Hbv rnase h purification and enzyme inhibitors

Abstract

Provided herein are methods for the obtention of an active HBV RNaseH preparation and its use in screening methods to identify potential inhibitors of the enzyme for possible use as therapeutic agents. Also provided are methods of treatment using agents identified according to the screen.

1 . A method isolating a hepatitis B virus (HBV) RNAseH comprising:
 (a) providing a cell that expresses said HBV RNAseH;   (b) lysing said cell by sonication to produce a lysate;   (c) applying said lysate to a nickel-agarose affinity chromatography column;   (d) eluting bound material from said column with imidazole; and   (e) removing imidazole from the eluate.   
     
     
         2 . The method of  claim 1 , wherein said cell is a cell infected with HBV. 
     
     
         3 . The method of  claim 1 , wherein said cell is a cell comprising an HBV RNAseH expression vector. 
     
     
         4 . The method of  claim 3 , wherein said cell is a prokaryotic cell. 
     
     
         5 . The method of  claim 4 , wherein said cell is an  Eschericia coli  cell. 
     
     
         6 . The method of  claim 3 , wherein said cell is a eukaryotic cell. 
     
     
         7 . The method of  claim 1 , wherein said imidazole is applied to said column at 100 mM to 500 mM. 
     
     
         8 . The method of  claim 1 , wherein removing imidazole comprise dialyzing the eluate into a buffered salt solution. 
     
     
         9 . The method of  claim 8 , wherein said salt solution contains, or has added thereto, glycerol and dithiothreitol. 
     
     
         10 . The method of  claim 1 , wherein the RNAseH is produced in an in vitro translation system. 
     
     
         11 . The method of  claim 1 , wherein lysing comprises sonication, lysozyme/detergent treatment, shearing or nitrogen rupture. 
     
     
         12 . A purified and active hepatitis B virus (HBV) RNAseH obtained by the process comprising:
 (a) providing a cell that expresses said HBV RNAseH;   (b) lysing said cell to produce a lysate;   (c) applying said lysate to a nickel-agarose affinity chromatography column;   (d) eluting bound material from said column with imidazole; and   (e) removing imidazole from the eluate.   
     
     
         13 . The RNAseH of  claim 12 , wherein said cell is a cell infected with HBV. 
     
     
         14 . The RNAseH of  claim 12 , wherein said cell is a cell comprising an HBV RNAseH expression vector. 
     
     
         15 . The RNAseH of  claim 12 , wherein said imidazole is applied to said column at 100 mM to 500 mM. 
     
     
         16 . The RNAseH of  claim 12 , wherein removing imidazole comprise dialyzing the eluate into a buffered salt solution. 
     
     
         17 . A purified hepatitis B virus (HBV) RNAseH composition retaining RNAseH activity and being substantially devoid of other enzyme activity, wherein said composition has (a) RNAseH activity in the presence 2-8 mM Mg 2+  and (b) no specific activity for RNA:DNA heteroduplexes below 90 mM NaCl. 
     
     
         18 . The RNAseH of  claim 17 , wherein the RNAseH is a genotype H RNAse H 
     
     
         19 . A method of screening a substance for hepatitis B virus (HBV) RNAseH inhibitory activity comprising:
 (a) providing a purified hepatitis B virus (HBV) RNAseH composition retaining RNAseH activity and being substantially devoid of other enzyme activity;   (b) contacting said HBV RNAseH composition with a candidate substance; and   (c) assessing whether the candidate substance inhibits activity of said HBV RNAseH composition.   
     
     
         20 . The method of  claim 19 , wherein assessing comprises a DNA oligonucleotide-directed RNA cleavage assay. 
     
     
         21 . The method of  claim 20 , wherein assessing comprises gel electrophoresis. 
     
     
         22 . The method of  claim 20 , wherein assessing comprises measuring fluorescent quenching from a dual-labeled substrate. 
     
     
         23 . The method of  claim 20 , wherein assessing comprises measuring soluble radioactivity following annealing of radiolabeled RNA to DNA. 
     
     
         24 . The method of  claim 20 , wherein assessing comprises measuring soluble fluorescence following annealing of radiolabeled RNA to DNA. 
     
     
         25 . The method of  claim 23 , wherein the RNA or DNA is immobilized on a surface. 
     
     
         26 . The method of  claim 19 , wherein the RNAseH is present at between about 0.5 ng/μl to about 100 ng/μl, or about 0.15 ng/μl to about 0.75 ng/μl. 
     
     
         27 . The method of  claim 19 , further comprising assessing activity of said HBV RNAseH composition in the absence of said candidate substance. 
     
