about 521 to about 540 consecutive amino acids, about 541 to about 560 consecutive amino acids, about 561 to about 576 consecutive amino acids, and any combination thereof. The fragment can be located within any portion of the sequence of SEQ ID NO: 2, 21, 22, 24 or 25.

  In another embodiment of the invention, the nucleic acid molecule has one or more conservative substitutions, non-conservative substitutions, or both amino acid substitutions made to the amino acid sequence of SEQ ID NO: 2, 21, 22, 24, or 25. Having a nucleotide sequence encoding a polypeptide having a defined amino acid sequence. For example, the amino acid substitution is amino acid numbers 107, 110, 115, 177, 179, 181, 182, 193, 194, 196, 197, 200, 201, 203, 204, 212, 213, 214, of the sequence of SEQ ID NO: 2. 215, 216, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 244, 253, and 256. One or more of these amino acid positions may be substituted.

  Another aspect of the present invention relates to a vector comprising any of the above nucleic acid molecules or a recombinant expression vector. Here, the vector is used to amplify DNA or RNA encoding HIV-RT / hTERT and / or to express DNA encoding HIV-RT / hTERT. Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (ie, fragments that can be integrated into the host genome by homologous recombination). It is not something. Preferred viral particles include, but are not limited to, adenovirus, parvovirus, herpes virus, pox virus, adeno-associated virus, Semliki Forest virus, vaccinia virus, and retrovirus. 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 pcDNA3 (Invitrogen), pSVL (Pharmacia Biotech), pSPORT vector, pGEM vector (Promega), pPROEX vector (LTI, Bethesda, MD), Bluescript vector (Stratagene), pQE vector, QQE vector, iQ (Invitrogen), and pYES2 (Invitrogen) and various Gateway expression plasmids (Life Technologies), but are not limited to these.

  A preferred expression vector is a replicable DNA wherein the DNA sequence encoding HIV-RT / hTERT is operably linked to appropriate regulatory sequences capable of effecting expression of HIV-RT / hTERT in an appropriate host. Is a construct. A DNA region is said to be operably linked if the DNA regions have a functional relationship to each other. For example, a promoter is operably linked to a coding sequence if the promoter regulates the transcription of the sequence. An amplification vector does not require an expression regulatory domain, and only requires a selection gene that facilitates replication in the host (usually conferred by the origin of replication) and recognition of the transformant. The need for introducing regulatory sequences into the expression vector may vary depending upon the host selected and the transformation method chosen. In general, regulatory sequences include transcriptional promoters, operator sequences that may be present to regulate transcription, sequences encoding appropriate mRNA ribosome binding, and sequences that regulate the termination of transcription and translation.

  Preferred vectors preferably contain a promoter that is recognized by the host organism. The promoter sequence of the present invention may be any of prokaryotes, eukaryotes or viruses. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage λ (The bacteria labmba, Hershey, AD, Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), the entire contents of Incorporated herein by reference), λII (Hendrix, RW, Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1980), the entire contents of which are incorporated herein by reference. ), Trp, recA, heat shock, E.I. E. coli lacZ promoter, SV40 early promoter (Benoist, et al. Nature, 1981, 290, 304-310, the entire contents of which are incorporated herein by reference). Other promoters include mouse mammary tumor virus promoter, human immunodeficiency virus promoter terminal repeat, 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 metallothionein promoter, but are not limited thereto.

  Other regulatory sequences can also be introduced into preferred vectors. Preferred examples of suitable regulatory sequences are the phage MS-2 replicase gene and the bacteriophage lambda gene cII Shine-Dalgarno. The Shine-Dalgarno sequence can be directly postfixed with DNA encoding HIV-RT / hTERT to express mature HIV-RT / hTERT protein.

  Furthermore, appropriate markers that allow screening of transformed host cells can be introduced into appropriate expression vectors. Transformation of a host is well known to those skilled in the art and is performed using any one of a variety of techniques described in Sambrook et al.

  The origin of replication can also be provided by the construction of a vector for introducing an exogenous origin or by the chromosomal replication mechanism of the host cell. The latter may be sufficient if the vector is integrated into the host cell chromosome. Alternatively, those skilled in the art can transform mammalian cells by a method of co-transforming a selectable marker and HIV-RT / hTERT DNA without using a vector containing a viral origin of replication. Examples of suitable markers are dihydrofolate reductase (DHFR) or thymidine kinase (see US Pat. No. 4,399,216).

  Nucleotide sequences encoding HIV-RT / hTERT are blunt or cohesive termini for ligation, restriction enzyme digestion to give the proper terminator, cohesive end filling if appropriate, undesired ligation Can be recombined with the vector DNA by conventional techniques such as alkaline phosphatase treatment to avoid ligation and ligation using an appropriate ligase. Techniques for such operations are disclosed in the above Sambrook et al. And are well known in the art. Methods for constructing mammalian expression vectors are described in, for example, Okayama et al. , Mol. Cell. Biol. , 1983, 3, 280, Cosman et al. , Mol. Immunol. , 1986, 23, 935, Cosman et al. , Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, the entire contents of which are incorporated herein by reference.

  Another aspect of the present invention relates to a transformed host cell having an expression vector comprising any of the above nucleic acid molecules. The nucleotide sequence is expressed when the expression vector is introduced into a suitable host cell. Vector introduction into host cells includes calcium phosphate transfection, DEAE-dextran transfection, transfection, microinjection, cationic lipid transfection, electroporation, transduction, scrape loading , By ballistic introduction, infection or other methods. Such a method is described in “Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: A LABOROR MAND, Lold Ed. N. Y. (1989) "is described in many standard laboratory manuals.

  Suitable host cells for expressing the polypeptides of the present invention include, but are not limited to, prokaryotes, yeasts, and eukaryotes. If a prokaryotic expression vector is used, any prokaryotic cell capable of expressing the cloned sequence will be suitable as a host cell. Suitable prokaryotic cells include, but are not limited to, bacteria of the genus Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, Staphylococcus. In a preferred embodiment of the invention, said prokaryotic host is E. coli. Coli. If a eukaryotic expression vector is used, any eukaryotic cell capable of expressing the cloned sequence will be suitable as a host cell. Preferably, the eukaryotic cell is a higher eukaryotic cell. 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 mouse 3T3 fibroblasts. It is not a thing. Propagating such cells in cell culture has become a routine operation (see Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973), the entire contents of which are incorporated herein by reference. ).

  Furthermore, yeast hosts can be used as host cells. Preferred yeast cells include, but are not limited to, Saccharomyces, Pichia, and Kluberomyces. Preferred yeast hosts are S. cerevisiae. cerevisiae and P. cerevisiae. pastoris. Preferred yeast vectors can contain an origin of replication sequence derived from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence, and a selectable marker gene. Yeast and E. coli. This includes shuttle vectors for replication in both E. coli.

Alternatively, insect cells can be used as host cells. In a preferred embodiment, the polypeptides of the present invention are expressed using a baculovirus expression system (Luckow et al., Bio / Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Relly). al. (Eds.), WH Freeman and Company, New York, 1992, and US Pat. No. 4,879,236, the entire contents of which are incorporated herein by reference). Furthermore, the MAXBAC ^™ complete baculovirus expression system (Invitrogen) can be used, for example, for production in insect cells.

  Another aspect of the invention relates to an isolated polypeptide encoded by the nucleic acid molecule. In a preferred embodiment of the invention, the isolated polypeptide has the amino acid sequence set forth in SEQ ID NO: 2, 21, 22, 24 or 25. Alternatively, the polypeptide is a fragment of the polypeptide of SEQ ID NO: 2, 21, 22, 24 or 25. Preferably, the fragment comprises about 5 to about 20 contiguous amino acids, about 21 to 40 contiguous amino acids, about 41 to about 60 contiguous amino acids, about 61 to 80 contiguous amino acids, about 81 to about 100 About 101 to about 120 contiguous amino acids, about 121 to about 140 contiguous amino acids, about 141 to about 160 contiguous amino acids, about 161 to about 180 contiguous amino acids, about 181 to about 200 About 201 to about 220 contiguous amino acids, about 221 to about 240 contiguous amino acids, about 241 to about 260 contiguous amino acids, about 261 to about 280 contiguous amino acids, about 281 to about 300 About 301 to about 320 consecutive amino acids Acid, about 321 to about 340 consecutive amino acids, about 341 to about 360 consecutive amino acids, about 361 to about 380 consecutive amino acids, about 381 to about 400 consecutive amino acids, about 401 to about 420 consecutive Amino acids, about 421 to about 440 consecutive amino acids, about 441 to about 460 consecutive amino acids, about 461 to about 480 consecutive amino acids, about 481 to about 500 consecutive amino acids, about 501 to about 520 consecutive Amino acids, about 521 to about 540 consecutive amino acids, about 541 to about 560 consecutive amino acids, about 561 to about 576 consecutive amino acids, and any combination thereof. Fragments can be placed within any portion of the sequence of SEQ ID NO: 2, 21, 22, 24 or 25.

