Neomycin (Molecular Biology)

Neomycin is a polycationic aminoglycoside produced by actinomycetes that was first isolated in 1949 from the soil-dwelling microbe Streptomyces fradiae by Waksman and Lechevalier (1). As isolated, neomycin is a mixture composed primarily of two stereoisomers, neomycin B and C (Fig. 1 ), both of which possess antibacterial activity. In addition, there is a third component, neomycin A, that is generated by the hydrolysis of neomycins B and C. There are a number of other antibiotics which fall into the same class as neomycin, including streptomycin, gentamycin, kanamycin, tobramycin, and netilmicin.

Figure 1. Neomycins A, B, and C. The structure of neomycin B is shown in full. Neomycin C is the same, except that a second neosamine C moiety replaces the neosamine B. Neomycin A is produced by hydrolysis of neomycins B and C and consists of just the neosamine C and deoxystreptamine units.

Neomycins A, B, and C. The structure of neomycin B is shown in full. Neomycin C is the same, except that a second neosamine C moiety replaces the neosamine B. Neomycin A is produced by hydrolysis of neomycins B and C and consists of just the neosamine C and deoxystreptamine units.

1. Clinical Uses

Neomycin has been used as both an oral and a topical antibiotic and is effective against most clinically important aerobic gram-negative bacteria. It has, however, only minor effects on Streptococci and gram-positive Bacilli. It was used originally to treat tuberculosis. This treatment was discontinued when it was realized that there were serious side effects (discussed below). Neomycin has also been given in combination with niacin to decrease serum cholesterol and lipoprotein A concentrations (2). Again, side effects have prevented it from being used clinically as a cholesterol-lowering agent. Neomycin sulfate is, however, widely used both as an over-the-counter topical agent for cuts and wounds and microbial skin infections and as a preparative treatment in bowel surgery.

2. Mechanisms of Toxicity

The side effects of neomycin administration in humans have included both nephrotoxicity and ototoxicity (involving the auditory and vestibular functions of the eighth cranial nerve). Neomycin has been found to inhibit both the renal and cochlear forms of ornithine decarboxylase, enzymes important for polyamine biosynthesis (3). Use of neomycin as a cholesterol-lowering drug was discontinued due to its side effects of ototoxicity and diarrhea (2). Neomycin also induces acute neuromuscular blockade, perhaps by displacing bound calcium and inhibiting the prejunctional release of the neurotransmitter acetylcholine (4).

3. Neoresistant Gene and Use in Molecular Biology

Although limited in its clinical usefulness, neomycin has in recent years become a major tool in molecular biology. Neomycin inhibits the initiation of protein biosynthesis by binding to the 30S ribosome, thereby blocking translation . The real usefulness of neomycin as a tool in molecular biology came with the discovery of the mammalian antibiotic resistance gene (NeoR) (5). This gene codes for an enzyme, aminoglycoside phosphotransferase, that catalyzes the phosphorylation of neomycin, gentamycin, and kanamycin, which inactivates them as inhibitors of protein synthesis. Expression of the NeoR gene, therefore, blocks the toxic effects of the aminoglycosides. Growth of transfected cells in the presence of neomycin allows for selection of cells that are expressing NeoR coupled to other genes of interest. NeoR and other drug-resistant genes are being considered for use in gene therapy to protect patients from the hematological side effects of chemotherapy (6). This new approach involves the use of a chimeric vector that fuses the antibiotic resistance genes with the genes of therapeutic value.

4. Interaction with Ribosomes and Protein Synthesis

Aminoglycosides such as neomycin B are bactericidal in that they prevent translation of messenger RNA by prokaryotic ribosomes. RNA molecules that recognize neomycin share a common stem-loop structural motif (7) (see RNA Structure). Recent studies have suggested that aminoglycosides inhibit protein synthesis by direct interaction with the ribosomal RNA (8). The nuclear magnetic resonance (NMR) structure of paromycin (a member of the neomycin family) bound to the A site of E. coli 16S rRNA has been analyzed. Aminoglycosides such as neomycin bind to the A site of rRNA (9), which is the entry site for aminoacyl tRNAs. Binding of the aminoglycoside interferes with protein synthesis by causing codon misreading (10). The translational block may arise from stabilization of a unique high-affinity conformation of rRNA in the tRNA-mRNA complex, thereby perturbing the accuracy of protein synthesis (11).

5. Interaction with Lipids

Neomycin also appears to inhibit the phosphatidyl inositol (PI) cycle (12, 13). Platelet-Derived Growth Factor (14), thrombin (12), and a number of other molecules that bind to certain surface receptors mediate their cellular effects through activation of phospholipase C (PLC), leading to the cleavage of phosphatidylinositol 4,5-bis-phosphate (PIP2). Neomycin B indirectly inhibits the activity of PLC. PLC normally cleaves PIP2 to generate 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG), which are involved in activation of the IP3 receptor and protein kinase C (PKC), respectively. Neomycin binds to PIP2 and blocks the cleavage of PIP2 by PLC. Because PIP2 is a cofactor in the activation of phospholipase D, neomycin also inhibits its activity and the subsequent production of phosphatidic acid (15).

6. Ion Channels and Calcium

Neomycin has been shown to inhibit a number of ion channels, including surface membrane Ca channels (16, 17), two intracellular Ca release channels, the ryanodine receptor (RyR) (18, 19), and the IP3 receptor (20). K+ channels, such as the ATP-sensitive K+ channel (21) and the Ca2+- activated maxi-K+ channel (22), also appear to be inhibited by neomycin.

The specific interaction of neomycin with an ion channel has probably been most thoroughly studied with the skeletal muscle Ca -release channel, also known as RYR1. Neomycin has been suggested to inhibit the Ca -release channel by enhancing its rate of inactivation (23), and it appears to bind to a location in the carboxy-terminal 20% of the molecule (19). There may actually be multiple binding sites for neomycin on RYR1 (18). Neomycin has been used as a tool to transfer a fluorescent label to its binding site on RYR1 to determine whether this region of the protein is involved in the conformational changes induced by various channel activators (24).

A specific binding motif for neomycin on the target proteins has not yet been identified. Neomycin may be binding to the binding sites for either Ca or Mg -ATP (25) on its target proteins. The identities of the actual binding sites and the specificities of these sites are unknown. Nevertheless, neomycin is thought to mediate its effect through direct interaction with ion channels and continues to be used as a tool to study ion channel properties.

7. Summary

Although of limited clinical value, neomycin has been an extremely valuable tool for basic science research. Its use as an inhibitor of ion channels and of the PI cycle has helped to elucidate the molecular events involved in these processes. The discovery of the antibiotic resistant genes has led to the widespread usage of neomycin and other aminoglycosides to select cells containing exogenous genes. The success of the neomycin selection technique in molecular biology and in the generation of transgenic mice has led to the possibility of using this approach in gene therapy (6).

Next post:

Previous post: