Switch-region: target and method for inhibition of bacterial RNA polymerase

Abstract

The invention provides a target and methods for specific binding and inhibition of RNA polymerase from bacterial species. The invention provides methods for identifying agents that bind to a bacterial RNA polymerase, and that inhibit an activity of a bacterial RNA polymerase, through interactions with a bacterial RNA polymerase homologous switch-region amino-acid sequence. Said methods comprise preparing a reaction solution comprising the compound to be tested and an entity containing a bacterial RNAP homologous switch-region amino-acid sequence, and detecting binding or inhibition. The invention has applications in control of bacterial gene expression, control of bacterial viability, control of bacterial growth, antibacterial chemistry, and antibacterial therapy.

1 . A method for identifying an agent that binds to a bacterial RNAP homologous switch-region amino-acid sequence in a first entity, comprising the steps of: (a) preparing a reaction solution including the agent to be tested and a first entity including a bacterial RNAP homologous switch-region amino-acid sequence; and (b) detecting at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to the homologous switch-region amino-acid sequence.  
     
     
         2 . The method of  claim 1  wherein the first entity is an intact bacterial RNAP.  
     
     
         3 . The method of  claim 1  wherein the first entity is a fragment of a bacterial RNAP.  
     
     
         4 . The method of  claim 1  wherein the first entity is  Escerichia coli  RNAP or a derivative thereof.  
     
     
         5 . The method of  claim 1  wherein the first entity is  Bacillus subtilis  RNAP or a derivative thereof.  
     
     
         6 . The method of  claim 1  further comprising the step of: assessing at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to a second entity that contains a derivative of a bacterial RNAP homologous switch-region amino-acid sequence having at least one substitution, insertion, or deletion.  
     
     
         7 . The method of  claim 6  wherein the second entity is a derivative of an intact bacterial RNAP.  
     
     
         8 . The method of  claim 6  wherein the second entity is a derivative of a fragment of a bacterial RNAP.  
     
     
         9 . The method of  claim 6  wherein the second entity is a derivative of  Escerichia coli  RNAP.  
     
     
         10 . The method of  claim 6  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
     
     
         11 . The method of  claim 1  further comprising comparison of: (a) at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to the first entity, and (b) at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to a eukaryotic RNAP derivative.  
     
     
         12 . The method of  claim 11  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
     
     
         13 . The method of  claim 11  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
     
     
         14 . A method for identifying an agent that inhibits an activity of a bacterial RNAP by binding to a bacterial RNAP homologous switch-region amino-acid sequence, comprising: (a) preparing a reaction solution comprising the agent to be tested and a first entity containing a bacterial RNAP homologous switch-region amino-acid sequence; and (b) detecting at least one of the presence, extent, concentration-dependence, or kinetics of inhibition of an activity of said first entity, wherein inhibition involves binding of the agent to the bacterial RNAP homologous switch-region amino-acid sequence.  
     
     
         15 . The method of  claim 14  wherein the first entity is an intact bacterial RNAP.  
     
     
         16 . The method of  claim 14  wherein the first entity is a fragment of a bacterial RNAP.  
     
     
         17 . The method of  claim 14  wherein first entity is  Escerichia coli  RNAP or a derivative thereof.  
     
     
         18 . The method of  claim 14  wherein the first entity is  Bacillus subtilis  RNAP or a derivative thereof.  
     
     
         19 . The method of  claim 14  wherein the activity is transcription initiation.  
     
     
         20 . The method of  claim 14  wherein the activity is transcription elongation.  
     
     
         21 . The method of  claim 14  wherein the activity is σ binding.  
     
     
         22 . The method of  claim 14  wherein the activity is DNA binding.  
     
     
         23 . The method of  claim 14  wherein the activity is open-complex formation.  
     
     
         24 . The method of  claim 14  wherein the activity is RNA synthesis.  
     
     
         25 . The method of  claim 14  further comprising the step of: assessing at least one of the presence, extent, concentration-dependence, or kinetics of the inhibition by the agent of the activity of a second entity that contains a derivative of a bacterial RNAP homologous switch-region amino-acid sequence having at least one substitution, insertion, or deletion.  
     
     
         26 . The method of  claim 25  wherein the second entity is a derivative of an intact bacterial RNAP.  
     
     
         27 . The method of  claim 25  wherein the second entity is a derivative of a fragment of a bacterial RNAP.  
     
     
         28 . The method of  claim 25  wherein the second entity is a derivative of  Escerichia coli  RNAP.  
     
     
         29 . The method of  claim 25  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
     
     
         30 . The method of  claim 25  wherein the activity is transcription initiation.  
     
     
         31 . The method of  claim 25  wherein the activity is transcription elongation.  
     
     
         32 . The method of  claim 25  wherein the activity is open-complex formation.  
     
     
         33 . The method of  claim 25  wherein the activity is DNA binding.  
     
     
         34 . The method of  claim 25  wherein the activity is open-complex formation.  
     
     
         35 . The method of  claim 25  wherein the activity is RNA synthesis.  
     
     
         36 . The method of  claim 25  wherein inhibition of an activity of the first entity and inhibition of an activity of the second entity are assessed sequentially.  
     
     
         37 . The method of  claim 25  wherein inhibition of an activity of the first entity and inhibition of an activity of the second entity are assessed simultaneously.  
     
     
         38 . The method of  claim 14  further comprising comparison of: (a) at least one of the presence, extent, concentration-dependence, or kinetics of inhibition by the agent of an activity of the first entity, and (b) at least one of the presence, extent, concentration-dependence, or kinetics of inhibition by the agent of an activity of a eukaryotic RNAP derivative.  
     
     
         39 . The method of  claim 38  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
     
     
         40 . The method of  claim 3   8  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
     
     
         41 . The method of  claim 14  wherein at least one of the presence, extent, concentration-dependence, or kinetics of inhibition by the agent of an activity of the first entity also is compared to at least one of the presence, extent, concentration-dependence, or kinetics of inhibition by an inhibitory compound specific to the bacterial RNAP homologous switch-region amino-acid sequence of an activity of the first entity.  
     
     
         42 . A method for identifying an agent that exhibits antibacterial activity by binding to a bacterial RNAP homologous switch-region amino-acid sequence, comprising: (a) preparing a reaction solution comprising the agent to be tested and a first bacterium containing a bacterial RNAP homologous switch-region amino-acid sequence; and (b) detecting inhibition of at least one of viability of the bacterium and growth of the bacterium, wherein inhibition involves binding of the agent to the bacterial RNAP homologous switch-region amino-acid sequence.  
     
     
         43 . The method of  claim 42  wherein the first bacterium is  Escerichia coli  or a derivative thereof.  
     
     
         44 . The method of  claim 43  wherein the first bacterium is a tolC strain of  Escerichia coli  or a derivative thereof.  
     
     
         45 . The method of  claim 44  wherein the first bacterium is a tolC rfa strain of  Escerichia coli  or a derivative thereof.  
     
     
         46 . The method of  claim 42  wherein the first bacterium is  Bacillus subtilis  or a derivative thereof.  
     
     
         47 . The method of  claim 42  further comprising the step of: assessing inhibition by the agent of at least one of viability of a second bacterium and growth of a second bacterium, said second bacterium containing a derivative of a bacterial RNAP homologous switch-region amino-acid sequence having at least one substitution, insertion, or deletion.  
     
     
         48 . The method of  claim 47  wherein the second bacterium is a derivative of  Escerichia coli.    
     
     
         49 . The method of  claim 48  wherein the second bacterium is a derivative of a tolC strain of  Escerichia coli.    
     
     
         50 . The method of  claim 49  wherein the second bacterium is a derivative of a tolC rfa strain of  Escerichia coli.    
     
     
         51 . The method of  claim 47  wherein the second bacterium is a derivative of  Bacillus subtilis.    
     
     
         52 . The method of  claim 47  wherein antibacterial activity against the first bacterium and antibacterial activity against the second bacterium are assessed sequentially.  
     
     
         53 . The method of  claim 47  wherein antibacterial activity against the first bacterium and antibacterial activity against the second bacterium are assessed simultaneously.  
     
     
         54 . The method of  claim 42  wherein antibacterial activity of the agent against the first bacterium also is compared to antibacterial activity of an inhibitory compound specific to the bacterial RNAP homologous switch-region amino-acid sequence against the first bacterium.  
     
     
         55 . A method for identifying an agent that binds to a bacterial RNAP homologous switch-region amino-acid sequence, comprising (a) preparing a reaction solution comprising the agent to be tested, a reference compound that binds to a homologous bacterial RNAP switch-region amino-acid sequence, and a first entity containing a bacterial RNAP homologous switch-region amino-acid sequence, and (b) detecting at least one of the presence, extent, concentration-dependence, or kinetics of competition by the agent for binding of the reference compound to the homologous switch-region amino-acid sequence.  
     
     
         56 . The method of  claim 55  wherein the first entity is an intact bacterial RNAP.  
     
     
         57 . The method of  claim 55  wherein the first entity is a fragment of a bacterial RNAP.  
     
     
         58 . The method of  claim 55  wherein the first entity is  Escerichia coli  RNAP or a derivative thereof.  
     
     
         59 . The method of  claim 55  wherein the first entity is  Bacillus subtilis  RNAP or a derivative thereof.  
     
     
         60 . The method of  claim 55  wherein the reference compound contains a detectable group.  
     
     
         61 . The method of  claim 55  wherein the detectable group contains a chromophore.  
     
     
         62 . The method of  claim 55  wherein the detectable group contains a fluorophore.  
     
