Patent application title: BRIDGE-HELIX CAP: TARGET AND METHOD FOR INHIBITION OF BACTERIAL RNA POLYMERASE
Inventors:
Richard H. Ebright (New Brunswick, NJ, US)
Richard H. Ebright (New Brunswick, NJ, US)
David Degen (New Brunswick, NJ, US)
Katherine Y. Ebright (New Brunswick, NJ, US)
Assignees:
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
IPC8 Class: AA61K3815FI
USPC Class:
514 29
Class name: Micro-organism destroying or inhibiting bacterium (e.g., bacillus, etc.) destroying or inhibiting cyclopeptide utilizing
Publication date: 2016-03-24
Patent application number: 20160082076
Abstract:
It has been discovered that the Sal target represents a new and promising
target for antibacterial drug discovery. The Sal target is distinct from
the rifamycin target and from the CBR703 target. This indicates that
antibacterial compounds that function through the Sal target should
exhibit no, or minimal, cross-resistance with rifamycins and CBR703. This
further implies that it should be possible to co-administer antibacterial
compounds that function through the Sal target together with a rifamycin,
together with CBR703, or together with both a rifamycin and CBR703, in
order to achieve additive or synergistic antibacterial effects and in
order to suppress or eliminate the emergence of resistance.Claims:
1. A method to treat a bacterial infection in a subject in need thereof,
comprising administering to the subject a first compound selected as
being an inhibitor of growth of a bacterium by binding to the
bridge-helix cap target of an RNA polymerase, and a second compound that
inhibits growth of a bacterium by binding to a site other than the
bridge-helix cap target of an RNA polymerase.
2. The method of claim 1, wherein the first compound is salinamide A.
3. The method of claim 1, wherein the second compound is a rifamycin or CBR703.
4. The method of claim 3, wherein the second compound is rifampin or CBR703.
5. The method of claim 1, further comprising administering a third compound that inhibits growth of a bacterium by binding to a site other than the bridge-helix cap target of an RNA polymerase.
6. The method of claim 1, wherein the first and second compounds are administered concurrently.
7. The method of claim 1, wherein the first and second compounds are administered sequentially.
8. The method of claim 5, wherein the third compound is administered concurrently with the first or second compound, or concurrently with the first and second compound.
9. The method of claim 5, wherein the third compound is administered sequentially.
10. A composition that comprises salinamide A and a rifamycin and/or CBR703.
11. The composition of claim 10 that comprises salinamide A and rifampin and/or CBR703.
12. The composition of claim 10 that comprises salinamide A and a rifamycin.
13. The composition of claim 12 that comprises salinamide A and rifampin.
14. The composition of claim 10 that comprises salinamide A and CBR703.
15. The composition of claim 10 that comprises salinamide A, a rifamycin, and CBR703.
16. The composition of claim 15 that comprises salinamide A, rifampin, and CBR703.
Description:
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This patent application is a divisional of U.S. application Ser. No. 14/006,035, which is a 35 U.S.C. §371 application of International Application No. PCT/US2012/029679, filed Mar. 19, 2012, which claims the benefit of U.S. Provisional Application Ser. No. 61/454,323, filed Mar. 18, 2011, now expired. The entire content of the applications referenced above are hereby incorporated by reference herein.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, is named 83503WO1.txt and is 29,392 bytes in size.
BACKGROUND
[0004] Bacterial infections remain among the most common and deadly causes of human disease. Multi-drug-resistant bacteria now cause infections that pose a grave and growing threat to public health. It has been shown that bacterial pathogens can acquire resistance to first-line and even second-line antibiotics. New approaches to drug development for treating bacterial infections are necessary, e.g., to combat the ever-increasing number of antibiotic-resistant pathogens.
SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION
[0005] We disclose here that the antibiotic salinamide A (Sal) inhibits bacterial RNA polymerase (RNAP) in vivo, that Sal kills bacteria by inhibiting RNAP, and how Sal inhibits RNAP. To determine whether Sal inhibits RNAP in vivo, effects of Sal on RNAP activity in vivo were assayed. Upon addition of Sal to bacteria, RNAP-dependent RNA synthesis decreased immediately, indicating that Sal inhibits RNAP in vivo. To determine whether Sal kills bacteria by inhibiting RNAP, spontaneous Sal-resistant mutants of were isolated, and genes for RNAP subunits in Sal-resistant mutants were amplified and sequenced. One hundred percent (39 of 39) of analyzed Sal-resistant mutants contained mutations in genes encoding RNAP subunits, indicating that Sal kills bacteria by inhibiting RNAP. Substitutions conferring Sal-resistance were obtained at positions 690, 697, 738, 748, 758, 763, 779, 780, 782, and 783 of Escherichia coli RNAP subunit and positions 569, 675, and 677 of Escherichia coli RNAP β-subunit. Additional Sal-resistant mutants were isolated following mutagenesis of genes encoding RNAP subunits. In these additional Sal-resistant mutants, substitutions conferring Sal-resistance were identified at positions 758, 780, and 782 of Escherichia coli RNAP (3' subunit and positions 561, 665, and 680 of Escherichia coli RNAP β subunit.
[0006] When mapped onto the structure of RNAP, the sites of substitutions conferring Sal-resistance formed a compact cluster. We designate the sites of substitutions conferring Sal-resistance as the "Sal target."
[0007] Determination of a crystal structure of Escherichia coli RNAP in complex with Sal showed that the Sal target is the binding site on RNAP for Sal, and that sites of substitutions conferring Sal-resistance correspond to RNAP residues of RNAP that contact, or are close to, Sal.
[0008] The Sal target does not overlap the targets of currently used antibiotics. Accordingly, Sal does not exhibit cross-resistance with currently used antibiotics, and coadministration of Sal with a currently used antibiotic results in an extremely low spontaneous resistance frequency. The Sal target represents an attractive new target for antibacterial drug discovery.
[0009] A subset of residues of the Sal target have no overlap with any previously described target of any previously described RNAP inhibitor: i.e., residues 690, 697, 758, and 763 of Escherichia coli RNAP β' subunit and residues 561, 569, 665, 675, 677, and 680 of Escherichia coli RNAP β subunit. In the structure of RNAP, these residues are located in proximity to the N-terminus of the RNAP-active-center bridge helix, in and near RNAP-active-center sub-regions termed the "β' F loop," the "β D2 loop," and the "β link region" (FIGS. 4A-C). We designate these residues as the "bridge-helix cap target."
[0010] In RNAP from Escherichia coli, the bridge-helix cap target comprises residues 690, 697, 758, and 763 of Escherichia coli RNAP β' subunit and residues 561, 569, 665, 675, 677, and 680 of Escherichia coli RNAP β subunit. In RNAP from other bacterial species, the bridge-helix cap target comprises the residues corresponding to, and alignable with, residues 690, 697, 758, and 763 of Escherichia coli RNAP β' subunit and residues 561, 569, 665, 675, 677, and 680 of Escherichia coli RNAP β subunit (FIGS. 4A-C). For example, in RNAP from Bacillus subtilis, the bridge-helix cap target comprises the residues corresponding to, and alignable with, residues 709, 716, 762, and 767 of Bacillus subtilis RNAP β' subunit and residues 517, 525, 623, 633, 635, and 638 of Bacillus subtilis RNAP β subunit (FIGS. 4A-C).
[0011] Accordingly, certain embodiments of the present invention provide
[0012] (i) a target for binding of a molecule to an RNA polymerase from a bacterial species, comprising at least one residue corresponding to, and alignable with, residues 561, 569, 665, 675, 677, and 680 of the β subunit and residues 690, 697, 758, and 763 of the β' subunit of RNA polymerase from Escherichia coli ("bridge-helix cap target");
[0013] (ii) a target for inhibition by a molecule of an RNA polymerase from a bacterial species, comprising at least one residue corresponding to, and alignable with, residues 561, 569, 665, 675, 677, and 680 of the β subunit and residues 690, 697, 758, and 763 of the β' subunit of RNA polymerase from Escherichia coli ("bridge-helix cap target");
[0014] (iii) a method to identify a molecule that binds to the target of i and ii comprising identification of a molecule that (a) binds to an RNA polymerase from a bacterial species, but (b) does not bind, or binds less well, to a derivative of an RNA polymerase from a bacterial species that has at least one amino acid substitution, deletion, or insertion, in the set of residues taught in i and ii;
[0015] (iv) a method to identify a molecule that inhibits an RNA polymerase from a bacterial species through the target of i and ii, comprising identification of a molecule that (a) inhibits an RNA polymerase from a bacterial species, but (b) does not inhibit, or inhibits less well, a derivative of an RNA polymerase from a bacterial species that has at least one amino acid substitution, deletion, or insertion, in the set of residues taught in i and ii;
[0016] (v) a method to identify a molecule that inhibits growth of a bacterium through the target of i and ii, comprising identification of a molecule that (a) inhibits growth of a bacterium, but (b) does not inhibit, or inhibits less well, a derivative of said bacterium that contains a derivative of an RNA polymerase that has at least one amino acid substitution, deletion, or insertion, in the set of residues taught in i and ii; and
[0017] (vi) a method to identify a molecule that binds to an RNA polymerase from a bacterial species through the target of i and ii, comprising (a) preparation of a first molecule that binds to the target of i and ii and that contains a detectable group, and (b) identification of a second molecule that competes with said first molecule for binding to an RNA polymerase from a bacterial species.
