JP4606841B2 - Anti-leishmaniasis and Chagas disease preventive / therapeutic agent by inhibition of novel quinol oxidase - Google Patents

Description

  The present invention relates to a novel quinol oxidase, and relates to a preventive / therapeutic agent for anti-leishmaniasis and Chagas disease by inhibiting the enzyme.

Leishmaniasis has been designated as one of the 10 major infectious diseases to be eradicated by the WHO. Even today, an estimated 12 million people are infected in 88 countries around the world.
This disease is caused by Leishmania protozoa as a pathogen and transmitted by the bite of the vector insect, the fly flies. WHO says that more than 20 species of the genus Leishmania infect humans. The disease is found in 22 countries on the new continent and in 66 countries on the old continent. In Europe, human infections have been reported in 16 countries including France, Italy, Greece, Malta, Spain and Portugal.

  Leishmaniasis is classified into a skin type, a mucous membrane type, and a visceral type, and is known to cause various symptoms. Visceral leishmaniasis is also called Karaazaar, and more than 90% is found in Bangladesh, Brazil, India, Sudan, etc., but in severe cases it is fatal. On the other hand, more than 90% of cutaneous leishmaniasis is recognized in Iran, Afghanistan, Syria, Saudi Arabia, Brazil and Peru.

  Anti-leishmaniasis treatments have traditionally included pentavalent antimony-containing preparations and diamidine-based drugs, both of which are highly toxic and the dosage form is intravenous and long-lasting. high. At present, protozoa that are resistant to these drugs have emerged, and the development of safer and more effective drugs is desired.

  On the other hand, Chagas disease is a disease first discovered in 1909 and caused by Trypanosoma cruzi which is a genus Protozoan genus Plasmodium plus module genus. The World Health Organization (WHO) has designated this disease as an infectious disease that should be overcome.

  Currently, the disease has been seen in 18 countries such as South America, Central America and South America, and 13,000 people die every year. Infected animals are found in many wild animals and livestock (dogs, cats), and there are human health insects.

  Infection of humans with Chagas disease is mediated by sub-family Triatominae. In both stages of larval, pupal and adult development, both males and females are infected at the stage of sucking blood, and infected turtles excrete large amounts of protozoa into the feces. The turtle defecation coincides with the timing of the stab, and the infected feces are dropped onto the skin near the bite wound. Therefore, after that, when a human scratches the skin or rubs the mucous membrane, especially the conjunctiva, a pathogen enters the body from the bite wound. Very rarely, it can be transmitted through placental infections, breast milk, blood transfusions, laboratory accidents, and carcasses of infected animals without the use of vector insects.

Symptoms of Chagas disease present with high fever, headache, and joint pain, can become severe without proper treatment, and can fall into a coma and eventually die.
As countermeasures for Chagas disease, attempts have been made so far for 1) countermeasures against turtles with insecticides, 2) prevention and treatment with drugs, but due to the emergence of insecticide-resistant turtles and drug resistance of Trypanosoma cruzi, so far No effective method has been found.

The inventors have so far, Trypanosoma brucei, a protozoan that causes African sleeping sickness, has an AOX enzyme, which is a quinol oxidase, as well as mitochondria in plants and yeast, and controls important energy metabolism. As a result, various substances such as ascofuranone have been discovered as enzyme inhibitors (Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4). On the other hand, it has been reported that quinol oxidase typified by AOX enzyme does not exist in protozoa, L. major, and T. cruzi that cause leishmaniasis and Chagas disease (Non-patent Document 1). . For this reason, it is considered that known AOX enzyme inhibitors cannot treat Leishmaniasis and Chagas disease, and there has been a need to develop new therapeutic agents.
Japanese Unexamined Patent Publication No. 9-13532 Japanese Patent Publication No. 56-25310 Japanese Patent Laid-Open No. 9-13532 Japanese Patent Application 2003-24643 JJ Hellemond et. Al; Ent. Microbiol., 45 (4), 426-430, 1998

  An object of the present invention is to provide a quinol oxidase which has been considered to be absent in L. major and T. cruzi, which are the cause of Leishmaniasis and Chagas disease. The present invention also inhibits a novel quinol oxidase in protozoa, L. major and T. cruzi, which are the cause of leishmaniasis and Chagas disease, thereby preventing and effectively preventing leishmaniasis and Chagas disease. It is an object to provide a therapeutic agent.

  The inventors have a specific insertion from the mitochondria of each protozoan newly causing leishmaniasis and Chagas disease, but unlike the known AOX enzyme, it has an EXXH motif that is an iron binding site. No new type of quinol oxidase was found. In addition, a novel quinol oxidase inhibitor was found and the present invention was completed.

That is, the present inventors have solved the above-described problem,
(A) the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) the deletion of one or more amino acids in the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. A protein having quinol oxidation activity, or (c) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; Or (d) hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, and has quinol oxidation activity A protein having quinol oxidation activity, comprising: an amino acid sequence encoded by DNA encoding the protein.

  The present invention further provides at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. A protein having quinol oxidation activity consisting of an amino acid sequence having amino acid sequence homology is also provided.

In order to solve the above-mentioned problems, the present inventors have further described (a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 with respect to the above-mentioned novel quinol oxidase of T. cruzi. A coding DNA; or (b) a protein having a deletion, substitution, or addition of one or more amino acids in the sequence described in SEQ ID NO: 2 or SEQ ID NO: 4, and having quinol oxidation activity Or (c) DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or (d) SEQ ID NO: 1 or SEQ ID NO: DNA comprising any one of: a DNA that hybridizes with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence according to 3 under a stringent condition and encodes a protein having quinol oxidation activity
Also provide.