     
         63 . The method of  claim 55  wherein the reference compound is a chromophore-labeledinhibitory compound specific to the bacterial RNAP homologous switch-region amino-acid sequence.  
     
     
         64 . The method of  claim 55  wherein the reference compound is a fluorophore-labeled inhibitory compound specific to the bacterial RNAP homologous switch-region amino-acid sequence.  
     
     
         65 . The method of  claim 55  further comprising measurement of FRET.  
     
     
         66 . The method of  claim 55  further comprising the step of: assessing at least one of the presence, extent, concentration-dependence, or kinetics of the binding of the agent to a second entity that contains a derivative of a bacterial RNAP homologous switch-region amino-acid sequence having at least one substitution, insertion, or deletion.  
     
     
         67 . The method of  claim 66  wherein the second entity is a derivative of an intact bacterial RNAP.  
     
     
         68 . The method of  claim 66  wherein the second entity is a derivative of a fragment of a bacterial RNAP.  
     
     
         69 . The method of  claim 66  wherein the second entity is a derivative of  Escerichia coli  RNAP.  
     
     
         70 . The method of  claim 66  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
     
     
         71 . The method of  claim 55  further comprising comparison of: (a) at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to the first entity, and (b) at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to a eukaryotic RNAP derivative.  
     
     
         72 . The method of  claim 71  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
     
     
         73 . The method of  claim 71  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
     
     
         74 . The method of  claim 55  wherein at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to the first entity is compared to at least one of the presence, extent, concentration-dependence, or kinetics of binding of an inhibitory compound specific to the bacterial RNAP homologous switch-region amino-acid sequence to the first entity.  
     
     
         75 . A method for identifying an agent that binds to a bacterial RNAP homologous switch-region amino-acid sequence, comprising at least one of computer docking and energy calculations with a first entity containing at least one residue of a bacterial RNAP homologous switch-region amino-acid sequence.  
     
     
         76 . The method of  claim 75  wherein the first entity is an intact bacterial RNAP.  
     
     
         77 . The method of  claim 75  wherein the first entity is a fragment of a bacterial RNAP.  
     
     
         78 . The method of  claim 75  wherein the first entity is an intact bacterial RNAP in complex with a compound specific for the switch-region target.  
     
     
         79 . The method of  claim 75  wherein the first entity is a fragment of a bacterial RNAP in complex with a compound specific for the switch-region target.  
     
     
         80 . The method of  claim 75  wherein first entity is  Escerichia coli  RNAP or a derivative thereof.  
     
     
         81 . The method of  claim 75  wherein the first entity is  Bacillus subtilis  RNAP or a derivative thereof.  
     
     
         82 . The method of  claim 75  wherein the first entity is  Thermus  sp. RNAP or a derivative thereof.  
     
     
         83 . The method of  claim 75  further comprising the step of: performing at least one of computer docking and energy calculations with a second entity containing at least one residue of a derivative of a bacterial RNAP homologous switch-region amino-acid sequence having at least one substitution, insertion, or deletion.  
     
     
         84 . The method of  claim 83  wherein the second entity is a derivative of an intact bacterial RNAP.  
     
     
         85 . The method of  claim 83  wherein the second entity is a derivative of fragment of a bacterial RNAP.  
     
     
         86 . The method of  claim 83  wherein the second entity is a derivative of an intact bacterial RNAP in complex with a compound specific for the switch-region target.  
     
     
         87 . The method of  claim 83  wherein the second entity is a derivative of a fragment of a bacterial RNAP in complex with a compound specific for the switch-region target.  
     
     
         88 . The method of  claim 83  wherein second entity is a derivative of  Escerichia coli  RNAP.  
     
     
         89 . The method of  claim 83  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
     
     
         90 . The method of  claim 83  wherein the second entity is a derivative of  Thermus  sp. RNAP.  
     
     
         91 . The method of  claim 83  wherein computational analysis with the first entity and computational analysis with the second entity are performed sequentially.  
     
     
         92 . The method of  claim 83  wherein computational analysis with the first entity and computational analysis with the second entity are performed simultaneously.  
     
     
         93 . The method of  claim 75  further comprising comparison of: (a) results of at least one of computer docking and energy calculations with the agent and a first entity, and (b) results of at least one of computer docking and energy calculations with the agent and a eukaryotic RNAP derivative.  
     
     
         94 . The method of  claim 93  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
     
     
         95 . The method of  claim 93  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
     
     
         96 . The method of  claim 93  wherein the eukaryotic RNAP derivative is a yeast RNAP derivative.  
     
     
         97 . The method of  claim 93  wherein the eukaryotic RNAP derivative is a yeast RNAP II derivative.  
     
     
         98 . The method of  claim 75  wherein results of at least one of computer docking and energy calculations with the agent and the first entity also are compared to results of at least one of computer docking and energy calculations with an inhibitory compound specific to the bacterial RNAP homologous switch-region amino-acid sequence and the first entity.