Tackling antimicrobial resistance through fragment-based drug discovery

“GCRF START has been pretty awesome, in the way it’s allowed us to help accelerate some truly cutting-edge South African science with the tools we get to develop and provide at Diamond.”

Professor Frank Von Delft, GCRF START Co-I at Diamond Light Source, UK

According to the World Health Organization (WHO), antimicrobial resistance is one of the top 10 global public health threats facing humanity. In South Africa, infections caused by Staphylococcus aureus and Mycobacterium tuberculosis are an all-too-common reality. While M. tuberculosis is the causative agent of tuberculosis (TB) which shows increasing prevalence of drug-resistance, S. aureus is one of the ESKAPE[1] pathogens [2] – a group of organisms that are leading causes for community- and hospital-acquired infections globally. ESKAPE pathogens are also notoriously difficult to treat and are resistant to many first line antibiotics. Considering the rise in antimicrobial-resistant organisms there is a desperate need to identify new antimicrobial compounds that work differently from those currently in clinical use.

Mr Warrick Sitzer, an MSc. student in the Strauss Laboratory at Stellenbosch University, South Africa, preparing bacterial colonies which will then be used to produce large amounts of his protein of interest. Photo credit: Blake Balcomb. ©Diamond Light Source

New and exciting drug discovery initiatives

With the GCRF START grant, the Strauss Laboratory has established new, cutting edge structural biology capabilities at Stellenbosch University in South Africa to collect data onsite and remotely from Diamond. This includes embarking on an exciting approach in drug discovery initiatives to identify new antimicrobial compounds, using the UK’s world class national synchrotron, Diamond Light Source (Diamond).

Research in the Strauss Laboratory is focused on understanding the biosynthetic pathway of the central metabolic cofactor coenzyme A (CoA)[3], as well as other enzymes that play a role in maintaining the redox balance in the human pathogen S. aureus. The Lab makes use of a number of chemical biology tools to develop novel agents for the selective inhibition of both drug-sensitive and drug-resistant strains of S. aureus by targeting enzymes involved in the biosynthesis and utilisation of CoA[4] and other enzymes associated with its resistance to oxidative stress.

While we would be keen to develop a new antimicrobial compound that could one day be used in a clinical setting, our current primary goal is to see if we can use small molecules to modulate the survival of S. aureus in the host–pathogen interface,” says Professor Erick Strauss, Group Leader at Stellenbosch University’s Strauss Laboratory in South Africa. “Ideally, such compounds would work synergistically with the body’s natural defences to ward off infections, thereby reducing the likelihood of antimicrobial resistance arising.”

“Through Diamond’s X-ray structure-accelerated, synthesis-aligned fragment medicinal chemistry facility, under guidance from GCRF START Co-Investigator, Prof Frank von Delft, we have been able to fast-track the identification of novel compounds that we are currently pursuing further as promising leads of such modulators of the survival of S. aureus”, adds GCRF START-funded Postdoctoral Research Fellow, Dr Blake Balcomb. “This is the first time that researchers from Stellenbosch University have used this cutting-edge technology in drug discovery initiatives.”

Diamond’s XChem workflow is geared towards automation and high-throughput screening of hundreds of compound fragments with automated structure determination of the protein of interest with each individually bound fragment, which is what enables a process that could normally take months to be fast-tracked to take just a few days.

“To explain the process in simple terms, it is like the protein represents a sponge and one by one individual compounds would be soaked into the sponge and only those compounds that have a natural propensity or chemical attraction to bind to the sponge would remain bound,” says Balcomb. “If one had to do this manually one by one it would take several months to go through each of the compounds and investigate if the compound fragment is bound to the protein of interest and, importantly, what orientation it is bound. With XChem one can obtain data and final results within a week!”

Aerial view of Diamond Light Source Ltd, Harwell Campus, UK. ©Diamond Light Source

Successful experiments and new collaborations

Researchers in the Strauss Laboratory have completed two successful XChem experiments on two separate S. aureus enzymes. One enzyme is involved in the biosynthesis of CoA and a second enzyme is involved in detoxification of a host derived antimicrobial. Through XChem they have collected data on >800 crystals and obtained >150 novel crystal structures all containing different fragment compounds.

“To mitigate the usual slow progression of fragment hits to promising drug leads we are pursuing two hits in two separate approaches,” explains Prof. Strauss. “The first approach, also developed by the Frank Von Delft team, is similar to the XChem workflow and is optimized and streamlined for automation and high-throughput analysis. This technique makes use of an Opentrons pipetting robot[5], which is used to perform automated multi-step parallel syntheses in a checkerboard format, therefore allowing one to do combinatorial synthesis and to make many similar compounds at the same time. The added benefit is there is no need to purify the end-products, as these are soaked directly into crystals of the target enzyme to determine their inhibition potential. These experiments again would feed back into the XChem workflow.”