[0018] (vii) a method to treat a bacterial infection in a subject in need thereof, comprising administering to the subject a first compound selected as being an inhibitor of growth of a bacterium by binding to the target of i and ii, and a second compound that inhibits growth of a bacterium by binding to a site other than the target of i and ii.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1. Structure of Sal.
[0020] FIGS. 2A-B. Measurement of effects of Sal on nucleic acid synthesis in vivo. (FIG. 2A) Experimental approach. (FIG. 2B) Effects of Sal on RNA (left) and DNA (right) synthesis in vivo.
[0021] FIGS. 3A-C. Isolation of spontaneous Sal-resistant mutants, followed by PCR-amplification and sequencing of RNAP-subunit genes. (FIG. 3A) Experimental approach. (FIG. 3B) Representative data, isolation of Sal-resistant mutants. (FIG. 3C) Representative data, PCR-amplification of rpoC (left) and rpoB (right) genes.
[0022] FIGS. 4A-C. Sal target: location in the amino acid sequence of RNAP β' and ρ subunits. (FIG. 4A, FIG. 4B and FIG. 4C) Amino acid sequence alignments for regions of E. coli RNAP β' subunit and β subunit containing single-residue substitutions that confer Sal-resistance (see Tables 2-3). Sequences for bacterial RNAP are in the top twenty rows of FIG. 4A, FIG. 4B and FIG. 4C (Sequences for bacterial RNAP are disclosed as SEQ ID NOS 5-64, respectively, in order of appearance. Sequences for bacterial RNAP are disclosed as SEQ ID NOS 74-113, respectively, in order of appearance.); sequences for human RNAP I, RNAP II, and RNAP III are in the bottom three rows of FIG. 4A, FIG. 4B and FIG. 4C (Sequences for human RNAP I, RNAP II, and RNAP III are disclosed as SEQ ID NOS 65-73, respectively, in order of appearance. Sequences for human RNAP I, RNAP II, and RNAP III are disclosed as SEQ ID NOS 114-119, respectively, in order of appearance.); sites of single-residue substitutions that confer Sal-resistance are boxed (with E. coli residue numbers); RNAP active-center sub-regions are indicated with black bars above the sequences. Species are as follows: E. coli (ECOLI), Haemophilus influenzae (HAEIN), Vibrio cholerae (VIBCH), Pseudomonas aeruginosa (PSEAE), Treponema pallidum (TREPA), Bordetella pertussis (BORPE), Xylella fastidiosa (XYLFA), Campylobacter jejuni (CAMJE), Neisseria meningitidis (NEIME), Rickettsia prowazekii (RICPR), Chlamydia trachomatis (CHLTR), Mycoplasma pneumoniae (MYCPN), Bacillus subtilis (BACSU), Staphylococcus aureus (STAAU), Mycobacterium tuberculosis (MYCTU), Synechocystis sp. PCC 6803 (SYNY3), Aquifex aeolicus (AQUAE), Deinococcus radiodurans (DEIRA), Thermus aquaticus (THEAQ), Thermus thermophilus (THETH), and Homo sapiens (HUMAN).
[0023] FIG. 5. Sal target: location in the three-dimensional structure of RNAP. Three-dimensional structure of RNAP showing the Sal target. RNAP is illustrated in a ribbon representation in gray. Sites of substitutions that confer Sal-resistance (see Tables 2-3) are illustrated in a space-filling representation. (*) The RNAP active-center Mg2+ ion is illustrated as a sphere (**). The structure shown is a crystal structure of T. thermophilus RNAP holoenzyme (8; (3'-subunit non-conserved region and a subunit omitted for clarity); correspondences between residues of E. coli RNAP and T. thermophilus RNAP are based on amino acid sequence alignments (see FIGS. 4A-C).
[0024] FIG. 6. Sal target: relationship between the Sal target, the rifamycin target, and the CBR703 target. Three-dimensional structure of RNAP showing the Sal target, the rifamycin target, and the CBR703 target. Sites of substitutions that confer Sal-resistance (*) (see Tables 2-3), rifamycin-resistance (***) (10-15), and CBR703-resistance (****) (16,17) are illustrated in space-filling representations. The RNAP active-center Mg2+ ion is illustrated as a sphere (**).
[0025] FIGS. 7A-B. Crystal structure of E. coli RNAP σ70 holoenzyme in complex with Sal. FIG. 7A shows electron density for Sal (#; 2σ Fo-Fc difference electron density for E. coli RNAP σ70 holoenzyme with Sal vs. E. coli RNAP holoenzyme without Sal). FIG. 7B shows, for comparison, sites of substitutions that confer Sal-resistance (*; see Tables 2-3). In each panel, the RNAP active-center Mg2+ ion is illustrated as a sphere (**).
DETAILED DESCRIPTION
[0026] It has been discovered that the bridge-helix cap target represents a new and promising target for antibacterial drug discovery. The bridge-helix cap target is distinct from the rifamycin target and from the CBR703 target. This implies that antibacterial compounds that function through the bridge-helix cap target should exhibit no, or minimal, cross-resistance with rifamycins and CBR703. This further implies that it should be possible to co-administer antibacterial compounds that function through the bridge-helix cap target together with a rifamycin, together with CBR703, or together with both a rifamycin and CBR703, in order to achieve additive or super-additive/synergistic antibacterial effects and in order to suppress or eliminate the emergence of resistance.
[0027] Accordingly, certain embodiments of the present invention provide a method to identify a molecule that binds to the bridge-helix cap target of an RNA polymerase from a bacterial species, comprising identifying a molecule that binds to an RNA polymerase from a bacterial species but binds substantially less to a derivative of an RNA polymerase from a bacterial species that has at least one amino acid substitution, deletion, or insertion of at least one residue corresponding to, or alignable with, residues 561, 569, 665, 675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 of the β' subunit of RNA polymerase from Escherichia coli.
[0028] Certain embodiments of the present invention provide a method to identify a molecule that inhibits an RNA polymerase from a bacterial species by binding to the bridge-helix cap target of the RNA polymerase, comprising identifying a molecule that inhibits an RNA polymerase from a bacterial species but inhibits substantially less a derivative of an RNA polymerase from a bacterial species that has at least one amino acid substitution, deletion, or insertion of at least one residue corresponding to, or alignable with, residues 561, 569, 665, 675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 of the β' subunit of RNA polymerase from Escherichia coli.
[0029] Certain embodiments of the present invention provide a method to identify a molecule that inhibits growth of a bacterium, comprising identifying a molecule that inhibits growth of a bacterium but inhibits growth substantially less of a derivative of said bacterium that has at least one amino acid substitution, deletion, or insertion of at least one residue corresponding to, or alignable with, residues 561, 569, 665, 675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 of the β subunit of RNA polymerase from Escherichia coli.
[0030] Certain embodiments of the present invention provide a method to identify a molecule that binds to an RNA polymerase from a bacterial species through the bridge-helix cap target of the RNA polymerase, comprising identifying a target molecule that competes with a first molecule for binding to an RNA polymerase from a bacterial species, wherein the first molecule preferentially binds to the bridge-helix cap target of the RNA polymerase.
[0031] In certain embodiments, the first molecule comprises a detectable group.
[0032] Certain embodiments of the present invention provide a method to treat a bacterial infection in a subject in need thereof, comprising administering to the subject a first compound selected as being an inhibitor of growth of a bacterium by binding to the bridge-helix cap target of an RNA polymerase, and a second compound that inhibits growth of a bacterium by binding to a site other than the bridge-helix cap target of an RNA polymerase.
[0033] In certain embodiments, the first compound is salinamide A.
[0034] In certain embodiments, the second compound is a rifamycin or CBR703.
[0035] In certain embodiments, the method further comprises administering a third compound that inhibits growth of a bacterium by binding to a site other than the bridge-helix cap target of an RNA polymerase.
[0036] In certain embodiments, the first and second compounds are administered concurrently.
[0037] In certain embodiments, the first and second compounds are administered sequentially.
[0038] In certain embodiments, the third compound is administered concurrently.
[0039] In certain embodiments, the third compound is administered sequentially. Certain embodiments of the present invention provide a composition (e.g., a pharmaceutical composition), or a kit, that comprises salinamide A and a rifamycin and/or CBR703.
[0040] In certain embodiments, the composition or kit comprises salinamide A and a rifamycin.
[0041] In certain embodiments, the composition or kit comprises salinamide A and CBR703.
[0042] In certain embodiments, the composition or kit comprises salinamide A, a rifamycin and CBR703.
[0043] Certain embodiments of the present invention provide a method to treat a bacterial infection in a subject in need thereof, comprising administering to the subject a composition described herein.
[0044] Certain embodiments of the present invention provide the use of a composition described herein to treat a bacterial infection.
[0045] Certain embodiments of the present invention provide an RNA polymerase, e.g., an isolated RNA polymerase, that has at least one amino acid substitution, deletion, or insertion of at least one residue corresponding to, or alignable with, residues 561, 569, 665, 675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 of the β' subunit of RNA polymerase from Escherichia coli.
