Structure–Activity Relationship of Flavin Analogues That Target the Flavin Mononucleotide Riboswitch

Abstract

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.

Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

“…The flavin mononucleotide (FMN) riboswitch is notable because it senses the cofactor FMN as well as the natural product roseoflavin, which acts as an antibacterial (23,127). This vulnerability has fostered efforts to target the JBC REVIEWS: Molecular recognition of HIV TAR RNA FMN riboswitch with novel antibiotics (19,21,22,25,26). Recent structure-guided design led to the discovery of compound BRX1555-an FMN analogue that binds with a K D of 39.0 Ϯ 0.7 nM based on in-line probing (Fig.…”

“…Recent structure-guided design led to the discovery of compound BRX1555-an FMN analogue that binds with a K D of 39.0 Ϯ 0.7 nM based on in-line probing (Fig. 4C) (25). The ligand has an in vitro EC 50 of 1.70 Ϯ 0.18 M in single-turnover transcription termination assays and an IC 50 of 0.49 Ϯ 0.09 M in bacterial growth inhibition assays (25).…”

“…4C) (25). The ligand has an in vitro EC 50 of 1.70 Ϯ 0.18 M in single-turnover transcription termination assays and an IC 50 of 0.49 Ϯ 0.09 M in bacterial growth inhibition assays (25).…”

“…These properties are suited for shape-specific recognition of small molecules or peptides and provide a basis to manipulate conformation or dynamics to alter downstream function. Several notable achievements accentuate such efforts, including the identification of inhibitors that target the following: cancerassociated miR-21; CUG repeats of myotonic dystrophy; riboswitches in pathogenic bacteria; and exon splicing in spinal muscular atrophy (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). These successes underscore the feasibility of sequence-specific targeting of RNAs to create research tools or as a means to treat human disease.…”

Advances that facilitate the study of large RNA structure and dynamics by nuclear magnetic resonance spectroscopy

“…Numerous studies have highlighted how complex structural folds in RNA molecules confer specific functions including riboswitches (Knappenberger, Reiss, & Strobel, ; Liberman et al, ; Peselis & Serganov, ; Vicens et al, ; B. Zhao, Guffy, Williams, & Zhang, ; also reviewed by Jones & Ferre‐D'Amare, ; Roth & Breaker, ), ribozymes (Chan et al, ; Costa, Walbott, Monachello, Westhof, & Michel, ; Meyer et al, ; L. A. Nguyen, Wang, & Steitz, ; Qu et al, ; Suslov et al, ; C. Zhao, Rajashankar, Marcia, & Pyle, ; also reviewed by Pyle, ; Ren, Micura, & Patel, ), and viral elements (Akiyama et al, ; Au et al, ; Imai, Kumar, Hellen, D'Souza, & Wagner, ; Keane et al, ). Additionally, recent studies have identified highly structured ncRNAs as important regulators in various cellular processes and linked misregulation of ncRNAs to important human diseases (Esteller, ).…”