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Antimicrobial Agents and Chemotherapy, November 2007, p. 4202-4204, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00181-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Vitro Antimycobacterial Spectrum of a Diarylquinoline ATP Synthase Inhibitor{triangledown}

Emma Huitric,1,2* Peter Verhasselt,3 Koen Andries,4 and Sven E. Hoffner1,2

Swedish Institute for Infectious Disease Control, Solna, Sweden,1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden,2 Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium,3 Tibotec N.V., Beerse, Belgium4

Received 7 February 2007/ Returned for modification 11 June 2007/ Accepted 13 August 2007


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ABSTRACT
 
The diarylquinoline R207910 is in clinical development for tuberculosis treatment. The MIC50 for 41 drug-susceptible and 44 multidrug-resistant Mycobacterium tuberculosis clinical isolates was 0.032 µg/ml. Out of 20 additional mycobacterial species, three were found to be naturally resistant to R207910 and were shown to exhibit a polymorphism in their atpE genes.


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TEXT
 
There is an urgent public health need for new drugs for the treatment of tuberculosis (TB). An effective TB control is hindered by the long and complex treatment which current drugs require. The situation is further complicated by the interaction with human immunodeficiency virus, the rise of multidrug-resistan TB (MDR TB; resistance to at least rifampin and isoniazid), and the recent emergence of extensively drug-resistant TB (XDR TB; MDR as well as resistance to any fluoroquinolone and at least one of the following injectable drugs: capreomycin, kanamycin, or amikacin) (12).

A number of promising compounds have recently been identified, of which some are in the first stages of clinical testing (1, 7, 11). The diarylquinoline R207910, currently in phase 2 of clinical development as TMC207, inhibits mycobacteria through the inhibition of the ATP synthase, an enzyme essential for energy homeostasis (1, 5). Being endowed with a new mechanism of action, R207910 has been shown to be active against a high number of TB isolates that are both susceptible and resistant to first- and second-line drugs, as well as against several nontuberculous mycobacteria (NTM) (1).

In this study we have further characterized the activity of R207910 against members of the genus Mycobacterium. We compared the MIC distribution of a large number of drug-susceptible (DS) and MDR TB clinical isolates. Such careful comparison is necessary to enable trials of the compound in MDR TB patients. To fully characterize the compound's antimycobacterial spectrum, we also determined the MICs of numerous NTMs, with a special emphasis on the clinically important Mycobacterium avium complex (MAC; M. avium and Mycobacterium intracellulare).

All TB and NTM strains were from the national Mycobacterium strain collection at the Swedish Institute for Infectious Disease Control. Eighty-five Mycobacterium tuberculosis clinical isolates were tested, among which several were from World Health Organization (WHO) panels for the external quality assessment of drug susceptibility testing and others were clinical isolates from patients of different geographical origins. Forty-four of the isolates were MDR (one being identified as XDR) and 41 were DS isolates, as confirmed with the BACTEC 460 radiometric reference system (10) at critical concentrations (rifampin, 2 µg/ml; isoniazid, 0.2 µg/ml; streptomycin, 4 µg/ml; and ethambutol, 5 µg/ml). The XDR isolate was resistant to amikacin at 1 µg/ml and ofloxacin at 2 µg/ml (12). At least three of the MDR isolates were of the Beijing genotype, a family of strains that is increasingly spreading and has been associated with MDR (3).

A total of 51 NTM isolates from 20 different species, as identified by their 16S rRNA (4), including both slow and rapid growers, were characterized (Table 1). Considering its clinical relevance and natural resistance to many antibiotics, an emphasis was placed on the MAC, with a total of 22 isolates, belonging to 13 serotypes, being included.


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  		TABLE 1. MIC range, MIC50, and MIC90for 85 M. tuberculosis clinical isolates and 20 NTM species (51 isolates in total)a

The MIC to R207910 was determined using a twofold dilution series of R207910 in Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose-catalase. A 96-stick replication system, enabling the simultaneous inoculation of up to 96 bacterial suspensions on large (14-cm) agar plates, was used. M. tuberculosis isolates were exposed to an R207910 concentration range of 0.002 to 0.256 µg/ml. Being more heterogeneous, NTM isolates were exposed to a wider range (0.03 to 8 µg/ml). Depending on the species' growth rate, MICs were determined 2 to 4 weeks after incubation at 36°C, 5% CO2. The MIC was defined as the first concentration at which no bacterial growth was visible.

We have previously shown, on a smaller number of DS and MDR TB isolates, that isolates are susceptible to at least 0.1 µg/ml of R20710 (1). The present study determined the MICs and their distribution among a larger number of isolates and confirms that there is indeed no difference between DS and MDR strains (Fig. 1 and Table 1). Of the 41 DS and 44 MDR M. tuberculosis strains, all but one showed an MIC of ≤0.06 µg/ml; MIC values ranged between 0.002 and 0.12 µg/ml, and the median MIC (MIC50) was 0.03 µg/ml. The XDR isolate was as susceptible as the DS isolates (MIC, 0.03 µg/ml), as were the three Beijing genotype isolates (MIC, 0.03 to 0.06 µg/ml). MICs were confirmed by testing 10 M. tuberculosis strains in the reference radiometric BACTEC 460 system (10) (data not shown).


