Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents

Abstract

DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

Fig 1

Computational docking of a pyrrole NMR fragment hit overlaid with ATP in the crystal structure of the S. aureus GyrB 24-kDa N-terminal domain. Hydrogen bonds between the adenine of ATP, Asp81, and a water molecule (red sphere) are shown with dotted lines. Carbons of the pyrrole NMR hit are colored green, while those of ATP are colored gray. Other atoms are shown in standard coloring. The GyrB protein is colored yellow and displayed as a ribbon structure, with the exception of key amino acid side chains labeled by sequence number.

Fig 2

Design of a compound library based on a pyrrole NMR hit. Me, methyl-; Et, ethyl-; iPr, isoproyl.

Fig 3

Chemical structures of representative pyrrolamides with identification numbers used in the text.

Fig 4

X-ray crystal structure of the S. aureus GyrB 24-kDa N-terminal domain in complex with the initial lead, pyrrolamide 1 (A), and a later series representative, pyrrolamide 2 (B). Carbons of the pyrrolamide inhibitors are colored green, while the other atoms are shown in standard coloring. The GyrB protein is colored yellow and displayed as a ribbon structure, with the exception of key amino acid side chains labeled by sequence number. Hydrogen bonds are shown with dotted lines, and a conserved water molecule is shown as a red sphere.

Fig 5

Efficacy of pyrrolamide 4 in an S. pneumoniae lung infection model in mice. Shown are the numbers of S. pneumoniae CFU recovered from the lungs of mice treated with 0, 80, 160, and 320 mg/kg of pyrrolamide 4 administered orally twice per day, every 12 h. The doses shown are the total daily dose. The error bars represent standard errors in the CFU measurements.