Patent Publication Number: US-2013231276-A1

Title: Combination Of An Aminoacyl-tRNA Synthetase Inhibitor With A Further Antibacterial Agent For Attenuating Multiple Drug Resistance

Description:
RELATED APPLICATIONS 
     This application is a continuation of International Application No. PCT/US2011/057119, which designated the United States and was filed on Oct. 20, 2011, published in English, which claims the benefit of U.S. Provisional Application Nos. 61/405,771, filed Oct. 22, 2010, and 61/426,289, filed on Dec. 22, 2010. The entire teachings of the above applications are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Many microorganisms have survived for many years by adapting to antimicrobial agents, which permits certain bacteria to resist antibiotics. Bacteria that exhibit multidrug resistance include, for example,  Staphylococci, Enterococci, Gonococci, Streptococci, Salmonella  and  Mycobacterium tuberculosis.  Multidrug resistance can lead to illness and death. Thus, there is a need for new and effective methods to attenuate multidrug resistance in a subject. 
     SUMMARY OF THE INVENTION 
     The invention is generally directed to methods of attenuating bacterial multidrug resistance. 
     In an embodiment, the invention is a method of attenuating bacterial multidrug resistance in a subject, comprising the step of administering an aminoacyl-tRNA synthetase inhibitor and an antibacterial agent to the subject, wherein the aminoacyl-tRNA synthetase inhibitor is distinct from the antibacterial agent and the aminoacyl-tRNA synthetase inhibitor is neither an isoleucyl-tRNA synthetase inhibitor nor a methionyl-tRNA synthetase inhibitor. 
     In another embodiment, the invention is a method of attenuating resistance to an antibacterial agent that has aminoacyl-tRNA synthetase inhibitor activity in a subject, comprising the step of administering a composition that includes an aminoacyl-tRNA synthetase inhibitor and an antibacterial agent to the subject, wherein the aminoacyl-tRNA synthetase inhibitor is distinct from the antibacterial agent and the aminoacyl-tRNA synthetase inhibitor is neither an isoleucyl-tRNA synthetase inhibitor nor a methionyl-tRNA synthetase inhibitor. 
     In another embodiment, the invention is a method of treating a subject with a multidrug resistant bacterial infection, comprising the step of administering an aminoacyl-tRNA synthetase inhibitor and an antibacterial agent to the subject, wherein the aminoacyl-tRNA synthetase inhibitor is distinct from the antibacterial agent. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  depicts the effect of indolmycin on the chloramphenicol sensitivity of four indolmycin-resistant strains of  S. coelicolor.    
         FIG. 2  depicts the effect of indolmycin on the erythromycin sensitivity of four indolmycin-resistant strains of  S. coelicolor.    
         FIG. 3  depicts the effect of indolmycin on the vancomycin sensitivity of four indolmycin-resistant strains of  S. coelicolor.    
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. 
     In an embodiment, the invention is a method of attenuating bacterial multidrug resistance in a subject, comprising the step of administering an aminoacyl-tRNA synthetase inhibitor and an antibacterial agent to the subject, wherein the aminoacyl-tRNA synthetase inhibitor is distinct from the antibacterial agent and the aminoacyl-tRNA synthetase inhibitor is neither an isoleucyl-tRNA synthetase inhibitor nor a methionyl-tRNA synthetase inhibitor. 
     In another embodiment, the invention is a method of attenuating resistance to an antibacterial agent that has aminoacyl-tRNA synthetase inhibitor activity in a subject, comprising the step of administering a composition that includes an aminoacyl-tRNA synthetase inhibitor and an antibacterial agent to the subject, wherein the aminoacyl-tRNA synthetase inhibitor is distinct from the antibacterial agent and the aminoacyl-tRNA synthetase inhibitor is neither an isoleucyl-tRNA synthetase inhibitor nor a methionyl-tRNA synthetase inhibitor. 
     “Attenuate,” as used herein with respect to bacterial multidrug resistance, means that the multidrug resistance is reduced, preferably to have a clinically beneficial outcome. Attenuate is also referred to as “decrease” with respect to bacterial multidrug resistance. 
     “Distinct,” as used herein in reference to the aminoacyl-tRNA synthetase inhibitor and the antibacterial agent used in the methods of the invention, means that the aminoacyl-tRNA synthetase inhibitor and the antibacterial agent are different compounds. 
     In another embodiment, the invention is a method of treating a subject with a multidrug resistant bacterial infection, comprising the step of administering an aminoacyl-tRNA synthetase inhibitor and an antibacterial agent to the subject, wherein the aminoacyl-tRNA synthetase inhibitor is distinct from the antibacterial agent. 
     The aminoacyl-tRNA synthetase inhibitor employed in the methods of the invention can be a natural product inhibitor, an analog of a natural product inhibitor, or pharmaceutically acceptable salts. Exemplary natural product inhibitors of aminoacyl-tRNA synthetases include mupirocin, borrelidin, furanomycin, granaticin, indolmycin, ochratoxin A, and cis-pentacin. The aminoacyl-tRNA synthetase inhibitors of the present invention include inhibitors of, for example, at least one member selected from the group consisting of isoleucyl (e.g., mupirocin, furanomycin), leucyl (e.g., granaticin), threonyl (e.g., borrelidin), phenylalanyl (e.g., ochratoxin A), tryptophanyl (e.g., indolmycin, chuangxinmycin), methionyl, prolyl (e.g., cis-pentacin) and lysyl tRNA synthetases. 
