Hijacking prenyl and geranyl transferases - A route to carry out click modifications and to enhance cellular permeability of peptides

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry

Abstract

Recent advances in biological research have allowed a better understanding of the causation of many diseases and identified new targets for therapy. An ideal drug should bind to a specific cellular target and have no affinity to others. One of the recently identified challenging targets for drug discovery is the protein-protein interactions that have been proved to be involved in many difficult-to-treat diseases e.g. immune disorders and cancer. These interactions are taking place along the extended surface of large proteins and thus are very challenging for small molecule drugs. Biological drugs e.g. antibodies are large molecules and can disrupt protein-protein interactions but cannot be administered orally and are very expensive. Macrocyclic peptides are an emerging class of drug candidates that have the ability to disrupt protein-protein interactions e.g. the immune-suppressant, cyclosporin that made transplant surgery possible. They are smaller in size and are very much cheaper than biologics. In contrast to their linear "non-cyclic" counterparts, they are more stable against enzymes and are semi-rigid to fit better with their targets much like a key fits into a lock. A limitation that hampers the development of many of these compounds is their low ability to cross cellular membranes and to reach intracellular targets. Several modified cyclic peptides are commonly found in medicinal natural products. These compounds were evolved via natural selection which is presumably driven by their pharmacological potency against specific molecular targets as well as their ability to reach these targets that is, by crossing one or more biological membranes. In nature, several modifications are introduced to cyclic peptides to enhance membrane permeability. These modifications aim to reduce the hydrophilic (polar) surface of the molecule by shielding with hydrophobic side chains thus the compound can easily diffuse through the hydrophobic (mainly lipid) cellular membranes. Ideally these modifications should be applied to specific sites to avoid a large reduction in water solubility or the change of three dimensional shape of the molecule with subsequent decrease in its ability to bind to its target. Recent research revealed how a large group of these modified cyclic peptides is made inside their hosts. In this project, I will identify and recruit new modifying biosynthetic enzymes that will add hydrophobic chemical groups such as prenyl and geranyl groups at specific sites in cyclic peptides. Making these modifications using chemical methods is very challenging, not eco-friendly and in most cases entails total re-synthesis which is time consuming. I will determine the structure and biochemical features of these enzymes to identify the key residues that underlie their activity and specificity. I will use these insights to engineer and generate enzyme variants with different residue specificity and ability to introduce other chemical groups. I will use chemical synthesis and the engineered enzymes to generate modified derivatives of bespoke bioactive cyclic peptides and test the effect of different modifications on membrane permeability and the three dimensional shape of the molecule that underlies target affinity. These data will help to generate a computational model to predict membrane permeability of bioactive cyclic peptides that will be invaluable for development of peptides into drugs.

Planned Impact

The proposed work has an impact on economy, society and environment.
Economic impact: The proposal will have an impact on industrial biotechnology and pharmaceutical industry sectors. Both industries are identified as crucial to the UK economy and are consistently at the top of industrial sectors in terms of trade surplus and number of employees, with around 73,000 people employed directly (Source: Association of British Pharmaceutical Industry). Currently, there is a global pharmaceutical interest in developing constrained / cyclic peptides as cheaper and equally potent alternatives to biologics. However, the poor ability of these compounds to cross cellular membranes has been identified as an impediment to their development as therapeutics. Introducing structural modifications to enhance permeability and balancing this with other physicochemical parameters that underlie pharmacodynamic and pharmacokinetic properties are very challenging and involve synthesis and testing of large number of variants which is time consuming. This project will give a market leadership to the UK as it provides novel enzymatic tools that can make challenging chemical modifications to peptides with high residue, regio- and stereo- specificities. Another important deliverable is a theoretical model to predict cellular permeability of cyclic peptides and modified derivatives. The project is multidisciplinary with elements of synthetic biology, peptide chemistry and theoretical chemistry. The EPSRC has considered synthetic biology of high strategic importance to the UK and has made a commitment to invest more in this area.
Societal Benefit: The project will benefit the society by providing trained workforce in biotechnology, synthetic biology and peptide therapeutics. School children and the general public will benefit from the planned outreach activities that will inspire children and increase the awareness of the public with synthetic biology. The project will provide novel enzymatic tools to modify peptides for enhanced cellular permeability and diversity. These compounds have underexploited potential in combating a range of diseases such as infection, cancer and autoimmune disorders. Introducing these modifications will allow exploring the potential of these compounds in tackling intracellular therapeutic targets and thus open new avenues for discovery and development of new drugs for critical diseases without currently available treatment options. The work will also generate a library of new biologically active molecules designed to disrupt key intracellular protein-protein interaction in Hippo signalling pathway involved in tumour development.
Environmental impact: The proposed research provides a green method to make challenging chemical transformations. The use of enzymes instead of toxic chemical reagents saves on energy required for production and is eco-friendly. In many occasions, enzymes can be immobilised and recycled and this saves on the amount of energy used in their production.

