Development and validation of new reagents and assays to exploit the final steps of peptidoglycan construction

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Millions of people die each year from bacterial infections and tens of millions suffer from the consequences of these infections. The discovery of the antibiotic penicillin once opened the door to treat these infections. It did this by stopping bacteria from making the polymer in the cell wall that holds them together. This polymer, called peptidoglycan (PG), is made up of an interlocking network of sugars and strings of amino acids (peptides). Specialised proteins (called penicillin-binding-proteins or PBPs, which are present in all bacteria) either have the ability to stitch together the sugar backbone and peptides. The construction of peptide cross-links by PBPs is famously the target inhibited by penicillin which stops cell wall construction and kills the bacterium. Penicillin has been an excellent antibiotic, not least because it targets multiple PBPs simultaneously within a bacterium.

Unfortunately, many bacteria are no longer killed by penicillin and other antibiotics that attack the production of peptidoglycan. Bacteria have changed by evading the action of these antibiotics by modifying the target PBPs and producing enzymes that degrade the antibiotic. We need to fight back and the strategy of exploring PBPs for new inhibitors is widely recognised as an important well validated option.

Progress in achieving this has been hampered by our inability to routinely synthesise the key chemical components that make this polymer. We can now do this at Warwick, and have an exceptional track record of providing reagents to study peptidoglycan biosynthesis to academia worldwide. Having studied how the precursors of these reagents are produced by enzymes in the PG pathway, we intend to exploit the opportunities we have discovered to develop completely new reagents with bespoke components. This is exciting for both the academic and industrial communities as we will become able to produce tailor-made intermediates for specific functions. For example, we can include radioactive sugars or amino acids, fluorescent labels, or modifying sugars or amino acids in ways which alter their ability to polymerise. These reagents will enable us, and the wider community, to explore fundamentally important unanswered questions about these targets andhow bacteria grow and control the production of peptidoglycan. We will be in a position to use these reagents to develop ambitious new assays, not only to characterise the activities of these targets, but also to explore the translation of these assays into formats for industry to use them in the search for completely new classes of inhibitors, overcoming current problems of resistance to penicillin and related antibiotics.

To achieve this we will use our academic expertise gathered over the past decade of funding with enzymologists, chemists, engineers, mathematicians and physicists, and use this in a new closer partnership with industry. This partnership will provide open access for us to develop the work more widely, to increase the platform of reagents we can produce, extend our capability into new assays to study the complex, difficult, final stages of peptidoglycan construction. All of this will work towards fundamentally new biological insights. It will also underpin opportunities to further develop these reagents and assays for use by industry. To do this we will have to refine current methods to scale up production and develop robust industry quality assays. Our partnership consists of scientists and technical support at Warwick University with complementary skills and specialist knowledge to acomplish these tasks, along with Astra Zeneca, who are committed to supporting open access to this new underpinning technology and helping to develop novel approaches to high throughput screens. This heralds an era where academics and industry can work closely together in the search for new antibiotics.

Technical Summary

Penicillin-binding- proteins (PBPS) involved in the construction of peptidoglycan (PG) are the target of important classes of antibiotics, the b-lactams and glycopeptides. Although resistance has arisen to these classes of antibiotic, the formation of peptide cross-links between the glycan backbone of PG by transpeptidation (TP) remains an excellent target for antimicrobial development. Advances have been made in understanding mechanisms underlying the activity of PBPs, however, assay development (let alone high throughput screening) for these enzymes has remained difficult, involving extensive product isolation and characterization.

We will extend our development and scale up of novel reagents and target enzymes. Reagents will encompass the chemical diversity that exists in PG structure across bacteria, including, non-cognate substitutions within the linear muramyl pentapeptide, the production of polymeric lipidII where required to prime reactions, and truncation of the C55 lipid tail (using C35 or C20 lipids etc.) to improve in assay solubility and assay function. Interestingly, the required acceptor transpeptidation substrate can be as small as a single amino acid with D-chirality around the alpha-carbon atom. This observation opens up the development of a range of novel more complex (linear/branched) acceptor molecules for use in assays to probe the TP acceptor site structurally and to format assays to identify novel non-lactam TP inhibitors.

These reagents will ultimately enable a systematic characterization of the transpeptidase activity of PBPs, the development of co-crystal structures, and provide the foundation for previously unavailable robust assays with direct readouts. Astra Zeneca will work with us to help scale up reagent production, format assays for use in high throughput screens, test sensitivity, and help validate these for industry though IP free access to trial compound libraries.

Planned Impact

1) Impacts: relate to the exploitation of new reagents and assays generated by this project enabling the kinetic and structural exploration of terminal stages of peptidoglycan (PG) biosynthesis. Specifically the targets of penicillin (penicillin-binding-proteins PBPs) which remain difficult to assay yet represent fundamental targets for research and exceptional targets for drug discovery to identify new classes of antiinfectives using these radically new, bespoke, assays.