     
         28 . The method of  claim 19 , wherein providing comprises:
 (a) providing a cell that expresses said HBV RNAseH;   (b) lysing said cell to produce a lysate;   (c) applying said lysate to a nickel-agarose affinity chromatography column;   (d) eluting bound material from said column with imidazole; and   (e) removing imidazole from the eluate.   
     
     
         29 . A method of inhibiting a hepatitis B virus (HBV) RNAseH comprising contacting said enzyme with a compound having the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is —OH; 
         R 2  is —OH; 
         R 3  is —OH or O; 
         R 4  is —OH; and 
         X is H, methyl, substituted or unsubstituted phenyl or piperidine; 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 5  is H, —OH or C 1 -C 6  alkoxy; 
         R 6  is H, O or substituted or unsubstituted phenyl; 
         R 7  is H, —OH or —COOH; 
         R 8  is —CH 3  or O; 
         R 9  is —OH or H; 
         R 10  is H, —OH, C 1 -C 4  alkyl optionally substituted with substituted or unsubstituted phenyl; 
         Y is O or NR 11 , wherein R 11  is branched or straight-chain C 1 -C 6  alkyl or hydroxyalkyl; or 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 11  is hydrogen, —OR 17 , hydroxy, or halo, wherein R 17  is C 1 -C 8  acyl, C 1 -C 8  alkyl, or a substituted version of either of these groups; 
         R 12  is hydrogen, hydroxy or halo; 
         R 13  and R 14  are each independently hydrogen, hydroxy, nitroso, C 1 -C 12  alkyl, C 1 -C 12  alkenyl, C 1 -C 12  alkynyl, C 1 -C 12  aryl, C 1 -C 12  aralkyl, C 1 -C 12  amido, a substituted version of any of these groups, or R 13  is taken together with R 14  as provided below; 
         R 15  is hydrogen, C 1 -C 12  alkyl, C 1 -C 12  alkenyl, C 1 -C 12  alkynyl, C 1 -C 12  aryl, C 1 -C 12  aralkyl, 
       
       
         
           
           
               
               
           
         
       
       or a substituted version of any of these groups; and
 R 16  is hydrogen, unsubstituted C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, or hydroxyl; 
 provided that when R 13  and R 14  are taken together as further defined by FORMULA IV: 
 
       
         
           
           
               
               
           
         
         R 18  and R 19  are each independently hydrogen, C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  alkynyl. 
       
     
     
         30 . The method of  claim 29 , wherein the compound is of FORMULA I and X is phenyl substituted with O or NR 12 R 13  at the para position, wherein R 12  and R 13  are H or —CH 3 . 
     
     
         31 . The method of  claim 29 , wherein the compound is of FORMULA II and R 6  is di- or tri-substituted phenyl. 
     
     
         32 . The method of  claim 29 , wherein the compound is of FORMULA II and R 10  is CH 2 R 14 , wherein R 14  is halogen-disubstituted phenyl. 
     
     
         33 . The method of  claim 29 , wherein the compound is of FORMULA III and R 11  is hydroxy. 
     
     
         34 . The method of  claim 29 , wherein the compound is of FORMULA III and at least one of R 12 , R 13 , R 14  or R 15  is C 1 -C 12 -alkyl. 
     
     
         35 . The method of  claim 29 , wherein the compound is of FORMULA IV and R 18  is C 1 -C 6  alkenyl. 
     
     
         36 . The method of  claim 29 , further comprising contacting said enzyme with a second inhibitor of RNAse enzyme activity. 
     
     
         37 . The method of  claim 36 , wherein said second inhibitor is a nucleoside analog. 
     
     
         38 . The method of  claim 29 , further comprising contacting said enzyme with said compound a second time. 
     
     
         39 . The method of  claim 29 , wherein said enzyme is located in a cell. 
     
     
         40 . The method of  claim 39 , wherein said cell is located in vitro. 
     
     
         41 . The method of  claim 39 , wherein said cell is located in a living subject. 
     
     
         42 . The method of  claim 41 , wherein said subject is a mammal infected with HBV. 
     
     
         43 . The method of  claim 42 , wherein said compound is administered intravenously, intra-arterially, orally, or subcutaneously. 
     
     
         44 . The method of  claim 42 , wherein said subject is further administered a second inhibitor of RNAse enzyme activity. 
     
     
         45 . The method of  claim 44 , wherein said second inhibitor is a nucleoside analog. 
     
     
         46 . The method of  claim 44 , wherein said second inhibitor is administered to said subject before or after said compound. 
     
     
         47 . The method of  claim 44 , wherein said second inhibitor is administered to said subject at the same time as said compound. 
     
