Impact of fgd1 and ddn Diversity in Mycobacterium tuberculosis Complex on In Vitro Susceptibility to PA-824

Abstract

PA-824 is a promising drug candidate for the treatment of tuberculosis (TB). It is in phase II clinical trials as part of the first newly designed regimen containing multiple novel antituberculosis drugs (PA-824 in combination with moxifloxacin and pyrazinamide). However, given that the genes involved in resistance against PA-824 are not fully conserved in theMycobacterium tuberculosiscomplex (MTBC), this regimen might not be equally effective against different MTBC genotypes. To investigate this question, we sequenced two PA-824 resistance genes (fgd1[Rv0407] andddn[Rv3547]) in 65 MTBC strains representing major phylogenetic lineages. The MICs of representative strains were determined using the modified proportion method in the Bactec MGIT 960 system. Our analysis revealed single-nucleotide polymorphisms in both genes that were specific either for several genotypes or for individual strains, yet none of these mutations significantly affected the PA-824 MICs (≤0.25 μg/ml). These results were supported byin silicomodeling of the mutations identified in Fgd1. In contrast, “Mycobacterium canettii” strains displayed a higher MIC of 8 μg/ml. In conclusion, we found a large genetic diversity in PA-824 resistance genes that did not lead to elevated PA-824 MICs. In contrast,M. canettiistrains had MICs that were above the plasma concentrations of PA-824 documented so far in clinical trials. AsM. canettiiis also intrinsically resistant against pyrazinamide, new regimens containing PA-824 and pyrazinamide might not be effective in treatingM. canettiiinfections. This finding has implications for the design of multiple ongoing clinical trials.

Mutations in Genes for the F 420 Biosynthetic Pathway and a Nitroreductase Enzyme Are the Primary Resistance Determinants in Spontaneous In Vitro -Selected PA-824-Resistant Mutants of Mycobacterium tuberculosis

“…The orthologs Rv1261c and Rv1558 may be functional analogs of Ddn (6), while other enzymes (Rv1155 and Rv2991) have been found to be structural analogs of FGD1 (3). In a pool of 65 PA-824-susceptible clinical isolates, Feuerriegel et al (35) identified five unique phylogenetic or neutral SNPs in fgd1. Accordingly, the FGD1 model elucidated in this study shows that these phylogenetic SNPs are dispersed outside the F 420 -binding site and are not found in the M. tuberculosis H37Rv resistant mutants selected here (Fig.…”

Determination of MIC Distribution and Epidemiological Cutoff Values for Bedaquiline and Delamanid in Mycobacterium tuberculosis Using the MGIT 960 System Equipped with TB eXiST

“…Similarly to the related drug PA-824, delamanid is a prodrug requiring activation by the mycobacterial F420 system, including the nitroreductase Ddn (Rv3547) (8)(9)(10). Delamanid resistance is thought to arise from mutations in the mycobacterial F420 genes (ddn, fgd1, fbiA, fbiB, and fbiC) associated with the prodrug's activation (8,11). The spontaneous rate of delamanid resistance has been reported to be as high as 6.44 ϫ 10 Ϫ6 to 4.19 ϫ 10 Ϫ5 , emphasizing the need to protect delamanid with other active anti-TB drugs during therapy (9).…”

Novel drugs against tuberculosis: a clinician's perspective

“…Mutations have been identified in fgd1 and ddn [57,62,63], with cross-resistance to 5-nitrothiophenes (a tuberculostatic compound) [64] as well as delamanid [53]. With minimal inhibitory concentration (MIC) around 0.15-0.3 μg·mL −1 [58,63], the activity of PA-824 is comparable to a combination of isoniazid and rifampicin in the continuation phase of a murine drug model [61].…”