Efflux Pump-Binding 4(3-Aminocyclobutyl)Pyrimidin-2-Amines Are Colloidal Aggregators

Biomolecules. 2023 Jun 16;13(6):1000. doi: 10.3390/biom13061000.

Abstract

Efflux pumps are a relevant factor in antimicrobial resistance. In E. coli, the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the bacterium. Therefore, small molecules interfering with efflux pump function are considered adjuvants for improving antimicrobial therapies. Several compounds targeting the periplasmic adapter protein AcrA and the efflux pump AcrB have been identified to act synergistically with different antibiotics. Among those, several 4(3-aminocyclobutyl)pyrimidin-2-amines have been shown to bind to both proteins. In this study, we intended to identify analogs of these substances with improved binding affinity to AcrA using virtual screening followed by experimental validation. While we succeeded in identifying several compounds showing a synergistic effect with erythromycin on E. coli, biophysical studies suggested that 4(3-aminocyclobutyl)pyrimidin-2-amines form colloidal aggregates that do not bind specifically to AcrA. Therefore, these substances are not suited for further development. Our study emphasizes the importance of implementing additional control experiments to identify aggregators among bioactive compounds.

Keywords: AcrA; E. coli; antimicrobial studies; assay interference; colloidal aggregators; dynamic light scattering; efflux pump inhibitor; efflux pumps; microscale thermophoresis; nano-differential scanning fluorimetry; virtual screening.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / metabolism
  • Membrane Transport Proteins* / metabolism
  • Multidrug Resistance-Associated Proteins / metabolism
  • Periplasm / metabolism

Substances

  • Membrane Transport Proteins
  • Escherichia coli Proteins
  • Anti-Bacterial Agents
  • AcrB protein, E coli
  • Multidrug Resistance-Associated Proteins

Grants and funding

This work was supported by the German Federal Ministry for Education and Research (01DQ20007) and the Indian Council of Medical Research (ICMR), New Delhi, India (AMR/INDO/GER/215/2019-ECD-II), as well as under the framework of the JPIAMR—Joint Programming Initiative on Antimicrobial Resistance by the German Federal Ministry for Education and Research (01KI1827A) and the Latvian State Education Development Agency (1.1.1.5/ERANET/19/03).