Target and method for inhibition of bacterial rna polymerase

Abstract

Target and method for inhibition of bacterial RNA polymerase disclosed are targets and methods for specific binding and inhibition of RNAP from bacterial species. Bacterial RNA polymerase RPOC_ECOLI (736) QIRQLAGMRGLM  (779) ARK RPOC_HAEIN (737) QIRQLAGMRGLM  (780) ARK RPOC_VIBCH (736) QIRQLAGMRGLM  (779) ARK RPOC_PSEAE (736) QIRQLAGMRGLM  (779) ARK RPOC_TREPA (703) QIRQLAGMRGLM  (746) ARK RPOC_BORBU (699) QIRQLAGMRGLM  (742) ARK RPOC_XYLFA (759) QIRQLAAMRGLM  (802) ARK RPOC_CAMJE (734) QISQLAAMRGLM  (777) ARK RPOC_NEIMA (738) QIKQLSGMRGLM  (781) ARK RPOC_RICPR (730) QIKQLGGMRGLM  (773) MRK RPOC_THEMA(1010) QVKQLAGIRGLM (1072) ARK RPOC_CHLTR (736) QLKQLGALRGLM  (779) ARK RPOC_MYCPN (820) NFTQLFGMRGLM  (874) ARK RPOC_BACSU (740) NFTQLAGMRGLM  (783) ARK RPOC_STAAU (744) NFTQLAGMRGLM  (787) ARK RPOC_MYCTU (813) QTRTLAGMKGLV  (856) ARK RPOC_SYNY3 (763) QVRQLVGMRGLM  (806) ARK RPOC_AQUAE (850) QIRQLAGMRGLM  (893) ARK RPOC_DEIRA(1052) QIRQLAGMRGLM (1095) ARK RPOC_TTHER(1034) QIRQLCGLRGLM (1077) ARK RPOC_THEAQ(1034) QIRQLCGMRGLM (1077) ARK Human RNA polymerases I, II, and III RPA1_HUMAN (908) NTMQISCLLGQI  (971) GRE RPB1_HUMAN (780) NISQVIAVVGQQ  (843) GRE RPC1_HUMAN (791) NISQMIACVGQQ  (854) GRE

1 . An analog of bacteriocidal -peptide microcin J25 (MccJ25) that (1) has an amino acid sequence that differs from that of MccJ25 in terms of at least one amino acid substitution, insertion, or deletion; and (2) that binds a bacterial RNAP and inhibits an activity of bacterial RNAP with a potency at least equal to that of MccJ25.  
   
   
       2 . An analog according to  claim 1  selected from the group consisting of [Lys 5 ]MccJ25, [Lys13]MccJ25, [Lys 15 ]MccJ25, and [Lys 17 ]MccJ25.  
   
   
       3 . An analog according to  claim 1  selected from the group consisting of [X-Lys 5 ]MccJ25, [X-Lys 13 3MccJ25, [X-Lys 15 ]MccJ25, and [X-Lys 17 ]MccJ25, where X contains a detectable group.  
   
   
       4 . An analog according to  claim 3  where the detectable group contains a chromophore.  
   
   
       5 . An analog according to  claim 3  where the detectable group contains a fluorophore.  
   
   
       6 . An analog according to  claim 3  where the detectable group is Cy3.  
   
   
       7 . An analog according to  claim 1  that also contains a detectable group.  
   
   
       8 . An analog according to  claim 7  where the detectable group contains a chromophore.  
   
   
       9 . An analog according to  claim 7  where the detectable group contains a fluorophore.  
   
   
       10 . An analog according to  claim 7  where the detectable group is Cy3.  
   
   
       11 . A method for identifying an agent that binds to a bacterial RNAP homologous secondary channel 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 secondary channel amino acid sequence; and (b) detecting at least one of the presence, extent, concentration-dependence, or kinetics of binding of the agent to the bacterial RNAP homologous secondary channel amino acid sequence.  
   
   
       12 . The method of  claim 11  wherein the first entity is an intact bacterial RNAP.  
   
   
       13 . The method of  claim 11  wherein the first entity is a fragment of a bacterial RNAP.  
   
   
       14 . The method of  claim 11  wherein the first entity is a derivative of  Escherichia coli  RNAP.  
   
   
       15 . The method of  claim 11  wherein the first entity is a derivative of  Bacillus subtilis  RNAP.  
   
   
       16 . The method of  claim 11  further comprising comparison of: (a) the binding of the agent to the first entity; and (b) the binding of the agent to a second entity that contains a derivative of a bacterial RNAP homologous secondary channel amino acid having at least one substitution, insertion, or deletion.  
   
   
       17 . The method of  claim 16  wherein the second entity is a derivative of an intact bacterial RNAP.  
   
   
       18 . The method of  claim 16  wherein the second entity is a derivative of a fragment of a bacterial RNAP.  
   
   
       19 . The method of  claim 16  wherein the second entity is a derivative of  Escherichia coli  RNAP.  
   
   
       20 . The method of  claim 16  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
   
   
       21 . The method of  claim 11  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.  
   
   
       22 . The method of  claim 21  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
   
   
       23 . The method of  claim 21  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
   
   
       24 . The method of  claim 11  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 MccJ25 to the first entity.  
   