     The aminoacyl-tRNA synthetase inhibitor employed in the methods of the invention can be a tryptophanyl-tRNA synthetase inhibitor (e.g., indolmycin or a pharmaceutically acceptable salt). The antibacterial agent employed in the methods of the invention can be at least one member selected from the group consisting of ampicillin, chloramphenicol, erythromycin, lincomycin, mupirocin and vancomycin or pharmaceutically acceptable salts. In some embodiments, the antibacterial agent employed can be at least one member selected from the streptogramin class of antibiotics. 
     Aminoacyl-tRNA synthetase inhibitors interfere with the biosynthesis of charged aminoacyl-tRNA substrates for protein synthesis. Many antibiotic resistance determinants are proteins. If a particular charged aminoacyl-tRNA is unavailable, then protein synthesis cannot be completed. Therefore, inhibition of aminoacyl-tRNA synthetases could lead to inhibition of the biosynthesis of resistance determinants. For example, by perturbing the biosynthesis of the tryptophan-rich proteins required for resistance to chloramphenicol, erythromycin and vancomycin, a tryptophanyl-tRNA synthetase inhibitor (e.g., indolmycin, chuangxinmycin) can suppress resistance to chloramphenicol, erythromycin, and vancomycin. 
     In an embodiment, the aminoacyl-tRNA synthetase inhibitor and the antibacterial agent can be co-administered to the subject. In another embodiment, the aminoacyl-tRNA synthetase inhibitor can be administered prior to administration of the antibacterial agent. In a further embodiment, the antibacterial agent is administered prior to the aminoacyl-tRNA synthetase inhibitor. 
     The methods of the invention can attenuate bacterial multidrug resistance to at least one bacteria selected from the group consisting of  Staphylococci, Enterococci, Gonococci, Streptococci, Salmonella  and  Mycobacterium tuberculosis.  In other embodiments, the methods of the invention can attenuate bacterial multidrug resistance to  Streptomyces.  In yet another embodiment, the methods of the invention can attenuate resistance to methicillin-resistant  Staphylococcus aureus  (MRSA). 
     An “effective amount,” also referred to herein as a “therapeutically effective amount,” when referring to the amount of a compound or composition (e.g., an antibacterial agent, an aminoacyl-tRNA synthetase inhibitor) is defined as that amount, or dose, of a compound or composition that, when administered to a subject, is sufficient for therapeutic efficacy (e.g., an amount sufficient to attenuate multidrug resistance, an amount sufficient to prevent a multidrug resistant bacterial infection). 
     The methods of the invention can be accomplished by the administration of the compounds of the invention (e.g., compositions including an antibacterial agent, an aminoacyl-tRNA synthetase inhibitor) to a subject (e.g., a human subject) by enteral or parenteral means. The route of administration can be by oral ingestion (e.g., tablet, capsule form) or intramuscular injection of the compound. Other routes of administration can include intravenous, intraarterial, intraperitoneal, or subcutaneous routes, nasal administration, suppositories and transdermal patches. The compounds employed in the methods of the invention can be administered in suitable excipients, including pharmaceutically acceptable salts. 
     In an embodiment, the compounds (e.g., an antibacterial agent, an aminoacyl-tRNA synthetase inhibitor) employed in the methods of the invention can be administered in a dose of between about 0.01 mg/kg to about 0.1 mg/kg; about 0.001 mg/kg to about 0.01 mg/kg; about 0.001 to about 0.05 mg/kg; about 0.1 mg/kg to about 1 mg/kg body weight; about 1 mg/kg to about 5 mg/kg body weight; or between about 5 mg/kg to about 15 mg/kg body weight. 
     The compounds can be administered in doses of about 0.1 mg, about 1 mg, about 2 mg, about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 80 mg, 100 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1400 mg, about 1600 mg or about 2000 mg, or any combination thereof. The compounds can be administered once a day or multiple (e.g., two, three, four, five) times per day. 
     EXEMPLIFICATION 
     There has been much interest in using combinations of antibacterial drugs to treat multidrug resistant pathogens and/or slow the rate at which drug resistance emerges (1-4).  Streptomyces coelicolor  is a non-pathogenic relative of the Gram-positive, human pathogen  Mycobacterium tuberculosis  (5).  S. coelicolor  is a useful model organism for studies of antibacterial resistance because because it is resistant to several antibiotics, including indolmycin, chloramphenicol, daptomycin, erythromycin, the streptogramins, and vancomycin (6-12). With the exception of daptomycin, the resistance of  S. coelicolor  to each of these antibiotics has been ascribed to the expression of one or more genes (6, 8-12). We assessed the effect of indolmycin, an antibiotic that inhibits bacterial tryptophanyl-tRNA synthetases (8-10, 13), on the viability of  S. coelicolor  when used in combination with other antibiotics to which the organism is resistant. 
     Changes in the susceptibility of  S. coelicolor  to three clinically used antibacterial drugs (chloramphenicol, erythromycin, and vancomycin) grown in the presence of indolmycin were assessed. The effects of antibiotic combinations were assessed in wild-type  S. coelicolor,  which has an auxiliary, indolmycin-resistant isoform of tryptophanyl-tRNA synthetase (TrpRS1). We also examined these combinations in three other indolmycin-resistant strains of  S. coelicolor —a trpRS2 null strain (B728) (8) and two strains lacking trpRS1 with resistance conferring point mutations in trpRS2 (B734 with TrpRS2 H48N and B735 with TrpRS2 H48Q) (9). These strains were selected because the strong antibacterial activity of indolmycin precluded testing of this hypothesis in the indolmycin-sensitive trpRS1 null strain of  S. coelicolor.  The effects were measured in terms of changes in the chloramphenicol, erythromycin, and vancomycin MICs of the four strains over a range of sub-lethal indolmycin concentrations. 
