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Antimicrobial Agents and Chemotherapy, September 2005, p. 3974-3976, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3974-3976.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Activities of Rifamycin Derivatives against Wild-Type and rpoB Mutants of Chlamydia trachomatis

Minsheng Xia,1 Robert J. Suchland,1 Joli A. Carswell,1 John Van Duzer,2 Debra K. Buxton,2 Kara Brown,2 David M. Rothstein,2 and Walter E. Stamm1*

Division of Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington 98195,1 ActivBiotics, Inc., Lexington, Massachusetts 024212

Received 1 February 2005/ Returned for modification 28 February 2005/ Accepted 25 April 2005


   ABSTRACT
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Rifalazil, a semisynthetic rifamycin, was shown previously to have exceptional potency against Chlamydia trachomatis (MIC of 0.00025 µg/ml). We therefore tested 250 additional rifamycin derivatives and identified 12 with activities that are eightfold more potent than that of rifalazil. These compounds also showed exceptional activities against rifampin-resistant strains that carry missense mutations in the rpoB gene. The antimicrobial potency and intracellular penetration of these agents suggest their potential in treatment of chlamydial infections.


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Rifampin, a rifamycin derivative, is one of the most active antimicrobial agents against the obligate intracellular pathogen Chlamydia trachomatis, but its tendency to select for resistant strains makes it less attractive than other drugs for routine treatment of chlamydial infections (3, 4, 10). Rifalazil (ABI-1648), a semisynthetic rifamycin which has potent activity against several clinically important bacterial pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Helicobacter pylori, Clostridium difficile, and Mycobacterium tuberculosis, has also been shown to be effective in cell culture against Chlamydia trachomatis and Chlamydia pneumoniae, with MICs in the range of 0.00025 µg/ml (5, 8, 9, 12). The antibacterial activities of rifamycin and rifamycin derivatives, including rifalazil, result from inhibition of the bacterial RNA polymerase by binding to its ß subunit encoded by the rpoB gene (1, 2). With regard to Chlamydia infections, rifalazil also has excellent tissue and cellular penetration and has had a good safety profile in human clinical trials to date (9). Unlike rifampin, interaction of rifalazil with the P450 system is insignificant, and thus it produces less interference with metabolism of other drugs (6, 7). Furthermore, rifalazil was shown recently to retain substantial activity against strains of C. trachomatis that are highly resistant to rifampin (12). These features prompted us to evaluate additional rifamycin derivatives that are closely related to rifalazil for potency against wild-type C. trachomatis, as well as against rifampin-resistant strains of C. trachomatis containing missense mutations in the rpoB gene (12).

Assays to determine the MICs of rifampin, rifalazil, and other rifamycin derivatives for C. trachomatis were performed as previously described (11). Briefly, C. trachomatis D/uw-3 was inoculated onto monolayers of McCoy cells in 96-well microtiter plates and centrifuged for 1 h. Immediately following centrifugation, wells were overlaid with growth medium containing serial twofold dilutions of the appropriate drug and incubated for 48 h. Wells were then fixed with methanol, labeled with fluorescein isothiocyanate-labeled anti-Chlamydia lipopolysaccharide monoclonal antibody, and visualized by fluorescence microscopy as described previously (11). The MIC was defined as the minimum drug concentration at which no C. trachomatis were detected while the organisms were exposed to the drug. To measure the minimum cidal concentration (MCC) of selected rifamycin derivatives, D/uw-3 was inoculated onto cell cultures exposed to the selected agents and incubated as described above. The cells were then subpassaged to drug-free cell cultures. The MCC for a given agent was defined as the minimum concentration at which no viable chlamydial organisms were detected in the first subpassage in drug-free cell cultures. The minimum concentrations at which no viable chlamydial organisms were detected in three consecutive passages in drug-free cell cultures (MCC3s) were also determined (11).

We determined the MICs of 250 rifamycin derivative compounds for C. trachomatis D/uw-3. Overall, most of these compounds had MICs ranging from 0.000016 µg/ml to 0.01 µg/ml. The distribution of the number of compounds with a particular MIC is shown in Fig. 1. For points of reference, the MICs of rifampin and rifalazil were determined to be 0.004 and 0.00025 µg/ml, respectively. The 12 most potent compounds among the 250 rifamycin derivatives tested had MICs of 0.000032 µg/ml, with one compound, ABI-1662, showing an MIC of 0.000016 µg/ml. These extremely potent agents are, thus, new rifamycin derivatives with significantly improved antimicrobial activities compared with those of rifampin and rifalazil.



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  		FIG. 1. Distribution of MIC of rifamycin derivatives for C. trachomatis D/uw-3.

