Mechanistic and Structural insights of interaction of beta-lactam inhibitors of multiple Ldts.
Lead Research Organisation:
UNIVERSITY OF OXFORD
Department Name: Interdisciplinary Bioscience DTP
Abstract
L,D-Transpeptidases are sporadically distributed in bacteria, and where present, mostly possess non-essential functions in peptidoglycan biosynthesis. Exceptions include Mycobacterium tuberculosis, M. abscessus and Clostridium difficile, where L,Dtranspeptidases have essential roles in peptidoglycan formation. The essential L,D-transpeptidase LdtMt2 in M. tuberculosis, which forms 3-3 cross-links in peptidoglycan, has been established as a promising target in developing TB
therapies in both mouse model and patient treatment. Faropenem, reportedly the strongest inhibitor of LdtMt2, fragments upon reaction with an active site cysteine residue to form a stable acyl-enzyme complex. Recently, we observed gradual fragmentation of ampicillin and oxacillin via scission of C5-C6 bond, following acylation of LdtMt2. This mechanism is not typical for the nucleophilic serine D,Dtranspeptidases. The S70C variant of the serine beta lactamase OXA-48 was also found to catalyse the analogous fragmentation of ampicillin and ticarcillin, which was not observed for wild-type OXA-48. We believe these differences originate from the different properties of the corresponding thioester and ester acyl-enzyme complex intermediates. We aim to investigate these fragmentations via mass spectrometry, NMR and X-ray crystallographic studies to probe the mechanistic differences of Cys and Ser nucleophiles and the associated inhibitor fragmentation. These data may allow for the design of new inhibitors that are liable to fragment and form a stable acyl-enzyme complex with LdtMt2.
therapies in both mouse model and patient treatment. Faropenem, reportedly the strongest inhibitor of LdtMt2, fragments upon reaction with an active site cysteine residue to form a stable acyl-enzyme complex. Recently, we observed gradual fragmentation of ampicillin and oxacillin via scission of C5-C6 bond, following acylation of LdtMt2. This mechanism is not typical for the nucleophilic serine D,Dtranspeptidases. The S70C variant of the serine beta lactamase OXA-48 was also found to catalyse the analogous fragmentation of ampicillin and ticarcillin, which was not observed for wild-type OXA-48. We believe these differences originate from the different properties of the corresponding thioester and ester acyl-enzyme complex intermediates. We aim to investigate these fragmentations via mass spectrometry, NMR and X-ray crystallographic studies to probe the mechanistic differences of Cys and Ser nucleophiles and the associated inhibitor fragmentation. These data may allow for the design of new inhibitors that are liable to fragment and form a stable acyl-enzyme complex with LdtMt2.
Publications
Chan HTH
(2021)
Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding.
in Chemical science
De Munnik M
(2019)
Targeting the Mycobacterium tuberculosis transpeptidase LdtMt2 with cysteine-reactive inhibitors including ebselen.
in Chemical communications (Cambridge, England)
El-Baba TJ
(2020)
Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.
in Angewandte Chemie (International ed. in English)
Lohans C
(2019)
Non-Hydrolytic ß-Lactam Antibiotic Fragmentation by l,d -Transpeptidases and Serine ß-Lactamase Cysteine Variants
in Angewandte Chemie International Edition
Lucic A
(2021)
Faropenem reacts with serine and metallo-ß-lactamases to give multiple products.
in European journal of medicinal chemistry
Malla TR
(2021)
Mass spectrometry reveals potential of ß-lactams as SARS-CoV-2 Mpro inhibitors.
in Chemical communications (Cambridge, England)
Redhead MA
(2021)
Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19.
in Scientific reports
Thun-Hohenstein STD
(2022)
Structure-Activity Studies Reveal Scope for Optimisation of Ebselen-Type Inhibition of SARS-CoV-2 Main Protease.