  In the present invention, at least 60%, more preferably at least 70%, more preferably at least 80%, and most preferably at least 90% of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 is used. DNA comprising a nucleotide sequence having nucleotide sequence homology and also encoding a protein having quinol oxidation activity is also provided.

  Furthermore, by inhibiting the function of the above-mentioned protein having quinol oxidation activity, it is possible to inhibit the growth of protozoa, L. major and T. cruzi, which cause leishmaniasis and Chagas disease, and as a result, leishmania Can be treated or prevented. That is, this invention provides the pharmaceutical composition for treating or preventing Leishmaniasis or Chagas disease containing a quinol oxidase inhibitor.

  The present invention further provides an antibody that binds to the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4. This antibody may be characterized by not binding to T. brucei-derived quinol oxidase, AOX.

  The present invention further provides a method for treating or preventing Leishmaniasis or Chagas disease containing a quinol oxidase inhibitor for the purpose of inhibiting the non-cytochrome respiratory chain and efficiently suppressing the growth of protozoa. A pharmaceutical composition is provided. In addition to the quinol oxidase inhibitor, the present invention further aims to inhibit not only the non-cytochrome respiratory chain but also the normal cytochrome respiratory chain at the same time, and more efficiently suppress the growth of protozoa, Provided is a pharmaceutical composition for treating or preventing Leishmaniasis or Chagas disease, further comprising an inhibitor for a normal cytochrome respiratory chain.

  The present invention can provide a novel quinol oxidase derived from L. major which is a causative protozoan of leishmaniasis and T. cruzi which is a causal protozoan of Chagas disease. The present invention also shows that inhibition of this novel quinol oxidase can treat and prevent Leishmaniasis and Chagas disease, and as a result, a pharmaceutical composition containing quinol oxidase can be provided.

BEST MODE FOR CARRYING OUT THE INVENTION

  In addition to the normal cytochrome respiratory chain, T. brucei, the causative protozoan of African trypanosomiasis, has a non-cytochrome respiratory chain that functions as a cyanide-resistant terminal oxidase (AOX). It is known that it can be adapted to the environment by skillfully using it. Based on the knowledge that quinol oxidase typified by AOX enzyme does not exist in L. major, which is the causative protozoa of Leishmaniasis, and T. cruzi, which is the causative protozoan of Chagas disease, We predicted that leishmaniasis and Chagas disease could be treated by inhibiting the respiratory chain, and tried to treat patients with Leishmaniasis and Chagas disease by administering atobacon, a cytochrome b inhibitor. . However, as a result, leishmaniasis and Chagas disease could not be sufficiently treated only by inhibiting the cytochrome respiratory chain.

  From this finding, the present inventors have found an unknown `` protein having quinol oxidation activity '' that receives electrons from ubiquinol in L. major, the causative protozoan of leishmaniasis, and T. cruzi, the causative protozoan of Chagas disease. Presumed that it exists and maintains the survival of L. major and T. cruzi by activating the non-cytochrome respiratory chain.

  Based on this assumption, the present inventors first used T. cruzi quinol oxidase belonging to the same genus as T. cruzi, the sequence of AOX (GenBank Accession no. AB070617), The institute for Genome Research (TIGR) eukaryotic project database (http://www.tigr.org/tdb/euk/) was searched. However, no similar sequence was searched.

  Therefore, using the Arabidopsis thaliana quinol oxidase, AOX sequence (GenBank accession no, AB003176), the TIGR database containing the partial sequence of T. cruzi (above) was subsequently searched again. As a result, a part of the sequence derived from T. cruzi was obtained as a sequence having 33% homology with AOX of Arabidopsis thaliana.

  Based on the obtained partial sequence derived from T. cruzi, 3′RACE and 5′RACE were performed to extend the 3 ′ end side and the 5 ′ end side of the obtained partial sequence, respectively. As a result, a DNA having a nucleotide sequence represented by SEQ ID NO: 1 consisting of 1101 nucleotides starting from the start codon was obtained. The protein encoded by this nucleotide sequence has the amino acid sequence shown in SEQ ID NO: 2.

  When the DNA having the nucleotide sequence represented by SEQ ID NO: 1 was analyzed in detail, no other species on the database showed strong homology to this base sequence. In addition, the GC content of this base sequence was 48%, which was a value that could be expressed in a normal gene expression system.

  Next, the protein having the amino acid sequence shown in SEQ ID NO: 2 was compared with the amino acid and nucleotide sequences of T. brucei quinol oxidase, AOX at the amino acid and nucleotide levels.

  In the present invention, a sequence comparison program used by those skilled in the art can be used for sequence homology analysis of amino acid or base sequences. For example, it can be determined by comparing with sequence information using the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997). Specifically, in the case of nucleotide sequence analysis, it is possible to input a query base sequence with the Nucleotide BLAST (BLASTN) program and collate it with base sequence databases such as GenBank, EMBL, DDBJ. In the case of amino acid sequence analysis, a Query BLAST (BLASTP) program can be used to input a query amino acid sequence and collate with amino acid sequence databases such as GenBank CDS, PDB, SwissProt, and PIR. The program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website. Various conditions (parameters) for homology search using the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values. Other programs used by those skilled in the art of sequence comparison can also be used.

  In the present invention, the sequence homology of amino acid or base sequence may be determined by visual inspection and mathematical calculation. Alternatively, the sequence homology of two protein sequences is based on the algorithm of Needleman and Wunsch (J. Mol Biol., 48: 443-453, 1970) and is available from the University of Wisconsin Genetics Computer Group (UWGCG). The determination may be made by comparing the sequence information using a computer program. Preferred default parameters for the GAP program include: (1) Scoring matrix, blosum62; as described in Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992); ) 12 gap weights; (3) 4 gap length weights; and (4) No penalty for end gaps.