 
Rifalazil has unusual properties that may contribute to its activity against intracellular infections, such as chlamydiae. These include exceptional tissue penetration and high intracellular concentrations of compound (9). Interestingly, rifalazil is poorly soluble in aqueous solutions at a neutral pH (~1 µg/ml [unpublished results]). It is possible that more water-soluble compounds might not partition as extensively inside mammalian cells and might therefore have less activity against C. trachomatis than rifalazil. In fact, the trend was that the most water-soluble compounds were the least potent in terms of antichlamydial activity. When solubility exceeded 10 µg/ml, compounds were uniformly less active against Chlamydia (Fig. 2). However, there was not a strict inverse relationship between solubility and activity against C. trachomatis; 5 of the 14 more soluble compounds had strong activities, including 3 compounds having at least as much activity as rifalazil. Importantly, for potent compounds that were poorly soluble, the solubility exceeded the MIC for C. trachomatis by a factor of at least 4,000-fold.



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  		FIG. 2. Solubilities of rifamycin derivatives in relation to MICs for C. trachomatis D/uw-3.

 
A panel of four C. trachomatis L2 mutants that are highly resistant to rifampin was then tested for susceptibility to identify potent compounds (12). These mutants all carried missense mutations in the rpoB gene (Table 1). Significantly, we found that all four mutants were susceptible to the most active compounds. The MICs of rifalazil ranged from 0.002 to 0.064 µg/ml, considerably lower than the MICs of rifampin, which ranged from 0.5 to 512. Among the selected rifamycin derivatives listed in Table 1, ABI-0046, ABI-1131, and ABI-1662 demonstrated overall improved MICs for the mutants, even compared to rifalazil.


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  		TABLE 1. Susceptibilities of C. trachomatis L2 rpoB mutants to rifampin, rifalazil, and seven other rifamycin derivatives

 
Besides measuring inhibitory potency, we also assessed these agents' bactericidal potency by determining the MCCs and MCC3s of rifalazil, ABI-1131, ABI-0204, and ABI-1662 (Table 2). These compounds demonstrated excellent potencies against C. trachomatis, especially compound ABI-1662.


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  		TABLE 2. Potency of selected rifamycin derivative agents measured by susceptibilities of C. trachomatis D/uw-3 in drug-treated cell cultures and subpassages in drug-free cell cultures

 
Structures of the most potent compound against both wild-type and mutant strains of C. trachomatis (ABI-1662), as well as that of another potent compound (ABI-1131) (13) and rifalazil, are shown in Fig. 3. Although it is difficult to derive structure-activity relationships at this time, it is clearly possible to make subtle modifications outside of the ansa ring that can increase potency in cell culture. If the ability of rifalazil to penetrate tissue is an important component of potency, the propensity of these compounds to penetrate mammalian cells may lead to potent in vivo activities, as with rifalazil (5).



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  		FIG. 3. Chemical structures of rifalazil, ABI-1131, and ABI-1662.

 
Compound ABI-0046 is noteworthy in that it is no more potent against wild-type C. trachomatis than rifalazil, but it demonstrated better potency against all four rpoB mutants tested. The attribute of having selectively more potent activities against rifampin-resistant mutant strains suggests possible differences in the affinities of these two compounds for mutant and wild-type RNA polymerases. This hypothesis should be tested.

In conclusion, our studies suggest that novel rifamycin derivatives, especially ABI-1662, ABI-1131, ABI-0046, and ABI-0204, are promising new antibiotics that have improved MICs, MCCs, and MCC3s for wild-type C. trachomatis. These compounds are also potent against rifampin-resistant strains, as indicated by their excellent MICs. Because of their extraordinary in vitro potencies against both the wild-type and rifampin-resistant variants of C. trachomatis, these compounds warrant further evaluation as to their usefulness for treatment of chlamydial infections in vivo. Further investigation may also illuminate the molecular mechanisms by which these potent compounds are effective against rifampin-resistant bacteria. Such studies may define whether the potencies they exhibit are due to a possible increased uptake and intracellular concentration, an improved interaction with the mutant polymerases compared to rifampin, or both.


   ACKNOWLEDGMENTS
 
This work was supported by a research grant from ActivBiotics, Inc., and a grant from the National Institutes of Health (AI31448).


   FOOTNOTES
 
* Corresponding author. Mailing address: 1959 NE Pacific Street, University of Washington, Box 356523, Seattle, WA 98195. Phone: (206) 616-4170. Fax: (206) 616-4898. E-mail: wes{at}u.washington.edu. Back


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Antimicrobial Agents and Chemotherapy, September 2005, p. 3974-3976, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3974-3976.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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