Cryle Lab research
About Associate Professor Max Cryle
In addition to his position in the Infection and Immunity Program at the Monash Biomedicine Discovery Institute, Associate Professor Max Cryle is an EMBL Australia Group leader in the Victorian Node, based in the Department of Biochemistry and Molecular Biology at Monash University and an associate investigator at the Australian Research Council Centre of Excellence in Advanced Molecular Imaging. After obtaining his PhD in chemistry from the University of Queensland in 2006, he moved to the Max Planck Institute for Medical Research in Heidelberg as a Cross Disciplinary Fellow of the Human Frontiers Science Program. He was subsequently awarded funding from the German Research Foundation (Deutsche Forschungsgemeinschaft) to establish his own group to investigate glycopeptide antibiotic biosynthesis as part of the Emmy Noether program. His group works at the boundary of chemistry and biology, where they apply a multidisciplinary approach including synthetic chemistry, biochemistry, structural biology and enzyme catalysis. In 2016 he joined EMBL Australia to continue his research into understanding the biosynthesis of important natural antibiotics and developing new antimicrobial agents, with his work supported by an NHMRC Career Development Fellowship since 2018.
1. Exploiting biocatalysis and chemical synthesis to generate new antibiotics
2. Redesigning antibiotic biosynthesis to enable production of modified antibiotics
3. Overcoming antimicrobial resistance by exploiting novel strategies to kill superbugs
Visit Associate Professor Cryle’s Monash research profile to see a full listing of current projects.
My group focus on antibiotic discovery: specifically, how to make new antibiotics to overcome the severe threat posed to modern medicine by antimicrobial resistance (e.g. MRSA).
To do this, we adopt a multi-disciplinary approach with a focus on the glycopeptide antibiotics (GPAs), which include the last resort clinical drugs vancomycin and teicoplanin. As these are highly complex molecules, as a society we depend on biosynthesis for their production. Thus, generating new variants of GPAs relies on our ability to understand and exploit the natural biosynthesis machinery, which centres on a fascinating enzymatic peptide assembly line known as a non-ribosomal peptide synthetase (NRPS) and the cyclisation of this NRPS precursor peptide into a rigid, active antibiotic through the action of Cytochrome P450 monooxygenases. My group investigates these enzymatic systems using a combination of approaches (chemical synthesis, structural biology, biochemistry, enzymatic catalysis & protein engineering) in order to reengineer these and produce new, more effective antibiotics and to exploit these enzymes as biocatalysts. Furthermore, my group is using our expertise with GPAs to identify new cellular targets for novel antimicrobial therapies as well as developing novel approaches to treat deadly antibiotic-resistant bacteria such as the ESKAPE pathogens, with our current focus on MRSA.
Figure 1. The structures of the two natural glycopeptide antibiotics in clinical use (A) as well as the general structures of other members of this antibiotic class (B). All these compounds are produced in bacteria and purified before their use in medicine.
Antibiotics are one of the most important discoveries in human health. These compounds – largely derived from compounds found in nature – have enabled many aspects of modern medicine that we call upon today. However, the early success of antibiotics has led to a serious underinvestment in identifying new antibiotics and antimicrobial targets: this means that we as a society are in dire need of new antibiotics. One of the difficulties in achieving this goal is that antibiotics are often highly complex molecules and we are restricted to natural compounds or modified forms of these compounds. If we are to develop new antibiotics, we will need to reengineer the natural enzymatic machinery that produces antibiotics – and this will only be possible once we understand the how these complex enzymatic machineries work.
Figure 2. Examples of effective – yet structurally complex – peptide-based antibiotics that all are produced naturally through non-ribosomal peptide synthesis.
Thus, we aim to understand the biosynthesis of one of the most important classes of clinically relevant antibiotics – the glycopeptide antibiotics, such as teicoplanin and vancomycin – in order to be able to reengineer this machinery and thus produce novel antibiotics. We use a range of techniques, including synthetic chemistry, biocatalysis, biochemistry and structural biology, to study these systems. The group also applies these techniques to develop novel antibiotic compounds and explore potential new targets for treating bacterial infections: here, with a focus on the serious bacterial pathogen Staphylococcus aureus.