  In the present invention, BLAST was used to search for sequence homology at the amino acid level and at the nucleotide level for a protein consisting of the amino acid sequence of SEQ ID NO: 2 and DNA consisting of the nucleotide sequence of SEQ ID NO: 1 encoding the protein. However, the one having the highest sequence homology between the protein of SEQ ID NO: 2 and the DNA of SEQ ID NO: 1 of the present invention was AOX, a conventionally known quinol oxidase, In the first place, it has only 28% and 27% homology with T. brucei AOX protein and DNA, and 27% and 17% respectively with A. thaliana AOX protein and DNA. It was found to have only homology. From these facts, it was found that the protein of SEQ ID NO: 2 and the DNA of SEQ ID NO: 1 did not have significant sequence homology with the known quinol oxidase, AOX, respectively.

  In addition, Arabidopsis thaliana, Cryptosporidium parvum, Aspergillus niger, Aspergillus niger, Chiony intestinalis, Chlamydomonas reinhardtii, cystecium, cyste Structures consisting of two EXXH motifs (where X is any amino acid) as an iron-binding domain in all species of AOX, including α, Proteobacterium (Novosphingobium aromaticivorans), and its genus Brustrypanosoma (T. brucei) However, in the DNA molecule having the nucleotide sequence shown in SEQ ID NO: 1, there was no site aligned with the known AOX as the EXXH motif.

  From the analysis of such nucleotide sequence and amino acid sequence, in the present invention, the protein comprising the amino acid sequence shown in SEQ ID NO: 2 obtained from T. cruzi, a protozoan causing Chagas disease, is structurally novel. It was expected to be an enzyme. Therefore, we next tried to specify the function of the protein consisting of the amino acid sequence of SEQ ID NO: 2.

  The DNA of SEQ ID NO: 1 incorporated into the expression vector is used to transform E. coli that cannot express endogenous terminal oxidases (cytochrome bo and cytochrome bd) due to lack of heme synthesis. The quinol oxidase activity measurement method using a spectrophotometer (Biochem. Biophys. Res. Commu., 313, 1044-1052, 2004) revealed that the protein consisting of the amino acid sequence of SEQ ID NO: 2 has activity as a quinol oxidase that oxidizes quinol. It was.

  Based on these findings, in one embodiment of the present invention, a novel protein having quinol oxidation activity is a protein having the amino acid sequence described in SEQ ID NO: 2 derived from T. cruzi, or a variant thereof. Thus, a protein having quinol oxidation activity can be provided.

  In the present invention, the term “variant” of the protein of SEQ ID NO: 2 refers to an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the target protein; An amino acid sequence encoded by a base sequence capable of hybridizing under stringent conditions between a base sequence encoding the amino acid sequence of and a complementary base sequence; or between the amino acid sequence of the target protein An amino acid sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% amino acid sequence homology, including a protein having quinol oxidation activity Means.

That is, the present invention more specifically,
(A) an amino acid sequence described in SEQ ID NO: 2; or (b) an amino acid sequence having a deletion, substitution, or addition of one or more amino acids in the sequence described in SEQ ID NO: 2. , A protein having quinol oxidation activity;
(C) an amino acid sequence encoded by a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1; or (d) a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1; A protein having quinol oxidation activity consisting of: an amino acid sequence encoded by DNA encoding a protein having a quinol oxidation activity and hybridizing under stringent conditions; or
Consisting of an amino acid sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% amino acid sequence homology with the amino acid sequence shown in SEQ ID NO: 2. A protein having quinol oxidation activity;
Can be provided.

In another embodiment of the present invention, a DNA encoding a novel protein (SEQ ID NO: 2) derived from T. cruzi having quinol oxidation activity (eg, SEQ ID NO: 1) or a variant encoding the variant thereof Body DNA can be provided. That is, in another aspect, the present invention relates to the above-described novel quinol oxidase of T. cruzi (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (b) SEQ ID NO A DNA comprising any one of: a DNA having a deletion, substitution or addition of one or more amino acids in the sequence described in 2 and having a quinol oxidation activity;
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1; or (d) DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions. A DNA that hybridizes and encodes a protein having quinol oxidation activity; or
DNA comprising a nucleotide sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% nucleotide sequence homology with the nucleotide sequence represented by SEQ ID NO: 1 A DNA encoding a protein having quinol oxidation activity;
Can also be provided.

  Here, for example, “(a) DNA encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2” has a degenerate relationship with DNA consisting of the nucleotide sequence of SEQ ID NO: 1. Those skilled in the art can easily understand that DNA having a nucleotide sequence is included.

  Here, “one or more” means preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5. In the case of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 of the present invention, “deletion”, “substitution”, and “added” occur in the amino acid sequence represented by SEQ ID NO: 2. Deletion ”,“ substitution ”,“ addition ”, wherein the“ deletion ”,“ substitution ”,“ addition ”protein is quinol, similar to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. It has something which has oxidation activity. Further, in the case of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, "deletion", "substitution", "added" means "deletion" that occurs in the nucleotide sequence shown in SEQ ID NO: 1, A protein that is a “substitution”, “addition”, and that is encoded by a nucleotide sequence that is “deletion”, “substitution”, “addition” is the same as the protein consisting of the amino acid sequence described in SEQ ID NO: , Which has quinol oxidation activity.

  For example, in the case of “substitution” of amino acids, substitution between amino acids having similar properties, for example, substitution from one hydrophobic amino acid to another hydrophobic amino acid, substitution from one hydrophilic amino acid to another hydrophilic amino acid Substitution that does not significantly change the function of the whole protein, such as substitution from one acidic amino acid to another acidic amino acid, or substitution from one basic amino acid to another basic amino acid, is included.

  In order to create a protein having such “deletion”, “substitution”, “addition”, or a base sequence having “deletion”, “substitution”, “addition” as described above, site-specific Use various methods known in the technical field of the present invention, such as mutagenesis (Site Directed Mutagenesis), random mutagenesis using mutagen treatment and PCR amplification mistakes (Random Mutagenesis), and cassette mutagenesis (Cassette Mutagenesis). Can do.

  As described above, a variant of a protein having quinol oxidation activity consisting of the amino acid sequence shown in SEQ ID NO: 2 includes a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a complementary DNA. And a protein having quinol oxidation activity, which consists of an amino acid sequence encoded by DNA that can hybridize under stringent conditions.

  In the present invention, stringent conditions mean that the target DNA is a DNA consisting of a nucleotide sequence encoding the protein shown in SEQ ID NO: 2 (for example, SEQ ID NO: 1) or a degenerate relationship therewith. It refers to conditions that allow specific hybridization with a specific base sequence. Hybridization conditions are determined in consideration of conditions such as temperature and ion concentration. In general, it is known that the higher the temperature and the lower the ion concentration, the higher the stringency level. Such stringent conditions can be set by those skilled in the art based on, for example, the description of Sambrook and Russel (Molecular Cloning: A Laboratory Manual, 3rd edition (2001)). As specific examples of these stringent conditions, it may be possible to use hybridization conditions such as 6 × SSC, 5 × Denhardt's, 0.1% SDS, 25 ° C. to 68 ° C., for example. In this case, the hybridization temperature is more preferably 45 ° C. to 68 ° C. (without formamide) or 25 ° C. to 50 ° C. (50% formamide).

  As described above, variants of the protein having quinol oxidation activity of the present invention include at least 60%, more preferably at least 70%, more preferably at least 80% between the amino acid sequence shown in SEQ ID NO: 2. %, Most preferably at least 90% of the amino acid sequence homologous protein comprising quinol oxidation activity. A variant of DNA encoding a protein having quinol oxidation activity of the present invention may also have at least 60%, more preferably at least 70%, more preferably at least 80% between the nucleotide sequence shown in SEQ ID NO: 1. %, Most preferably at least 90% of the DNA comprising a nucleotide sequence with nucleotide sequence homology, and DNA encoding a protein having quinol oxidation activity.

  Further, in the present invention, a cDNA derived from Leishmania major, a protozoan causing leishmaniasis, based on the protein of SEQ ID NO: 2 or the sequence of the DNA of SEQ ID NO: 1 encoding the same. To obtain DNA having the nucleotide sequence set forth in SEQ ID NO: 3, which encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, and this protein is also quinol oxidized. It became clear to have activity.

Based on this finding, in another aspect of the present invention,
(A) the amino acid sequence described in SEQ ID NO: 4; or (b) the amino acid sequence having one or more amino acid deletions, substitutions, or additions in the sequence described in SEQ ID NO: 4. , A protein having quinol oxidation activity;
(C) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3; or (d) DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3. A protein having quinol oxidation activity consisting of: an amino acid sequence encoded by DNA encoding a protein having a quinol oxidation activity and hybridizing under stringent conditions; or
Consisting of an amino acid sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, and most preferably at least 90% amino acid sequence homology with the amino acid sequence shown in SEQ ID NO: 4. A protein having quinol oxidation activity;
Can be provided.

The present invention further relates to this novel Leishmania quinol oxidase: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (b) in the sequence set forth in SEQ ID NO: 4, Or a DNA encoding a protein having a deletion, substitution, or addition of a plurality of amino acids and having a quinol oxidation activity;
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3; or (d) DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions. A DNA that hybridizes and encodes a protein having quinol oxidation activity; or
DNA comprising a nucleotide sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% nucleotide sequence homology with the nucleotide sequence represented by SEQ ID NO: 3. A DNA encoding a protein having quinol oxidation activity;
Can also be provided.

  In the present invention, a peptide characteristic of T. cruzi protein (SEQ ID NO: 2) or L. major protein (SEQ ID NO: 4) having quinol oxidation activity based on the results of these sequence analyses. Antibodies were raised against the region. That is, in one embodiment of the present invention, an antibody that binds to the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4 can be provided. In this embodiment, the obtained antibody is preferably characterized in that it does not bind to T. brucei-derived quinol oxidase, AOX, more preferably quinol oxidation derived from T. cruzi or L. major. It is characterized by not binding to AOX, a quinol oxidase derived from all species (including T. brucei), except for proteins having activity.

  The antibody according to the present invention can be obtained by using Delves (Antibody Production: Essential Techniques (1997)), Howard and Bethell (Basic Methods) using the protein of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof prepared as described above. in Antibody Production and Characterization (2000)), Kontermann and Dubel (Antibody Engineering: Springer Lab Manual (2001)) and the like.

  In order to prepare an antibody that reacts with the protein of SEQ ID NO: 2 or SEQ ID NO: 4 but does not react with the known quinol oxidase, AOX of other species, the sequence analysis results show that SEQ ID NO: 2 Alternatively, it is preferred to use as an immunogen a polypeptide consisting of an amino acid sequence characteristically present in SEQ ID NO: 4 but not present in the amino acid sequences of other species of AOX. The polypeptide region used in the present invention may be anywhere as long as it is not similar as a result of sequence alignment. For example, a polypeptide consisting of amino acids 308 to 321 of SEQ ID NO: 2 (SEQ ID NO: 5 ) Can be used as an immunogen.

The antibody of the present invention may be obtained as a polyclonal antibody or a monoclonal antibody.
Polyclonal antibodies can be obtained from sera collected by immunizing a suitable immunized animal with the protein of SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment thereof. Rabbits, sheep, goats, guinea pigs, mice and the like are generally used as immunized animals, but are not limited thereto.

  A monoclonal antibody is obtained by recovering antibody-producing cells of an animal immunized with the above-mentioned protein of SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment thereof, and cell-fusioning it with an appropriate fusion partner such as myeloma cells. By obtaining a hybridoma cell, culturing and cloning a clone producing an antibody having the necessary activity in a medium suitable for growth, and culturing the cloned hybridoma cell under a suitable condition, whereby the culture supernatant is obtained. Can be obtained from. Furthermore, antibodies can also be produced by growing the hybridoma cells thus obtained in the peritoneal cavity of mammals. As the immunized animal, for example, a mouse, a nude mouse, a rat, or a chicken is preferable. The antibody thus obtained can be purified using general isolation and purification methods such as centrifugation, dialysis, salting out with ammonium sulfate, ion exchange chromatography, gel filtration, affinity chromatography and the like. .

The antibody of the present invention that can be obtained as described above can be used for purification, detection, activity inhibition, and the like of a protein having quinol oxidation activity. The antibody of the present invention can be used after F (ab ′) ₂ fragment or Fab ′ fragment by a known method. When the antibody of the present invention is used for detection of a protein having DNA repair promoting activity, a radioisotope (eg, ³⁵ S or ³ H), an enzyme (eg, horseradish peroxidase), or an appropriate affinity It can be labeled with a ligand (eg, avidin-biotin).

  Based on these findings, the inventors of the present invention inhibit leishmaniasis and Chagas disease by inhibiting a novel quinol oxidase having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. I thought that I could be able to prevent and treat, and I applied Ascofuranone, a known AOX inhibitor against T. cruzi in vitro. As a result, it was revealed that the growth of T. cruzi was significantly inhibited by ascofuranone. This is because a novel quinol oxidase having the amino acid sequence shown in SEQ ID NO: 2 is inhibited by ascofuranone, resulting in inhibition of the non-cytochrome respiratory chain of T. cruzi, resulting in inhibition of T. cruzi growth. It is thought that it was done.

  Therefore, in one embodiment of the present invention, the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is used for the purpose of inhibiting non-cytochrome respiratory chain and efficiently suppressing protozoan growth. A pharmaceutical composition containing a quinol oxidase inhibitor for preventing or treating leishmaniasis and Chagas disease can be provided by inhibiting the novel quinol oxidase.

  In the present invention, any known quinol oxidase inhibitor can be used as long as it can inhibit T. cruzi quinol oxidase, and is not limited to ascofuranone. In the present invention, compounds that can be used as quinol oxidase inhibitors include ascofuranone or ascofuranone derivatives, ascochlorin, salicylhydroxamic acid (SHAM), n-propyl gallate, and the like. It is not limited. In the present invention, ascofuranone is preferably used as the quinol oxidase inhibitor.

  Ascofuranone that can be used in the present invention has the following chemical formula:

It is a compound which has a structure shown by these.
As an ascofuranone derivative that can be used in the present invention, for example, compounds described in PCT / JP2004 / 15390 can be exemplified. In particular,

(Where
X represents a hydrogen atom or a halogen atom,
R ¹ is a hydrogen atom or — (C _n H _2n ) —R ′ (n is an integer of 1 to 5, R ′ is a hydrogen atom or a group COOR ′ substituted with any one of n carbon atoms ', Where R''represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, or -COR''' (R '''is substituted with a pyridyl group or an alkyl group having 1 to 4 carbon atoms. Mean a phenyl group having an amino group, a phenoxyalkyl group having a halogen atom on the carbon atom of the benzene ring, or an alkoxy group having 1 to 4 carbon atoms or an alkoxycarbonyl group having 1 to 4 carbon atoms on the carbon atom of the benzene ring. Represent)
R ² represents a hydrogen atom or an alkyl group having 1 to 7 carbon atoms,
R ³ represents -CHO or -COOH, and
R ⁴ represents —CH═CH— (CH ₂ ) _p —CH ₃ (wherein p represents an integer of 1 to 12), or —CH (OH) — (CH ₂ ) _q —CH ₃ (wherein , Q represents an integer of 1 to 13), —CH (OH) —CH ₂ —CH (CH ₃ ) — (CH ₂ ) ₂ —CH═C (CH ₃ ) ₂ , —CH═CH—CH (CH _{3) - (CH 2) 3} -CH (CH 3) 2, - (CH 2) 2 -CH (CH 3) - (CH 2) 3 -CH (CH 3) 2 or, - (CH _{2) 8} - Represents CH ₃ )
Or a compound represented by the following formula:

A compound represented by
Those optical isomers and their pharmaceutically acceptable salts can be used as ascofuranone derivatives in the present invention.

In the present invention, examples of the compound of the present invention include the following compounds:
(I) Compound A. In the above formula (I),
X represents a hydrogen atom,
R ¹ represents a hydrogen atom,
R ² represents an alkyl group having 1 to 4 carbon atoms,
R ³ represents -CHO, and
R ⁴ represents -CH (OH)-(CH ₂ ) _q -CH ₃ (wherein q represents an integer of 1 to 12), the optical isomer thereof, and their A pharmaceutically acceptable salt;
(Ii) Compound B. In the above formula (I),
X represents a halogen atom,
R ¹ represents a hydrogen atom,
R ² represents an alkyl group having 1 to 4 carbon atoms,
R ³ represents -CHO, and
R ⁴ represents -CH (OH)-(CH ₂ ) _q -CH ₃ (wherein q represents an integer of 1 to 12), the optical isomer thereof, and their A pharmaceutically acceptable salt;
(Iii) Compound C. In the above formula (I),
X represents a hydrogen atom or a halogen atom,
R ¹ represents a hydrogen atom,
R ² represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ³ represents -CHO, and
_2. The compound according to claim 1, wherein R ⁴ represents —CH═CH— (CH ₂ ) _p —CH ₃ (wherein p represents an integer of 1 to 12), optical isomers thereof, and pharmaceuticals thereof Top acceptable salt.

