ABSTRACT
The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral ß-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Because of its essential function in many Gram-negative bacteria, the LPS transport pathway is an interesting target for the development of new antibiotics. A family of macrocyclic peptidomimetics was discovered recently that target LptD and inhibit LPS transport specifically in Pseudomonas spp. The related molecule Murepavadin is in clinical development for the treatment of life-threatening infections caused by P. aeruginosa. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we characterized the binding site by cross-linking to a photolabeling probe. We used a hypothesis-free mass spectrometry-based proteomic approach to provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a ß-jellyroll domain and an N-terminal insert domain characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacterial Outer Membrane Proteins/metabolism , Peptidomimetics/metabolism , Pseudomonas aeruginosa/chemistry , Bacterial Outer Membrane Proteins/chemistry , Binding Sites , Gram-Negative Bacteria/drug effects , Lipopolysaccharides/metabolism , Periplasm , Protein Domains , Protein TransportABSTRACT
Manufacturing highly potent antibody-drug conjugates (ADCs) is a demanding task-combining conventional organic synthesis with biotechnological manufacturing. Hence a series of new and unique engineering and chemistry challenges have to be addressed to support clinical trials and commercial manufacturing. These include the development of reliable processes leading to uniform product properties, as well as establishment of ADC-specific analytical methods and safe strategies for handling cytotoxic compounds. This review focuses on process development and scale-up for the production of ADCs and highlights the most important features in such a process.
Subject(s)
Biotechnology/methods , Immunoconjugates/chemistry , Immunoconjugates/isolation & purification , Biotechnology/economics , Biotechnology/instrumentation , HumansABSTRACT
Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.
