Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance.
As increasing numbers of bacteria become resistant to antibiotics, new drugs are needed to fight bacterial infections. To develop new antibacterial drugs, researchers need to understand how existing antibiotics work. There are many ways to kill bacteria, but one of the most effective is to target an enzyme called bacterial RNA polymerase. If bacterial RNA polymerase is prevented from working, bacteria cannot synthesize RNA and cannot survive.
GE23077 (GE for short) is an antibiotic produced by bacteria found in soil. Although GE stops bacterial RNA polymerase from working, and thereby kills bacteria, it does not affect mammalian RNA polymerases, and so does not kill mammalian cells. Understanding how GE works could help with the development of new antibacterial drugs.
Zhang et al. present results gathered from a range of techniques to show how GE inhibits bacterial RNA polymerase. These show that GE works by binding to a site on RNA polymerase that is different from the binding sites of previously characterized antibacterial drugs. The mechanism used to inhibit the RNA polymerase is also different.
The newly identified binding site has several features that make it an unusually attractive target for development of antibacterial compounds. Bacteria can become resistant to an antibiotic if genetic mutations lead to changes in the site the antibiotic binds to. However, the site that GE binds to on RNA polymerase is essential for RNA polymerase to function and so cannot readily be changed without crippling the enzyme. Therefore, this type of antibiotic resistance is less likely to develop.
In addition, the newly identified binding site for GE on RNA polymerase is located next to the binding site for a current antibacterial drug, rifampin. Zhang et al. therefore linked GE and rifampin to form a two-part (‘bipartite’) compound designed to bind simultaneously to the GE and the rifampin binding sites. This compound was able to inhibit drug-resistant RNA polymerases tens to thousands of times more potently than GE or rifampin alone.