  In addition to the above, the following compounds can also be mentioned as examples.

A compound represented by
Furthermore, in the present invention, not only the non-cytochrome respiratory chain is inhibited by the quinol oxidase inhibitor as described above, but also the normal cytochrome respiratory chain is simultaneously inhibited to suppress the growth of protozoa more efficiently. For this purpose, a pharmaceutical composition for more efficiently preventing and treating leishmaniasis and Chagas disease by further containing an inhibitor for the cytochrome respiratory chain is provided. In the present invention, for example, atovacon and antimycin can be used as an inhibitor for the cytochrome respiratory chain. More preferably, atobacon is used.

  In the remainder of the description, examples are given for the purpose of illustrating the invention in more detail. The description of this example is not intended to limit the scope of the invention, but merely to explain the invention in more detail.

Example 1: Isolation of DNA from T. cruzi and analysis of sequence (1) Search of database based on AOX of Arabidopsis thaliana
Using the sequence of T. brucei quinol oxidase, AOX (GenBank Accession no. AB070617) belonging to the same genus as T. cruzi, a database TIGR containing a partial sequence of T. cruzi (http: // www. tigr.org/tdb/euk/) was searched, but a sequence similar to T. brucei quinol oxidase, AOX, was not found in the partial sequence of T. cruzi.

Therefore, TIGR was similarly searched using the Arabidopsis thaliana quinol oxidase, AOX sequence (GenBank accession no, AB003176). As a result, a part of the TIGR accession no, t_cruzi | chr_0 | 1047053504147 | 4932 sequence was obtained as a sequence having 33% homology with AOX of Arabidopsis thaliana.
(2) Analysis of the obtained fragment sequence From the results of sequence analysis using the above-mentioned database TIGR, a pair of PCR primers for fragment extension, an oligonucleotide having the sequence of SEQ ID NO: 6 as a forward primer, Then, oligonucleotides having the sequence of SEQ ID NO: 7 were prepared and used as reverse primers.

  PCR was carried out for 1 minute at 94 ° C using T. cruzi (Tulahuen strain) cDNA as a template and the oligonucleotides of SEQ ID NO: 6 and 7 described above as primers, and then dissociated at 94 ° C. The reaction is 15 seconds; annealing at 55 ° C. for 30 seconds; and extension reaction at 74 ° C. for 1 minute. This is performed as 35 cycles, and finally at 74 ° C. for 1 minute. KOD dash (Toyobo) was used. Here, cDNA of T. cruzi (Tulahuen strain) was prepared according to its protocol using Amersham's You-prime 1st strand synthesis beads using oligo dT (18) primer.

(3) Sequence extension at 3'RACE and 5'RACE The sequence obtained in (2) above was suggested to be part of the coding region. Since both sides are absent, i.e., there are no start and stop codons, then using the sequence portion amplified by PCR as described above in (2), 5 well known in the art. By performing the 'RACE method and the 3'RACE method, it was planned to obtain the entire coding region containing the sequence fragment described above in (2).

In the present invention, the 5′RACE method was performed according to the 5′RACE system of Invitrogen. Using 5'RACE primer (SEQ ID NO: 8), SuperScript ^TM II RT enzyme (Invitrogen) was used for reverse transcription to synthesize first strand cDNA, and then 3 'of the first strand cDNA. A poly C sequence was added to the end using Terminal deoxinucleotidyl transferase (TdT) enzyme (Invitrogen). Using this cDNA as a template, 5'RACE primer (SEQ ID NO: 8) and 5'RACE adapter primer (ggccacgcgt cgactagtac gggiigggii gggiig (I is inosine); SEQ ID NO: 12) that can bind to poly C PCR was performed under the same conditions as described in (2) to obtain the target 5 ′ end.

  The 3′RACE method was then performed according to the Invitrogen 3′RACE system. That is, first, a reverse strand reaction was performed using oligo (dT) with an adapter sequence at the 5 ′ end (3′RACE adapter primer; ggccacgcgt cgactagtac tttttttttt ttttttt; SEQ ID NO: 13) to perform first transcription cDNA Then, using the first strand cDNA as a template, the primer for 3'RACE (SEQ ID NO: 9) and the adapter primer for 3'RACE, under the same conditions as the PCR described in (2), PCR was performed to obtain the desired 3 ′ end.

(4) Acquisition of full-length sequence By performing 3'RACE and 5'RACE as described in (3) above, the full-length sequence of the target gene derived from T. cruzi was obtained. A sequence of 5 ′ and 3 ′ ends of this full-length sequence is estimated by analyzing the sequences registered in the TIGR database, and a pair of primers for amplifying the full-length sequence, SEQ ID NO as a forward primer : 10, and SEQ ID NO: 11 as a reverse primer was prepared.

  Obtained in (3) using the forward primer (SEQ ID NO: 10) prepared in this way and the reverse primer (SEQ ID NO: 11) to obtain the full-length DNA of SEQ ID NO: 1. Using the sequence as a template, the same conditions as the PCR described in (2) (after 1 cycle at 94 ° C for 1 minute, [dissociation reaction at 94 ° C for 15 seconds; annealing at 55 ° C for 30 seconds; Then, the extension reaction at 74 ° C. was performed for 1 minute]. This was performed as 35 cycles, and finally the reaction was performed at 74 ° C. for 1 minute.

