Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives

Abstract

The invention relates to a novel polypeptide vitamin K epoxide recycling polypeptide (VKORC1) as a target for coumarin and its derivatives. The invention further provides methods for identifying coumarin derivatives, and also claims VKORC1 polypeptides and VKORC1 nucleic acids containing a sequence abnormality associated with a VKORC1 associated deficiency such as warfarin resistance, wherein the VKORC1 polypeptides and VKORC1 nucleic acids can be used for diagnosing these deficiencies. Moreover, the invention relates to methods for identifying coumarin derivatives usable in pest control of rodents.

1 . A vitamin K epoxide recycling polypeptide (VKORC1) as a target for coumarin and its derivatives in mammals comprising a polypeptide sequence selected from the group consisting of:
 (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;   (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);   (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and   (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity.   
     
     
         2 . A VKORC1 nucleic acid comprising a nucleic acid sequence selected from the group consisting of:
 (a) a nucleic acid sequence coding for the VKORC1 polypeptide of  claim 1 ;   (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 2, 13, and 18;   (c) a nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid sequence defined in (a) or (b), wherein the nucleic acid sequence codes for a polypeptide having VKORC1 activity;   (d) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in (a), (b) or (c), and wherein the nucleic acid sequence codes for a polypeptide having VKORC1 activity; and   (e) a fragment of the nucleic acid sequence defined in (a), (b), (c) or (d), wherein the fragment codes for a polypeptide having VKORC1 activity.   
     
     
         3 . A fusion protein comprising
 (a) the VKORC1 polypeptide of  claim 1  or a polypeptide encoded by the VKORC1 nucleic acid of  claim 2 , and   (b) a heterologeous part.   
     
     
         4 . A vector comprising the VKORC1 nucleic acid of  claim 2 . 
     
     
         5 . The vector of  claim 4 , wherein the vector is an expression vector. 
     
     
         6 . The vector of  claim 4 , wherein the vector is a knock-out gene construct. 
     
     
         7 . A host cell containing the VKORC1 nucleic acid of  claim 2 . 
     
     
         8 . The host cell of  claim 7 , wherein the host cell is a non-human embryonic stem cell. 
     
     
         9 . A transgenic non-human mammal, wherein the transgenic mammal contains the host cell of  claim 8 . 
     
     
         10 . A DNA or a RNA probe directed against the VKORC1 nucleic acid of  claim 2 . 
     
     
         11 . A PCR primer directed against the VKORC1 nucleic acid of  claim 2 , preferably a PCR primer selected from the group consisting of a PCR primer according to SEQ ID No. 53 to 69, and 70. 
     
     
         12 . A small interfering RNA molecule (siRNA) or a short hairpin RNA (shRNA) directed against the VKORC1 nucleic acid of  claim 2 , preferably a siRNA selected from the group consisting of SEQ ID No. 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, and 50. 
     
     
         13 . An antisense RNA or DNA directed against the VKORC1 nucleic acid of  claim 2 . 
     
     
         14 . An RNA-aptamere directed against the VKORC1 polypeptide of  claim 1 , wherein the RNA-aptamere exerts an effect on the activity of the VKORC1 polypeptide. 
     
     
         15 . An antibody or a fragment thereof, which specifically recognizes and binds the VKORC1 polypeptide of  claim 1 . 
     
     
         16 . A method of producing a VKORC1 polypeptide comprising the steps of:
 (I) providing a host cell having been introduced the VKORC1 nucleic acid of  claim 2 ;   (II) expressing the VKORC1 polypeptide in the host cell; and   (III) isolating the VKORC1 polypeptide from the host cell.   
     
     
         17 . A method of identifying a coumarin derivative which exerts an effect onto the activity of VKORC1 polypeptide of  claim 1  comprising the steps of:
 (I) providing a host cell having been introduced the VKORC1 nucleic acid or a vector containing the VKORC1-nucleic acid; 
 (II) expressing the VKORC1 polypeptide in the host cell; 
 (III) administering a candidate coumarin derivative; 
 (IV) determining the activity of VKORC1 polypeptide (candidate activity value); 
 (V) comparing the candidate activity value with a control activity value; and 
 (VI) identifying the candidate coumarin derivative as a coumarin derivative exerting an effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value. 
 