Figure 1
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  		FIG. 1. MIC distribution of 132 R207910-susceptible mycobacterial strains. Eighty-five isolates were M. tuberculosis clinical isolates (44 MDR), 22 belonged to the MAC, and 25 were of other NTM species. M. tuberculosis isolates were exposed to an R207910 concentration range of 0.002 to 0.25 µg/ml, while all NTM isolates were exposed to a range of 0.03 to 8 µg/ml. Four NTM isolates had MIC levels between 4 to 8 µg/ml and are not included in the figure.

R207910 exhibited a strong inhibitory effect against the majority of NTM species tested (8/20 species having MICs of ≤0.03 µg/ml; Fig. 1 and Table 1), with no difference seen between rapid- and slow-growing species. Mycobacterium fortuitum exhibited a 10-fold higher MIC than was earlier reported (1), possibly reflecting an intraspecies susceptibility variation known for certain NTM species (9). Combined with the earlier reported (1) activity against other NTM species (e.g., Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium smegmatis), the present data demonstrate that the diarylquinoline is endowed with a remarkably broad antimycobacterial spectrum.

M. avium is a major cause of opportunistic infections in immunosuppressed patients, especially among human immunodeficiency virus-infected individuals, and its natural resistance to several antibiotics makes it a difficult infection to treat (6). In this study, all 22 MAC strains had MICs of ≤0.25 µg/ml (Table 1), and the MIC50 was 0.03 µg/ml. These results are in line with earlier reported values for seven MAC strains (1) and are promising considering the clinical need for new therapeutic drugs against MAC infections.

An interesting finding was that three NTM species (Mycobacterium xenopi, Mycobacterium novocastrense, and Mycobacterium shimoidei) had significantly higher MICs and were considered naturally resistant to R207910 (Table 1). It was reported by Petrella et al. that M. xenopi's high MIC (4 µg/ml) could probably be attributed to the polymorphism seen at amino acid 63 in the species' atpE gene (8). We therefore investigated whether a similar polymorphism existed in the two additional species.

By using degenerate primers (TGTAYTTCAGCCARGCSATGG and CCGTTSGGDABGAGGAAGTTG), the genomic atpE gene of these three naturally resistant species was amplified by PCR and directly sequenced, and the sequences' prearranged proteins were compared to the atpE protein sequence of wild-type (Swiss-Prot accession no. Q10598) and resistant mutant M. tuberculosis (EMBL accession no. AJ865377) (Fig. 2).


Figure 2
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  		FIG. 2. Sequence alignment of atpE protein sequences from sensitive and resistant M. tuberculosis and from M. xenopi, M. novocastrense, and M. shimoidei. *, corresponding residue in resistant M. smegmatis is mutated to V; {blacktriangledown}, resistance-causing mutation in M. tuberculosis (corresponding residue is A in all sensitive mycobacteria); WT, wild type; R, resistant.

As for M. xenopi (8), the alanine at position 63 is replaced by a methionine in M. shimoidei and M. novocastrense. In all other known atpE sequences of sensitive mycobacteria, this alanine is conserved (data not shown). Interestingly, in one set of in vitro-generated resistant M. tuberculosis mutants, the alanine at this same position is replaced by a helix-breaking proline (1, 8).

Based on structural studies of M. tuberculosis ATP synthase, R207910 is thought to have a binding pocket around amino acid residue 61, a residue known to be extremely well conserved in atpE proteins of many organisms and believed to be instrumental for the proton transport that drives ATP synthesis (2). As already suggested, the polymorphism giving a bulkier methionine at residue 63 might indeed interfere with R207910's access to its target at residue 61, thus giving these species natural resistance to R207910 (2, 8). This is again supported by the occurrence of mutations in residue 63 among in vitro-generated M. tuberculosis mutants.

Our study demonstrates that R207910 is endowed with a strong antimycobacterial spectrum that includes susceptible and resistant TB, MAC, and a wide range of other NTM isolates. The mycobacterial spectrum of R207910, as we know to date, is in full agreement with the polymorphisms seen in the atpE gene coding for the c protein of ATP synthase. The broad antimycobacterial spectrum of R207910 makes it an interesting drug candidate, not only to treat tuberculosis, but potentially also for other clinically relevant mycobacterial infections.


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ACKNOWLEDGMENTS
 
This work has been supported by Johnson & Johnson Pharmaceutical Research and Development.


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FOOTNOTES
 
* Corresponding author. Mailing address: Swedish Institute for Infectious Disease Control, Department of Bacteriology, S-171 82 Solna, Sweden. Phone: 46 8 4572473. Fax: 46 8 301797. E-mail: emma.huitric{at}smi.ki.se Back

{triangledown} Published ahead of print on 20 August 2007. Back


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Antimicrobial Agents and Chemotherapy, November 2007, p. 4202-4204, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00181-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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