Exploration and exploitation of the enzymology of PBP driven peptidoglycan

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences

Abstract

Millions of people die each year from bacterial infections and tens of millions suffer from non-fatal disease. The discovery of penicillin opened the door to treat these infections with antibiotics. Penicillin is one of the most important antibiotics in the world and works by stopping bacteria making the polymer in the cell wall that holds them together. This polymer, calle dpeptidoglycan, is made up of an interlocking network of sugars and strings of amino acids (peptides). Specialised proteins (called PBPs), with the ability to stitch together these sugars and peptides are the targets inhibited by penicillin, stopping cell wall synthesis and killing the bacterium. However, after all this time no one really knows how these PBPs work in making cell wall.

It is now more important then ever that we understand this mechanism. Many important bacteria are now no longer killed by penicillin - they have altered their PBPs so that they can still make cell wall but prevent penicillin from inhibiting these proteins. The group at Warwick has brought together a team from microbiology, biochemistry, chemistry and structural biology to crack this problem. They have, for the first time, the combined tools and new chemical reagents (made at Warwick) to look in minute detail how PBPs work and how antibiotic resistance has developed at the molecular level within these proteins.

This ability at Warwick has interested Pharmaceutical companies looking to design new antibiotics to kill bacteria that are resistant to penicillin. Prolysis, perhaps the premier bacterial drug discovery company within the UK and Europe, has linked up with the team at Warwick as collaborators to make their extensive company computing resources, robotics, and chemical library, available for use with this new ability in the quest for novel life saving antibiotics.

Technical Summary

The terminal stages of bacterial cell wall (peptidoglycan) biosynthesis is driven by a family of specialised transpeptidases better known as penicillin-binding proteins or PBPs (some of which also possess additional transglycosylase domains). These enzymes are important, being the targets for penicillin and other b-lactam antibiotics. However, PBPs have evolved genetic variants with low affinities for these antibiotics, rendering them therapeutically ineffective. For Streptococcus pneumoniae homologous recombinaitons to PBPs 1A, 2X and 2B has resulted in changes to amino acid composition and transpeptidase conformation. Furthermore, the cell wall composition of highly resistant strains indicates that these PBPs have to process modified substrate.
Important as they are, almost nothing is understood about how these proteins interact with their natural substrate, lipid II, a disaccharide conjugated to a pentapeptide and linked to a C55 undecaprenyl lipid carrier, during cell wall assembly (PBPs cross linking the disaccharide and peptide components of lipid II). The previous inability to easily synthesise this substrate has substantially hindered progress.
At Warwick we have recently developed and validated a continuous PBP assay based upon the natural substrate. We are able to synthesise; lipid II (and its MurMN mediated (Ser Ala ) peptide variants found in highly resistant strains) in quantity at high purity ( 98%); lipid I and its MurNAC peptide precursors; soluble S. pneumoniae PBPs, 1A (with transglycosylase and transpeptidase activity) , and 2X and 2B (with transpeptidase activity) from susceptible and resistant strains; site directed PBP mutants; and the reagents to establish a high throughput continuous kinetic analysis of these PBPs.
This long awaited breakthrough will enable a thorough kinetic and mechanistic analysis of amino acid alterations identified as being important in resistance. We will also be able to investigate; the impact PBP and stem peptide alterations have upon the kinetics of high and low affinity PBPs; and the interplay of transglycosylase and transpeptidase activity in substrate utilisation.
In addition to examining at the molecular level the evolution of penicillin resistance in S. pneumoniae, we, with our drug discovery collaborators, Prolysis, will use our PBP assay to establish a high throughput screen to identify novel non-b-lactam PBP inhibitors.
The natural substrate will also enable us to undertake co-crystallisation studies of enzymes and substrate, to better determine the binding and mechanism of action of these enzymes, and so help in the rational identification of novel inhibitors as part of the drug discovery programme.