     
     
         18 . The method of  claim 17 , wherein the control activity value is determined by a method comprising the steps of:
 (A) providing a host cell according to step (I);   (B) expressing the VKORC1 polypeptide in the host cell; and   (C) determining the activity of VKORC1 polypeptide (control activity value).   
     
     
         19 . The method of  claim 17 , wherein the determined activity of VKORC1 polypeptide is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different activity value is a candidate activity value which is significantly higher than the control activity value. 
     
     
         20 . The method of  claim 17 , wherein at least one additional compound is introduced into the host cell, which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase. 
     
     
         21 . A method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity, comprising the steps of:
 (I) providing a cell expressing the VKORC1 polypeptide of  claim 1 , which VKORC1 polypeptide has at least one sequence abnormality;   (Il) administering coumarin or a derivative thereof to the cell;   (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and   (IV) comparing the sequence abnormality activity value with the control sequence activity value,   
       wherein a significant deviation of the sequence abnormality activity value from the control sequence activity value is indicative that the sequence abnormality of the VKORC1 polypeptide conveys the coumarin effect exerted onto VKORC1 polypeptide. 
     
     
         22 . The method of  claim 21 , wherein the control sequence activity value is determined by a method comprising the steps of:
 (I) providing a cell expressing the VKORC1 polypeptide of  claim 1 ;   (II) administering coumarin or a derivative thereof to the cell;   (III) determining the activity of the VKORC1 polypeptide (control sequence activity value).   
     
     
         23 . The method of determining of  claim 21 , wherein the determined activity is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different value is a sequence abnormality activity value which is significantly higher than the control sequence activity value. 
     
     
         24 . The method of  claim 21 , wherein at least one additional compound is introduced into the cell which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase. 
     
     
         25 . The VKORC1 polypeptide of  claim 1 , wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide. 
     
     
         26 . The VKORC1 polypeptide of  claim 25 , wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C. 
     
     
         27 . A VKORC1 nucleic acid selected from the group consisting of:
 (a) a nucleic acid coding for the VKORC1 polypeptide of  claim 25  or  26 ,   (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 3, 4, 5, 6, 7, 14,and 94,   (c) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in (a) or (b), and wherein the nucleic acid sequence codes for the polypeptide of  claim 25  or  26 .   
     
     
         28 . A vector containing the VKORC1 nucleic acid of  claim 27 . 
     
     
         29 . A DNA or a RNA probe directed against the VKORC1 nucleic acid of  claim 27 . 
     
     
         30 . A PCR primer directed against the VKORC1 nucleic acid of  claim 27 . 
     
     
         31 . An antibody or a fragment thereof, which specifically recognizes and binds a VKORC1 polypeptide of  claim 25  or  26 . 
     
     
         32 . A transgenic non-human mammal wherein the mammal contains a stem cell containing the VKORC1 nucleic acid of  claim 27  or the vector according to  claim 28 . 
     
     
         33 . A diagnostic comprising a compound selected from the group consisting of a VKORC1 nucleic acid of  claim 27 , a DNA or the RNA probe directed against the VKORC1 nucleic acid of  claim 27 , a PCR primer directed against the VKORC1 nucleic acid of  claim 27 , and an antibody or a fragment thereof, which specifically recognizes and binds the VKORC1 polypeptide (VKORC1) as a target for coumarin and its derivatives in mammals comprising a polypeptide sequence selected from the group consisting of:
 (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;   (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);   (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and   (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity;
 wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide; or 
 wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide; and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C. 
   
     
     
         34 . A method of diagnosing a VKORC1 associated deficiency in a patient comprising the steps of:
 (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and   (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality in a nucleic acid sequence coding for the VKORC1 polypeptide of  claim 1  or in an amino acid sequence of the VKORC1 polypeptide;   
       wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency; preferably warfarin resistance. 
     