  In another preferred embodiment of the invention, the polypeptide has an amino acid sequence homologous to the sequence SEQ ID NO: 2, 21, 22, 24 or 25 or a fragment thereof as described above. It should be understood that proteins that are homologous to the polypeptides encoded by the nucleotide sequences described herein and have substantially the same biological properties, ie variants, are also encompassed by the present invention. Variant forms may be characterized, for example, by amino acid insertions, deletions or substitutions. In this regard, at least about 70% sequence homology, at least about 80% sequence homology, preferably about 90% sequence homology with the sequence of SEQ ID NO: 2, 21, 22, 24 or 25, more preferably about Variant forms having amino acid sequences with 95% sequence homology, most preferably about 98% sequence homology are contemplated to be encompassed by the present invention. Preferred homologous polypeptides have at least one conservative amino acid substitution compared to SEQ ID NO: 2, 21, 22, 24 or 25. An amino acid “insertion”, “substitution” or “deletion” is a change relative to or within an amino acid sequence. Permissible changes to an amino acid sequence can be made synthetically or systematically by peptide insertion, deletion or substitution of nucleotides present in the HIV-RT / hTERT nucleic acid sequence using recombinant DNA technology. This can be determined experimentally. Modification of a naturally occurring amino acid sequence can be performed by any of a number of known techniques. For example, 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), US Pat. Nos. 4,518,584 and 4,737,462 (the entire contents of each reference are incorporated herein by reference). Mutations can be introduced at specific sites in a polynucleotide encoding a polypeptide by procedures well known to those skilled in the art, such as oligonucleotide-directed mutagenesis.

  The HIV-RT / hTERT variants of the invention will preferably exhibit substantially the same biological activity as the naturally occurring HIV-RT / hTERT polypeptide. “Shows substantially the same biological activity as a naturally occurring HIV-RT / hTERT polypeptide” may have a conservatively substituted sequence of an HIV-RT / hTERT variant within the scope of the present invention. That is, one or more amino acid residues of the 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 substituting amino acids with residues having similar physicochemical properties, as described above. Other HIV-RT / hTERT variants that can retain substantially the same biological activity as HIV-RT / hTERT are variants in which amino acid substitutions are made in regions outside the functional region of the chimera.

  Other preferred variants of the chimeric molecules described herein consist of telomerase reverse transcriptase (TERT) from non-human organisms and reverse transcriptase from organisms other than HIV-1. The chimeric molecule hTERT / HIV-2 reverse transcriptase is a specific example of such a variant.

  The polypeptide to be expressed in the host cell may be a fusion protein comprising a region derived from a heterologous protein. Such a region can be introduced, for example, to allow secretion, to improve the stability of the polypeptide, or to facilitate purification of the polypeptide. For example, a sequence encoding an appropriate signal peptide can be introduced into an expression vector. The signal peptide DNA sequence (secretory leader) can be fused in-frame to the polynucleotide sequence so that the polypeptide is translated as a fusion protein with the signal peptide. A signal peptide that exerts a function in the target host cell promotes extracellular secretion of the polypeptide. The signal sequence will preferably be cleaved from the polypeptide when the polypeptide is secreted from the cell. Thus, a fusion protein in which the N-terminus of HIV-RT / hTERT is fused to a carrier peptide can be prepared.

  Many of the available peptides used for such functions allow the fusion protein to selectively bind to the binding pair. Suitable binding pairs include glutathione-S-transferase (GST) domains that are readily and uniformly purified by affinity chromatography on an immobilized glutathione column. The protein can then be used directly with the GST domain attached. Alternatively, the GST fusion protein is constructed so as to have a protease cleavage site between the GST domain and the protein so that the GST domain is completely removed by digestion with a protease such as thrombin or blood coagulation factor Xa.

  Alternatively, many vectors have the advantage of carrying an extension of histidine residues that can be expressed at the N-terminus or C-terminus of the target protein. Thus, the target protein can be recovered by metal chelate chromatography. Nucleotide sequences encoding restriction sites for enterokinase, factor X, or proteolytic enzymes such as procollagenase or thrombin can be used to cleave the fusion protein to yield a mature HIV-RT / hTERT protein. -Can be placed immediately before the RT / hTERT sequence. Other examples of fusion pairs include yeast factor I, honeybee melatin leader in sf9 insect cells, 6-His tag, thioredoxin tag, hemagglutinin tag, protein A IgG binding domain, OmpA signal sequence tag, It is not limited to these. As will be appreciated by those skilled in the art, binding pairs that recognize and bind peptides include metal ions (eg, metal affinity columns), antibodies or fragments thereof, any protein or peptide that binds peptides (FLAG tag). Etc.) can be any molecule or compound.

  Alternatively, the polypeptide may be E. coli without providing a signal sequence. It can also be expressed in E. coli. In this case, the polypeptide is not secreted into the medium and can be purified from the cytosol as an insoluble inclusion body. We have described HIV-RT / hTERT as E. coli as described in the Examples section. It was successfully expressed at an extremely high level (100 mg / liter) as an insoluble inclusion body in Coli. The chimeric molecule thus obtained can be purified using protein purification techniques well known to those skilled in the art. In one stage, these techniques use a crude fraction that separates the cellular environment into a peptide and a non-peptide fraction. After separating a protein from another protein, the protein of interest can be further purified using chromatography or electrophoresis techniques to achieve partial or complete purification (ie, purification to a uniform state). Particularly suitable analytical methods for preparing pure peptides are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, isoelectric focusing. A particularly efficient method for purifying peptides is FPLC (fast protein liquid chromatography), sometimes HPLC.

  In general, "purified" refers to an enzyme preparation that has been subjected to fractionation to remove various other components and that substantially retains its biological activity in which the composition is expressed. I will. When using the term “substantially purified”, the notation is about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% of the protein present in the composition. % Or more, about 95% or more, the preparation in which the enzyme constitutes the main component of the composition.

  In light of this disclosure, various methods for quantifying the purity of an enzyme will be known to those skilled in the art. These include, for example, measuring the specific activity of the active fraction or assessing the amount of polypeptide within the fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a fraction is to determine the specific activity of the fraction, compare it to the specific activity of the original extract, and evaluate the purity (in this specification by “purification factor”). Is to seek. The units actually used to represent the magnitude of activity will, of course, vary depending on the assay technique selected for the purification and whether the expressed protein or peptide exhibits detectable activity. Let's go.

  Various techniques suitable for use in protein purification will be well known to those skilled in the art. These include, for example, ammonium sulfate fractionation, PEG, using an antibody or the like or precipitation by heat denaturation, followed by centrifugation, ion exchange, gel filtration, reverse phase, hydroxylapatite, chromatography step such as affinity chromatography, It includes performing isoelectric focusing, gel electrophoresis, a combination of these and other techniques. As is generally known in the art, the order in which the various purification steps are performed can be altered, or some steps can be omitted and substantially purified protein Or it seems to be still a suitable method for preparing peptides.

  It is known that the mobility of polypeptides can change (sometimes significantly) when the SDS / PAGE conditions are different (Capaldi et al., 1977). Thus, it will be appreciated that under different electrophoresis conditions, the apparent molecular weight of a purified or partially purified expression product may vary.

  High performance liquid chromatography (HPLC) is characterized by excellent peak resolution and very rapid separation. This is achieved by using very fine particles and high pressure to maintain a sufficient flow rate. Separation can be performed in minutes or at most about 1 hour. Furthermore, since the particles are very small and closely packed so that the void volume accounts for a small percentage of the bed volume, only a very small amount of sample is required. Further, since the band is narrow and the sample hardly dilutes, the concentration of the sample may not be so high.

  Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography based on molecular size. The theory underlying gel chromatography is that a column made of fine particles of inert material containing small pores allows molecules to pass through or around the pores, depending on the size of the molecule. For this purpose, large molecules and small molecules are separated. The only factor that determines the flow rate is size, unless the material that makes up the particles adsorbs molecules. For this reason, as long as the shape is relatively constant, the molecules are eluted from the column so that the size decreases sequentially. Gel chromatography is very good at separating molecules of different sizes because the separation does not depend on any other factors such as pH, ionic strength, temperature. Furthermore, since there is virtually no adsorption and there is little zone spread, the elution volume is simply correlated with molecular weight.

  Affinity chromatography is a chromatographic technique based on the specific affinity between the substance to be isolated and the molecule to which it can specifically bind. This is a receptor-ligand type interaction. The column material is synthesized by covalently bonding one of the binding pairs to an insoluble matrix. The column material can then specifically adsorb the substance from the solution. Elution is performed by changing to conditions that do not cause binding (changing pH, ionic strength, temperature, etc.).

  We have established an efficient purification protocol that performs gel filtration after affinity chromatography, as specifically described in the experimental section. Such a purification protocol yields a dimeric chimeric enzyme. The purified enzyme preparation is refolded by dilution and the regenerated chimera is dialyzed and concentrated.

  Depending on the organism that expresses HIV-RT / hTERT, the chimeric enzyme may be subjected to other post-translational modifications such as glycosylation, acylation, sialylation and the like. HIV-RT / hTERT expressed in yeast, insect, or mammalian expression systems may then be similar or significantly different in molecular weight and glycosylation pattern from HIV-RT / hTERT polypeptide. In some cases. Of course, expression of HIV-RT / hTERT in a bacterial expression system will result in unglycosylated HIV-RT / hTERT.

  The following embodiments of the invention relate to several methods using the polypeptides and nucleic acids of the invention.

  One aspect of the invention is a method for identifying a compound that binds to either HIV-RT / hTERT or a nucleic acid molecule encoding HIV-RT / hTERT, comprising HIV-RT / hTERT or HIV-RT / hTERT. A method comprising: contacting a nucleic acid molecule encoding a compound with the compound; and determining whether the compound binds a nucleic acid molecule encoding HIV-RT / hTERT or HIV-RT / hTERT. To do. Binding to the chimeric protein can be measured by an isothermal titration calorimeter (ITC). In this method, the heat of binding released when the compound is bound to the protein can be directly measured by compensating for thermal changes in a temperature-controlled reaction vessel. This method is representative as a useful tool for evaluating the binding specificity and selectivity of a test compound to an HIV-RT / hTERT chimera, and provides an important index for screening telomerase inhibitors. give.

  Alternatively, gel shift assays, western blots, radiolabeled competition assays, expression cloning using phage, co-fractionation by chromatography, coprecipitation, cross-linking, interaction trap / two-hybrid analysis, southwestern analysis, ELISA, etc. Binding can be measured by binding assays that are well known to those skilled in the art and are described, for example, in “Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, the entire contents of which are hereby incorporated by reference. ) ”. Compounds to be screened (which may include compounds believed to bind nucleic acid molecules encoding HIV-RT / hTERT or HIV-RT / hTERT) include extracellular, intracellular, biological or chemical sources It is not limited to the molecule | numerator which has this. The HIV-RT / hTERT polypeptide or polynucleotide used in such tests may be released in solution, attached to a solid support, supported on the cell surface, or present within the cell. One skilled in the art can measure, for example, the formation of complexes between HIV-RT / hTERT and the compound being investigated. Alternatively, one skilled in the art can examine the decrease in complex formation between HIV-RT / hTERT and its substrate caused by the compound under investigation.

  Another aspect of the present invention is a method for identifying a compound that inhibits the reverse transcriptase activity of HIV-RT / hTERT, wherein the compound is contacted with HIV-RT / hTERT, wherein said compound is HIV-RT / measuring whether to inhibit the reverse transcriptase activity of hTERT. The activity in the presence of the test compound is compared with the activity in the absence of the test compound. If the activity of the sample containing the test compound is lower than the activity in the sample in which no test compound is present, the compound will have inhibitory activity.

  The present invention is particularly useful for screening compounds by using HIV-RT / hTERT in a variety of arbitrary drug screening techniques. Compounds to be screened (which may include compounds thought to modulate HIV-RT / hTERT activity) include molecules with extracellular, intracellular, biological or chemical origins, including It is not limited. The HIV-RT / hTERT chimeric enzyme used in such tests can take any form, but is released in solution, attached to a solid support, supported on the cell surface, or intracellularly. Preferably it is present. One skilled in the art can measure, for example, the formation of complexes between HIV-RT / hTERT and the compound being investigated. Alternatively, one skilled in the art can examine the reduction in complex formation between HIV-RT / hTERT and the substrate of HIV-RT / hTERT caused by the compound being investigated.

  The activity of the HIV-RT / hTERT polypeptides of the present invention can be determined by general RT activity measurements.

  Typically, this assay is based on detecting the incorporation of radioactivity into an RNA / DNA hybrid that can be precipitated by trichloroacetic acid (TCA). For radioactivity detection, β-radionucleotide scintillation fluid is used.

  Alternatively, a modification containing an antigenic epitope or a structure with high affinity for a given ligand (eg, 5-bromo-deoxy-uridine triphosphate or digoxigenin labeled dUTP) instead of a radiolabeled base Nucleotides are used. The presence of these epitopes or structures in the RNA / DNA hybrid is revealed by an antibody or ligand conjugated to an ELISA enzyme. The amount of bound ELISA enzyme is then measured by a secondary enzyme test.

  Another way to demonstrate RT activity is to use a series of specific probes to detect newly synthesized cDNA. This enzymatic reaction utilizes a heteropolymeric RNA molecule having a 20 base oligonucleotide primer complementary to the RNA sequence near the 5 'end. During the RT reaction, a complete cDNA strand is produced. After hydrolysis of the template RNA, the cDNA hybridizes with two different oligonucleotide probes (a capture probe and a detection probe, respectively). Capture probes are used to bind the cDNA to the wells in the microtiter plate. The detection probe is conjugated to horseradish peroxidase and a colorimetric reaction occurs.

  In the present invention, two different activity assays are specifically disclosed. The first assay is an assay aimed at confirming the presence or absence of reverse transcriptase activity (ie, a qualitative assay), whereas the second assay is a reverse transcriptase of the chimeric molecule of the present invention. An assay intended to measure activity (ie, a quantitative assay).

For qualitative purposes, two commercially available reverse transcriptase PCR kits were used (Titan ^™ One Tube RT-PCR kit available from Roche and Access RT-PCR kit available from Promega).

Since three different enzymes are used, the Titan ^™ RT-PCR System provides sensitivity and reproducibility in RT-PCR in one tube. The Titan ^™ system takes advantage of the thermal stability of AMV reverse transcriptase for the cDNA synthesis process to reduce secondary structure. AMV is also highly progressive and gives a full-length transcript compared to the RNase H negative M-MuLV reverse transcriptase mutant. In the final PCR step, the Titan system uses an Expand High Fidelity enzyme blend with improved fidelity and high yield.

  The Access RT-PCR System is designed to perform reverse transcription (RT) and polymerase chain reaction (PCR) amplification of specific target RNAs derived from either total RNA or mRNA. This system uses AMV Reverse Transcriptase (AMV RT) from Avian Myeloblastosis Virus for 1st strand DNA synthesis, and uses thermostable Tfl DNA Polymerase from Thermus flavus for 2nd strand cDNA synthesis and DNA amplification. ing. The Access RT-PCR system is equipped with an optimized single buffer system that allows sensitive detection of RNA transcripts without the need to add buffer between the reverse transcription and PCR amplification steps. This simplifies operation and reduces the possibility of sample contamination. Furthermore, since the reverse transcriptase performance at the time of temperature increase (48 ° C.) in the AMV / Tfl 5x Reaction Buffer AMV is improved, the problem of encountering secondary structure in RNA is minimized.

  Details of both these assays are described in the Examples section. For quantitative purposes, three commercially available reverse transcriptase assay kits (Retrosys (R), available from Innovagen, Reverse Transscriptase Assay, Chemiluminescent, available from Roche, Quan-T-RT (R), available from Amersham Bioscience )It was used. The first two are ELISA non-radioactive assays and Quan-T-RT® is a radioactive assay. In the following, two non-radioactive assays are described.

  In summary, Retrosys® is a 96-well microtiter plate that uses covalently linked riboadenosine homopolymer in a well and 5-bromodeoxyuridine 5′-triphosphate (BrdUTP) as dNTPs. Non-radioactive reverse transcriptase assay. The entire assay, including quantitative detection of incorporated BrdU, performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibody and colorimetry is performed in a single well. The assay can be used with various types of cell culture materials. The results are discussed in the example section.

  Reverse Transscriptase Assay, Chemiluminescent is another non-radioactive 96-well microtiter plate reverse transcriptase assay, based on the synthesis of DNA via reverse transcriptase using a poly (A) template and oligo (T) primers. The reaction mixture contains digoxigenin and biotin labeled TTP along with unlabeled TTP, all of which are incorporated into the same newly synthesized DNA molecule. The incorporated biotin serves to immobilize DNA on a microplate coated with streptavidin, and then the incorporated digoxigenin is specifically detected using an anti-digoxigenin antibody conjugated with peroxidase. Cleavage of the substrate by peroxidase produces a chemiluminescent signal that is quantified using a chemiluminescent microplate reader. This ELISA type assay makes it possible to quantify the synthesized DNA as an indicator of RT activity, and can be used for various types of cell culture materials. The results are discussed in the example section.

  In a preferred aspect of the invention, a method of screening for a compound that inhibits the reverse transcriptase activity of HIV-RT / hTERT comprises contacting said compound with HIV-RT / hTERT in the presence of a suitable substrate, and HIV. Assaying a decrease in RT / hTERT reverse transcriptase activity.

  “Inhibitor” simply means any reagent, drug, or chemical that is capable of inhibiting reverse transcriptase activity in vitro or in vivo. Such inhibitors contact the chimeric molecule of the present invention with a candidate inhibitor and measure the level of reverse transcription activity in the presence or absence of the inhibitor or in the presence of various amounts of inhibitor. Can be easily identified using standard screening protocols. Thus, not only can useful inhibitors be identified, but the optimal level of such inhibitors can be measured in vitro for further investigation in vivo.

  If an organic compound is identified, it is used as the “lead” compound. The design of pharmaceutically active mimetics of known compounds is a well-known approach to developing medicines based on such “lead compounds”. Mimic design, synthesis, and testing are generally used to avoid randomly screening large numbers of molecules for target properties. Furthermore, the structural data obtained by analysis of the deduced amino acid sequence encoded by the DNA of the present invention has higher specificity and is therefore useful for designing new drugs with higher pharmacological potency. is there.

  Thus, computer modeling can be used to obtain a predicted tertiary structure based on the protein of the present invention. Thus, a novel enzyme inhibitor based on the predicted structure of HIV-RT / hTERT can be designed.

  In certain embodiments, the novel molecule identified by the screening methods of the invention is a small molecular weight organic molecule, where the composition or pharmaceutical composition may be prepared for oral consumption, such as in a tablet. it can. A composition or pharmaceutical composition comprising a compound identified by the screening methods described herein can be any route of administration including oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal (these (But not limited to)). The nature of the carrier or other ingredient will depend on the particular route of administration and the particular embodiment of the invention to be administered. Examples of useful techniques and protocols in this regard are described in particular in “Remington's Pharmaceutical Sciences, 16TH edition Nosol, A (ed.), 1980”, the entire contents of which are incorporated herein by reference. To do.

  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 the weight or condition of the human or animal, the route of administration of the compound and the like. When treating humans or animals, a compound of about 0.5 mg / kg body weight to 500 mg / kg body weight can be administered. Treatment is typically administered in smaller doses and is continued until the desired treatment result is observed.

  The compounds identified by the screening methods described herein have a variety of pharmaceutical uses, for example to treat or prevent uncontrolled cell growth such as cancer cells and tumor growth. Can be used. In certain embodiments, the molecules of the invention are used for gene therapy. For an overview of gene therapy operations, see, for example, “Anderson, Science, 1992, 256, 808-813” (the entire contents of which are incorporated herein by reference).

  It will be apparent that the invention may be practiced otherwise than as specifically described herein. Many modifications and variations of the present invention are possible in light of the teachings herein, and such modifications and variations are also within the scope of the invention. In particular, those skilled in the art can design any chimeric construct based on combining various animal-derived telomerase reverse transcriptases (TERT) with any reverse transcriptase for the purpose of identifying telomerase inhibitors. And could be assumed to be built. In fact, the sequence homology between the active sites of TERTs from various organisms is extremely high (see FIG. 4, Nakamura, Science, 1997, 277, 955-959), among others, Homo sapiens, Mus musculus, Mesotricetus auratus, X There is very high homology between the sequences of laevis. Thus, for the purpose of identifying telomerase inhibitors, it is described in the claims of this patent and in accordance with the methods exemplified for HIV-RT / hTERT chimeras in combination with reverse transcriptase, particularly HIV RT sequences. However, constructing a chimeric enzyme containing any of these other TERT active site sequences is only a slight variation of the claimed method and is therefore claimed in this patent. It will be obvious to those skilled in the art that they are included in Examples of other chimeric constructs encompassed by the present invention include chimeric molecules based on HIV-2 reverse transcriptase and human telomerase reverse transcriptase (hTERT) catalytic sites.

  The invention is further illustrated by the following examples which are set forth for the purpose of clarifying the invention. These examples are not intended to limit the scope of the invention and should not be so construed. All references cited herein are incorporated by reference in their entirety.

Design and cloning of HIV-RT / hTERT chimeras In the following description, the numbers for the residues of HIV-1 RT (amino acid residues 1 to 560, SEQ ID NO: 6) and hTERT (amino acid residues 1 to 1132, SEQ ID NO: 4) Are assigned according to the published protein / DNA sequence (HIV-1 RT: nucleotide coding sequence 2550-4229, SEQ ID NO: 5, hTERT: nucleotide coding sequence 56-3454, SEQ ID NO: 3).

1.1 Construction of basic chimera First, a chimera construct is prepared based on HIV-1 RT (SEQ ID NO: 6). HIV-1 RT DNA (SEQ ID NO: 5) was cloned into pKK-223-3 (Amersham-Pharmacia), (Sharma et al. [1991] Biotechnol. Appl. Biochem. 14, 69-81). In alignment with the sequence alignment of hTERT and HIV-1 RT, based on the crystal structure of HIV-1 RT, three sequence portions of hTERT that form the catalytic site were identified (625 to 630, 706 to 722, 862 to 941, (See FIG. 3). This corresponds to 103 hTERT residues out of a total of 576 residues present in the chimeric protein (SEQ ID NO: 2) (18%). To replace these three sequence portions into the HIV-1 RT expression vector, the standard PCR method was used to encode the coding sequence for the entire catalytic site of HIV-1 RT spanning amino acid residues 63 to 241. Replaced by unique restriction sites (NcoI, ClaI, SalI and XhoI).

  The hTERT coding sequence corresponding to 75 amino acids from residue 867 to residue 941 having the sequence listed below was obtained by standard PCR and introduced between the SalI and XhoI sites.

VD DFLLVTPHLTHAKTFFLTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAVVQMPAHGLFPWCGLLLDTRT LE (restriction site is underlined).
Subsequently, a mixed HIV-1 RT / hTERT coding sequence in which amino acid residues 121 to 178 of HIV-1 RT are followed by residues 862 to 869 of hTERT is obtained by standard PCR, and the NcoI and SalI sites Introduced in between. This sequence is as follows.

PW DEDFRKYTAFTIPSINNETPIGIRYQYNVLPQGWKGSPAIFQSSMTKILEEPFRKQNPDLLLLRL VD (SEQ ID NO: 8; hTERT sequence is bold, restriction sites are underlined).
Finally, mixed HIV-RT / hTERT coding sequences were synthesized, amplified by standard PCR methods and introduced between the ClaI and NcoI sites. This coding sequence consists of the dipeptide isoleucine-aspartic acid preceding amino acids 626 to 630 of hTERT followed by amino acids 72 to 103 of HIV-1 RT, followed by amino acids 707 to 721 of glycine and hTERT, followed by Corresponds to the following sequence followed by tryptophan.