  When this PCR product was sequenced (ABI9700), it was found that the DNA obtained in (3) had the nucleotide sequence of SEQ ID NO: 1, and based on this DNA, the amino acid sequence of SEQ ID NO: 2 was It was revealed that the protein possessed was encoded.

Example 2: Isolation of a similar protein from Leishmania major Next, in order to obtain a similar protein from L. major, the forward shown in Example 1 using L. major (MHOM / SU173 / 5ASKH strain) cDNA as a template. Amplify the full-length sequence by PCR using the primer (SEQ ID NO: 10) and reverse primer (SEQ ID NO: 11) under the same conditions as described in Example 1 (2) Tried. here,
When this PCR product was sequenced (ABI9700), it was found that the DNA amplified in this example had the nucleotide sequence of SEQ ID NO: 3, and based on this DNA, the amino acid sequence of SEQ ID NO: 4 was It was revealed that the protein possessed was encoded. L. major (MHOM / SU173 / 5ASKH strain) cDNA was prepared according to the protocol using Amersham's You-prime 1st strand synthesis beads using oligo dT (18) primer.

Example 3: Analysis of the full-length sequence The DNA of SEQ ID NO: 1 and the protein of SEQ ID NO: 2 obtained in Example 1 and the similar protein derived from L. major obtained in Example 2 were conventionally used. Comparison was made with the sequences of various known quinol oxidases, AOX. Both the comparison of the nucleotide sequence of DNA and the comparison of the amino acid sequence of proteins were performed using the BLAST program.

  In this example, the molecules used for comparison were AOX derived from T. brucei, including Arabidopsis thaliana, Cryptosporidium parvum, Aspergillus niger, Aspergillus niger, Ciona intestinalis, and Chlamydomonas. It was a known AOX protein derived from Chlamydomonas reinhardtii), Dictyostelium discoideum, and α-Proteobacterium (Novosphingobium aromaticivorans) and the DNA encoding it.

  As a result, the sequence homology between the protein of SEQ ID NO: 2 and the DNA of SEQ ID NO: 1 of the present invention is 28% and 27% of the T. brucei AOX protein and DNA, respectively. It was only found to have homology and only 27% and 17% homology with the A. thaliana AOX protein and DNA, respectively.

  The amino acid sequences of these different species of AOX were then aligned with the amino acid sequence of SEQ ID NO: 2 from T. cruzi and the amino acid sequence of SEQ ID NO: 4 from L. major, but were conserved. There is no characteristic sequence, for example, the iron binding site that was completely conserved in species other than T. cruzi and L. major, the structure of “EXXH motif-EXXH motif” is also the amino acid sequence of SEQ ID NO: 2. And was not present in the amino acid sequence of SEQ ID NO: 4.

  Here, as shown in FIG. 1, the amino acid sequence of SEQ ID NO: 2 derived from T. cruzi and the amino acid sequence of SEQ ID NO: 4 derived from L. major are the amino acids of AOX of T. brucei considered to be the most closely related species. The result of alignment with the sequence is shown. As is clear from this, no sequence homology was observed. In this alignment, the part indicated by the two arrowheads indicates the “EXXH motif-EXXH motif” that was completely conserved in species other than T. cruzi and L. major.

  On the other hand, when the amino acid sequence of SEQ ID NO: 2 derived from T. cruzi obtained for the first time in the present invention was compared with the amino acid sequence of SEQ ID NO: 4 derived from L. major, 96.7% sequence homology was found. In addition, when the nucleotide sequence of SEQ ID NO: 1 from T. cruzi was compared with the nucleotide sequence of SEQ ID NO: 3 from L. major, 98% sequence homology was found.

Example 4: Preparation of antibody Of SEQ ID NO: 2 obtained in Example 1, a peptide consisting of amino acids 308 to 321 (SEQ ID NO: 5) was used as an immunogen at a concentration of 100 µg / ml. After 21 days, the animals were boosted with the same immunogen at a concentration of 100 μg / ml, and blood was collected after 53 days, and antiserum was obtained from the blood.

Subsequently, the IgG class was purified from the antiserum using an affinity column (peptide Sepharose bead column).
FIG. 2 shows the result of fluorescent staining of T. cruzi in vitro using this antibody. In Fig. 2, the image stained with an antibody shows that a site completely different from the nucleus (part stained with DAPI) is stained, while T. cruzi is mitochondrially specific. It was found that almost the same site as that stained with Mitotracker (Molecular Probe), which is a chemical reagent, was stained. Therefore, when the stained image with Mitotracker and the stained image with the antibody were superimposed, it became clear that this antibody recognized the exact same site (Fig. 2).

Example 5: Functional analysis of a novel protein derived from T. cruzi In this example, the effect of the protein having the amino acid sequence of SEQ ID NO: 2 obtained in Example 1 was examined.

  First, SEQ ID NO: 2 incorporated into pCR T7 / NT-TOPO vector (Invitrogen) against E. coli strain (FN102 strain) that cannot express endogenous terminal oxidase (cytochrome bo and cytochrome bd). Was attempted to be transduced and expressed.

  When this E. coli was plated in the presence and absence of 100 μM IPTG, only the former showed growth of E. coli colonies (FIG. 3). In other words, E. coli that cannot express endogenous terminal oxidase cannot grow in normal media, but a new protein derived from T. cruzi (SEQ ID NO: 2) whose expression was induced by IPTG is functionally expressed in E. coli. It was shown to complement the terminal oxidase of quinol oxidase.