in ChemMedChem
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011224/1 | 30/09/2015 | 31/03/2024 | |||
| 1945345 | Studentship | BB/M011224/1 | 30/09/2017 | 30/03/2022 | Tika Malla |
| NE/W502728/1 | 31/03/2021 | 30/03/2022 | |||
| 1945345 | Studentship | NE/W502728/1 | 30/09/2017 | 30/03/2022 | Tika Malla |
| Description | 1) I am working with penicillin, one of the most commonly prescribed antibiotics. I have unearthed two new mechanisms by which bacteria could develop resistance against it. Not only does not this explain why penicillin are not good inhibitors for mycobaterium bacteria but also allows us to postulate how other bacteria can acquire resistance against penicillin by adapting similar mechanisms. Discovering such mechanisms help us design better inhibitors to combat this future resistance mechanism with time largely on our side. 2) Structural studies on how the target protein for tuberculosis protein interacts with its natural substarte inc ell. Limited progress has been obtained as the unatural peptides have very low yield on synthesis and substrate harvested from bacterial cells directly remain too impure to use for crystallography studies. 3) Our collalorations with different group has also enabled us to suggest medicinal plants could potentially act as a supplement during treatment against tuberculosis. The work remains still active. |
| Exploitation Route | Fictionalization of some compounds can be used for other proteins that share the active site residues cysteines and tested for inhibitory potential or as probes for functional studies. |
| Sectors | Agriculture Food and Drink Chemicals Pharmaceuticals and Medical Biotechnology |
| Description | Garlic extracts for probing active molecules for TB treatment |
| Organisation | University of Ghana |
| Country | Ghana |
| Sector | Academic/University |
| PI Contribution | For centuries, garlic has been cited as a medicinal plant in scriptures and passed down the generation as a plant with healing properties. Dr. Dacquah from University of Ghana had previously identified garlic extracts and some isolated compounds from it had antimicrobial activity against Mycobacterium cells, the causative agent of tuberculosis. At a time when bacteria are predicted to completely grow resistance to antibiotics in the next 40 years, search for new antibiotics has deemed to be absolutely crucial. We formed a collaboration where we tested inhibitory potential of garlic and onion extracts against L,D-transpeptidases, the essential enzymes that crosslink the cell wall in the peptidoglycan layer of mycobacterium cell. Based on this finding new disulphide based compounds were synthesized and are currently being studies structurally. We have obtained a 2.7 Angstorm resolution of crystal structure with the compound in the active site. As disulphides are less specific, these compounds are used as a starting material for designing new cysteine probes. |
| Collaborator Contribution | Our partner, Dr. Dacquah identified and prepared the garlic and onion extracts from eight different varities and travelled to the UK with it. We optimized an existing assay to test the inihibitory potency and performed Mass Spec analysis with the compounds. |
| Impact | 1) Small molecules that selectively target cysteine residues 2)Design of new probes for cysteines 2) Food based treatment against tuberculosis The project is multidisciplinary in nature as it undertakes research on ancient medicine from anthropological sources, conducting biological studies with these plant extracts and bacteria and eventually using synthetic chemistry to design molecules that are widely prevalent in these extracts. |
| Start Year | 2019 |
| Description | Macrocyclic inhibitors of Mpro |
| Organisation | University of Tokyo |
| Country | Japan |
| Sector | Academic/University |
| PI Contribution | I screened the peptidic inhibitors and suggested the modification of the original peptide which led to the development of more potent Mpro inhibitors. |
| Collaborator Contribution | Synthesis of macrocyclic peptide using RaPid method |
| Impact | Accepted Manuscript Nat. Chem. |
| Start Year | 2020 |
| Description | Plasmid for E.coli L,D-transpeptidases |
| Organisation | Newcastle University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | I am actively working on the expression of L,D-transpeptidases from Mycobacterium. We thought it would be worth looking into L,D-transpeptidases from another bacteria i.e. E.coli as they share the substrate. Currently, I am working to optimize the expression of these plasmids using in-house media and expression cell lines. |
| Collaborator Contribution | Dr. Winkle from New Castle University has synthesized and obtained an expression protocol for these E. coli proteins and shared this with out use. As one of the two porteins were inactive, we are looking to optimize the methods to obtain activity. |
| Impact | Open and accessible research with shared resources |
| Start Year | 2019 |
| Description | Sulfonic acids as potential drugs for LD-transpeptidases |
| Organisation | Indian Institute of Technology Kharagpur |
| Country | India |
| Sector | Academic/University |
| PI Contribution | Using the monooxygenase enzyme prepared by Dr. Singh, we prepared sulfonic acids and tested their inhibitory potential against L,D-transpeptiases. |
| Collaborator Contribution | Our collaborators at IIT, Kharagpur, purified and expressed EthA protein that convert uric acid into sulfonic acids. |
| Impact | Sulfonic acids are not able to bind to the active site of L,D-transpeptidases. It was multidisciplinary in approach as a monoxygenase enzyme was being exploratively investigated for its unidentified biological role in India was proposed by us in its use to produce chemicals which could be tested as drugs for another tuberculosis target protein. |
| Start Year | 2019 |