[0046] Certain embodiments of the present invention provide a bacterium, e.g., an isolated bacterium, that comprises an RNA polymerase that has at least one amino acid substitution, deletion, or insertion of at least one residue corresponding to, or alignable with, residues 561, 569, 665, 675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 of the β' subunit of RNA polymerase from Escherichia coli.
[0047] Provided herein are targets and methods for specific binding and inhibition of RNA polymerase from bacterial species. Embodiments of the invention have applications in control of bacterial gene expression, control of bacterial growth, antibacterial chemistry, and antibacterial therapy.
[0048] Certain embodiments of the invention include: a new target (the bridge-helix cap target) and associated assay methods for inhibition of bacterial RNA polymerase, inhibition of bacterial RNA synthesis and inhibition of bacterial growth. Antibacterial compounds that function through the new target should exhibit minimal or no cross-resistance with antibacterial compounds that function through previously disclosed targets. It should be possible to co-administer antibacterial compounds that function through the new target together with antibacterial compounds that function through previously disclosed targets, to achieve additive antibacterial effects and to suppress the emergence of resistance.
[0049] Certain embodiments of the invention will now be illustrated by the following non-limiting Example.
Example 1
Identification of the Target of the Antibiotic Salinamide A
[0050] Salinamide A (Sal) is a bicyclic depsipeptide antibiotic, comprising seven amino acids and two non-amino-acid residues (1,2; FIG. 1). Sal is produced by Streptomyces sp. CNB-091, a marine bacterium isolated from the surface of the jellyfish Cassiopeia xamachana (1-3), and also by Streptomyces sp. NRRL 21611, a soil bacterium (4). Sal exhibits antibacterial activity against bacterial pathogens including Streptococcus pneumoniae and S. pyogenes (1,2).
[0051] Sal has been reported to inhibit bacterial RNA polymerase (RNAP), the enzyme responsible for bacterial RNA synthesis, in vitro (IC50=0.5 μM; 4). However, it has not previously been determined whether RNAP is the functional cellular target of Sal.
[0052] The hypothesis of this research was that RNAP is the functional cellular target of Sal. Corollaries to this hypothesis were that Sal should inhibit RNAP in vivo, that mutations conferring resistance to Sal should occur in RNAP-subunit genes, and that mapping of sites of substitutions conferring resistance to Sal onto the three-dimensional structure of RNAP should define residues of RNAP important for function of Sal. The objectives of this research were to determine whether Sal inhibits RNAP in vivo, to determine whether Sal kills bacteria by inhibiting RNAP, and to determine how Sal inhibits RNAP.
[0053] The results of this research show that Sal inhibits RNA synthesis in vivo (FIGS. 2A-B) and show that mutations in RNAP-subunit genes result in Sal-resistance (FIGS. 3A-C; Tables 1-3), thereby demonstrating that RNAP is the functional cellular target of Sal. In addition, the results of this research show that transcription inhibition by Sal requires a compact determinant--the "Sal target"--adjacent to and overlapping the RNAP active center and adjacent to but not overlapping the targets of the RNAP inhibitors rifamyins and CBR703 (FIG. 5; Tables 2-5). The Sal target comprises RNAP β'-subunit residues 690, 697, 738, 748, 758, 763, 775, 779, 780, 782, and 783 and RNAP β-subunit residues 561, 569, 665, 675, 677, and 680. Finally, determination of a crystal structure of E. coli RNAP in complex with Sal shows that the Sal target is the binding site on RNAP for Sal, and that sites of substitutions conferring Sal-resistance correspond to RNAP residues of RNAP that contact or are close to Sal (FIGS. 7A-B).
[0054] The Sal target is located adjacent to, and partly overlaps, the RNAP active center (FIGS. 4A-C, 5, 6 and 7A-B). It is inferred that Sal most likely inhibits RNAP by inhibiting RNAP active-center function.
[0055] Most sites of substitutions conferring Sal-resistance involve amino acids that are conserved in RNAP from a broad range of bacterial species (FIGS. 4A-C, upper rows in each panel); this is consistent with, and accounts for, the observation that Sal inhibits RNAP from a broad range of bacterial species (4). Nine of the sites of substitutions conferring Sal-resistance--β' residues 690, 697, 738, 775 and 779, and β residues 569, 675, 677, and 680--are not conserved in human RNAP I, RNAP II, and RNAP III (FIGS. 4A-C, bottom three rows in each panel); this is consistent with, and accounts for, the observation that Sal does not inhibit human RNAPs (4).
[0056] Mapping of substitutions conferring Sal-resistance onto the three-dimensional structure of a transcription elongation complex comprising RNAP, DNA, RNA, and a nucleoside triphosphate (9) indicates that the Sal target does not overlap the RNAP active-center Mg2+ ion and does not overlap RNAP residues that interact with the DNA template, the RNA product, and the nucleoside triphosphate substrate. It is inferred Sal most likely inhibits RNAP active-center function allosterically, through effects on RNAP conformation, and not through direct interactions with RNAP residues that mediate bond formation, product binding, and substrate binding.
[0057] The Sal target overlaps RNAP active-center sub-regions that have been designated as the "μ' bridge helix hinge N" (BH-HN), the "β' F loop," the "β link region," and the "β D2 loop" (FIGS. 4A-C; active-center sub-region nomenclature as in 18,19). Fully 20 of the 24 identified substitutions conferring Sal-resistance map to these RNAP active-center sub-regions (FIGS. 4A-C; Tables 2-3). The BH-HN may undergo conformational changes coupled to, and essential for, the nucleotide-addition cycle in RNA synthesis, and the F-loop, and possibly also the link region and the D-2-loop, may coordinate these conformational changes (18,19). It is inferred herein that Sal most likely inhibits RNAP active-center function by inhibiting BH-HN hinge-opening and/or hinge-closing.
[0058] Sal is the first RNAP inhibitor that has been inferred to function through effects on BH-HN conformational cycling. Accordingly, Sal will find use as a research tool for dissection of mechanistic and structural aspects of BH-HN conformational cycling.
[0059] Relationship Between the Sal Target and the Rifamycin Target.
[0060] The Sal target is located adjacent to, but does not overlap, the target of the rifamycin antibacterial agents (e.g., rifampin, rifapentine, rifabutin, and rifalazil), which are RNAP inhibitors in current clinical use in antibacterial therapy (FIG. 6). Consistent with the lack of overlap between the Sal target and the rifamycin target, Sal-resistant mutants are not cross-resistant to the rifamycin rifampin (Table 4), and rifamycin-resistant mutants are not cross-resistant to Sal (Table 5). The absence of overlap and the absence of cross-resistance indicates that it should be possible to co-administer Sal and a rifamycin in order to achieve additive or superadditive antibacterial activities and in order to suppress the emergence of resistance. Consistent with this inference, co-administration of Sal and the rifamycin rifampin results in an extremely low, effectively negligible, spontaneous resistance frequency: <2×10-11(<1/200 the spontaneous resistance frequency of Sal alone; <1/500 the spontaneous resistance frequency of rifampin alone; Table 6). This is important in view of the fact that susceptibility to spontaneous resistance is the main limiting factor in the clinical use of rifamycins (15,21,22).
[0061] Relationship Between the Sal Target and the CBR703 Target.
[0062] The Sal target also is located adjacent to, but does not overlap, the target of CBR703, an RNAP inhibitor under investigation for clinical use in antibacterial therapy (FIG. 6). Consistent with the lack of overlap between the Sal target and the CBR703 target, Sal-resistant mutants exhibit no cross-resistance to CBR703 (Table 4). In fact, the majority of Sal-resistant mutants exhibit hypersensitivity to CBR703, and half exhibit high-level hypersensitivity to CBR703 (i.e., are at least four times more sensitive to CBR703 than the wild-type parent strain; Table 4). The absence of overlap, the absence of cross-resistance, and the presence of hypersensitivity, indicate that it should be possible to co-administer Sal and CBR703 in order to achieve additive or superadditive antibacterial activities and in order to suppress the emergence of resistance. Consistent with this inference, co-administration of Sal and CBR703 results in an extremely low, effectively negligible, spontaneous resistance frequency: <2×10-11 (<1/200 the spontaneous resistance frequency of Sal alone; <1/10 the spontaneous resistance frequency of CBR703 alone; Table 7).
[0063] Results
[0064] Sal Inhibits RNA Synthesis In Vivo.
[0065] To determine whether Sal inhibits RNAP in vivo, effects of Sal on RNA synthesis in vivo were assayed, and, as a control, effects of Sal on DNA synthesis in vivo were assayed. Sal was added to cultures of E. coli D21f2tolC growing in media containing either a radioactively labeled RNA-synthesis precursor ([14C]-uracil) or a radioactively labeled DNA synthesis precursor ([14C]-thymidine); aliquots were removed and mixed with trichloroacetic acid (TCA) 0, 5, 10, and 15 min thereafter to lyse cells and precipitate nucleic acids; TCA-precipitated nucleic acids were collected by vacuum filtration; and radioactivity in TCA-precipitated nucleic acids was quantified (FIG. 2A). It was observed that, upon addition of Sal to bacterial cultures, RNA synthesis essentially stopped (FIG. 2B, left panel). The effect was rapid; it was observed at the earliest time point tested (5 min; FIG. 2B, left panel). The effect was specific; addition of Sal to bacterial cultures had no effect on DNA synthesis (FIG. 2B, right panel). It is concluded that Sal inhibits RNA synthesis in vivo, and it is inferred that Sal inhibits RNAP in vivo.