Publications

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Colombano A (2023) Chemoenzymatic Late-Stage Modifications Enable Downstream Click-Mediated Fluorescent Tagging of Peptides. in Angewandte Chemie (International ed. in English)

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Houssen WE (2019) Peptide Cyclization Catalyzed by Cyanobactin Macrocyclases. in Methods in molecular biology (Clifton, N.J.)

 
Description 1- We have identified a new enzyme that can introduce a lipophilic chemical group called prenyl group to the arginine and homoarginine amino acids. We have showed that we can use this enzyme as a reagent to modify the structures of peptides containing these residues. Introduction of lipophilic groups into peptides should enhance their ability to penetrate lipid cellular membranes. This work has been published in ChemComm.
2- We have synthesized a library of novel unnatural co- factors for a group of enzymes called prenyltransferases. These enzymes catalyze the transfer of a prenyl group from a co-factor donor to specific amino acid in peptides. We have found and engineered some of these enzymes to accept unnatural co-factors in which the prenyl group is changed to a functional group that can be easily modified by a technique called click chemistry. Incorporation of these clickable functional groups in peptides enables further conjugation with fluorescent tags and antibodies. We have published a paper in a top chemistry journal (Angew Chem Int Ed) and are discussing with the University of Aberdeen's Technology Transfer Office the potential of filing a patent on new ideas /unpublished work in this direction.
3- We have produced few enzymatically prenylated peptides that are in silico designed to inhibit the SARS-CoV-2 virus protease which is one most validated target for this virus. Testing of the activity of these is underway.
4- We have studied a role of a chaperone protein in facilitating the entry of SARS-CoV-2 and possibly other lethal viruses into cells. We have designed peptides that inhibit the interaction of the spike protein with this target based on the structure of SARS-CoV-2 spike protein. We have solid data to prove the role of this chaperone protein and we are preparing a manuscript to disseminate our findings.
5- We have identified and chemically characterized twelve previously undescribed scorpion venom peptides and tested their activity in inhibiting SARS-CoV-2 spike -ACE2 interaction. This interaction is a very challenging therapeutic target for drug design. We have identified some active peptides and we synthesised the most active one and tested its anti-SARS-CoV-2 activity. the peptide shows an IC50 of 200nM. the work has been published in Peptides.
6- We have designed a library of peptides that could inhibit the critical interaction of YAP/TAZ and TEAD. These are the downstream effectors of the Hippo pathway which control the size of our organs. YAP/TAZ interaction with TEAD has been associated with the development of different cancers. We are carrying NMR analysis of these compounds and will send them for testing soon to our collaborators.
Exploitation Route I am discussing with some companies a partnership to further develop the enzymatic based technology we developed to allow tagging, labeling and conjugation of peptides.
Sectors Chemicals

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Societal impact: this project has benefitted the society by providing trained workforce in biotechnology, synthetic biology and peptide therapeutics. One PDRA has received training on peptide synthesis and has secured a job in Almac group (Edinburgh) as peptide chemist after a competitive interview process. Two PhD students are now in their final year of study. One PhD student has been graduated last year and secured a PDRA position at University of Bournemouth.
Impact Types Societal

Policy & public services

 
Description Conduct a science-based peer-reviewed fact-finding study on the uses of digital Sequence information to help the ongoing discussion at the UN on the regulations to access the genetic resources and the fair and equitable sharing of benefits arising from their utilization
Geographic Reach Multiple continents/international 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
URL https://www.cbd.int/doc/c/fef9/2f90/70f037ccc5da885dfb293e88/dsi-ahteg-2020-01-03-en.pdf
 
Description Analysis of biological small molecule mixtures using multiple modes of mass spectrometric fragmentation coupled with new bioinformatics workflows
Amount £810,134 (GBP)
Funding ID BB/X019802/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2023 
End 07/2024
 
Description Design and Chemoenzymatic Generation of Anti-SARS-CoV-2 Modified Cyclic Peptides
Amount £89,000 (GBP)
Organisation Royal Embassy of Saudi Arabia 
Sector Public
Country United States
Start 09/2022 
End 09/2026
 
Description Development of novel modified scorpion venom peptides as anti-SARS-CoV-2 virus.
Amount £48,550 (GBP)
Organisation Government of Scotland 
Department Scottish Funding Council
Sector Public
Country United Kingdom
Start 09/2020 
End 07/2021
 
Description Downstream processing and structure confirmation of chemoenzymatically produced macrocycles
Amount £102,000 (GBP)
Organisation IBioIC 
Sector Academic/University
Country United Kingdom
Start 09/2021 
End 09/2025
 
Description Floor standing cooled vibration free incubator for protein crystals 100L
Amount £8,862 (GBP)
Organisation ScotCHEM 
Sector Academic/University
Start 05/2022 
End 07/2023
 