ID KPDGLRKLVDFRENKRTQDFWEVQLGIPHPAGLKKKNGYFVKVDVTGAYDTI PW (SEQ ID NO: 9; hTERT sequence is shown in bold, restriction sites are underlined).

Because of the low expression yield with pKK-223-3, the chimeric construct thus obtained (construct 1 in Table 1 below, which encodes the polypeptide sequence of SEQ ID NO: 10) was obtained using standard PCR methods. And cloned into the pET-20b vector (Novagen) using NdeI and HindIII restriction sites to give construct 2 of SEQ ID NO: 11. As a result of this cloning step, the new N-terminus contains a hexahistidine-tag, followed by residue 1 of HIV-1 RT. The C-terminus has 7 residues (residues of SEQ ID NO :) due to restriction sites to be translated (ie, HindIII, NtoI and XhoI) and an added hexahistidine tag (residues 600-605 of SEQ ID NO: 11). 593 to residue 599). The entire coding sequence was confirmed by nucleotide sequence analysis of both strands using an Applied Biosystems 373A DNA Autosequencer. This construct was later modified (ΔCys mutant, construct 3, SEQ ID NO: 12) by introducing four specific point mutations and replacing all four cysteine residues with serine residues. Mutation was repeated in four steps using the Quikchange ^™ site-directed mutagenesis kit (Stratagene) according to the manufacturer's specifications. Nucleotide sequence analysis of both strands using an Applied Biosystems 373A DNA Autosequencer confirmed the presence of the mutation and the integrity of the entire coding sequence.
Based on construct 3, a series of constructs with varying N- and C-termini were prepared using standard PCR methods (constructs 4-7, SEQ ID NOs: 13-16). Point construct N103K (numbered according to HIV-1 RT) was found in constructs 3-7. A further point mutation R125G was found in construct 7, which was corrected by performing site-directed mutagenesis using the Qukchange ^™ kit as per manufacturer's specifications. All constructs were verified by nucleotide sequence analysis of both strands using an Applied Biosystems 373A DNA Autosequencer. Table 1 below lists the first group of constructs of the constructed HIV-RT / hTERT chimeras (residue number assignments are made according to HIV-1 RT and HIV-1 RT residues Is bold).

In addition, for comparison, four HIV-1 RT constructs (constructs 8-11, SEQ ID NOs: 17-20) were prepared using standard PCR methods.

1.2 Construction of the modified chimera In addition to the three sequence portions of hTERT inserted into the HIV-1 RT sequence as described above, we also added 15 additional HIV-1 RT sequences. An amino acid followed by a pre-introduced glycine residue at the transition point from the HIV-RT to the hTERT sequence corresponds to the corresponding residue derived from hTERT (RTFVLRVRAQDPPPEL) (corresponding to residues 691 to 706 of SEQ ID NO: 4) ). The basis for this modification is that the sequence is in close proximity to the allosteric site for HIV-1 RT inhibition, known as the non-nucleoside reverse transcriptase inhibition (NNRTI) site. Since the two sequences differ considerably in this region (especially in the C-terminal direction of this sequence portion), replacing the LKKKNG sequence with PPPEL may improve the presentation of the hTERT active site in the chimeric protein There is.

  The added hTERT coding sequence was introduced by two synthetic oligonucleotides each containing an 8 amino acid coding sequence and a SacI restriction site. Using this set of primers in a standard PCR reaction allowed amplification of the entire vector excluding the coding sequence for 16 modified amino acids. The amplified PCR was purified using a PCR purification kit (Qiagen), followed by digestion with SacI restriction enzyme and ligation again to obtain a modified chimeric construct. The modified chimera (SEQ ID NO: 21) thus obtained is represented as construct 12 in Table 3 below.

  The specific activity of reverse transcriptase of the modified chimera was 10-30 times higher than that of the basic chimera as measured using reverser transcriptase assay, chemiluminescent (Roche).

1.3 Construction of basic and modified chimeras as GST-fusion proteins Using the commercially available pGEX-6p2 vector (Amersham Biosciences), the basic chimeric protein and modification as a C-terminal fusion to glutathione-S-transferase (GST) A chimeric protein was expressed. The GST fusion system has been demonstrated to increase the amount of soluble protein for proteins that are almost insoluble when expressed alone. E. Since both basic and modified chimeras are insoluble proteins when expressed in E. coli, It was investigated whether it could be an improvement that could express soluble chimeras in E. coli.

In the pGEX-6p2 vector with a slight modification containing a unique NdeI restriction site 3 base pairs (encoding leucine) downstream of the BamHI restriction site that marks the first restriction site of the multiple cloning site of pGEX-6p2. The entire coding sequence of the basic chimera and modified chimera was inserted. Two chimeric pET-20b constructs were digested using NdeI and NotI restriction enzymes simultaneously. The obtained DNA fragment corresponding to the coding sequence of the chimeric protein was separated using 1% agarose gel electrophoresis, cut out from the gel, and purified using Gel Extraction Kit (Qiagen). The modified pGEX-6p2 vector was digested using the same restriction enzyme combination (ie, NdeI, NotI) and purified as described above for the chimeric coding sequence insert. The two GST fusion constructs thus obtained (constructs 13 and 14 in Table 3 below, SEQ ID NOs: 22 and 23, respectively) are amino acid sequences GP L HM KHHHHHHH even after cleavage of the N-terminus of the chimeric enzyme by Precision protease. GP (restriction sites are underlined: 2 × ApaI, 1 × NdeI; chimera-derived proline residue 1 is shown in bold) (residue 227 of SEQ ID NOs: 22 and 23) To 241).

  To remove this added sequence, the modified chimeric pGEX-6p2 construct was subjected to mutagenesis to remove the added ApaI restriction site outside the coding sequence of the chimeric enzyme. Subsequently, the modified chimeric pGEX-6p2 introduced with the mutation was digested with ApaI to remove the His tag sequence and ligated again. The final construct (No. 15 in Table 3 below, SEQ ID NO: 24) expresses a modified GST chimera that retains only a single glycine residue on the N-terminus of the enzyme after Precision cleavage.

When the GST fusion expression system is used, both the basic chimeric enzyme and the modified chimeric enzyme are partially soluble and can be purified using glutathione-sepharose resin (Amersham-Biosciences). The soluble GST fusion chimeric enzyme has a specific activity that is 10 times higher than the refolded enzyme expressed without GST fusion.

1.4 Mutation of one amino acid near the active site in the modified GST chimera A single point mutation of the modified GST chimera construct 14 was performed by changing the amino acid 931 of hTERT from serine to cysteine. In fact, cysteine is an amino acid that is naturally present at this position in the hTERT sequence. However, in order to facilitate purification, serine variants were prepared prior to this when all four cysteines present in the chimeric protein were repeatedly replaced with serine.

  Cysteine 931 may have an important role for enzyme activity and inhibitor specificity because of the proximity of the active site after achieving soluble expression of the chimeric protein using the GST fusion system It was considered. Therefore, the original cysteine residue was reintroduced into the modified GST chimeric protein. This single amino acid substitution contains an NcoI / XhoI restriction fragment from the modified GST chimeric construct 14 and an NcoI / XhoI restriction fragment from the intermediate construct generated on the basis of construct 2 (containing the last two cysteines). But the first two cysteines are mutated to serine). This intermediate construct was chosen because the NcoI / XhoI restriction fragment of construct 2 contained two coding sequences for cysteine (amino acids 896 and 931) and only the latter cysteine should have been mutated.

The modified GST chimeric cysteine mutant (construct 16 in Table 4, SEQ ID NO: 25) was not different in expression and purification compared to the original construct 14. Its enzymatic activity was equivalent to the modified GST chimera expressed using construct 14.

Expression of HIV-RT / hTERT chimeras E. coli using pET-20 and pGEX-6p2 constructs. E. coli BL21 (DE3) cell line (Novagen) and transformed into E. coli using the pKK-233 construct. E. coli DH5α cell line (Life Technologies) was transformed. In 2xYT (Life Technologies) broth supplemented with 100 μg / ml carbenicillin, the newly transformed bacteria were grown at 37 ° C. until OD600 was 0.8, and 1 mM isopropylthiogalactopyranoside ( IPTG) for 2 to 4 hours, collected by centrifugation at 7000 rpm for 15 minutes, and the pellet was stored at -20 ° C until use.