     
         35 . The method of  claim 34 , wherein the amplified DNA encodes at least a partial sequence of the VKORC1 polypeptide according to SEQ ID No. 1 and wherein the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C. 
     
     
         36 . The method of  claim 34 , wherein the amplified DNA is analyzed by a technique selected from the group consisting of PCR-based analysis, restriction digestion analysis, and DNA sequencing analysis. 
     
     
         37 . A method of diagnosing a VKORC1 associated deficiency in a patient comprising the steps of:
 (I) providing a sample obtained from the patient; and   (II) detecting a VKORC1 polypeptide having a sequence abnormality in the sample using the antibody of  claim 31 ,   
       wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency. 
     
     
         38 . The method of  claim 37 , wherein the sample is analyzed by a technique selected from the group consisting of immunohistochemical detection, immunoblotting, preferably Western blotting, and ELISA. 
     
     
         39 . A method of identifying a coumarin derivative which exerts an inhibitory effect onto the activity of a VKORC1 polypeptide having at least one sequence abnormality, comprising the steps of:
 (I) providing a cell expressing the VKORC1 polypeptide according to  claim 25  or  26 , preferably a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 14, and 94;   (II) administering a candidate coumarin derivative to the cell;   (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and   (IV) comparing the sequence abnormality activity value with the control sequence activity value,   (V) identifying the candidate coumarin derivative as the coumarin derivative exerting an inhibitory effect onto the activity of a VKORC1 polypeptide, if the administration of the candidate coumarin derivative results in a sequence abnormality activity value which is significantly lower than the control sequence activity value.   
     
     
         40 . A method of identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of:
 (I) providing a warfarin-resistant rodent;   (II) administering a candidate coumarin derivative to the rodent;   (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value);   (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value;   (V) identifying the candidate coumarin derivative as a toxicologically effective coumarin derivative, provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value.   
     
     
         41 . A method of identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of:
 (I) providing a warfarin-resistant rodent;   (II) administering a candidate coumarin derivative to the rodent;   (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value);   (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value;   (V) identifying the candidate coumarin derivative as a toxicologically effective coumarin derivative, provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value;   
       wherein the warfarin-resistant rodent is a rodent transgenic for VKORC1 polypeptide of  claim 1 , wherein the VKORC1 polypeptide contains at least one sequence abnormality, which causes warfarin resistance, preferably a polypeptide encoded by the sequence according to SEQ ID No. 14. 
     
     
         42 . The method of  claim 40 , wherein VKORC1 polypeptide is the polypeptide according to SEQ ID No. 12 and the sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), and Y139C (416 A>G).). 
     
     
         43 . A composition for killing rodents:comprising a rodenticidally effective amount of the coumarin derivatives identified by the method according to  claim 39 . 
     
     
         44 . Use of the PCR primers according to SEQ ID No. 88 to 91 for determining whether or not a rat has a warfarin resistance genotype in a sample obtained from a rat. 
     
     
         45 . The vector of  claim 5 , wherein the vector is a knock-out gene construct. 
     
     
         46 . A host cell containing the vector of any one of  claims 4  through  6  and  45 . 
     
     
         47 . The host cell of  claim 46 , wherein the host cell is a non-human embryonic stem cell. 
     
     
         48 . A transgenic non-human mammal, wherein the transgenic mammal contains the host cell of  claim 47 . 
     
     
         49 . A method of producing a VKORC1 polypeptide comprising the steps of:
 (I) providing a host cell having been introduced a vector containing the VKORC1 nucleic acid of any one of  claims 4  through  6  and  46 ;   (II) expressing the VKORC1 polypeptide in the host cell; and   (III) isolating the VKORC1 polypeptide from the host cell.   
     
     
         50 . A composition for killing rodents, comprising a rodenticidally effective amount of the coumarin derivatives identified by the method according to  claim 40 . 
     
     
         51 . A composition for killing rodents, comprising a rodenticidally effective amount of the coumarin derivatives identified by the method according to  claim 41 .