     Indolmycin markedly suppresses the resistance of  S. coelicolor  to chloramphenicol (a bacteriostatic protein synthesis inhibitor than binds to the ribosome), erythromycin (a macrolide antibiotic), and vancomycin (a glycopeptide antibiotic). The fact that indolmycin suppresses resistance to at least three different antibacterial drugs (each having a different mechanism of action) is notable and potentially valuable. It suggests that indolmycin can be useful even if the bacterium to be killed is resistant to it or other antibiotics, provided that the proteins conferring resistance require tryptophan for their biosynthesis. 
     Indolmycin can be used to kill multi-drug resistant strains that are sensitive to indolmycin. However, when the inevitable resistance to indolmycin emerges, indolmycin may still be useful as an adjuvant to other antibacterial drugs to which a pathogenic bacterium is resistant. This utility may be due to the ability of indolmycin to suppress other mechanisms of drug resistance by interfering with the biosynthesis of resistance determinants. 
     Aminoacyl-tRNA synthetase inhibitors have emerged as a useful class of antibacterial drugs. The prototypical member of this class is the clinically used drug mupirocin, which is an inhibitor of isoleucyl-tRNA synthetase. Another aminoacyl-tRNA synthetase inhibitor that has attracted attention is indolmycin. This antibiotic, derived from  Streptomyces griseus  ATCC 12648, is a competitive inhibitor of bacterial tryptophanyl-tRNA synthetases. Although indolmycin is not presently used in the clinic, its potent activity against methicillin-resistant  Staphylococcus aureus  (MIC is 0.5 μg/mL) and  Helicobacter pylori  (MIC is 0.016 μg/mL) has renewed interest in its clinical utility. 
     Resistance to indolmycin has been reported. Many indolmycin-resistant bacterial species harbor auxiliary isoforms of tryptophanyl-tRNA synthetase that are insensitive to indolmycin. In  Streptomyces coelicolor  (a non-pathogenic relative of  Mycobacterium tuberculosis ), transcription of the gene encoding an auxiliary, indolmycin-resistant isoform of tryptophanyl-tRNA synthetase (trpRS1) is induced by indolmycin. The induced expression of trpRS1 was confirmed by analytical two-dimensional gel electrophoresis and peptide-mass fingerprinting of soluble protein isolated from  S. coelicolor  grown in media containing 40 μg/mL indolmycin. In the same experiment, the abundance of several proteins was notably reduced. These observations were intriguing because  S. coelicolor  is highly resistant to indolmycin (MIC is about&gt;500 μg/mL) and did not exhibit any apparent growth defects in the experiment. Proteins whose synthesis was negatively affected by indolmycin have multiple tryptophan residues. 
     Indolmycin may reduce the availability of charged tryptophanyl-tRNA for protein synthesis even though the resistance gene trpRS1 is expressed. TrpRS1 is weakly inhibited by the antibiotic (Ki=about 900 nM) (19). Indolmycin may perturb the synthesis of antibacterial drug resistance determinants with multiple tryptophan residues and consequently affect the susceptibility of  S. coelicolor  to the corresponding antibacterial drugs.  S. coelicolor  is a useful model organism for studies of multi-drug resistant bacteria because it is resistant to several clinically used antibacterial drugs, including ampicillin, chloramphenicol, erythromycin, lincomycin, mupirocin, and vancomycin, via multiple genetic determinants. Erythromycin, chloramphenicol, and vancomycin resistance determinants have multiple tryptophan residues. 
       Streptomyces coelicolor  strains were grown on DIFCO Nutrient Agar supplemented with indolmycin (0-100 μg/mL) and one of the following antibacterial agents: chloramphenicol (10-80 μg /mL), erythromycin (1-110 μg/mL), and vancomycin (30-140 μg/mL). The strains were grown on the media at 30° C. for 48 hours, after which point growth was assessed visually. 
     Vancomycin was purchased from Sigma Chemical Co. and a 50 mg/ml stock solution was created by dissolving the solid vancomycin in dH 2 0. Chloramphenicol was purchased from Sigma Chemical Co., and a 25 mg/ml stock solution was created by dissolving the chloramphenicol in 100% ethanol. Erythromycin was purchased from Sigma Chemical Co., and a 50 mg/ml stock solution was created by dissolving the erythromycin in 100% ethanol. Ochratoxin A and borrelidin can be purchased from Sigma Chemical Co. Indolmycin was chemically synthesized according to established procedures as described, for example, by Hasuoka, A., et. al.,  Chem. Pharm. Bull.  49:1604-1608 (2001). 
     A 50 mg/mL (active enantiomer) stock solution of indolmycin was prepared by dissolving the indolmycin in DMSO. The concentrations of each stock solution reflect the actual concentration of the pharmacologically active substance. Aliquots of the stock solutions were added directly to molten, sterile DIFCO Nutrient Agar prior to solidification.  Streptomyces coelicolor  spores were directly spread onto the surface of each DNA plate and the plates were incubated at 30° C. for about 48 hours. Growth was assessed visually after the incubation period. Minimal inhibitory concentrations (MICs) reflect the drug concentrations at which no  Streptomyces coelicolor  growth was observed. 
