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   (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) a VKORC1 polypeptide and   (b) a heterologeous part.   
   
   
       4 . A vector comprising the VKORC1 nucleic acid of  claim 2 . 
   
   
       5 - 6 . (canceled) 
   
   
       7 . A host cell containing the VKORC1 nucleic acid of  claim 2 . 
   
   
       8 . (canceled) 
   
   
       9 . A transgenic non-human mammal, wherein the transgenic mammal contains the host cell of  claim 7 . 
   
   
       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 - 20 . (canceled) 
   
   
       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;   (II) 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 - 24 . (canceled) 
   
   
       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 ,   (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 .   
   
   
       28 - 32 . (canceled) 
   
   
       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 a 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 - 36 . (canceled) 
   
   
       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 an antibody that specifically recognizes and binds a VKORC1 polypeptide of  claim 25 ,   wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency.   
   
   
       38 . (canceled) 
   
   
       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 , 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 - 48 . (canceled) 
   
   
       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  claim 4 ;   (II) expressing the VKORC1 polypeptide in the host cell; and   (III) isolating the VKORC1 polypeptide from the host cell.   
   
   
       50 - 51 . (canceled)