The potential beneficiaries of the impacts are: the resurging UK and global academic research community exploring bacterial cell wall biosynthesis, cell division and drug discovery; national and international pharmaceutical industries; UK capacity building (skills and business) for this Strategically Important and Vulnerable Area of UK Bioscience Expertise, international collaborations; public and understanding of science (PUS) and government policy.

2) Engagement: Training and skills into the development and use of assays and reagents will extend beyond the PDRAs and technician employed by LINK by inviting at least UK (6), EU (5), US (2) and CA (6) research groups to i) research workshop yr 1 (of about 50-100 people) including members of the £20M regional development agency funded Science City Research Alliance (SCRA) in Translational Medicine between Warwick and Birmingham, and ii) smaller experimental workshops yr 2 (~10 - 20 people from as many groups as possible). Workshop i) will include a competition and prize for the best PUS poster(s) these will be used within host institutions and available via a NEW PUS section of the well used BACWAN web site.

Workshop i) will also include invitations to: UK and global industry ( AZ, Basilea, CBV, Cubist, GSK, Novacta, Novartis); Health Protection Agency; NHS clinical microbiologists; representatives from Antibiotic Action; TSB; Warwick Corporate Affairs and Communications teams. Together these constituencies will help to produce media outputsduring yr 1 (post workshop) and yr 2, such as an iCAST video, inform PUS and academic groups to better engage with pharmaceutical industry, influence national and international government policy of the need to support drug discovery activities, specifically teams of academic industry partnerships.

We will help convene 1 additional international workshop to promote policy for funding and to attract industry investment and showcase activities at MRCT ELRIG drug discovery each year (planned UK/Canada meeting with academics, industry, HPA, government representatives and national funding agencies at the Canadian Embassy).

Applications for reagents and assays will be developed across UK, EU, US and CA academic research groups and in consultation with AZ and other pharma, exploitation and commercialisation will be guided by Warwick Ventures (technology transfer and IP protection) and Warwick Corporate Affairs. Identification of applications will be helped by the planned workshops.

Additional funding and training will be sought through BBSRC/MRC CASE awards (yr 1), Follow on funding (yr 2) and the TSB (yr 2) or earlier (into yr 1) if rapid progress is made with reagents and the identification of an industry partner to progress applications.

If this LINK is awarded CGD will apply, yr 1, for a BBSRC Flexible Interchange Programme (FLIP) for secondment allowing closer engagement with industry, and industry focussed research groups in the field, to improve and extend these relationships, identify current applications (and specifications) for industry and future needs. The aim being to foster long term collaboration and investment. Additionally, reagents and assays developed by this LINK will be promoted through 3 national and international industry outsourcing conventions such as Bio Trinity, Bio Chicago, Bio Europe

Community: Posters and media presentations will be used in outreach to at least 2 local schools and Cafe Scientifique B'ham
 
Description New understanding about how the targets of penicillin work and how we might begin to develop wholly new types of antibiotic to escape current problems of antibiotic resistance

We have developed a 2 new assays to look for chemicals that inhibit bacterial growth and may become 21c alternatives to penicillin
Exploitation Route Exploitation with wider industry within the UK and worldwide

Have subsequently won a £100k Pathfinder Award with Wellcome Trust working with an Astra Zeneca spin out company to exploit these new findings.
Sectors Education,Pharmaceuticals and Medical Biotechnology

 
Description Development of new reagents and assays to better understand the mechanism of the targets of penicillin and to develop new high throughput screens for antibiotic discovery Establishment of a first in class assay for bifunctional penicillin-binding-proteins PBPs Translation into industry standard HTS format Z'>0.8
First Year Of Impact 2015
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description All Party Parliamentary Group on Antimicrobial Resistance
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
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 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 Coventry General Charities
Amount £40,000 (GBP)
Organisation General Charity of the City of Coventry 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2014 
End 01/2015
 
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 Wellcome Trust Innovation Award
Amount £100,000 (GBP)
Funding ID 109676/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 06/2017
 
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 
 
Description Astra Zeneca global screening platform 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Knowledge transfer, biological insight and assay development
Collaborator Contribution Advice, knowledge transfer, training and access to high throughput screening platforms and chemical libraries
Impact Novel assay development and assay validation Hit screening underway
Start Year 2013
 
Description Developing new reagents to underpin pharmaceutic research 
Organisation Vita-Salute San Raffaele University
Country Italy 
Sector Academic/University 
PI Contribution Provision of reagents as biological standards to help inform assay development
Collaborator Contribution Discussion and insight into screening strategies and fundamental science underpinning antibiotic discovery
Impact knowledge transfer
Start Year 2014
 
Description Entasis collaboration 
Organisation Entasis Therapeutics
Country United States 
Sector Private 
PI Contribution Biochemistry and assay development
Collaborator Contribution Assay validation and hit characterisation insight
Impact Successful award of Wellcome Trust Pathfinder award
Start Year 2015
 