Description Making thiazolines and oxazolines: Expanding the enzymatic repertoire to incorporate other 5- and 6-membered heterocyclic rings in peptides
Amount £102,300 (GBP)
Organisation IBioIC 
Sector Academic/University
Country United Kingdom
Start 09/2020 
End 09/2024
 
Description Marine Biodiversity as Sustainable Resource of Disease-Suppressive Microbes and Bioprotectants for Aquaculture and Crop Diseases (MARBLES)
Amount € 7,496,896 (EUR)
Organisation European Commission H2020 
Sector Public
Country Belgium
Start  
 
Description Phosphodiesterase 1 signalsomes: the utility of peptide disruptors for pulmonary arterial hypertension.
Amount £110,000 (GBP)
Organisation East of Scotland BioScience (EastBio) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2023 
End 09/2026
 
Title Full transcriptome data of the scorpion Androctonus amoreuxi 
Description Full transcriptome data of the scorpion Androctonus amoreuxi venomous gland. The data can be used to predict the sequence of venom peptides that could be produced by this scorpion. GEO. Accession number: GSE221799 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact We used the above data as well as high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV- 2 spike protein and inhibition of the spike RBD - human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117-1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD - hACE2 binding inhibition assay. the results was published in Peptides (2024) 173, 171139 
URL https://www.ncbi.nlm.nih.gov/geo/
 
Description Present my research to public (visitors of the Institute of Medical Sciences at university of Aberdeen 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Th Institute of Medical Sciences at university of Aberdeen opened its doors to general public to visit and see our research on 10 Sept. 2022. During this event I have showed our visitors the power of synthetic biology as I have prepared plates with engineered bacterial cells expressing colored proteins. I used these to draw nice pictures on the agar plate. I then explained to the public how we engineer bacterial cells to produce useful products like enzymes or novel medicines. I also had models for protein 3D structures and showed the visitors how I designs drugs to prevent protein-protein interactions underlying disease states.
Year(s) Of Engagement Activity 2022
 
Description Present my research to public at 2022 PeckaKucha Night 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I have presented my research findings and methodology to public audience at PeckaKucka night event held at The Aberdeen Arts Centre on 28th Nov, 2022. This was followed by questions and answer session. the talk has been then recorded and uploaded to the PeckaKucka website (https://www.pechakucha.com/)
Year(s) Of Engagement Activity 2022
URL https://www.pechakucha.com/
 
Description Public lecture on my research based on invitation from the Royal Society of Chemistry North East local section 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I was invited by the RSC North East local section to deliver a talk to the public entitled "Animal Venoms - Friend or Foe" on 7th Nov 2023 and it was well-received
Year(s) Of Engagement Activity 2023
URL https://www.abdn.ac.uk/ncs/events/19674/
 
Description Webinar on digital sequence information on genetic resources organised by the ABS Capacity Development initiative and The UN Secretariat of the Convention on Biological Diversity 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact I was invited to speak in a webinar organised by the ABS Capacity Development Initiative and The UN Secretariat of the Convention on Biological Diversity to provide a technical overview of the production, distribution and use of digital sequence information (DSI) on genetic resources. The event was well attended and 50% of participants were Government representatives while 19% were academics and the remaining were general public and indigenous organisations. The aim of the webinar is to increase the public awareness with the use of DSI on genetic resources in different fields and to help the ongoing discussions at the UN on the fair sharing of benefits arising from their utilization. My research that is supported with EPSRC fellowship award involves heavy use of DSI to discover novel enzymes that can catalyse challenging chemical transformations in an efficient and eco-friendly way.
Year(s) Of Engagement Activity 2020
URL https://www.cbd.int/article/dsi-webinar-series-2020
 
Description interview for local and international news - generate a video on my research 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I have produced a short video on the efforts in my lab to develop modified cyclic peptides that can prevent the SARS-CoV-2 virus entry to cell. The video was published as part University news (https://www.abdn.ac.uk/news/15459/) (https://www.youtube.com/watch?v=iU1nOr84zwg) and has attracted the attention of many radio and TV channels and newspaper worldwide. Many of them have contacted me for interviews.
These include
The Metro, Daily Record, The Independent, Press and Journal, Evening Express, Daily Star, and Wales Online
BBC Aberdeen, Northsound, Radio Borders, Tay FM, Moray Firth Radio, West Sound, Clyde 1, Forth 2 and Original 106.
International: The Jerusalem Post (Israel) and Youm7 (Egypt)
Year(s) Of Engagement Activity 2021
URL https://www.abdn.ac.uk/news/15459/
 
Description podcast 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact 2. A podcast describing my background and current research
Year(s) Of Engagement Activity 2023
URL https://open.spotify.com/episode/2FqAjWLuerwridRdM3Sjwa?si=7d005e7dc10641c6