Fig 1. XCHEM Pipeline. ©Diamond Light Source[6]

In a second approach, the team has initiated a new collaboration with Dr Nir London at the Weizmann Institute of Science to develop compounds that target proteins covalently (to form an irreversible attachment to proteins). This approach is also based on a high-throughput setup that screens several fragments that contain specific reactive groups. The results of the most reactive fragments are then again fed back into the XChem workflow, whereby one would be able to visualise the compound-protein complex. All of these findings help aid in the development of potent and specific compounds that could be assessed further in the drug discovery pipeline and in turn, the discovery of novel antimicrobials.

In addition to Diamond’s XChem facilities, the Strauss Laboratory also has regular access to Diamond’s MX instruments through a dedicated South African BAG . This BAG access is a highly effective means in which a consortium of other scientists can work together to share a full beamtime shift for data collection. Through the BAG access the Strauss Laboratory, has on average, sent three to four shipments of samples per year to Diamond, and has so far solved eight novel crystal structures.

Capacity building and peer training in structural biology

Alongside the cutting-edge science, the GCRF START grant has enabled the Strauss Laboratory to invest in training and capacity development in techniques like fragment screening. Dr Blake Balcomb, formally trained in structural biology, and Dr Anton Hamann originally trained as an organic chemist but now a GCRF START grant funded Postdoctoral Fellow skilled in protein X-ray crystallography, have participated in two separate CCP4 workshops as well as training on the use of XChem facilities.  These skills they pass on in their research group, as well as cascade to other research groups at Stellenbosch that show interest in structural biology as a research tool.

The scientists have, in addition, transferred skills to several students and Postdoctoral students in the Strauss Laboratory, including Dr Koketso Mogwera, who is now a Post-doc at H3D at the University of Cape town (UCT), Tim Kotze (PhD), Warrick Sitzer (MSc), Karli Bothma (MSc) and Nicholas Herbert (BSc Hons), now an MSc. student at the AHRI.  

Dr Koketso Mogwera at the Strauss Laboratory, Stellenbosch University, South Africa. Photo credit: Blake Blacomb. ©Diamond Light Source

The GCRF START grant has also enabled the Strauss Laboratory to call on expertise and initiate new collaborations with scientists in other African universities. For example, MSc student Warrick Sitzer is currently investigating the structure of one of the enzymes involved in CoA biosynthesis using CryoEM and is co-supervised by GCRF START Co-I, Dr Jeremy Woodward, from the University of Cape Town.

Warrick Sitzer reports below on the training and mentoring he received,

As an MSc. student, learning about CryoEM has given me a deeper appreciation on how it is used in structural biology research as opposed to other major techniques in the field such as X-ray crystallography and NMR spectroscopy. I find that the scientific progress made in detector technology and software algorithms to unravel difficult or complex biomolecular structures at near atomic resolution without the need of crystallization to be very exciting. The ability to determine these biomolecular complexes in their native state opens up a door to a room full of possibilities that may contribute significantly to structure-based drug discovery.”

Describing the impact that GCRF START has had on his research, Anton said,

The GCRF START grant has opened a new field for me in medicinal chemistry and has introduced me to protein crystallography. Through the GCRF START grant, I had the privilege to carry out crystallographic fragment screening experiments at Diamond’s XChem facility, which has accelerated my research in developing novel antibiotics for S. aureus. Thanks to START, I am now a better and more developed medicinal chemist.

Commenting on the capacity building achievements supported by the GCRF START grant, Frank von Delft said,

“While a lot of Diamond’s facilities for macromolecular crystallography can be offered through remote access, our XChem facility must be attended in person; and it is only thanks to the GCRF START grant that those persons include South Africans. What’s been equally exciting is seeing those researchers being able to use those results to accelerate not only their science, but their careers.”

GCRF START PDRA, Blake Balcomb, at Stellenbosch University in South Africa with Master’s student Karli Bothma. ©Diamond Light Source

Read about the UN’s Sustainable Development Goals for Health and Wellbeing here

[1] Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species

[2] Front Microbiol. 2019 Apr 1;10:539. doi: 10.3389/fmicb.2019.00539. eCollection 2019

[3] Strauss E. Coenzyme A Biosynthesis and Enzymology, in Comprehensive Natural Products II Chemistry and Biology Vol. 7 (Eds. Lew Mander & Hung-Wen Liu) 351-410 (Elsevier, 2010).

[4] Front. Cell. Infect. Microbiol., 15 December 2020 | https://doi.org/10.3389/fcimb.2020.605662

[5] https://www.opentrons.com/

[6] Source: https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening/The-XChem-Pipeline.html