Ultimately, this research will enable the development of novel antibiotic therapies as well as unlock new pathways to treat serious bacterial infections in the clinic.
Antibiotic Biosynthesis via Megaenzyme Synthases
Non-ribosomal peptide synthesis is one of the major sources of medically active compounds, in particular compounds with antibiotic activity. Non-ribosomal peptide synthesis is performed by very large, modular enzymatic assemblies known as non-ribosomal peptide synthetases (NRPSs), which are able to synthesise peptides completely independently of the ribosome.
Figure 3.Non-ribosomal peptide synthesis exemplified by the biosynthesis of the glycopeptide antibiotic teicoplanin. During peptide synthesis, each module of the peptide assembly line inserts one amino acid into the growing peptide chain, which is passed down the assembly line from left to right. Once the peptide is on the final module, this is then made into its final, active conformation by the actions of the Oxy enzymes and removed from the assembly line to be further modified into its final structure. Each of the circled letters in the assembly line represent one individual enzymatic domain, making the final peptide synthesis machinery very large and complex.
This allows NRPSs to synthesise peptides from a much greater pool of building blocks than standard protein synthesis and also allows NRPS machines to generate cyclic compounds, compounds with unnatural stereochemistry and a further array of modifications. Given that many current antibiotics are either natural products produced by NRPS machines in bacteria or modified forms of the natural compounds, there is great interest in understanding the biochemical and structural basis for peptide synthesis by NRPS machines. This is particularly due to the possibility of reengineering these molecular assembly lines to enable the production novel antibiotics. However, to date such reengineering attempts have had limited success, which stems largely from our incomplete knowledge of these complex systems.
Figure 4. An example of how non-ribosomal synthesis works in a modular fashion. Here, the machinery that makes teicoplanin is divided into the 7 modules on 4 separate proteins (Tcp9-12). The largest protein, Tcp11, is the shown divided into the different enzymatic domains that make up the protein. Repeating domains are found in each module, with the A-domains responsible for amino acid selection, C-domain responsible for peptide bond formation and CP-domains acting as carriers to move the peptide intermediates to all the different catalytic domains during synthesis. The selectivity of each catalytic domain and the addition of option domains (such as E-domains) then leads to the tremendous variety of non-ribosomal peptides observed, with one specific peptide made by one assembly line.
My group uses in vitro techniques to understand the biosynthetic machinery behind the glycopeptide antibiotics (GPAs), which includes the clinical drugs vancomycin and teicoplanin. Our interest in GPAs is due both to our reliance on their commercial production via in vivo bacterial biosynthesis and the complex nature of their biosynthesis. We have made a number of significant discoveries concerning GPA biosynthesis and are continuing our structural and biochemical investigations into these systems, with the overall goal of being able to redesign this machinery to produce novel compounds for commercial application through their production in bacteria.
Beyond GPA biosynthesis, we are also exploring interesting examples of non-ribosomal peptide synthesis to identify novel catalytic potential in these machineries. This includes the identification and characterisation of novel biosynthesis pathways, the structural analysis of important catalytic complexes and even the analysis of examples of such systems from higher eukaryotes. An example of this work is our recent characterisation of a novel domain from a non-ribosomal peptide synthesis pathway that plays an important role in neurotransmitter regulation in Drosophila.
Figure 5. Recruitment of essential Cytochrome P450 enzymes that perform the side chain crosslinking during the biosynthesis of the glycopeptide antibiotics is mediated by the X-domain. We have shown, using a combination of chemical, biochemical and structural approaches that this recruitment process is essential for the cyclisation of glycopeptide antibiotics to occur, and more importantly that we can then use this knowledge to make and test new antibiotics in the laboratory.
Development of Biocatalysts
Cytochrome P450 enzymes (P450s) are highly powerful oxidative enzymes that perform a wide range of oxidation reactions in nature. P450 are able to combine their ability to catalyse chemically challenging transformations together with high degrees of regioselectivity and stereoselectivity, which has made these enzymes of great interest as potential biocatalysts. During our investigations into P450s involved in antibiotic biosynthesis, we have uncovered several examples where these P450s show great potential for use as biocatalysts to perform synthetically challenging transformations both in vitro and in vivo. We are pursuing the development of several of these routes to produce new medically important bioactive compounds.