Description Exploring peptidoglycan polymerization 
Organisation Cubist Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Provision of commercially unavailable reagents and knowhow to assist in assay development
Collaborator Contribution iterative feedback on assay development and outcome
Impact Have obtained letters of support from Cubist for recent grant applications and developed a good working relationship with their senior scientists
Start Year 2013
 
Description New assays and reagents to exploit peptidoglycan biosynthesis 
Organisation AstraZeneca
Department Research and Development AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Development of new reagents and assays
Collaborator Contribution Insight into establishment of high throughput screening approaches, pitfalls and access to equipment
Impact Wider collaboration with other global pharma, new grant applications, increased industrial understanding, training and skills for researchers and students
Start Year 2013
 
Description Peptidoglycan flux and inhibition 
Organisation Basilea Pharmaceutica
Country Switzerland 
Sector Private 
PI Contribution Developing new insight into the ealy stages of peptidoglycan biosynthesis and how protein protein interaction may impact upon ability to inhibit these reactions
Collaborator Contribution Historical insight and approaches to targeting these enzymes
Impact Knowledge transfer
Start Year 2012
 
Description Underpinning technology to exploit peptidoglycan biosynthesis 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution Insight into the assembly of the cytoplasmic phase of peptidoglycan biosynthesis
Collaborator Contribution Insight into defence requirements and work leading up to the project
Impact too early - completing initial contracting process
Start Year 2014
 
Description Warwick Oxford Chemistry Collaboration 
Organisation University of Oxford
Department Department of Paediatrics
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing underpinning assays to help inform antibiotic development approaches.....................................
Collaborator Contribution Chemistry input and design
Impact Multidisciplinary
Start Year 2014
 
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 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 O'Neil AMR review 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact In December we published our first report1
showing that infections caused by
drug-resistant pathogens are one of the biggest health problems the world faces
today. Bacteria and other pathogens have always evolved to resist the new drugs
that modern medicine uses to combat them. But in recent years the rise in drug
resistance has been a particular worry, especially the emergence of antibioticresistant
superbugs. Unless action is taken to address this huge global issue, our
conservative estimate is that it will cost the world an additional 10 million lives
a year by 2050, more than the number of people currently dying from cancer
annually. It will also have a cumulative cost of 100 trillion USD, more than one
and a half times annual world GDP today, or roughly the equivalent to losing the
UK economy from global output every year.
We now turn our attention to how this problem can be tackled. This paper is the
first in a series that works towards global and sustainable solutions. There are
many angles to the problem that we will need more time to consider. In particular,
the focus of our next paper, due to be published in the spring, will be how to
stimulate the market for companies to invest in and develop new antimicrobials and
diagnostics, which is not fully addressed here. There we will assess potential 'push'
and 'pull' incentives to encourage the development of new antimicrobial drugs,
and set out our proposals for action by policy makers. In later papers we will also
focus on important issues such as the use of antibiotics in agriculture and potential
alternatives to antimicrobials
Year(s) Of Engagement Activity 2015
URL https://amr-review.org/sites/default/files/Report-52.15.pdf
 
Description Pew Road Map for Antibiotic Discovery 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact The Pew Charitable Trusts convened a multidisciplinary group of leading industry and academic experts to
identify the key scientific roadblocks to antibiotic discovery and consulted with numerous other public and
private sector stakeholders to develop a Scientific Roadmap for Antibiotic Discovery. The roadmap outlines a
concrete approach-both a scientific plan and organizational structure to support this research-that would lay a
foundation for the sustained and diversified discovery and development of new antibiotics and therapies over the
coming decades.The report's key findings show a need for:
• A targeted approach to tackle the basic scientific barriers impeding antibiotic discovery and development.
• A better understanding of how to overcome the cellular defenses of drug-resistant Gram-negative bacteria,
which cause some of the most difficult-to-treat infections.
• Generation of new chemical matter designed for antibiotic discovery.
• Tools and methodologies to evaluate promising alternatives to traditional antibiotic use.
• A framework for sharing information, expertise, and materials across the research community to foster
innovative science and spur the discovery of novel antibacterial therapies.
Year(s) Of Engagement Activity 2016
URL http://www.pewtrusts.org/~/media/assets/2016/05/ascientificroadmapforantibioticdiscovery.pdf
 
Description School visit 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 20 questions panel followed by open discussion around - what is a scientist

Invited to attend further activities by the schools career department
Year(s) Of Engagement Activity 2014
 
Description Trustee for a regional science charity MLS 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Development and distribution of new literature

Inspired fund raising
Year(s) Of Engagement Activity 2013
URL http://www.charitychoice.co.uk/medical-and-life-sciences-research-fund-25719
 
Description University open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Discussion around training and careers in microbiology an biomedical science

Inspired about microbiology
Year(s) Of Engagement Activity 2014