[0066] Sal-Resistant Mutants Map to RNAP-Subunit Genes.
[0067] To determine whether Sal kills bacteria by inhibiting RNAP, spontaneous Sal-resistant mutants were isolated, and genes for RNAP subunits in Sal-resistant mutants were PCR-amplified and sequenced (FIG. 3A). Spontaneous Sal-resistant mutants were isolated by plating high-density cultures of E. coli D21f2tolC (˜3×109 cells per plate) on agar containing Sal and identifying rare resistant colonies (FIG. 3B). For each Sal-resistant mutant, genomic DNA was prepared, and the genes for the largest RNAP subunit and the second-largest RNAP subunit--rpoC encoding RNAP β' subunit and rpoB encoding RNAP β subunit were PCR-amplified and sequenced (FIG. 3C).
[0068] Spontaneous Sal-resistant mutants were isolated with a frequency of ˜4×10-9 (Table 1). A total of 39 Sal-resistant mutants were isolated, PCR-amplified, and sequenced (Table 1). Strikingly, one hundred percent (39 of 39) of sequenced Sal-resistant mutants were found to contain mutations in genes for RNAP subunits: 31 were found to contain single mutations in rpoC, 1 was found to contain a double mutation in rpoC, and 7 were found to contain single mutations in rpoB (Table 1). It is concluded that a single substitution in an
[0069] RNAP-subunit gene, either rpoC or rpoB, is sufficient to confer Sal-resistance, and it is inferred that RNAP is the functional cellular target for Sal.
TABLE-US-00001 TABLE 1 Sal-resistant mutants: summary statistics frequency of spontaneous mutation to Sal-resistance ~4 × 10-9 number of Sal-resistant isolates 39 number of Sal-resistant isolates containing single-substitution 31 mutations in rpoC number of Sal-resistant isolates containing multiple- 1 substitution mutations in rpoC number of Sal-resistant isolates containing single-substitution 7 mutations in rpoB number of Sal-resistant isolates containing multiple- 0 substitution mutations in rpoB percentage of Sal-resistant isolates containing mutations in 100 RNAP-subunit genes
[0070] Sal-Resistant Mutants Define Residues of RNAP Important for Function of Sal.
[0071] A total of 20 different substitutions conferring Sal-resistance were identified (Table 2). Substitutions were obtained at 11 sites in RNAP β' subunit (residues 690, 697, 738, 748, 758, 763, 779, 780, 782, and 783) and at 3 sites in RNAP β subunit (residues 569, 675, and 677) (Table 2; FIGS. 4A-C).
[0072] Seven additional Sal-resistant mutants were isolated following mutagenesis of rpoC and rpoB. Substitutions conferring Sal-resistance were obtained at 3 sites in RNAP β subunit (residues 561, 665, and 680) and 3 sites in RNA β' subunit (residues 758, 780, and 782) (Table 3; FIGS. 4A-C).
[0073] In the three-dimensional structure of RNAP, the sites of substitutions conferring Sal-resistance formed a tight cluster (the "Sal target"; see FIG. 5). The identified Sal target was located immediately adjacent to, and partly overlapped, the RNAP active center. The Sal target was located adjacent to, but did not overlap, the targets of RNAP inhibitors in current use in antibacterial therapy, rifamycins (see FIGS. 6; and 10-15), and the target of an RNAP inhibitor under investigation for use in antibacterial therapy, CBR703 (see FIGS. 6; and 16,17).
[0074] The dimensions of the identified Sal target were ˜35 Åט18 Åט12 Å, and thus the identified Sal target was sufficiently large to be able to encompass Sal (˜16 Åט12 Åט10 Å). Based on the resistance properties and the size of the Sal target, it was inferred that the Sal target most likely was the binding site for Sal on RNAP.
TABLE-US-00002 TABLE 2 Spontaneous Sal-resistant mutants: sequences and properties number of amino acid independent resistance level substitution isolates (MIC/MIC.sub.wild-type)a rpoC (RNAP β' subunit) 690 Asn→Asp 2 16 697 Met→Valb 3 >16 738 Arg→Cys 2 >16 738 Arg→His 1 >16 738 Arg→Pro 2 >16 738 Arg→Ser 1 >16 748 Ala→Glu 2 16 758 Pro→Ser 1 16 763 Phe→Cys 4 16 775 Ser→Ala 1 8 779 Ala→Thr 2 >16 779 Ala→Val 5 >16 780 Arg→Cys 2 >16 782 Gly→Ala 2 782 Gly→Cys 1 783 Leu→Arg 1 >16 rpoB (RNAP β subunit) 569 Ile→Ser 2 >16 675 Asp→Ala 2 >16 677 Asn→His 1 >16 677 Asn→Lys 2 >16 aMIC with wild-type rpoC and wild-type rpoB was 0.024 μg/ml. bOne isolate was a double-substitution mutant: 697 Met→Val; 1054 Thr→Ala.
TABLE-US-00003 TABLE 3 Induced Sal-resistant mutants: sequences and properties number of amino acid independent resistance level substitution isolates (MIC/MIC.sub.wild-type)a rpoC (RNAP β' subunit) 758 Pro→Thr 1 4 780 Arg→Cys 2 8 782 Gly→Cys 1 8 rpoB (RNAP β subunit) 561 Ile→Ser 1 2 665 Ala→Glu 1 8 680 Leu→Met 1 4 aAssayed as D21f2toIC pRL663 and D21f2toIC pRL706 derivatives; MIC with wild-type rpoC and wild-type rpoB was 0.098 μg/ml. bOne isolate was a double-substitution mutant: 697 Met→Val; 1054 Thr→Ala.
[0075] Crystal Structures Confirm Identification of Residues of RNAP Important for Function of Sal.
[0076] Crystal structures were determined for E. coli RNAP σ70 holoenzyme in the presence of Sal (resolution=4.2 Å) and in the absence of Sal (resolution=4.0 Å) (FIGS. 7A-B). Comparison of electron density maps for E. coli RNAP σ70 holoenzyme in the presence of Sal to E. coli RNAP σ70 holoenzyme in the absence of Sal revealed unambiguous difference density attributable to Sal (FIG. 7A). The difference density was located in the Sal target (FIG. 7A) and was in contact with or close to sites of substitutions conferring Sal resistance are obtained (FIG. 7B). The results establish that the Sal target is the binding site on RNAP for Sal, and that sites of substitutions conferring Sal-resistance correspond to RNAP residues of RNAP that contact or are close to Sal.
[0077] Resistance Levels of Sal-Resistant Mutants.
[0078] Resistance to Sal was quantified using broth microdilution assays. All analyzed mutants exhibited at least 2-fold resistance to Sal (Tables 2,3). Thirteen mutants exhibited >16-fold resistance to Sal.
[0079] Cross-Resistance Levels of Sal-Resistant Mutants.
[0080] Cross-resistance to previously characterized small-molecule inhibitors of RNAP was quantified by use of broth microdilution assays. The Sal-resistant mutants exhibited no cross-resistance with rifampin and no cross-resistance with CBR703 (Table 4). Indeed, two Sal-resistant mutants exhibited moderate, 2-fold, hypersensitivity to rifampin, and ten Sal-resistant mutants exhibited moderate to high-level, 2-fold to >4-fold, hypersensitivity to CBR703 (mutants with resistance levels <1 in Table 4).
TABLE-US-00004 TABLE 4 Sal-resistant mutants: absence of cross-resistance to rifampin and CBR703 cross-resistance level amino acid (MIC/MIC.sub.wild-type)a substitution rifampin CBR703 rpoC (RNAP β' subunit) 690 Asn→Asp 1 ≦0.25b 697 Met→Val 0.5 0.5 738 Arg→Cys 1 ≦0.25 738 Arg→Pro 1 ≦0.25 738 Arg→Ser 1 1 748 Ala→Glu 1 ≦0.25 763 Phe→Cys 1 ≦0.25 779 Ala→Thr 1 1 779 Ala→Val 1 ≦0.25 780 Arg→Cys 1 0.5 782 Gly→Ala 782 Gly→Cys rpoB (RNAP β subunit) 569 Ile→Ser 1 1 675 Asp→Ala 0.5 ≦0.25 677 Asn→His 1 1 677 Asn→Lys 1 ≦0.25 aMIC with wild-type rpoC and wild-type rpoB was 0.098 μg/ml for rifampin and 6.25 μg/ml for CBR703. bValues <1 indicate that the substitution conferred hypersensitivity to the inhibitor.
[0081] Cross-Resistance Levels of Rifamycin-Resistant Mutants.
[0082] More than 70% of clinical isolates of rifamycin-resistant Mycobacterium tuberculosis contain β Asp516→Val, β His526→Asp, β His526→Tyr, or β Ser531→Leu substitutions (15). Derivatives of E. coli D21f2tolC containing the corresponding rifamycin-resistant mutations were obtained from laboratory stocks, and cross-resistance to Sal was assessed by use of broth microdilution assays. The rifamycin-resistant mutants exhibited no cross-resistance to Sal (Table 5).