Publications

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Majce V (2013) Crystallization and preliminary X-ray analysis of a UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (PaMurF) from Pseudomonas aeruginosa. in Acta crystallographica. Section F, Structural biology and crystallization communications

 
Description All Party Parliamentary Group on Antimicrobial Resistance
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Attendance at All Parliamentary Group for AMR
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact Help shaped national AMR policy
 
Description Canada/UK AMR Workshop
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a national consultation
 
Description Lessons to be learnt from pharma A summary of an interactive one-day symposium about discovery and development of new antibacterial drugs 1 Lessons to be learnt from pharma about discovery and development of new antibacterial drugs
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description MRC strategic review on antimicrobial resistance
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description Accelerate CHNUK AMR discovery: Establishing joint China/UK training and research platforms enabling highthroughput fragment based inhibitor discovery
Amount £1,000,000 (GBP)
Funding ID MR/P007503/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 05/2019
 
Description BBSRC LINK award
Amount £464,000 (GBP)
Funding ID BB/K017268/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 09/2015
 
Description BaCWAN2
Amount £264,000 (GBP)
Funding ID G0701400 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2008 
End 10/2010
 
Description CIHR /MRC team grant
Amount £1,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2012 
End 01/2016
 
Description MRC AMR theme 1 collaboration award
Amount £3,200,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2015 
End 09/2020
 
Description Project grant
Amount £589,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2011 
End 10/2014
 
Description UK CAN BaCWAN
Amount £60,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 09/2011
 
Description Wellcome Trust Pathfinder Award
Amount £100,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 03/2017
 
Description flexible interchange partnership FLIP
Amount £150,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 02/2016
 
Title New assays for penicillin binding proteins 
Description First in class continuous quantitative bifunctional PBP assay 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Use in HTS assay with Astra Zeneca 
 
Title peptidoglycan pathway intermediates 
Description Capacity to synthesise peptidoglycan intermediates at high purity and semi-commercial quantities 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Access of reagents to the wider community - supplied over 40 research groups worldwide 
 
Description MRCT 
Organisation MRC-Technology
Department MRCT Centre for Therapeutics Discovery (CTD)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provision of reagents and assays, developing structural information
Collaborator Contribution Pump priming drug discovery research and further grant applications to MRC
Impact DPFS grant applications
Start Year 2008
 
Description Novacta 
Organisation Novacta Biosystems Ltd
Country United Kingdom 
Sector Private 
PI Contribution updating company on our assay development and screening capabilities
Collaborator Contribution Indistrial focussed advice
Impact Novacta became industrial advisors as part of our MRC programme grant application and Mike Dawson from Novacta still performs this function
 
Description Prolysis 
Organisation Prolysis Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have assisted in compound screen and assay development
Collaborator Contribution Intellectual input and advice relating to direction in two translational grant applications
Impact Prolysis have been involved in the development of a large transnational programme grant and lloyd Czaplewski still remains in close contact advising us from and industrial pharma perspective
 
Description Ramoplanin induced pertubation of pneumococcal peptidoglycan 
Organisation Pfizer Ltd
Department Vicuron Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Advice
Collaborator Contribution assisted the company in pure paration of cell wall intermediates
Impact assisted in pureparation of cell wall intermediates
 
Title Synthesis of peptidoglycan intermediates 
Description Optimisation of peptidoglycan pathway intermediates synthesis and purification 
IP Reference  
Protection Protection not required
Year Protection Granted 2006
Licensed No
Impact Development of a synthesis facility to provide thes eintemediates to the global research community and the fundamental platform for several UK and international peptidoglycan networks
 
Company Name Antimicrobial Discovery Solutions Ltd 
Description Reagents, assay development and services 
Year Established 2015 
Impact N/A
 
Description ANTRUK Antibiotic Research UK 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Establishment of new charity and new fundraising campaign

regional and national media interest
Year(s) Of Engagement Activity 2014
URL http://www.antibioticresearch.org.uk
 
Description Getting to grips with antibiotic resistance 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact The School of Life Sciences at Warwick hosted members of the public and press for a presentational evening with talks, lab tours and demonstrations in an event entitled: "Getting to grips with antibiotic resistance" This event was held in antibiotic awareness week in November 2016 and hosted around 90 visitor
Year(s) Of Engagement Activity 2016
URL http://www2.warwick.ac.uk/fac/sci/lifesci/outreach/next/
 
Description MRC Flemming video 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact 100000. Short film presentation linking our current MRC funded research to the groundbreaking work by alexander Flemming

to be released later this year as part of the MRC celebrations
Year(s) Of Engagement Activity 2013
 
Description Regional news papers 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Contribution to regional news

Further contacts from regional and national media
Year(s) Of Engagement Activity 2006,2007
 
Description Warwick University iCAST 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Communication to UK and global audience

Further media contacts
Year(s) Of Engagement Activity 2008