Blocking Bacterial Naphthohydroquinone Oxidation and ADP-Ribosylation Improves Activity of Rifamycins against Mycobacterium abscessus

Abstract

Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat M. abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti- M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against M. tuberculosis . Here, we asked i) why is rifabutin more active against M. abscessus than rifampicin, and ii) why is rifabutin’s anti- M. abscessus activity poorer than its anti-TB activity. Comparative analysis of naphthoquinone versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase Arr Mab like rifampicin, preventing it from achieving the nanomolar activity it displays against M. tuberculosis . Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule’s naphthoquinone core plus a modification that blocks ADP-ribosylation at its C23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively-resistant naphthoquinone core and C25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.

In vitro activity of rifabutin against Mycobacterium abscessus, clinical isolates

“…18,27,28 On the other hand, rifabutin has been shown to contain a less readily oxidisable naphthoquinone core, while other rifamycins like rifampicin contain a naphthohydroquinone core that is highly susceptible to oxidisation in M. abscessus. 29 Patients infected with the rough M. abscessus usually exhibit worse clinical characteristics and are at a higher risk of exacerbated lung disease. 30 Such patients may be in critical need of timely and effective clinical intervention.…”

“…One possible explanation is that M. abscessus expresses an ADP‐ribosyltransferase which inactivates rifamycins, with a much lower affinity for rifabutin 18,27,28 . On the other hand, rifabutin has been shown to contain a less readily oxidisable naphthoquinone core, while other rifamycins like rifampicin contain a naphthohydroquinone core that is highly susceptible to oxidisation in M. abscessus 29 …”

Drug development challenges in nontuberculous mycobacterial lung disease: TB to the rescue

“…Optimization of these approved drug classes to increase bactericidal activity and circumvent intrinsic or acquired resistance is an underexploited low-hanging fruit strategy with fast-track potential. The rational design and synthesis of rifabutin analogs that overcome intrabacterial metabolism has generated molecules with 50- to 100-fold increased potency against M. abscessus ( Ganapathy et al, 2021b ; Lan et al, 2022 ).…”

Why Matter Matters: Fast-Tracking Mycobacterium abscessus Drug Discovery

“…Drug repurposing has also been proposed to quickly bolster treatment options for patients [ 19 , 20 , 21 ]. Drug repositioning could restore functionality to drug classes inactivated by M. abscessus intrinsic drug resistance mechanisms [ 22 , 23 ]. In addition to these efforts, de novo M. abscessus drug discovery should be pursued.…”