  Furthermore, in order to perform more detailed biochemical analysis, partial purification of a novel protein derived from T. cruzi was performed as follows. By inducing the expression of a new T. cruzi protein (SEQ ID NO: 2) in the presence of 1 mM IPTG, disrupting E. coli by sonication, and performing ultracentrifugation (200,000 g, 1 hr) The membrane fraction and the cytoplasm fraction were separated. The membrane fraction and cytoplasm fraction obtained in this way were electrophoresed using SDS-PAGE and the antibody (polyclonal antibody) obtained in Example 4 was used to express the SEQ Identification of ID NO: 2 protein was performed by Western blotting. The results are shown in FIG. In FIG. 4, lane 1 shows a sample of cytoplasmic fraction of recombinant E. coli, lane 2 shows a sample of membrane fraction of recombinant E. coli, and lane 3 shows a sample of membrane fraction of E. coli recombined with an empty vector as a negative control. Indicates.

As a result, a signal reacting with the antibody was found only in the membrane fraction derived from recombinant Escherichia coli in Lane 2. The molecular weight of this protein was 45 kDa, which was consistent with the calculated value.
Furthermore, taking advantage of the feature that the absorbance at 278 nm increases when quinone increases, quinol oxidation activity was measured by monitoring with a spectrophotometer. The results are shown in FIG.

As shown in FIG. 5, when the membrane fraction was added to the reaction solution, an increase in absorbance was detected, and when 5 nM ascofuranone was added thereto, the increase in absorbance decreased to the background level. That is, it was revealed that a novel protein derived from T. cruzi localized in the membrane fraction has quinol oxidase activity, and that activity is inhibited by ascofuranone. The IC ₅₀ value at this time was considered to be 5 nM or less.

Example 6: Action of a quinol oxidase inhibitor on the growth of T. cruzi In this example, ascofuranone, which is a quinol oxidase inhibitor, was administered to T. cruzi in vitro, The action of furanone was investigated.

In vitro, human neuroglioma cells GA-1 were infected with 10 ⁴ T. cruzi (Tulahuen strain) protozoa per well of a 24-well plastic plate. After infection was established (2 days later), a known quinol oxidase inhibitor was used. A certain ascofuranone and atobacon, a cytochrome b inhibitor, were added to the culture solution, and the ability to inhibit the growth of T. cruzi protozoa was examined.

  As a result, disappearance of protozoa from the culture broth could be confirmed by the combined use of 50 μM Ascofuranone and 5 μM Atobacon. Since no protozoa appeared in the culture solution even at 7 days after administration, it was judged to have been treated. This effect was not observed with either Ascofuranone or Atobakon alone.

  The present invention can provide a novel quinol oxidase derived from L. major which is a causative protozoan of leishmaniasis and T. cruzi which is a causal protozoan of Chagas disease. The present invention also shows that inhibition of this novel quinol oxidase can treat and prevent Leishmaniasis and Chagas disease, and as a result, a pharmaceutical composition containing quinol oxidase can be provided.

Figure 1 compares the amino acid sequence of SEQ ID NO: 2 from T. cruzi and the amino acid sequence of SEQ ID NO: 4 from L. major with the amino acid sequence of AOX, a known quinol oxidase from T. brucei. FIG. FIG. 2 shows that the antibody prepared in Example 4 specifically binds to mitochondria. Fig. 3 shows the case in which expression is induced by IPTG when E. coli strain (FN102 strain) that cannot express endogenous terminal oxidase (cytochrome bo and cytochrome bd) is transduced with SEQ ID NO: 2. It is a figure which shows that colon_bacillus | E._coli grows only in this. FIG. 4 is a diagram showing that the protein of SEQ ID NO: 2 is specifically distributed in the membrane fraction when expressed in E. coli by Western blotting. Figure 5 shows that the absorbance at 278 nm, which increases with increasing quinone, was reduced to background level by adding 5 nM ascofuranone, so that the T localized in the membrane fraction was reduced. FIG. 3 shows that a novel protein derived from cruzi (SEQ ID NO: 2) has quinol oxidase activity, and that activity is inhibited by ascofuranone.

Claims (10)

  (A) the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) the deletion of one or more amino acids in the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. A novel quinol oxidase comprising: an amino acid sequence having a substitution or addition;   (C) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or (d) from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. A novel quinol oxidase comprising: an amino acid sequence which is hybridized with a DNA having a nucleotide sequence complementary to said DNA and a DNA encoding a protein having a quinol oxidation activity, which hybridizes under stringent conditions.   The novel quinol oxidase according to claim 1 or 2, wherein the amino acid sequence is derived from a protozoan causing leishmaniasis and Chagas disease.   (A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) in the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, A DNA comprising any one of: a DNA having a plurality of amino acid deletions, substitutions or additions and encoding a protein having quinol oxidation activity.   (C) DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or (d) complementary to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. A DNA comprising any one of: a DNA that hybridizes with a DNA comprising a nucleotide sequence under stringent conditions and encodes a protein having quinol oxidation activity.   6. The DNA according to claim 4 or 5, wherein the DNA is derived from a protozoan that causes leishmaniasis and Chagas disease.   An antibody that binds to a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.   The antibody according to claim 7, which does not bind to quinol oxidase AOX (GenBank Accession no. AB070617) derived from T. brucei.   It is a quinol oxidase inhibitor for the prevention and treatment of leishmaniasis and Chagas disease by inhibiting a novel quinol oxidase having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. A pharmaceutical composition comprising ascofuranone.   10. The pharmaceutical composition according to claim 9, wherein the composition further comprises atobacon, which is an inhibitor of the cytochrome respiratory chain, to more efficiently prevent and treat leishmaniasis and Chagas disease.