TABLE-US-00005 TABLE 5 Rifampin-resistant mutants: absence of cross-resistance to Sal amino acid cross-resistance level substitution (MIC/MIC.sub.wild-type)a rpoB (RNAP β subunit) 516 Asp→Val 1 526 His→Asp 1 526 His→Tyr 1 531 Ser→Leu 1 aMIC with wild-type rpoB is 0.024 μg/ml.
[0083] Co-Administration of Sal and Rifampin Reduces Spontaneous Resistance to Undetectable Levels.
[0084] Spontaneous resistance frequencies were determined by plating E. coli D21f2tolC on LB agar (7) containing Sal at 4×MIC, rifampin at 4×MIC, or both, and counting numbers of colonies after 24 h at 37° C. The results in Table 6 show that the spontaneous resistance frequencies for Sal alone, rifampin alone, and Sal co-administered with rifampin were, respectively, 4×10-9, 1×10-8, and undetectable (<2×10-11).
TABLE-US-00006 TABLE 6 Spontaneous resistance frequencies for Sal, rifampin, and co-administered Sal and rifampin spontaneous resistance compound frequency Sal 4 × 10-9 Rif 1 × 10-8 Sal + Rif <2 × 10-11
[0085] Co-Administration of Sal and CBR703 Reduces Spontaneous Resistance to Undetectable Levels.
[0086] Spontaneous resistance frequencies were determined by plating E. coli D21f2tolC on LB agar (7) containing Sal at 4×MIC, CBR703 at 4×MIC, or both, and counting numbers of colonies after 24 h at 37° C.
[0087] The results in Table 7 show that the spontaneous resistance frequencies for Sal alone, CBR703 alone, and Sal co-administered with CBR703 were, respectively, 4×10-9, 2×10-10, and undetectable (<2×10-11).
TABLE-US-00007 TABLE 7 Spontaneous resistance frequencies for Sal, CBR703, and co-administered Sal and CBR703 spontaneous resistance compound frequency Sal 4 × 10-9 CBR703 .sup. 2 × 10-10 Sal + CBR703 <2 × 10-11
[0088] Materials and Methods
[0089] Sal.
[0090] Sal was isolated as in reference 1.
[0091] Measurement of Nucleic Acid Synthesis In Vivo.
[0092] Macromolecular synthesis assays were performed essentially as in reference 5. Escherichia coli D21f2tolC (a strain with cell-envelope defects resulting in increased susceptibility to antibiotics, including Sal; 6) was cultured in 10 ml M5T broth (5) at 37° C. with shaking until OD600=0.4-0.8, and cultures were diluted with pre-warmed M5T broth to OD600=0.167. Aliquots (90 μl) were dispensed into wells of a 96-well plate, were supplemented with 7 μl pre-warmed 6 ρCi/ml [14C]-uracil or [14C]-thymidine, were supplemented with 3 μl 0.048 μg/ml Sal in methanol (yielding a final Sal concentration two times the minimal inhibitory concentration) or 3 μl solvent blank, and were incubated at 37° C. with shaking. At time points 0, 5, 10, and 15 min after the addition of Sal or solvent blank, rows of samples were transferred to a second 96-well plate, containing 100 μl ice-cold 10% trichloroacetic acid (TCA) in each well, and the second plate was incubated on ice. One hour after the final time point, TCA precipitates were collected by filtration onto glass-fibre filters (Filtermat A; Perkin-Elmer, Inc.; pre-rinsed twice with 5% TCA), washed twice with 5% TCA, washed three times with water, and washed twice with 10% ethanol, using a Packard FilterMate 196 cell harvester with an OmniFilter upper head assembly (Perkin-Elmer, Inc.). Filters were dried under a heat lamp, wrapped in a single layer of plastic wrap, and exposed to a phosphorimager screen for 16-18 h. Radioactivity was quantified using a Typhoon Variable Mode Imager and ImageQuant v5 (Molecular Dynamics, Inc.).
[0093] Isolation of Sal-Resistant Mutants: Spontaneous Sal-Resistant Mutants.
[0094] E. coli D21f2tolC (6) was cultured to saturation in 5 ml LB broth (7) at 37° C., cultures were centrifuged, and cell pellets (˜3×109 cells) were re-suspended in 50 μl LB broth and plated on LB agar (7) containing 1.2 μg/ml Sal (a concentration four times the minimal concentration required to prevent growth of wild-type cells under these conditions), and incubated 24-48 h at 37° C. Sal-resistant mutants were identified by the ability to form colonies on this medium and were confirmed by re-streaking on the same medium.
[0095] Isolation of Sal-Resistant Mutants: Induced Sal-Resistant Mutants.
[0096] E. coli Random mutagenesis of rpoC in plasmid pRL663 and rpoB in plasmid pRL706 was performed as in 24, mutagenenized plasmids were passaged in E. coli XL1-Blue (Stratgene, Inc.) as in 24, mutagenized plasmids were introduced by transformation into E. coli D21f2tolC as in 24, and transformants cells) were applied to LB-agar plates containing 1 μg/ml Sal, 200 μg/ml ampicillin, and 1 mM IPTG, and plates were incubated 16-24 h at 37° C. Sal-resistant mutants were identified by the ability to form colonies on this medium and were confirmed by re-streaking on the same medium.
[0097] PCR-Amplification and Sequencing of RNAP-Subunit Genes of Sal-Resistant Mutants.
[0098] For spontaneous Sal-resistant mutants, rpoC and rpoB genese were PCR-amplified and sequenced as follows: Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega, Inc.; procedures as specified by the manufacturer) and was quantified by measurement of UV-absorbance (procedures as in 7). The rpoC gene and the rpoB gene were PCR-amplified in reactions containing 0.2 ng genomic DNA, 0.4 μM forward and reverse oligonucleotide primers
TABLE-US-00008 (5'-AGGTCACTGCTGTCGGGTTAAAACC-3' (SEQ ID NO: 1) and 5'-TGACAAATGCTCTT TCCCTAAACTCC-3' (SEQ ID NO: 2) for rpoC; 5'-GTTGCACAAACTGTCCGCTCA ATGG-3' (SEQ ID NO: 3) and 5'-TCGGAGTTAGCACAATCCG CTGC-3' (SEQ ID NO: 4) for rpoB), 5 U Taq
DNA polymerase (Genscript, Inc.), and 800 μM dNTP mix (Agilent/Stratagene, Inc.) (initial denaturation step of 5 min at 94° C.; 30 cycles of 30 s at 94° C., 45 s at 55° C., and 4.5 min at 72° C.; final extension step of 10 min at 72° C.). PCR products containing the rpoC gene (4.3 kB) or the rpoB gene (4.1 kB) were isolated by electrophoresis on 0.8% agarose (procedures as in 7), extracted from gel slices using the Gel/PCR DNA Fragments Extraction Kit (IBI Scientific, Inc.; procedures as specified by the manufacturer), and submitted to High-Throughput Sequencing Solutions (Seattle Wash.) for sequencing (Sanger sequencing; eight sequencing primers per gene).
[0099] For each induced Sal-resistant mutants, plasmid DNA was isolated and submitted to High-Throughput Sequencing Solutions (Seattle Wash.) for sequencing (Sanger sequencing; eight sequencing primers per gene).
[0100] Quantitation of Resistance to Sal.
[0101] Resistance to Sal was quantified by performing broth microdilution assays. Single colonies were inoculated into 3 ml LB broth, and incubated 3-6 h at 37° C. with shaking. Diluted aliquots (2×104 cells in 98 μl LB broth; concentrations determined using OD600=1 for 109 cells) were dispensed into wells of a 96-well plate, were supplemented with 2 μl of a 2-fold dilution series of Sal in methanol (final concentrations of 1.56, 0.390, 0.195, 0.0975, 0.0488, 0.0244, 0.0122, and 0.00609 μg/ml) or 2 μl of a solvent blank, and were incubated 16 h at 37° C. with shaking. The minimum inhibitory concentration (MIC) was defined as the lowest tested concentration of Sal that inhibited bacterial growth by ≧95%.
[0102] Quantitation of Cross-Resistance to Rifampin and CBR703.
[0103] Cross-resistance levels were determined analogously to resistance levels, using 0.12-0.78 μg/ml of rifampin (Sigma, Inc.) and 0.39-25 μg/ml of CBR703 (Maybridge, Inc.).
[0104] Molecular Modeling.
[0105] Sites of substitutions conferring Sal-resistance were mapped onto a crystal structure of Thermus thermophilus RNAP holoenzyme (8; PDB accession code 1L9U) and a crystal structure of the T thermophilus transcription elongation complex (RNAP in complex with DNA, RNA, and a nucleoside triphosphate; 9; PDB accession code 205J). Correspondences between residues of E. coli RNAP and T. thermophilus RNAP were based on amino acid sequence alignments (25; FIGS. 4A-C).
REFERENCES
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[0107] 2. Moore, B., Trischman, J., Dieter Seng, D., Kho, D., Jensen, P., and Fenical, W. (1999) Salinamides: antiinflammatory depsipeptides from a marine streptomycete. J. Org. Chem., 64, 1145-1150.