     Initially, the impact of indolmycin on chloramphenicol resistance in  S. coelicolor  was tested. Chloramphenicol is an antibiotic used in the treatment of eye infections. In  S. coelicolor,  chloramphenicol resistance is conferred by two major facilitator superfamily efflux pumps, Cm1R1 and Cm1R2 (7). Since each of these membrane proteins has seven tryptophan residues, indolmycin may perturb their biosynthesis and, thus affect chloramphenicol susceptibility in  S. coelicolor.  The chloramphenicol MIC of all four strains was reduced in media supplemented with indolmycin (Table 1,  FIG. 1 ). The most dramatic effect was observed in wild-type  S. coelicolor,  where the chloramphenicol MIC was reduced 10-fold. 
     Next, the impact of indolmycin on erythromycin resistance in  S. coelicolor  was tested. Erythromycin is a macrolide antibiotic and its semi-synthetic derivatives are used in clinical medicine for treatment of a variety of bacterial infections. The erythromycin resistance phenotype of  S. coelicolor  has been ascribed to a gene (SC06090) encoding a glycosyl transferase with 9 tryptophan residues (5). Indolmycin may negatively affect the biosynthesis of the erythromycin resistance determinant. Co-administration of indolmycin with erythromycin profoundly affected the viability of all four strains (Table 2,  FIG. 2 ). Strain B735 was found to be 50-times more sensitive to erythromycin in the presence of about 100 μg/mL indolmycin. 
     Finally, the effect of indolmycin on vancomycin resistance in  S. coelicolor  was assessed. Vancomycin is a glycopeptide antibiotic that is widely known as the “drug of last resort” in the treatment of infections caused by multidrug-resistant bacteria. As such, vancomycin resistance is a concern and has been the subject of extensive study (14).  S. coelicolor  has the characteristic vancomycin resistance cassette consisting of genes encoding a two-component regulatory system and enzymes that convert D-Ala-D-Ala termini of muropeptides to D-Ala-D-Lac (12). All 7 of the gene products encoded by this cassette have tryptophan residues (e.g., the membrane-bound histidine kinase VanS has 4 tryptophan residues and the D-Ala-D-Ala dipeptidase VanX has 6 tryptophan residues). Given the tryptophan content of the van gene products, indolmycin may make the  S. coelicolor  strains susceptible to vancomycin. Interestingly, indolmycin affected vancomycin susceptibility in only two of the four strains (Table 3,  FIG. 3 ). In media supplemented with about 100 μg/mL indolmycin, strains M600 and B735 were 1.6- and 4-times more sensitive to vancomycin, respectively. 
     In conclusion, indolmycin can suppress three different drug resistance phenotypes of a multi-drug resistant bacterium. Although it has potential as an antibacterial drug (15-18), indolmycin is not clinically used at present. In strains where resistance to indolmycin is innate or in strains where it emerged by point mutations (8-10), indolmycin could be used as an adjuvant to other antibacterial drugs to which a bacterium is resistant. An explanation for the apparent indolmycin-induced changes in the drug MICs may be partial inhibition of indolmycin-resistant tryptophanyl-tRNA synthetases, which could perturb the biosynthesis of resistance determinants that are rich in tryptophan residues. This explanation is supported by the observation that the indolmycin K i  of the resistant tryptophanyl-tRNA synthetase (TrpRS1) in  S. coelicolor  is 900 nM (19); thus, this isoform is likely to be inhibited by the antibiotic. Further, proteins in  S. coelicolor  mediating chloramphenicol, erythromycin, and vancomycin resistance are rich in tryptophan residues (Table 4). 
     While differing degrees of resistance to indolmycin may explain the extent to which the antibiotic affects the antibacterial MICs of specific strains, differences in gene expression, enzymatic activity, and/or protein stability could be used to explain the finding that indolmycin affects the MICs of certain drugs more than others. In any case, the suppression of multiple drug resistance phenotypes by indolmycin is noteworthy in the context of combination therapies (1-4). Combinations of antibacterial drugs to treat multidrug resistant pathogens and/or slow the rate at which drug resistance emerges have been reported (2, 3, 10, 15). For example, for AUGMENTIN®, a β-lactam inhibitor (potassium clavulanate) is used in conjunction with a β-lactam drug (20). As an adjuvant, indolmycin has a broader spectrum than potassium clavulanate because it presumably perturbs the biosynthesis of several resistance determinants. Since many drug resistance determinants (e.g., membrane-bound efflux pumps) in bacteria are rich in tryptophan residues, tryptophanyl-tRNA synthetase inhibitors like indolmycin could be used at sub-lethal concentrations to suppress multi-drug resistance phenotypes. 
     As described herein, indolmycin in combination with at least one antibacterial agent, can attenuate bacterial multidrug resistance to indolmycin, chloramphenicol, erythromycin and vancomycin. Methods of the invention may have the advantage for use if the bacteria to which drug resistance exists or will develop requires particular amino acids for resistance. For example, a tryptophanyl-tRNA synthetase inhibitor can be used in combination with an antibacterial agent to attenuate bacterial multidrug resistance against bacterial that require tryptophan for biosynthesis of proteins for multidrug resistance or growth. 
                     TABLE 1                  The Effect of Indolmycin on Chloramphenicol Sensitivity                         Chloramphenicol MIC (μg/ml)                                             0 μg/ml   10 μg/ml   25 μg/ml   50 μg/ml   75 μg/ml   100 μg/ml       Strain   Indolmycin   Indolmycin   Indolmycin   Indolmycin   Indolmycin   Indolmycin               M600   80   35   25   20   15   10       B728   60   40   30   30   20   20       B734   50   50   50   50   45   40       B735   50   50   50   30   20   15                    
All  Streptomyces  strains were grown on Difco Nutrient Agar at 30° C. The MICs were assessed after 48 hours.
 
                     TABLE 2                  The Effect of Indolmycin on Erythromycin Sensitivity                         Erythromycin MIC (μg/ml)                                             0 μg/ml   10 μg/ml   25 μg/ml   50 μg/ml   75 μg/ml   100 μg/ml       Strain   Indolmycin   Indolmycin   Indolmycin   Indolmycin   Indolmycin   Indolmycin                                                 M600   110   40   30   15   10   5       B728   70   50   30   20   10   5       B734   70   40   30   20   10   5       B735   50   10   10   5   5   1                    
All  Streptomyces  strains were grown on Difco Nutrient Agar at 30° C. The MICs were assessed after 48 hours.
 
                     TABLE 3                  The Effect of Indolmycin on Vancomycin Sensitivity                         Vancomycin MIC (μg/ml)                                             0 μg/ml   10 μg/ml   25 μg/ml   50 μg/ml   75 μg/ml   100 μg/ml       Strain   Indolmycin   Indolmycin   Indolmycin   Indolmycin   Indolmycin   Indolmycin                                                 M600   110   110   110   110   110   70       B728   130   130   130   130   130   130       B734   140   140   140   140   140   140       B735   120   120   120   120   120   30                    
All  Streptomyces  strains were grown on Difco Nutrient Agar at 30° C. The MICs were assessed after 48 hours.
 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Erythromycin, chloramphenicol, and vancomycin resistance 
               
               
                 determinants in  S. coelicolor  and their tryptophan content 
               
            
           
           
               
               
               
            
               
                 Gene 
                   
                 Number of 
               
               
                 Number 
                 Description 
                 Tryptophan Residues 
               
               
                   
               
               
                 SCO7526 
                 cmlR1, chloramphenicol efflux 
                 7 
               
               
                   
                 pump 
               
               
                 SCO7662 
                 cmlR2, chloramphenicol efflux 
                 7 
               
               
                   
                 pump 
               
               
                 SCO6090 
                 Putative erythromycin 
                 9 
               
               
                   
                 glycosyltransferase 
               
               
                 SCO3589 
                 vanS, probable two component 
                 4 
               
               
                   
                 sensor kinase 
               
               
                 SCO3596 
                 vanX, probable D-alanine; 
                 6 
               
               
                   
                 D-alanine dipeptidase 
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     1. Chait, R., et. al.,  Nature  446:668-671 (2007). 
     2. Golan, D. E. et al. (eds).  Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.  (Lippincott Williams &amp; Wilkins, Philadelphia, 2005) 
     3. Keith, C. T., et. al.,  Nat. Rev. Drug Discov.  4:71-8 (2005). 
     4. Torella, J. P., et. al.,  PLoS Computational Biology  6: e1000796 (2010). 
     5. Bentley, S. D., et. al.,  Streptomyces coelicolor. Nature  417:141-147 (2002). 
     6. Kieser, T., et. al., Practical  Streptomyces  Genetics. Norwich: John Innes Foundation (2000). 
     7. Vecchione, J. J., et. al.,  Antimicrob. Agents Chemother.  53:4673-4677 (2009). 
     8. Vecchione, J. J., et. al.,  J. Bacteriol.  190:6253-6257 (2008). 
     9. Vecchione, J. J. et. al.,  Antimicrob. Agents Chemother.  53:3972-3980 (2009). 
     10. Vecchione, J. J. et. al.,  J. Bacteriol.  192:3565-3573 (2010). 
     11. Folcher, M., et. al.,  J. Biol. Chem.  276:1479-1485 (2001). 
     12. Hong, H.-J., et. al.,  Mol. Microbiol.  59: 1107-1121 (2004). 
     13. Werner, R. G., et. al.,  Eur. J. Biochem.  68: 1-3 (1976). 
     14. Hong, H. J., et. al.,  Adv. Exp. Med. Biol.  631: 200-13 (2008). 
     15. Kim, S., et. al.,  Appl. Microbiol. Biotechnol.  61:278-288 (2003). 
     16. Hurdle, J. G., et. al.,  J. Antimicrob. Chemother.  54:549-552 (2004). 
     17. Hurdle, J. G., et. al.,  Antimicrob. Agents Chemother.  49:4821-4833 (2005). 
     18. Kanamaru, T., et. al.,  Antimicrob. Agents Chemother.  45:2455-2459 (2001). 
     19. Kitabatake, M., K. et. al.,  J. Biol. Chem.  277:23882-23887 (2002). 
     20. Stein, G. E. et. al.,  Clin. Pharmacy  3:591-599 (1984). 
     The teachings of all of the references cited herein are hereby incorporated by reference in their entirety. 
     While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.