   
       25 . A method for identifying an agent that inhibits an activity of a bacterial RNAP by binding to a bacterial RNAP homologous secondary channel amino acid sequence, comprising: (a) preparing a reaction solution comprising the agent to be tested and a first entity containing a bacterial RNAP homologous secondary channel amino acid sequence; and (b) detecting the 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 homologous bacterial RNAP secondary channel amino acid sequence.  
   
   
       26 . The method of  claim 25  wherein the first entity is an intact bacterial RNAP.  
   
   
       27 . The method of  claim 25  wherein the first entity is a fragment of a bacterial RNAP.  
   
   
       28 . The method of  claim 25  wherein the first entity is a derivative of  Escherichia coli  RNAP.  
   
   
       29 . The method of  claim 25  wherein first entity is a derivative of  Bacillus subtilis  RNAP.  
   
   
       30 . The method of  claim 25  wherein the activity is RNA synthesis.  
   
   
       31 . The method of  claim 25  wherein the activity is NTP uptake.  
   
   
       32 . The method of  claim 25  wherein the activity is pyrophosphate release.  
   
   
       33 . The method of  claim 25  wherein the activity is abortive-RNA release.  
   
   
       34 . The method of  claim 25  wherein the activity is edited-RNA release.  
   
   
       35 . The method of  claim 25  wherein the activity is transcriptional pausing.  
   
   
       36 . The method of  claim 25  wherein the activity is transcriptional arrest.  
   
   
       37 . The method of  claim 25  wherein the activity is Gre-factor binding.  
   
   
       38 . The method of  claim 25  further comprising comparison of: (a) at least one of the presence, extent, concentration-dependence, or kinetics of the 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 the inhibition by the agent of an activity of a second entity that contains a derivative of a bacterial RNAP homologous secondary channel amino acid having at least one substitution, insertion, or deletion.  
   
   
       39 . The method of  claim 38  wherein the second entity is a derivative of an intact bacterial RNAP.  
   
   
       40 . The method of  claim 38  wherein the second entity is a derivative of a fragment of a bacterial RNAP.  
   
   
       41 . The method of  claim 38  wherein the second entity is a derivative of  Escherichia coli  RNAP.  
   
   
       42 . The method of  claim 38  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
   
   
       43 . The method of  claim 38  wherein the activity is RNA synthesis.  
   
   
       44 . The method of  claim 38  wherein the activity is NTP uptake.  
   
   
       45 . The method of  claim 38  wherein the activity is pyrophosphate release.  
   
   
       46 . The method of  claim 38  wherein the activity is abortive-RNA release.  
   
   
       47 . The method of  claim 38  wherein the activity is edited-RNA release.  
   
   
       48 . The method of  claim 38  wherein the activity is transcriptional pausing.  
   
   
       49 . The method of  claim 38  wherein the activity is transcriptional arrest.  
   
   
       50 . The method of  claim 38  wherein the activity is Gre-factor binding.  
   
   
       51 . The method of  claim 38  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.  
   
   
       52 . The method of  claim 51  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
   
   
       53 . The method of  claim 51  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
   
   
       54 . The method of  claim 25  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 MccJ25 of an activity of the first entity.  
   
   
       55 . A method for identifying an agent that binds to a bacterial RNAP homologous secondary channel 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 secondary channel amino acid sequence, and a first entity containing a bacterial RNAP homologous secondary channel 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 secondary channel 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 a derivative of  Escherichia coli  RNAP.  
   
   
       59 . The method of  claim 55  the first entity is a derivative of  Bacillus subtilis  RNAP.  
   
   
       60 . The method of  claim 55  wherein the reference compound contains a detectable group.  
   
   
       61 . The method of  claim 60  wherein the detectable group contains a chromophore.  
   
   
       62 . The method of  claim 60  wherein the detectable group contains a fluorophore.  
   
   
       63 . The method of  claim 55  wherein the reference compound is MccJ25.  
   
   
       64 . The method of  claim 55  wherein the reference compound is a MccJ25 derivative.  
   
   
       65 . The method of  claim 55  wherein the reference compound is a chromophore-labelled MccJ25 derivative.  
   
   
       66 . The method of  claim 55  wherein the reference compound is a fluorophore-labelled MccJ25 derivative.  
   
   
       67 . The method of  claim 55  wherein the reference compound is selected from the group consisting of [Cy3-Lys 5 ]-MccJ25 and (Cy3-Lys 13 ]-MccJ25.  
   
   
       68 . The method of  claim 55  further comprising measurement of FRET.  
   
   
       69 . The method of  claim 55  further comprising comparison of: (a) at least one of the presence, extent, concentration-dependence, or kinetics of the binding of the agent to the first entity; and (b) 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 secondary channel amino acid having at least one substitution, insertion, or deletion.  
   
   
       70 . The method of  claim 69  wherein the second entity is a derivative of an intact bacterial RNAP.  
   
   
       71 . The method of  claim 69  wherein the second entity is a derivative of a fragment of a bacterial RNAP.  
   
   
       72 . The method of  claim 69  wherein the second entity is a derivative of  Escherichia coli  RNAP.  
   
   
       73 . The method of  claim 69  wherein the second entity is a derivative of  Bacillus subtilis  RNAP.  
   
   
       74 . The method of  claim 69  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.  
   
   
       75 . The method of  claim 74  wherein the eukaryotic RNAP derivative is a human RNAP derivative.  
   
   
       76 . The method of  claim 74  wherein the eukaryotic RNAP derivative is a human RNAP II derivative.  
   
   
       77 . 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 MccJ25 to the first entity.