Assessment of Small Molecule Permeability through Mycomembrane in Living Mycobacteria

Author:
Liu, Zichen, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Advisor:
Pires, Marcos, AS-Chemistry (CHEM), University of Virginia
Abstract:

Current developments of antibiotic resistance in Mycobacterium tuberculosis (Mtb) create a global threat to human health care and an urgent need for the development of novel antimycobacterial agents. Mycobacterium tuberculosis (Mtb) processes unique cell envelope which is considered the first line of intrinsic resistance against small molecule antibiotics, owing to its poor permeability to small molecules. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. Nonetheless, there is limited information on the types of molecular scaffolds that can readily permeate past the mycomembrane of mycobacteria. To address this, we describe a novel assay that combines metabolic tagging of the peptidoglycan scaffold with cyclooctyne moieties as Click chemistry handles, which sits directly beneath the mycomembrane, and a fluorescent labeling chase step, to measure the relative permeation of small molecules by reversely compare the available Click chemistry handles left by the small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in Mycobacterium smegmatis and Mtb. A small panel of molecules was tested initially, and we found a large range in the permeability profile of molecules in larger libraries. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly discovered compounds into mycobacteria. The methods described, which do not require genetic manipulation, can be generally adopted to other species for which envelope permeability is treatment-limiting.

Recently, the discovery of ClpC1P1P2 complex targeting antimycobacterial peptides highlighted the essentiality for the peptides to be cyclic with backbone N-methylation to cross the mycobacterial cell envelope and reach their intracellular targets. While it has been demonstrated beneficial for peptide drugs to be N-methylated to cross the mammalian cell membrane, there is limited information about whether the N-methylation method can help the peptides readily penetrate the mycomembrane. We have further demonstrated the versatility of assay operating in different conditions. A library of backbone N-methyl short peptides was established to systematically assess the effect of N-methylation on peptide permeability through the mycomembrane. Additionally, the permeability and antimycobacterial activity of an antimicrobial peptide were found to be enhanced by N-methylation. We project that the assay provided a platform for better understanding of the permeability of peptide libraries crossing the mycomembrane, facilitating novel antimycobacterial drug discovery.

Besides, we demonstrate that the assay can be adopted in high throughput screening of libraries with hundreds of azido compounds. We believe that this assay can be easily applied to screening libraries of thousands of compounds. The common structural features in the compounds with relatively higher permeability can be used to facilitate the development of antimycobacterial therapeutics or potentiate existing antimycobacterial drugs. However, the main drawback of the assay screening thousands of molecules lies in the unevenness of the reactivity for the different small molecules to have Click chemistry with the embedded strained alkyne handles on the peptidoglycan. Strain-promoted azide-alkyne cycloaddition reaction had been widely used in a wide range of fields. In chemical biology, azide compounds are used as chemical reporters in biomolecules to track their translocation, function and interactions with each other in their native environment. Due to the relatively low concentration of these labeled molecules in biological systems, efforts have been put in the development of the cyclooctyne counterpart with higher reactivity and at the same time maintaining their stability. However, the reactivity profiles for the azido compounds are limited. While a general trend of lower reactivity associates with aryl azides reacting with benzoannulated cyclooctynes, their kinetics are boosted sometimes with aliphatic cyclooctyne bicyclo[6.1.0]non-4-yne, BCN. Besides, even within the aliphatic azides, the tertiary azides suffer from steric hindrance reacting with benzoannulated cyclooctynes. We developed an approach to screen the relative reactivity profiles of hundreds of azido compounds in a high throughput manner, on both dibenzocyclooctyne and BCN, the two most used cyclooctynes in chemical biology field.

Degree:
PHD (Doctor of Philosophy)
Keywords:
Mycobacteria, Membrane Permeability , Click Chemistry
Language:
English
Issued Date:
2024/07/31