Purification of Chimeric Construct 3.1 Purification of Insoluble HIV-RT / hTERT Chimera The following protocol was used for all chimeric constructs and HIV-1 RT constructs except the GST fusion construct. Unless otherwise noted, all steps were performed at room temperature. If the protein contained cysteine residues (constructs 1, 2, 8, 9, 10, 11), all buffers were supplemented with 3 mM β-mercaptoethanol. The cell pellet obtained from 1 liter culture is resuspended in 30 ml lysis buffer (20 mM Na ₂ HPO ₄ , 500 mM NaCl, 5 mM imidazole, pH 7.8) on ice, followed by using an electric homogenizer (PBI). And homogenized. Cells were mechanically disrupted using a French Press (Spectronic) and the lysate was centrifuged at 20,000 rpm for 30 minutes at 4 ° C. Since the protein was expressed as insoluble inclusion bodies, it was exclusively contained in the pellet after centrifugation. Therefore, the supernatant was discarded, and the surface of the pellet was washed with 20 ml of Milli-Q water by gently shaking the centrifuge tube. The pellet was homogenized in 20 ml of Milli-Q water using an electric homogenizer (PBI) and centrifuged at 20,000 rpm for 30 minutes at 4 ° C. Finally, using an electric homogenizer (PBI), the washed white pellet containing the chimeric protein was transferred into 10 ml of denaturing buffer A (6M guanidinium hydrogen chloride in phosphate buffered saline (PBS) pH 7.8). ). The homogenized suspension was stirred overnight at room temperature using a magnetic stirrer. After extracting the protein from the pellet, the supernatant was centrifuged at 20,000 rpm for 30 minutes at 4 ° C. The clear supernatant was loaded onto a 1.5 × 10 cm column containing 3 ml of ProBond resin (Invitrogen), previously washed with 10 ml of Milli-Q water and equilibrated with 10 ml of denaturing buffer A. After loading the protein, it was first washed with 10 ml denaturation buffer and then with 10 ml denaturation buffer A containing 30 mM imidazole. After these two washes, the column was washed with denaturing buffer B (8M urea, 30 mM imidazole, 10 mM hydroxylamine, PBS, pH 7.8). Finally, the protein was eluted in 10 ml elution buffer (8M urea, 500 mM imidazole, 10 mM hydroxylamine, PBS, pH 6.9). If the protein contains cysteine residues (constructs 1, 2, 8, 9, 10, 11), DTT was added from the 1M stock solution to the elution fraction so that the final concentration was 10 mM DTT. The eluted protein was analyzed by 12.5% SDS-PAGE using a PhasSystem (Amersham-Pharmacia). The protein was concentrated to a concentration of 3-7 mg / ml using an Ultrafree®-15 30K concentrator (Millipore). After filtration using Steriflip (TM) (Millipoer), SuperLoad (R) 200 gel filtration column (Amersham-Pharmacia) was added to Superdex (R) 200 gel filtration column (Amersham-Pharmacia) using HiLoad (R) 26/60 AKTA-Explorer (Amersham-Pharmacia). Loaded. If the protein contains cysteine residues (constructs 1, 2, 8, 9, 10, 11), column buffer supplemented with 10 mM DTT (8M urea, 10 mM hydroxylamine, PBS, pH 6.9) The column was equilibrated. Chimera or HIV-1 RT containing fractions were analyzed by 12.5% SDS-PAGE and the protein concentration of the fractions was measured using Coomassie (R) Plus Protein Assay Reagent (Pierce). Pure fractions were snap frozen in liquid nitrogen and stored at −80 ° C. until use.

Example 3.2 Purification of Soluble GST-HIV-RT / hTERT Chimera The following protocol was used for the GST fusion chimera construct. Unless otherwise noted, all steps were performed at 4 ° C. The cell pellet obtained from 1 liter of culture was resuspended in 30 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM DTT, 50 μM PMSF) on ice, followed by electric homogenizer (PBI). And homogenized. Cells were mechanically disrupted using a French Press (Spectronic) and the lysate was centrifuged at 20,000 rpm for 30 minutes at 4 ° C. Since the GST fusion protein was expressed in a partially soluble state, the supernatant was subsequently loaded onto a gravity column containing 5 ml Glutathion-Sepharose ™ 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. Subsequently, the protein was eluted using 15 ml of 50 mM Tris-HCl pH 8.0, 5 mM DTT, 10 mM glutathione.

  The eluted protein was concentrated to reach a final volume of 1 ml using an Ultrafree concentrator (Millipore) with a cut-off value of 30 kDa. After filtration using Steriflip (TM) (Millipoer), the purified protein solution was loaded onto Superose (TM) 12 gel filtration column (Amersham-Pharmacia) using AKTA-Explorer (Amersham-Pharmacia). The column was equilibrated with column buffer (50 mM Tris-HCl, 5 mM DTT pH 8.0). Fractions containing the GST chimera were analyzed by 12.5% SDS-PAGE, the protein concentration of the fractions was measured using Coomassie (R) Plus Protein Assay Reagent (Pierce) and stored at 4 ° C .

Protein Refolding The following protocol was used for all chimeric and HIV-1 RT protein samples. Unless otherwise noted, all steps were performed at 4 ° C. If the protein contained cysteine residues (constructs 1, 2, 8, 9, 10, 11), 10 mM DTT was added to the refolding buffer. Dissolved protein fraction frozen, refolding buffer (440 mM sucrose, 550 mM _{L-arginine, 2.2mM MgCl 2, 2.2 mM CaCl} 2, 264mM NaCl, 11mM KCl, 55mM Tris-HCl, pH 8.2) Alternatively, the denatured protein was refolded by diluting 20 times in Milli-Q water. Following refolding, the protein was dialyzed overnight against refolding buffer or Milli-Q water. Reconstitute proteins from two different size subunits p67 and p53 (chimera) or p66 and p51 (HIV-1 RT), respectively, and then mix equimolar amounts of two independently expressed and purified protein samples And stirred for 30 minutes before refolding. Prior to dialysis, the protein was concentrated using an Ultrafree®-15 30K concentrator (Millipore).

Assays for Reverse Transcriptase Activity Various concentrations of refolded and dialyzed protein samples were used in the five different commercial reverse transcriptase assay formats described herein below.

Example 5.1 Titan (TM) One Tube RT-PCR Kit (Roche)
The Titan (TM) One Tube RT-PCR kit uses a two-step RT-PCR technique in which a reverse transcriptase reaction is first performed 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 the RNA template. During the subsequent PCR cycling step, the complementary DNA product is amplified in order to visualize the amplified DNA product on an agarose gel containing ethidium bromide. The Titan (TM) One Tube RT-PCR kit contains an enzyme mixture containing AMV reverse transcriptase and Expand (TM) High Fidelity System DNA polymerase. Qualitative evaluation of HIV-RT / hTERT chimera activity was performed using positive control components (human control RNA: MS2 carrier RNA, human β-actin upstream and downstream primers, and K562 total RNA) attached to the kit. It was. For all reactions except the control reaction, HIV-RT / hTERT chimera (construct 2) or HIV with Expand (TM) High Fidelity System DNA polymerase instead of Titan (TM) RT-PCR enzyme mix (Roche) -1 RT (construct 8) protein was used. As a positive control reaction, a Titan (TM) enzyme mix was used, and as a negative control reaction, Expand (TM) High Fidelity System DNA polymerase was used without adding any reverse transcriptase. Others performed reaction preparation and thermocycling protocol according to manufacturer's specifications.

Results The RT-PCR reaction product was subjected to electrophoresis on a 1% agarose gel (see FIG. 2). In addition to the positive control reaction (AMV RT), an amplified 324 bp β-actin DNA band was seen during the reaction with recombinant HIV-RT / hTERT chimera or recombinant IV-1 RT, respectively. The negative control without reverse transcriptase did not produce any amplified DNA product.

Conclusion This qualitative experiment clearly confirmed that the HIV-RT / hTERT chimera possesses reverse transcriptase activity. This activity of the chimeric protein can be used for quantitative inhibition assays.