[0108] 3. Moore, B. and Seng, D. (1998) Biosynthesis of the bicyclic depsipeptide salinamide A in Streptomyces sp. CNB-091: origin of the carbons. Tetrahedron Lett. 39, 3915-3918.
[0109] 4. Miao, S., Anstee, M., LaMarco, K., Matthew, J., Huang, L., and Brasseur, M. (1997) Inhibition of bacterial RNA polymerases: peptide metabolites from the cultures of Streptomyces sp. J. Nat. Prod. 60, 858-861.
[0110] 5. King, A. and Wu, L. (2009) Macromolecular synthesis and membrane perturbation assays for mechanisms of action studies of antimicrobial agents. Curr. Protoc. Pharmacol. 47:13A.7.1-13A.7.23.
[0111] 6. Fralick, J., and Burns-Keliher, L. (1994). Additive effect of tolC and rfa mutations on the hydrophobic barrier of the outer membrane of E. coli K-12. J. Bacteriol. 176, 6404-6406.
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[0114] 9. Vassylyev, D., Vassylyeva, M., Zhang, J., Palangat, M., Artsimovitch, I., and Landick, R. (2007). Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163-168.
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[0116] 11. Ovchinnikov, Y., Monastyrskaya, G., Guriev, S., Kalinina, N., Sverdlov, E., Gragerov, A., Bass, I., Kiver, I., Moiseyeva, E., Igumnov, V., et al. (1983). RNA polymerase rifampicin resistance mutations in Escherichia coli: sequence changes and dominance. Mol. Gen. Genet. 190, 344-348.
[0117] 12. Jin, D. J., and Gross, C. (1988). Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J. Mol. Biol. 202, 45-58.
[0118] 13. Severinov, K., Soushko, M., Goldfarb, A., and Nikiforov, V. (1993). Rifampicin region revisited: new rifampicin-resistant and streptolydigin-resistant mutants of the β subunit of Escherichia coli RNA polymerase. J. Biol. Chem. 268, 14820-14825.
[0119] 14. Campbell, E., Korzheva, N., Mustaev, A., Murakami, K., Nair, S., Goldfarb, A., and Darst, S. (2001). Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104, 901-912.
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[0122] 17. Wang, X., Sineva, E., and Ebright, R., personal communication.
[0123] 18. Weinzierl, R. (2010) The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain. BMC Biol. 8:134.
[0124] 19. Hein, P. and Landick, R. (2010) The bridge helix coordinates movements of modules in RNA polymerase. BMC Biol. 8:141.
[0125] 20. Baquero, M.-R., Nilsson, A., Turrientes, M., Sandvang, D., Galan, J. C., Mart nez, J. L., Frimodt-Moller, N., Baquero, F., and Andersson, D. (2004) Polymorphic mutation frequencies in Escherichia coli. J. Bacteriol., 186, 5538-5542.
[0126] 21. Chopra, I. (2007). Bacterial RNA polymerase: a promising target for the discovery of new antimicrobial agents. Curr. Opin. Investig. Drugs 8, 600-607.22. Villain-Guillot, P., Bastide, L., Gualtieri, M. & Leonetti, J. (2007) Progress in targeting bacterial transcription. Drug Discov. Today 12, 200-208.23. Tan, L. and Ma, D. (2008) Total synthesis of salinamide A: a potent anti-inflammatory bicyclic depsipeptide. Angew. Chem. Int. Ed. 47, 3614-3617.
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[0129] All publications cited herein are incorporated herein by reference. While in this application certain embodiments of invention have been described, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that certain of the details described herein may be varied without departing from the basic principles of the invention.
[0130] The use of the terms "a" and "an" and "the" and similar terms in the context of describing embodiments of invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. In addition to the order detailed herein, the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments of invention and does not pose a limitation on the scope of the invention unless otherwise specifically recited in the claims. No language in the specification should be construed as indicating that any non-claimed element as essential to the practice of the invention.
Sequence CWU
1
1
119125DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 1aggtcactgc tgtcgggtta aaacc
25226DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2tgacaaatgc tctttcccta aactcc
26325DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 3gttgcacaaa ctgtccgctc aatgg
25423DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4tcggagttag cacaatccgc tgc
23514PRTEscherichia coli 5Ala Ala
Ala Asn Asp Arg Val Ser Lys Ala Met Met Asp Asn 1 5
10 67PRTEscherichia coli 6Ala Gln Ile Arg Gln
Leu Ala 1 5 761PRTEscherichia coli 7Gly Leu Met
Ala Lys Pro Asp Gly Ser Ile Ile Glu Thr Pro Ile Thr 1 5
10 15 Ala Asn Phe Arg Glu Gly Leu Asn
Val Leu Gln Tyr Phe Ile Ser Thr 20 25
30 His Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys
Thr Ala Asn 35 40 45
Ser Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Ala Gln 50
55 60 814PRTHaemophilus influenzae 8Ala Ala Ala
Asn Glu Arg Val Ala Lys Ala Met Met Glu Asn 1 5
10 97PRTHaemophilus influenzae 9Ala Gln Ile Arg
Gln Leu Ala 1 5 1061PRTHaemophilus influenzae
10Gly Leu Met Ala Arg Pro Asp Gly Ser Ile Ile Glu Thr Pro Ile Thr 1
5 10 15 Ala Asn Phe Arg
Glu Gly Leu Asn Val Leu Gln Tyr Phe Ile Ser Thr 20
25 30 His Gly Ala Arg Lys Gly Leu Ala Asp
Thr Ala Leu Lys Thr Ala Asn 35 40
45 Ser Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Ala Gln
50 55 60 1114PRTVibrio cholerae
11Ala Ser Thr Asn Asp Arg Val Ala Lys Ala Met Met Glu Asn 1
5 10 127PRTVibrio cholerae 12Ala Gln
Ile Arg Gln Leu Ala 1 5 1361PRTVibrio cholerae
13Gly Leu Met Ala Arg Pro Asp Gly Ser Ile Ile Glu Thr Pro Ile Thr 1
5 10 15 Ala Asn Phe Lys
Glu Gly Leu Asn Val Leu Gln Tyr Phe Ile Ser Thr 20
25 30 His Gly Ala Arg Lys Gly Leu Ala Asp
Thr Ala Leu Lys Thr Ala Asn 35 40
45 Ser Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Ala Gln
50 55 60 1414PRTPseudomonas
aeruginosa 14Ser Lys Ala Asn Asp Glu Val Ser Lys Ala Met Met Ala Asn 1
5 10 157PRTPseudomonas
aeruginosa 15Ala Gln Ile Arg Gln Leu Ala 1 5
1661PRTPseudomonas aeruginosa 16Gly Leu Met Ala Lys Pro Asp Gly Ser Ile
Ile Glu Thr Pro Ile Thr 1 5 10
15 Ala Asn Phe Arg Glu Gly Leu Asn Val Leu Gln Tyr Phe Ile Ser
Thr 20 25 30 His
Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Thr Ala Asn 35
