Redesign of Rifamycin Antibiotics to Overcome ADP-Ribosylation-Mediated Resistance

Abstract

Rifamycin antibiotics are a valuable class of antimicrobials for treating infections by mycobacteria and other persistent bacteria owing to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is seriously compromised by a novel resistance mechanism, namely, rifamycin inactivation by ADP-ribosylation. Using a structure-based approach, we rationally redesign rifamycins through strategic modification of the ansa-chain to block ADP-ribosylation while preserving on-target activity. Validated by a combination of biochemical, structural, and microbiological studies, the most potent analogs overcome ADP-ribosylation, restored their intrinsic low nanomolar activity and demonstrated significant in vivo antibacterial efficacy. Further optimization by tuning drug disposition properties afforded a preclinical candidate with remarkable potency and an outstanding pharmacokinetic profile.

Keywords: Antibiotic Resistance; Antibiotics; Drug Design; Mycobacterium Abscessus; Rifamycin.

Figure 1

Rifabutin is inactivated in M. abscessus. Arr of M. abscessus catalyzes the formation of ADP‐ribosyl‐oxocarbenium intermediate from NAD⁺ and the consecutive ADP‐ribosylation on C23‐OH.

Figure 2

Homology‐model‐guided rifamycin design depicts C‐25 modification blocking interaction with Arr _Mab . A) The structure of the archetypical compound 25‐O‐benzoyl rifabutin (5 a), where the C25 acetyl group of rifabutin is replaced by a benzoyl group. B) The overall structure of Arr _Mab homology model complexed with the ADP‐ribosyl‐oxocarbenium intermediate (blue) and 5 a (cyan). C) Proposed binding modes of rifabutin (magenta) and 5 a (cyan) to the Arr _Mab homology model. The distance between C23‐OH and oxocarbenium‐C1′ is measured and shown as a purple dashed line.

Scheme 1

Synthesis of 25‐O‐acyl rifabutin analogs: a) 2,2‐dimethoxypropane, CSA, acetone, room temperature, 2 h, 68 %; b) K₂CO₃, MeOH, 50 °C, 48 h, 59 %; c) RC(O)OC(O)R, DMAP, 1,2‐dichloroethane, room temperature or 50 °C, 72–96 h; d) RCOOH, pivaloyl chloride, triethylamine, DMAP, DCM, 0 °C to room temperature, 4 h; e) CSA, MeOH, room temperature, 0.5 h; f) NaOH, ZnCl₂, MeOH, room temperature, overnight, 71 %. For 5 a–5 h and 5 j–5 o, the yields were 7–72 % over two steps (c or d, and then e); For 5 i, the yield was 6 % with procedure c from 25‐O‐desacetyl rifabutin 6. CSA=camphorsulfonic acid.

Figure 3

A) The active site structure of 5 a (orange) bound to RNAP _Mtb . Residues involved in important hydrogen bonds are shown as cyan sticks. H‐bonds are shown as purple dashed lines. B) C‐25 benzoate (C25‐OBz) of 5 a (orange) and F439 (cyan) form a π‐stacking interaction. C) C‐25 benzoate of 5 a (orange) is positioned in a cleft formed by F439 and R173. The cleft is demonstrated in blue.

Figure 4

In vitro characterization of ADP‐ribosylation of rifamycins using overexpressed Arr _Mab . All the peaks were identified by MS (Table S5). The retention time difference of the peaks is labelled. Rifabutin was fully converted into the ADP‐ribosyl adduct upon incubation in a 40‐minute time course. By contrast, no Arr _Mab ‐catalyzed transformation was observed for the synthetic compound 5 a under the same incubation condition.

Figure 5

Analog 5 j exhibited bactericidal in vivo efficacy at 10 mg kg⁻¹. Animals infected with M. abscessus underwent drug treatment for 10 consecutive days. Drugs were administered once daily to groups of 6 mice per study group. At 11 days postinfection, organ homogenates were plated on agar to determine the bacterial load. Results were analyzed using one‐way analysis of variance (ANOVA) multicomparison and Dunnett's posttest. *, P<0.05; **, P<0.01; ***, P<0.001. D1: Day‐1. D11: Day‐11. CLR: clarithromycin. RBT: rifabutin.