US2003191083A1PendingUtilityA1
RNase P polypeptides, polynucleotides, and methods using their mechanisms of action

Assignee: SMITHKLINE BEECHAM CORPPriority: May 7, 1999Filed: Apr 15, 2003Published: Oct 9, 2003
Est. expiryMay 7, 2019(expired)· nominal 20-yr term from priority
Inventors:Michael J. Gress Lisa Hegg Hu Li Joseph Juseop Park
A61K 39/00G01N 33/56911G01N 2500/00G01N 2333/315G01N 2333/31C07K 14/31C12Q 1/689C12N 9/22
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References
Claims
Abstract

This invention relates to a novel bacterial ribonucleoprotein complex and the component parts thereof. More specifically, this invention relates to RNase P RNA isolated from Staphylococcus aureus and the use of RNase P RNA in screens for the identification of antimicrobial compounds and to the use of such compounds in therapy.
Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . An antagonist that inhibits or an agonist that activates an activity of an RNA selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO: 3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO: 3, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).    
     
     
         2 . A method for the treatment of an individual having need to inhibit or activate RNAseP RNA or holoenzyme comprising the steps of: administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agonist that activates an activity of a RNA selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO: 3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO: 3, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2,5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271((bulged A).    
     
     
         3 . A method for the treatment of an individual infected with a bacteria comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agonist that activates an activity of a RNA selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO: 3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO: 3, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0-01% NP40,400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).    
     
     
         4 . The method of  claim 3  wherein said bacteria is selected from the group consisting of: a member of the genus Staphylococcus,  Staphylococcus aureus,  a member of the genus Streptococcus, and  Streptococcus pneumoniae.    
     
     
         5 . A method for the treatment of an individual having need to inhibit or activate RNaseP RNA or holoenzyme comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agoinist that activates an activity of RNaseP RNA or holoenzyme selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).    
     
     
         6 . A method for the treatment of an individual infected with a bacteria comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or an agonist that activates that activates an activity of RNaseP selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).    
     
     
         7 . The method of  claim 6  wherein said bacteria is selected from the group consisting of: a member of the genus Staphylococcus,  Staphylococcus aureus,  a member of the genus Streptococcus, and  Streptococcus pneumoniae.    
     
     
         8 . A method for the treatment of an individual infected by  Streptococcus pneumoniae  comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits or anagonist that activates an activity of  Streptococcus pneumoniae  RNAseP RNA or holoenzyme selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).    
     
     
         9 . An antagonist that inhibits an activity of a polypeptide selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO: 3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO: 3, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).    
     
     
         10 . A method for the treatment of an individual having need to inhibit RNaseP RNA or holoenzyme comprising the steps of administering to the individual an antibacterially effective amount of an antagonist that inhibits an activity of a polypeptide selected from the group consisting of: an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO: 3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO: 3: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213A203, 204, and A169, 292-294 (L15.2), 271(bulged A).    
     
     
         11 . A method for inhibiting an activity of RNAseP RNA or holoenzyme comprising the steps of contacting a composition comprising said polypeptide with an effective amount of an antagonist that inhibits an activity of RNAseP, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).    
     
     
         12 . A method for inhibiting an activity of RnaseP RNA or holoenzyme, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 nM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).    
     
     
         13 . The method of  claim 12  wherein said bacteria is selected from the group consisting of: a member of the genus Staphylococcus,  Staphylococcus aureus,  a member of the genus Streptococcus, and  Streptococcus pneumoniae.    
     
     
         14 . A method for inhibiting a growth of bacteria comprising the steps of contacting a composition comprising bacteria with an antibacterially effective amount of an antagonist that inhibits an activity of RNAseP RNA or holoenzyme, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).    
     
     
         14 . The method of  claim 13  wherein said bacteria is selected from the group consisting of: a member of the genus Staphylococcus,  Staphylococcus aureus,  a member of the genus Streptococcus, and  Streptococcus pneumoniae.    
     
     
         15 . A method for inhibiting a RNaseP RNA or holoenzyme comprising the steps of contacting a composition comprising bacteria with an antibacterially effective amount of an antagonist that inhibits an activity of RNaseP, wherein said activity is selected from the group consisting of: 
   Staphylococcus aureus  RNaseP K m =53±4 nM determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP k cat =3.4±0.1 min −1  determined in a reaction in 1× buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNA phe  as a substrate;      Staphylococcus aureus  RNaseP RNA binding isotherm of K d =8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP40, 400 mM NH 4 OAc, 10 mM MgCl 2 , and 5% glycererol;      Staphylococcus aureus  RNaseP V1 cleavage of nucleotides selected from the group consisting of: 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294; and      Staphylococcus aureus  RNase T2 cleavage of nucleotides selected from the group consisting of: 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177; and    said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of: 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).    
     
     
         16 . The method of  claim 15  wherein said bacteria is selected from the group consisting of: a member of the genus Staphylococcus,  Staphylococcus aureus,  a member of the genus Streptococcus, and  Streptococcus pneumoniae.
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