40 45 Ser Gly Tyr Leu Thr Arg
Arg Leu Val Asp Val Ala Gln 50 55
60 1714PRTTreponema pallidum 17Ser Lys Thr Ser Glu Glu Leu Thr Ser
Leu Met Met Glu Thr 1 5 10
187PRTTreponema pallidum 18Asn Gln Ile Arg Gln Leu Ala 1
5 1961PRTTreponema pallidum 19Gly Leu Met Ala Lys Pro Ser Gly
Asp Ile Ile Glu Leu Pro Ile Arg 1 5 10
15 Ser Asn Phe Lys Glu Gly Leu Asn Val Ile Glu Phe Phe
Ile Ser Thr 20 25 30
Asn Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Thr Ala Asp
35 40 45 Ala Gly Tyr Leu
Thr Arg Arg Leu Val Asp Ile Ala Gln 50 55
60 2014PRTBorrelia burgdorferi 20Leu Lys Thr Asn Glu Glu Leu
Thr Asn Lys Met Met Glu Ile 1 5 10
217PRTBorrelia burgdorferi 21Asn Gln Ile Arg Gln Leu Ala 1
5 2261PRTBorrelia burgdorferi 22Gly Leu Met Ala Lys
Thr Ser Gly Asp Ile Ile Glu Leu Pro Ile Ile 1 5
10 15 Ser Asn Phe Lys Glu Gly Leu Ser Val Ile
Glu Phe Phe Ile Ser Thr 20 25
30 Asn Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Thr Ala
Asp 35 40 45 Ala
Gly Tyr Leu Thr Arg Arg Leu Val Asp Ile Ala Gln 50
55 60 2314PRTXylella fastidiosa 23Ser Arg Thr Asn
Glu Arg Ile Ala Lys Ala Met Met Asp Thr 1 5
10 247PRTXylella fastidiosa 24Gln Gln Ile Arg Gln Leu
Ala 1 5 2561PRTXylella fastidiosa 25Gly Leu Met
Val Arg Pro Asp Gly Ser Ile Ile Glu Thr Pro Ile Lys 1 5
10 15 Ala Asn Phe Arg Glu Gly Leu Ser
Val Gln Glu Tyr Phe Asn Ser Thr 20 25
30 His Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys
Thr Ala Asn 35 40 45
Ser Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Thr Gln 50
55 60 2614PRTCampylobacter jejuni 26Lys Ser Thr
Asn Asn Val Leu Ser Lys Glu Met Met Lys Leu 1 5
10 277PRTCampylobacter jejuni 27Ala Gln Ile Ser
Gln Leu Ala 1 5 2861PRTCampylobacter jejuni 28Gly
Leu Met Thr Lys Pro Asp Gly Ser Ile Ile Glu Thr Pro Ile Ile 1
5 10 15 Ser Asn Phe Arg Glu Gly
Leu Asn Val Leu Glu Tyr Phe Ile Ser Thr 20
25 30 His Gly Ala Arg Lys Gly Leu Ala Asp Thr
Ala Leu Lys Thr Ala Asn 35 40
45 Ala Gly Tyr Leu Thr Arg Lys Leu Ile Asp Val Ala Gln
50 55 60 2914PRTNeisseria
meningitidis 29Gly Arg Ala Gly Asp Lys Ile Ala Lys Ala Met Met Asp Asn 1
5 10 307PRTNeisseria
meningitidis 30Ala Gln Ile Lys Gln Leu Ser 1 5
3161PRTNeisseria meningitidis 31Gly Leu Met Ala Lys Pro Asp Gly Ser Ile
Ile Glu Thr Pro Ile Thr 1 5 10
15 Ser Asn Phe Arg Glu Gly Leu Thr Val Leu Gln Tyr Phe Ile Ala
Thr 20 25 30 His
Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Thr Ala Asn 35
40 45 Ser Gly Tyr Leu Thr Arg
Arg Leu Val Asp Val Thr Gln 50 55
60 3214PRTRickettsia prowazekii 32Ser Arg Cys Thr Asp Arg Val Ala
Asn Asp Met Met Lys Glu 1 5 10
337PRTRickettsia prowazekii 33Gln Gln Ile Lys Gln Leu Gly 1
5 3461PRTRickettsia prowazekii 34Gly Leu Met Thr Lys Ser
Asn Gly Gln Ile Ile Gln Thr Pro Ile Ile 1 5
10 15 Ser Asn Phe Lys Glu Gly Leu Thr Glu Phe Glu
Cys Phe Asn Ser Ala 20 25
30 Asn Gly Met Arg Lys Gly Gln Ile Asp Thr Ala Leu Lys Thr Ala
Ser 35 40 45 Ser
Gly Tyr Leu Thr Arg Lys Leu Val Asp Val Ala Gln 50
55 60 3514PRTChlamydia trachomatis 35Thr Glu Val
Ser Asp Leu Leu Ser Asn Ala Leu Tyr Ser Glu 1 5
10 367PRTChlamydia trachomatis 36Ser Gln Leu Lys
Gln Leu Gly 1 5 3761PRTChlamydia trachomatis
37Gly Leu Met Ala Lys Pro Asn Gly Ala Ile Ile Glu Ser Pro Ile Thr 1
5 10 15 Ser Asn Phe Arg
Glu Gly Leu Thr Val Leu Glu Tyr Ser Ile Ser Ser 20
25 30 His Gly Ala Arg Lys Gly Leu Ala Asp
Thr Ala Leu Lys Thr Ala Asp 35 40
45 Ser Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Ala Gln
50 55 60 3814PRTMycoplasma
pneumoniae 38Asn Gly Val Lys Glu Lys Val Ser Ser Glu Ile Gln Asp Leu 1
5 10 397PRTMycoplasma
pneumoniae 39Ser Asn Phe Thr Gln Leu Phe 1 5
4072PRTMycoplasma pneumoniae 40Gly Leu Met Ser Lys Ser Phe Asn Tyr Glu
Arg Asn Asn Gln Ser Lys 1 5 10
15 Ile Ile Lys Asp Thr Ile Glu Val Pro Ile Lys His Ser Phe Leu
Glu 20 25 30 Gly
Leu Thr Ile Asn Glu Tyr Phe Asn Ser Ser Tyr Gly Ala Arg Lys 35
40 45 Gly Met Thr Asp Thr Ala
Met Lys Thr Ala Lys Ser Gly Tyr Met Thr 50 55
60 Arg Lys Leu Val Asp Ala Thr His 65
70 4114PRTBacillus subtilis 41Ser Ala Ala Lys Asp Val
Ile Gln Gly Lys Leu Met Lys Ser 1 5 10
427PRTBacillus subtilis 42Ser Asn Phe Thr Gln Leu Ala 1
5 4361PRTBacillus subtilis 43Gly Leu Met Ala Asn Pro
Ala Gly Arg Ile Ile Glu Leu Pro Ile Lys 1 5
10 15 Ser Ser Phe Arg Glu Gly Leu Thr Val Leu Glu
Tyr Phe Ile Ser Thr 20 25
30 His Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Thr Ala
Asp 35 40 45 Ser
Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Ala Gln 50
55 60 4414PRTStaphylococcus aureus 44Thr Asp Ala
Lys Asp Gln Ile Gln Gly Glu Leu Met Gln Ser 1 5
10 457PRTStaphylococcus aureus 45Ser Asn Phe Thr
Gln Leu Ala 1 5 4661PRTStaphylococcus aureus
46Gly Leu Met Ala Ala Pro Ser Gly Lys Ile Ile Glu Leu Pro Ile Thr 1
5 10 15 Ser Ser Phe Arg
Glu Gly Leu Thr Val Leu Glu Tyr Phe Ile Ser Thr 20
25 30 His Gly Ala Arg Lys Gly Leu Ala Asp
Thr Ala Leu Lys Thr Ala Asp 35 40
45 Ser Gly Tyr Leu Thr Arg Arg Leu Val Asp Val Ala Gln
50 55 60 4714PRTMycobacterium
tuberculosis 47Lys Glu Ala Thr Asp Glu Val Gly Gln Ala Leu Arg Glu His 1
5 10 487PRTMycobacterium
tuberculosis 48Thr Gln Thr Arg Thr Leu Ala 1 5
4961PRTMycobacterium tuberculosis 49Gly Leu Val Thr Asn Pro Lys Gly Glu
Phe Ile Pro Arg Pro Val Lys 1 5 10
15 Ser Ser Phe Arg Glu Gly Leu Thr Val Leu Glu Tyr Phe Ile
Asn Thr 20 25 30
His Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Arg Thr Ala Asp
35 40 45 Ser Gly Tyr Leu
Thr Arg Arg Leu Val Asp Val Ser Gln 50 55
60 5014PRTSynechocystis sp. 50Asn Gly Thr Ser Glu Glu Leu Lys
Asp Gln Val Val Val Asn 1 5 10
517PRTSynechocystis sp. 51Ser Gln Val Arg Gln Leu Val 1
5 5261PRTSynechocystis sp. 52Gly Leu Met Ala Asp Pro Gln Gly
Glu Ile Ile Asp Leu Pro Ile Lys 1 5 10
15 Thr Asn Phe Arg Glu Gly Leu Thr Val Thr Glu Tyr Val
Ile Ser Ser 20 25 30
Tyr Gly Ala Arg Lys Gly Leu Val Asp Thr Ala Leu Arg Thr Ala Asp
35 40 45 Ser Gly Tyr Leu
Thr Arg Arg Leu Val Asp Val Ser Gln 50 55
60 5314PRTAquifex aeolicus 53Ser Glu Ala Thr Asn Leu Val Ser
Lys Ala Met Phe Glu Glu 1 5 10
547PRTAquifex aeolicus 54Asp Gln Ile Arg Gln Leu Ala 1
5 5561PRTAquifex aeolicus 55Gly Leu Met Ala Lys His Ser Gly
Glu Phe Ile Glu Thr Pro Ile Ile 1 5 10
15 Ser Asn Phe Arg Glu Gly Leu Ser Val Leu Glu Tyr Phe
Ile Ser Thr 20 25 30
Tyr Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Thr Ala Phe
