Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects

Abstract

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUGexp). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUGexp binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1–(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUGexp RNA in nuclear foci, owing to reduced turnover of the CUGexp transcript. By comparison, the monomer did not induce CUGexp accumulation in cells and was more effective at rescuing a CUGexp-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

“…Only a few small molecules of natural origin have been described, such as neomycin B (21) and lomofungin (27). To our knowledge, our study is the first to focus on small molecules of natural origin and represents the first screening of natural extracts in DM1 drug discovery.…”

“…A variety of small molecules have been described that inhibit the (CUG) n -MBNL1 complex and improve DM1-associated molecular defects in vitro and in some cases also in vivo. Several approaches were successful in identifying small molecules, such as screening of known nucleic acid binders (21), rational design of small molecules based on the structure of (CUG) n RNA (22), rational design of oligomers of (CUG) n RNA binders by modular assembly (23,24), combinatorial chemistry (25,26), and high-throughput screening (27,28). …”

Redoxal, an inhibitor of de novo pyrimidine biosynthesis, augments APOBEC3G antiviral activity against human immunodeficiency virus type 1

“…Lomofungin, a natural product compound first isolated from the soil-dwelling Gram-positive bacteria Streptomyces lomodensis , was previously shown to inhibit RNA synthesis in yeast, fungi, and Gram-positive and Gram-negative bacteria (Cano et al, 1973; Johnson and Dietz, 1969; Klo et al, 1973). Lomofungin was subsequently identified as an inhibitor of botulinum neurotoxin light chain protease (Eubanks et al, 2010) and as a potential therapeutic for myotonic dystrophy type 1 (Hoskins et al, 2014a). Redoxal is a synthetic compound originally identified by a computer algorithm as an inhibitor of dihydroorotate dehydrogenase (DHODH), an essential enzyme in the de novo pyrimidine synthesis pathway (Fig.…”

“…Lomofungin is a natural product originally isolated from bacteria. While the specific cellular targets of lomofungin have not been identified, its inhibition of RNA synthesis is well characterized (Cano et al, 1973; Johnson and Dietz, 1969; Klo et al, 1973) and it has also been shown to inhibit the botulinum neurotoxin light chain protease (Eubanks et al, 2010) and was identified as a potential therapeutic for myotonic dystrophy type 1 (Hoskins et al, 2014b). However, its therapeutic potential is limited by cytotoxicity, demonstrated in the present study as well as prior studies.…”

RNA Structures as Mediators of Neurological Diseases and as Drug Targets

“…Finally, phase I and II clinical trials of a gapmer targeting mutant DMPK mRNA has been recently initiated (Isis Pharmaceuticals, 2014). A number of small molecule probes have also been developed for targeting r(CUG) exp that displace MBNL1 and improve downstream defects (Arambula et al, 2009; Childs-Disney et al, 2012a, 2012b, 2013; Hoskins et al, 2014; Jahromi et al, 2013a, 2013b; Parkesh et al, 2012). These compounds were either identified from screening, designed from the structure of r(CUG) repeats, or designed from privileged RNA motif-small molecule interactions including modularly assembled compounds thereof.…”