Doctoral Themes

Design and synthesis of membrane-disrupting sugar-based prodrugs with potential antimicrobial activity against multidrug-resistant Gram negative bacteria

Supervisors

Ana Marta Matos, amamatos@fc.ul.pt

Rodrigo Almeida, rfalmeida@fc.ul.pt

Registration Institution

Faculdade de Ciências, Universidade de Lisboa

Project description

Antimicrobial resistance is one of the most urgent public health concerns of our time, with Gram-negative bacterial strains being the most difficult types of pathogens to target due to the presence of two lipid bilayers in their cell envelope: the inner (IM) and the outer membrane (OM). These membranes are separated by the periplasm, a subcellular compartment containing the peptidoglycan cell wall and enzymes that are responsible for its degradation. Most hydrophobic and large antibiotics are unable to cross the OM, which constitutes a major obstacle for drug uptake in Gram-negative bacteria. There is no widely accepted set of principles for the design of antimicrobial agents with the appropriate physicochemical characteristics to pass through the OM; however, it is consensual that highly polar small molecules are more likely to enter the periplasm via porins. Moreover, positively charged compounds can potentially cause cation displacement, which distabilizes the lipopolysaccharide (LPS) in the OM (Domalaon et al. 2018).

This exploratory project will focus on the design and synthesis of alkyl disaccharide prodrugs with the appropriate structure and physicochemical characteristics to (1) pass through the OM; (2) be cleaved by periplasm enzymatic machinery, namely peptidoglycan glycosidases (PGGs); and (3) to cause bacterial cell lysis by interacting with IM lipids once the pharmacophore is released in the periplasm. To achieve these goals:

a. Analogues of the peptidoglycan MurNAc-GlcNAc repeating unit will be synthesized. An alkyl chain, which is known to interact with phosphatidylethanolamine (PE) when linked to a sugar moiety at alpha configuration (Dias et al. 2018), will be introduced in the reducing end of the disaccharide.

b. Chemical modifications in this lead scaffold will be conducted towards the generation of an extensive compound library, which will allow for the establishment of structure-activity relationships regarding for IM-disrupting activity and the ability to undergo PGG-mediated hydrolysis.

c. For each prodrug, the corresponding pharmacophore will be synthesized as well, to allow for compound screening through leakage assays with intraliposomal carboxyfluorescein, as previously described (Dias et al. 2018).

d. LPS affinity will also be determined for cationic prodrugs via cadaverine (BC) displacement assay (Wood et al. 2004). Other biophysical experiments destined to have a deeper understanding of how drugs and prodrugs interact with the IM and the OM, respectively, will also be conducted.

Compound design and synthesis will take place at the Carbohydrate Chemistry Lab of CQE, under the supervision of Ana Marta de Matos, while membrane interaction assays will be conducted at the Molecular Biophysics Lab of CQE, under supervision of Prof. Rodrigo Almeida. The development and optimization of an assay able to estimate PGG hydrolysis will be carried out at the Department of Organic Chemistry, University of Sevilla (Spain), under the coordination of Prof. Óscar López for a total period of at least 4 months.

With this multidisciplinary project, the PhD student will receive training in the areas of Organic Chemistry, Carbohydrate Chemistry, Medicinal Chemistry, Membrane Biophysics and Enzymology. The best compounds will be tested in multidrug-resistant Gram-negative bacteria for the assessment of porin passage through the OM and minimum inhibitory concentration (MIC) determination, in a collaboration with Dr. Francisco Dionísio from C3EC-FCUL.

References: Dias C, Pais JP, Nunes R, et al. Nat Commun. 2018;9(1):4857. DOI: 10.1038/s41467-018-06488-4. Domalaon R, Idowu T, Zhanel GG, et al. Clin Microbiol Rev. 2018;31(2):e00077-17. DOI: 10.1128/CMR.00077-17. Wood SJ, Miller KA, David SA. Comb Chem High Throughput Screen. 2004;7(3):239-429. DOI: 10.2174/1386207043328832.

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