Example 5.2 Access RT-PCR System (Promega)
The Access RT-PCR kit uses a two-step RT-PCR technique in which a reverse transcriptase reaction is first performed, 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 the RNA template. During the subsequent PCR cycling step, the complementary DNA product is amplified in order to visualize the amplified DNA product on an agarose gel containing ethidium bromide. The Access RT-PCR kit contains an enzyme mixture containing AMV reverse transcriptase and Tfl DNA polymerase. Qualitative evaluation of HIV-RT / hTERT chimera activity was carried out using the positive control component (E. coli carrier rRNA, kanamycin resistance gene upstream and downstream primers, as well as the kanamycin resistance gene mRNA) attached to the kit. . For all reactions except the control reaction, an HIV-RT / hTERT chimera (construct 2) or HIV with Tfl DNA polymerase instead of AMV reverse transcriptase and Tfl DNA polymerase (attached separately to the kit) -1 RT (construct 8) protein was used. As positive control reaction, AMV reverse transcriptase and Tfl DNA polymerase were used, and as negative control reaction, Tfl DNA polymerase was used without adding reverse transcriptase at all. Others performed reaction preparation and thermocycling protocol according to manufacturer's specifications.

Results The RT-PCR reaction product was subjected to electrophoresis on a 1% agarose gel. In addition to the positive control reaction (AMV RT), there was a band of amplified 323 bp kanamycin resistance gene DNA in the reaction with recombinant HIV-RT / hTERT chimera or recombinant IV-1 RT, respectively. . The negative control without reverse transcriptase did not produce any amplified DNA product.

Conclusion This qualitative experiment clearly confirmed that the HIV-RT / hTERT chimera possesses reverse transcriptase activity. This activity of the chimeric protein can be used for quantitative inhibition assays.

Example 5.3 Retrosys (TM) RT Activity Kit (Innovagen)
By this colorimetric ELISA type assay, the reverse transcriptase starting from the oligo (dt) ₂₂ primer and the immobilized poly (A) template by subsequently incorporating bromodeoxyuridine triphosphate (BrdUTP) detected with a specific antibody Quantification of DNA synthesis via H. (Henric et al., [1996] Biotechnol. Appl. Biochem. 23, 95-105). BrdU specific antibodies are conjugated to alkaline phosphatase. P-Nitrophenyl phosphate, which increases absorption upon cleavage, is a substrate for this phosphatase. Finally, the 405 nm absorbance of the sample is quantified using a 96 well plate reader. Instead of HIV-1 RT attached to the kit, HIV-RT / hTERT chimera (construct 2) or HIV-1 RT (construct 8) protein was added to all reactions except the control reaction. As a positive control reaction, HIV-1 RT (Innovagen) attached to the kit was added, and in the negative control reaction, no enzyme was added. Others performed reaction preparation and incubation protocol according to manufacturer's specifications.

Results In this commercially available colorimetric enzyme-linked immunosorbent assay (ELISA), reverse transcriptase activity is measured using absorption proportional to enzyme activity. It was revealed that the activity of the partially purified HIV-RT / hTERT chimera was lower on the order of 1500 times compared to the recombinant HIV-RT.

Conclusion The HIV-RT / hTERT chimera is functionally active as a reverse transcriptase. Using this ELISA type assay, it is possible to quantify the enzyme activity of the recombinant HIV-RT / hTERT chimera. Furthermore, when HIV-RT / hTERT chimeras are used as active enzymes, this type of assay can be used to determine the inhibitory concentration (IC50) of a candidate telomerase inhibitor.

Example 5.4 Reverse Transscriptase Assay Chemiluminescent (Roche)
This chemiluminescent reverse transcriptase assay utilizes DNA synthesis via reverse transcriptase using a poly (A) template and oligo (T) ₁₅ primer.

  The reaction mixture contains optimized digoxigenin and biotin labeled TTP and unlabeled TTP. In the first step, all three thymidine species are incorporated into the same newly synthesized DNA molecule by reverse transcriptase. In the second step, the incorporated biotin moiety binds the DNA onto a streptavidin-coated microplate. In the third step, subsequently, the incorporated digoxigenin moiety is specifically detected using an anti-digoxigenin antibody conjugated with peroxidase. In the last step, the chemiluminescent peroxidase substrate and the signal enhancer luminol / 4-iodophenol are added. Cleavage of the substrate via peroxidase results in a chemiluminescent signal that is quantified using a chemiluminescent microplate reader. This ELISA type assay makes it possible to quantify the synthesized DNA as an indicator of RT activity, and HIV-1 RT with known activity is used as an internal standard.

  Instead of the HIV-1 RT attached to the kit, the HIV-RT / hTERT chimera or HIV-1 RT protein was added to all reactions except the control reaction. As a positive control reaction, HIV-1 RT (Roche) attached to the kit was added, and no enzyme was added to the negative control reaction. Others performed reaction preparation and incubation protocol according to manufacturer's specifications.

Results In this commercially available colorimetric enzyme-linked immunosorbent assay (ELISA), reverse transcriptase activity is measured using chemiluminescence proportional to enzyme activity. It was revealed that the activity of pure and homogeneous HIV-RT / hTERT chimera was lower on the order of 800 times compared to recombinant HIV-RT.

Conclusion The HIV-RT / hTERT chimera is functionally active as a reverse transcriptase. Using this type of assay allows the quantification of the enzyme activity of the recombinant HIV-RT / hTERT chimera. Furthermore, when HIV-RT / hTERT chimeras are used as active enzymes, this type of assay can be used to determine the inhibitory concentration (IC50) of a candidate telomerase inhibitor.

Example 5.5 Quan-T-RT assay, (Amersham-Biosciences)
This radioactive reverse transcriptase assay uses a poly (A) template and oligo (T) ₁₅ primer and utilizes DNA synthesis via reverse transcriptase.

The reaction mixture contains ³ H-TTP with an optimized ratio and unlabeled TTP. First, both thymidine species are incorporated into the same DNA molecule newly synthesized based on a biotinylated oligo (dT) primer by reverse transcriptase. Finally, the incorporated radioactivity is quantified with streptavidin-coated SPA beads using a microplate reader. This radioactive SPA type assay makes it possible to quantify the synthesized DNA as an indicator of RT activity, and HIV-1 RT with known activity is used as an internal standard.

  HIV-RT / hTERT chimera or HIV-1 RT protein was added to all reactions except the control reaction. As a positive control reaction, HIV-1 RT (Amersham Biosciences) was added, and in the negative control reaction, no enzyme was added. Otherwise, the reaction preparation and incubation protocol were performed according to the manufacturer's specifications.

Results This commercially available radioactive SPA assay measures reverse transcriptase activity utilizing incorporated radioactivity proportional to enzyme activity.

Conclusion The HIV-RT / hTERT chimera is functionally active as a reverse transcriptase. Using this type of assay allows the quantification of the enzyme activity of the recombinant HIV-RT / hTERT chimera. Furthermore, when an HIV-RT / hTERT chimera is used as the active enzyme, this type of assay can be used to determine the inhibitory concentration (IC ₅₀ ) of a candidate telomerase inhibitor.

Binding experiments using microcalorimetry. Using an isothermal titration calorimeter (ITC), an HIV-RT / hTERT chimera of a series of compounds consisting of nucleosides, nucleoside analogs, reverse transcriptase inhibitors, and telomerase inhibitor analogs And binding to HIV-1 RT. All binding experiments were performed at 10 ° C. using HIV-RT / hTERT chimera (construct 3) or HIV-1 RT (construct 8). TTP (SIGMA) was used as a representative nucleoside and the nucleoside analog AZT (SIGMA) was used as a known HIV-1 RT inhibitor. Nevirapine was used as an example of a non-nucleoside reverse transcriptase inhibitor (NNRTI). Compound R645176 (Aldrich) is a commercially available homologue of the potent telomerase inhibitor BIBR-1532 (Damm, EMBO Journal, 2001, 20, 6958-6968).

  Except for AZT, all compounds were tested for binding of HIV-RT / hTERT chimera and HIV-1 RT, respectively. AZT binding was measured only for the HIV-RT / hTERT chimera.

Results The dissociation constant KD observed for the compound binding the HIV-RT / hTERT chimera was exclusively in the low micromolar range. Compounds TTP, AZT, R645176 showed similar dissociation constants KD for binding of HIV-RT / hTERT chimera and HIV-RT. When bound to HIV-1 RT, the dissociation constant KD in the low nanomolar range was measured for nevirapine.

Analysis Nucleoside TTP and nucleoside analog AZT bind to HIV-RT / hTERT chimeras and HIV-RT with similar affinity. This expected behavior indicates that the active sites of both proteins share a common three-dimensional structure. Nevirapine, a specific HIV-1 reverse transcriptase inhibitor, shows a much higher affinity for HIV-1 RT (1000 times) compared to the HIV-RT / hTERT chimera. Because the amino acid composition of the active site of HIV-RT / hTERT chimeras is significantly different from HIV-1 RT, specific HIV-1 RT inhibitors are compared to HIV-1 RT compared to HIV-1 RT chimeras. The selectivity for is significantly reduced.

FIG. 1 is a schematic diagram of an HIV-RT / hTERT chimera. The HIV-1 RT sequence region (1-63, 72-103, 121-178, 241-560) is drawn in black, whereas the hTERT sequence region (625-630, 706-722, 862-941). ) Is drawn in white. The two lines below the schematic diagram represent the length of the two subunits (p53: 1-430 and p67: 1-560) of the present heterodimer protein. FIG. 2 is a 1% agarose gel electrophoresis showing the reverse transcriptase activity of the chimera of the present invention. The experimental RT-PCR reaction product described in Example 5.1 was subjected to electrophoresis on a 1% agarose gel. Amplified 324 bp β-actin DNA bands were used for positive control reaction (lane 1: AMV RT), recombinant HIV-RT / hTERT chimera (lane 2) or recombinant HIV-1 RT (lane 3), respectively. Seen during the reaction. The negative control (lane 4) with no reverse transcriptase added did not yield any amplified DNA product. The figure which aligned HIV-RT / hTERT chimera arrangement | sequence with hTERT and HIV-1 RT. Three modified regions present in the HIV-RT / hTERT chimera are juxtaposed with the corresponding sequences of hTERT and HIV-1 RT.

  # Number of residues of HIV-RT / hTERT chimera relative to residue 1 of HIV-1 RT.

  * Residues in the restriction sites are underlined.

Abbreviations used:
Human telomerase reverse transcriptase: hTERT
HIV-RT / hTERT chimera: CHIM
HIV-1 reverse transcriptase: HIV-1
The figure which juxtaposed the hTERT active site sequence obtained from various organisms compared with HIV-1 RT. Conserved sequence motifs are highlighted in gray. Residues that are identical between the sequences of Homo sapiens, Mus musculus, Mesotricetus auratus, Xenopus laevis are boxed.

Abbreviations used:
HIV-1 reverse transcriptase: HIV-1
Telomerase reverse transcriptase: Homo sapiens: Hs; Mus musculus: Mm; Mesocricetus auratus: Ma; Xenopus laevis: Xl; Schizosaccharomyces pombe: Sp; Arabidopsis thaliana: At; Oryza sativa: Os; Euplotes aediculatus: Ea; Oaxtric Paramecium caudatum: Pc; Cryptosporidium parvum: Cp; Tetrahymena thermophila: Tt.

Claims (37)

a) the sequence of SEQ ID NO: 1, 26, 27, 28 or 29, or a fragment thereof,
b) a sequence complementary to at least a portion of the sequence of SEQ ID NO: 1, 26, 27, 28, or 29,
c) the sequence of SEQ ID NO: 1, 26, 27, 28 or 29, or a sequence homologous to a fragment thereof,
d) a sequence encoding a polypeptide having the sequence of SEQ ID NO: 2, 21, 22, 24 or 25, or a fragment thereof;
e) a sequence encoding a polypeptide having an amino acid sequence homologous to the sequence SEQ ID NO: 2, 21, 22, 24 or 25, or a fragment thereof
An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of:   2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is DNA.   2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is RNA.   The nucleic acid molecule of claim 2, wherein the nucleotide sequence has the sequence of SEQ ID NO: 1, 26, 27, 28, or 29.   An expression vector comprising the nucleic acid molecule of claim 1.   6. The expression vector of claim 5, wherein the nucleic acid molecule has the sequence of SEQ ID NO: 1, 26, 27, 28, or 29.   6. The vector of claim 5, wherein the vector is a plasmid.   8. The vector of claim 7, wherein the vector is selected from the group consisting of pET-20b, pGEX-6p2, and pKK-223-3.   6. The vector of claim 5, wherein the vector is a viral particle.   10. The vector of claim 9, wherein the vector is selected from the group consisting of adenovirus, parvovirus, herpes virus, pox virus, adeno-associated virus, Semliki Forest virus, vaccinia virus, and retrovirus.   The nucleic acid molecule is simian virus 40 promoter, mouse mammary tumor virus promoter, terminal repeat sequence of human immunodeficiency virus promoter, Maloney virus promoter, cytomegalovirus immediate early promoter, Epstein-Barr virus promoter, Rous sarcoma virus promoter, human actin promoter 6. The vector of claim 5, operably linked to a promoter selected from the group consisting of: a human myosin promoter, a human hemoglobin promoter, a human muscle creatine promoter, and a human metallothionein promoter.   A host cell transformed with the vector of claim 5.   The transformed host cell of claim 12, wherein the cell is a bacterial cell.   The bacterial cell is E. coli. 14. The transformed host cell of claim 13, which is E. coli.   The transformed host cell of claim 12, wherein the cell is yeast.   The yeast is S. cerevisiae. 16. The transformed host cell of claim 15, which is cerevisiae.   The transformed host cell of claim 12, wherein the cell is an insect cell.   The insect cells are S. cerevisiae. 18. A transformed host cell according to claim 17, which is frugipeda.   The transformed host cell of claim 12, wherein the cell is a mammalian cell.   20. The transformed host cell of claim 19, wherein the cell is selected from the group consisting of Chinese hamster ovary cells, HeLa cells, African green monkey kidney cells, human 293 cells, and mouse 3T3 fibroblasts. A method for producing a polypeptide having the sequence of SEQ ID NO: 2, or a homologue or fragment thereof,
a) introducing the recombinant expression vector of claim 5 into a host cell;
b) growing the host cell under conditions to express the polypeptide;
c) recovering the polypeptide from the host cell;
With a method.   22. The method of claim 21, wherein the host cell is lysed and the polypeptide is recovered from the lysate of the host cell.   23. The method of claim 21, wherein the polypeptide is recovered by purifying media from the host cell without lysing the host cell.   An isolated polypeptide encoded by the nucleic acid molecule of claim 1.   25. The polypeptide of claim 24, wherein the polypeptide sequence has the sequence of SEQ ID NO: 2.   25. The polypeptide of claim 24, wherein the polypeptide has an amino acid sequence homologous to SEQ ID NO: 2.   27. The polypeptide of claim 26, wherein said sequence homologous to the sequence of SEQ ID NO: 2 has at least one conservative amino acid substitution compared to SEQ ID NO: 2.   25. The polypeptide of claim 24, wherein the polypeptide has a fragment of SEQ ID NO: 2. A method for identifying a compound that binds HIV-RT / hTERT comprising:
a) contacting HIV-RT / hTERT with the compound;
b) measuring whether the compound binds HIV-RT / hTERT;
With a method.   30. The method of claim 29, wherein the binding of the compound to HVI-RT / hTERT is measured by a protein binding assay.   32. The method of claim 30, wherein the protein binding assay is performed based on an isothermal titration calorimeter. A method for identifying a compound that binds a nucleic acid molecule encoding HIV-RT / hTERT comprising:
a) contacting said nucleic acid molecule encoding HIV-RT / hTERT with a compound;
b) measuring whether the compound binds to the nucleic acid molecule;
With a method.   35. The method of claim 32, wherein binding is measured by a gel shift assay. A method for identifying a compound that inhibits the reverse transcriptase activity of HVI-RT / hTERT, comprising:
a) contacting HIV-RT / hTERT with the compound;
b) measuring whether the reverse transcriptase activity of HIV-RT / hTERT is inhibited;
With a method.   30. A compound identified by the method of claim 29.   35. A compound identified by the method of claim 32. 35. A compound identified by the method of claim 34.

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