Negamycin

Nonsense mutations caused by the presence of an in-frame premature termination codon (PTC) account for ~10% of gene lesions that together cause over 1800 inherited human diseases. One approach to treating genetic diseases that stem from PTCs is selective promotion of translational readthrough in a PTC using 'readthrough compounds' that can lead to partial restoration of full-length functional protein expression. (+)-Negamycin, a natural dipeptide-like antibiotic, may restore some dystrophin expression in the skeletal muscles of mice with Duchenne muscular dystrophy, and this compound has been recognized as a potential therapeutic agent for diseases caused by nonsense mutations. In an effort to develop new candidate molecules with improved activities, we established the efficient total synthesis in eight steps of (+)-negamycin using both achiral and chiral starting material. These routes provided a deamino derivative with in vivo readthrough activity with potential for long-term treatment. In a separate approach, we discovered two natural negamycin analogs, 3-epi-deoxynegamycin and its leucine derivative, which are potent readthrough compounds effective against nonsense mutations of eukaryotes but not prokaryotes. These compounds fail to display antimicrobial activity. More potent derivatives, whose structure is derived from 3-epi-deoxynegamycin, were identified and their chemistry is discussed in this review.

Negamycin is a natural pseudodipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of the proton-dependent oligopeptide transporter DtpD. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions points to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multifaceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

We report a novel negamycin derivative TCP-1109 (13x) which serves as a potent readthrough drug candidate against nonsense-associated diseases. We previously demonstrated that TCP-112 (7), a nor-compound of native 3-epi-deoxynegmaycin, showed a higher readthrough activity than (+)-negamycin. In the present study, we performed a structure-activity relationship (SAR) study of compound 7 focused on its 3-amino group in an effort to develop a more potent readthrough compound. Introduction of a variety of natural or unnatural amino acids to the 3-amino group gave us the more potent derivative 13x which has about four times higher readthrough activity than 7 in a cell-based assay using a premature termination codon of TGA derived from Duchenne muscular dystrophy. The activity was dose-dependent and relatively selective for TGA. However, the activities for TAG and TAA were also higher than those of (+)-negamycin and 7. Moreover, compound 13x showed significant cell-based readthrough activity for several nonsense mutations derived from other nonsense-associated diseases. It is suggested that 13x has the potential to be a readthrough drug useful for the treatment of many kinds of nonsense-associated diseases.