35 40 45 Ala Gly Tyr Leu
Thr Arg Arg Leu Val Asp Val Ala Gln 50 55
60 5614PRTDeinococcus radiodurans 56Asn Asn Thr Thr Asp Ala
Val Lys Asp Ala Val Phe Glu Asn 1 5 10
577PRTDeinococcus radiodurans 57Gln Gln Ile Arg Gln Leu Ala
1 5 5861PRTDeinococcus radiodurans 58Gly Leu Met
Ala Arg Pro Asp Gly Ser Thr Ile Glu Val Pro Ile Arg 1 5
10 15 Ala Ser Phe Arg Glu Gly Leu Thr
Val Leu Glu Tyr Phe Ile Ser Thr 20 25
30 His Gly Ala Arg Lys Gly Gly Ala Asp Thr Ala Leu Arg
Thr Ala Asp 35 40 45
Ser Gly Tyr Leu Thr Arg Lys Leu Val Asp Val Ala His 50
55 60 5914PRTThermus aquaticus 59Thr Glu Thr
Thr Glu Lys Val Thr Gln Ala Val Phe Lys Asn 1 5
10 607PRTThermus aquaticus 60Gln Gln Ile Arg Gln
Leu Cys 1 5 6161PRTThermus aquaticus 61Gly Leu
Met Gln Lys Pro Ser Gly Glu Thr Phe Glu Val Pro Val Arg 1 5
10 15 Ser Ser Phe Arg Glu Gly Leu
Thr Val Leu Glu Tyr Phe Ile Ser Ser 20 25
30 His Gly Ala Arg Lys Gly Gly Ala Asp Thr Ala Leu
Arg Thr Ala Asp 35 40 45
Ser Gly Tyr Leu Thr Arg Lys Leu Val Asp Val Ala His 50
55 60 6214PRTThermus thermophilus 62Thr Glu
Thr Thr Glu Lys Val Thr Gln Ala Val Phe Lys Asn 1 5
10 637PRTThermus thermophilus 63Gln Gln Ile
Arg Gln Leu Cys 1 5 6461PRTThermus thermophilus
64Gly Leu Met Gln Lys Pro Ser Gly Glu Thr Phe Glu Val Pro Val Arg 1
5 10 15 Ser Ser Phe Arg
Glu Gly Leu Thr Val Leu Glu Tyr Phe Ile Ser Ser 20
25 30 His Gly Ala Arg Lys Gly Gly Ala Asp
Thr Ala Leu Arg Thr Ala Asp 35 40
45 Ser Gly Tyr Leu Thr Arg Lys Leu Val Asp Val Thr His
50 55 60 6514PRTHomo sapiens 65Asn
Met Ile Asp Leu Lys Phe Lys Glu Glu Val Asn His Tyr 1 5
10 667PRTHomo sapiens 66Val Asn Thr Met
Gln Ile Ser 1 5 6781PRTHomo sapiens 67Gly Gln Ile
Glu Leu Glu Gly Arg Ser Thr Pro Leu Met Ala Ser Gly 1 5
10 15 Lys Ser Leu Pro Cys Phe Glu Pro
Tyr Glu Phe Thr Pro Arg Ala Gly 20 25
30 Gly Phe Val Thr Gly Arg Phe Leu Thr Gly Ile Lys Pro
Pro Glu Phe 35 40 45
Phe Phe His Cys Met Ala Gly Arg Glu Gly Leu Val Asp Thr Ala Val 50
55 60 Lys Thr Ser Arg
Ser Gly Tyr Leu Gln Arg Cys Ile Ile Lys His Leu 65 70
75 80 Glu 6814PRTHomo sapiens 68Glu Asn
Gln Val Asn Arg Ile Leu Asn Asp Ala Arg Asp Lys 1 5
10 697PRTHomo sapiens 69Ile Asn Ile Ser Gln
Val Ile 1 5 7081PRTHomo sapiens 70Gly Gln Gln Asn
Val Glu Gly Lys Arg Ile Pro Phe Gly Phe Lys His 1 5
10 15 Arg Thr Leu Pro His Phe Ile Lys Asp
Asp Tyr Gly Pro Glu Ser Arg 20 25
30 Gly Phe Val Glu Asn Ser Tyr Leu Ala Gly Leu Thr Pro Thr
Glu Phe 35 40 45
Phe Phe His Ala Met Gly Gly Arg Glu Gly Leu Ile Asp Thr Ala Val 50
55 60 Lys Thr Ala Glu Thr
Gly Tyr Ile Gln Arg Arg Leu Ile Lys Ser Met 65 70
75 80 Glu 7114PRTHomo sapiens 71Glu Ala Leu
Ile Leu Lys Glu Leu Ser Val Ile Arg Asp His 1 5
10 727PRTHomo sapiens 72Ile Asn Ile Ser Gln Met
Ile 1 5 7381PRTHomo sapiens 73Gly Gln Gln Ala Ile
Ser Gly Ser Arg Val Pro Asp Gly Phe Glu Asn 1 5
10 15 Arg Ser Leu Pro His Phe Glu Lys His Ser
Lys Leu Pro Ala Ala Lys 20 25
30 Gly Phe Val Ala Asn Ser Phe Tyr Ser Gly Leu Thr Pro Thr Glu
Phe 35 40 45 Phe
Phe His Thr Met Ala Gly Arg Glu Gly Leu Val Asp Thr Ala Val 50
55 60 Lys Thr Ala Glu Thr Gly
Tyr Met Gln Arg Arg Leu Val Lys Ser Leu 65 70
75 80 Glu 747PRTEscherichia coli 74Gly Pro Asn
Ile Gly Leu Ile 1 5 759PRTEscherichia coli 75Glu
His Asp Asp Ala Asn Arg Ala Leu 1 5
767PRTHaemophilus influenzae 76Gly Pro Asn Ile Gly Leu Ile 1
5 779PRTHaemophilus influenzae 77Glu His Asp Asp Ala Asn Arg
Ala Leu 1 5 787PRTVibrio cholerae 78Gly
Pro Asn Ile Gly Leu Ile 1 5 799PRTVibrio cholerae
79Glu His Asp Asp Ala Asn Arg Ala Leu 1 5
807PRTPseudomonas aeruginosa 80Gly Pro Asn Ile Gly Leu Ile 1
5 819PRTPseudomonas aeruginosa 81Glu His Asp Asp Ala Asn Arg
Ala Leu 1 5 827PRTTreponema pallidum
82Gly Pro Asn Ile Gly Leu Ile 1 5 839PRTTreponema
pallidum 83Glu His Asp Asp Ala Asn Arg Ala Leu 1 5
847PRTBorrelia burgdorferi 84Gly Pro Asn Ile Gly Leu Ile 1
5 859PRTBorrelia burgdorferi 85Glu His Asn Asp Ala Asn
Arg Ala Leu 1 5 867PRTXylella fastidiosa
86Gly Pro Asn Ile Gly Leu Ile 1 5 879PRTXylella
fastidiosa 87Glu His Asp Asp Ala Asn Arg Ala Leu 1 5
887PRTCampylobacter jejuni 88Gly Gln Asn Ile Gly Leu Ile 1
5 899PRTCampylobacter jejuni 89Glu His Asp Asp Ala
Asn Arg Ala Leu 1 5 907PRTNeisseria
meningitidis 90Gly Pro Asn Ile Gly Leu Ile 1 5
919PRTNeisseria meningitidis 91Glu His Asp Asp Ala Asn Arg Ala Leu 1
5 927PRTRickettsia prowazekii 92Gly Gln Asn
Ile Gly Leu Ile 1 5 939PRTRickettsia prowazekii
93Glu Asn Asp Asp Ala Asn Arg Ala Leu 1 5
947PRTChlamydia trachomatis 94Gly Pro Asn Ile Gly Leu Ile 1
5 959PRTChlamydia trachomatis 95Glu His Asp Asp Ala Asn Arg Ala
Leu 1 5 967PRTMycoplasma pneumoniae 96Gly
Met Asn Ile Gly Leu Ile 1 5 979PRTMycoplasma
pneumoniae 97Glu Asn Asp Asp Ser Ala Arg Ala Leu 1 5
987PRTBacillus subtilis 98Gly Pro Asn Ile Gly Leu Ile 1
5 999PRTBacillus subtilis 99Glu Asn Asp Asp Ser Asn Arg
Ala Leu 1 5 1007PRTStaphylococcus aureus
100Gly Pro Asn Ile Gly Leu Ile 1 5
1019PRTStaphylococcus aureus 101Glu Asn Asp Asp Ser Asn Arg Ala Leu 1
5 1027PRTMycobacterium tuberculosis 102Gly
Pro Asn Ile Gly Leu Ile 1 5 1039PRTMycobacterium
tuberculosis 103Glu His Asp Asp Ala Asn Arg Ala Leu 1 5
1047PRTSynechocystis sp. 104Gly Pro Asn Ala Gly Leu Ile 1
5 1059PRTSynechocystis sp. 105Glu His Asp Asp Ala
Asn Arg Ala Leu 1 5 1067PRTAquifex
aeolicus 106Gly Gln Asn Ile Gly Leu Val 1 5
1079PRTAquifex aeolicus 107Glu His Asp Asp Ala Asn Arg Ala Leu 1
5 1087PRTDeinococcus radiodurans 108Gly Ala Asn
Ile Gly Leu Ile 1 5 1099PRTDeinococcus
radiodurans 109Glu His Asp Asp Ala Asn Arg Ala Leu 1 5
1107PRTThermus thermophilus 110Gly Ala Asn Ile Gly Leu Ile 1
5 1119PRTThermus thermophilus 111Glu His Asp Asp
Ala Asn Arg Ala Leu 1 5 1127PRTThermus
aquaticus 112Gly Ala Asn Ile Gly Leu Ile 1 5
1139PRTThermus aquaticus 113Glu His Asp Asp Ala Asn Arg Ala Leu 1
5 1147PRTHomo sapiens 114Gly Glu Pro Cys Gly Leu
Met 1 5 1159PRTHomo sapiens 115Asp His Asn Gln
Ser Pro Arg Asn Met 1 5 1167PRTHomo
sapiens 116Gly His Ala Val Gly Leu Val 1 5
1179PRTHomo sapiens 117Asp His Asn Gln Ser Pro Arg Asn Thr 1
5 1187PRTHomo sapiens 118Gly Glu Ala Cys Gly Leu Val 1
5 1199PRTHomo sapiens 119His His Asn Gln Ser Pro
Arg Asn Thr 1 5
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