     
         48 . The method of  claim 41 , wherein said subject has previously received a first-line HBV therapy. 
     
     
         49 . The method of  claim 48 , wherein said HBV has developed resistance to said first-line HBV therapy. 
     
     
         50 . The method of  claim 42 , wherein said subject is administered interferon or pegylated interferon. 
     
     
         51 . A method of inhibiting a hepatitis B virus (HBV) RNAseH comprising contacting said enzyme with a compound having the formula selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         52 . The method of  claim 51 , further comprising contacting said enzyme with a second inhibitor of RNAse enzyme activity. 
     
     
         53 . The method of  claim 52 , wherein said second inhibitor is a nucleoside analog. 
     
     
         54 . The method of  claim 51 , further comprising contacting said enzyme with said compound a second time. 
     
     
         55 . The method of  claim 51 , wherein said enzyme is located in a cell. 
     
     
         56 . The method of  claim 55 , wherein said cell is located in vitro. 
     
     
         57 . The method of  claim 55 , wherein said cell is located in a living subject. 
     
     
         58 . The method of  claim 57 , wherein said subject is a mammal infected with HBV. 
     
     
         59 . The method of  claim 58 , wherein said compound is administered intravenously, intra-arterially, orally, or subcutaneously. 
     
     
         60 . The method of  claim 58 , wherein said subject is further administered a second inhibitor of RNAse enzyme activity. 
     
     
         61 . The method of  claim 60 , wherein said second inhibitor is a nucleoside analog. 
     
     
         62 . The method of  claim 60 , wherein said second inhibitor is administered to said subject before or after said compound. 
     
     
         63 . The method of  claim 60 , wherein said second inhibitor is administered to said subject at the same time as said compound. 
     
     
         64 . The method of  claim 57 , wherein said subject has previously received a first-line HBV therapy. 
     
     
         65 . The method of  claim 64 , wherein said HBV has developed resistance to said first-line HBV therapy. 
     
     
         66 . The method of  claim 57 , wherein said subject is administered interferon or pegylated interferon. 
     
     
         67 . A composition comprising purified hepatitis B virus RNAse H in a storage medium that retains at least 50% of initial RNAse H activity for at least 5 hours at 0° C. 
     
     
         68 . The composition of  claim 67 , wherein the composition has a temperature below minus 50° C. 
     
     
         69 . The composition of  claim 67 , wherein the storage medium maintains reducing conditions. 
     
     
         70 . The composition of  claim 69 , wherein the storage medium comprises dithiothreitol (DTT). 
     
     
         71 . The composition of  claim 67 , wherein the storage medium is buffered to a pH in the range of about pH 6.6 to about pH 8.5. 
     
     
         72 . The composition of  claim 67 , wherein the storage medium is buffered to a pH in the range of about pH 7.2 to about pH 7.8. 
     
     
         73 . The composition of  claim 67 , further comprising NaCl at a concentration in the range of about 300 mM to about 1M. 
     
     
         74 . The composition of  claim 67 , stored in liquid nitrogen. 
     
     
         75 . The composition of  claim 74 , stored in at least 5 containers each having a volume of less than 1 ml. 
     
     
         76 . The method of  claim 19 , wherein the HBV RNAse H composition is a buffered aqueous solution having a NaCl concentration of in the range of about 120 mM to about 240 mM. 
     
     
         77 . The method of  claim 76 , wherein the HBV RNAse H composition has a NaCl concentration in the range of about 170 mM to about 210 mM 
     
     
         78 . The method of  claim 19 , wherein the HBV RNAse H composition comprises Mg +2  ions. 
     
     
         79 . The method of  claim 19 , wherein the HBV RNAse H composition comprises Mn +2  ions. 
     
     
         80 . The method of  claim 19 , wherein the HBV RNAse H composition comprises between about 0.05% to about 1% of a nonionic surfactant, by volume. 
     
     
         81 . The composition of  claim 67 , wherein said RNAse H is present in the range of about 0.1 ng/μl to about 1.0 ng/μl. 
     
     
         82 . The composition of  claim 67 , wherein said RNAse H is present in the range of about 0.1 ng/μl to about 0.5 ng/μl. 
     
     
         83 . The composition of  claim 67 , wherein said RNAse H is present at about 0.5 ng/μl. 
     
     
         84 . The composition of  claim 67 , wherein said RNAse H is present in range of about 0.01% to about 0.05% of total protein. 
     
     
         85 . The composition of  claim 67 , wherein said RNAse H is present in the range of about 0.02% to about 0.025% of total protein.