Figure 6. Exploration and exploitation of Cytochrome P450s from glycopeptide antibiotic biosynthesis to perform in vitro peptide crosslinking – our established pipeline for producing novel compounds using the combination of enzymes and chemical synthesis (left). Some examples of modified antibiotics that can be produced using this pipeline that are currently undergoing testing (right).
Selective and Controlled Protein Modification
Being able to control the modification of proteins and to target these modifications selectively is key to investigating protein function both biochemically and structurally. In particular, the ability to introduce non-standard groups such as fluorophores, trap specific intermediate states and to control protein states using external signals has been demonstrated to be of great importance in these endeavours. My group is interested in developing and applying tools to allow for the generation of specifically labelled proteins and protein fusions in vitro. This encompasses the use of selective protein modification and ligation via chemical or enzymatic means, as well exploitation of enzymes responsible for inserting post-translational protein modifications and the incorporation of unnatural amino acids directly into proteins via stop codon suppression. We apply these tools in house to understand complex antibiotic biosynthesis pathways and help other groups to apply these techniques to other complex enzymatic systems.
Antibiotic Development and Target Identification
Antibiotics are arguably one of the greatest achievements in modern medicine and underpin the majority of current clinical therapies. However, the rapid development of bacterial resistance to most of our available antibiotics has left society facing the very real prospect of a return to a pre-antibiotic era. We are in desperate need of new antibiotic agents as well as new targets for antibiotic therapy - this is especially important for serious pathogens such as Staphylococcus aureus, which now has developed into strains that are resistant against virtually all of our available therapies. We are seeking to develop new antibiotics that have novel mechanisms to overcome serious bacterial infections and are also engaged in identifying new targets to help us overcome these bacterial infections. Our work is also interested in exploring the interactions of antibiotics and their bacterial targets together with the effects that these have on host immune function, with the goal of overcoming the ability of bacteria to evade our immune system and allow more rapid resolution of bacterial infections.
Figure 7. Imaging of one of our novel antibiotic compounds binding to its target – the bacteria MRSA.
Our group utilises many techniques, with our research combining aspects of chemistry, biochemistry, structural biology, immunology and microbiology. Specific examples of this includes:
-synthesis of chemical probes
-solid phase peptide synthesis
-protein expression and purification
-targeted protein modification
-enzymology and biocatalysis
-protein x-ray crystallography and cryo-electron microscopy
-immune cell recruitment and imaging
-whole cell bacterial assays
Our work targets bacterial pathogens, with our current focus on MRSA. We work closely with Professor Anton Peleg to utilise his unique collection of clinically resistant bacterial strains to allow us to specifically target the most relevant and challenging clinical pathogens for antibiotic development.
We collaborate with many scientists and research organisations around the world. Some of our more significant national and international collaborators are listed below. Click on the map to see the details for each of these collaborators (dive into specific publications and outputs by clicking on the dots).
Dr PD Evi Stegmann - Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Germany
Professor Roderich Süssmuth - Institute of Chemistry, Technische Universität Berlin, Germany
Professor Søren Bak - Department of Plant Biology and Biotechnology, University of Copenhagen, Denmark
Professor Nadine Ziemert - Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Germany
Professor Gerry Wright - Department of Biochemistry and Biomedical Sciences, McMaster University, Canada
Professor Greg Challis - Department of Chemistry, Warwick University, United Kingdom
Assistant Professor Louise Charkoudian - Department of Chemistry, Haverford College, United States of America
Dr Manuela Tosin - Department of Chemistry, Warwick University, United Kingdom
Professor Daniel Irimia - Harvard Medical School, United States of America
Professor Anton Peleg, Department of Microbiology, Monash University/ Alfred Hospital
Professor Richard Payne, Department of Chemistry, University of Sydney
Associate Professor Colin Jackson, Department of Chemistry, Australian National University
Professor James De Voss, School of Chemistry and Molecular Biosciences, University of Queensland
Associate Professor Stephen Bell, School of Chemistry & Physics, University of Adelaide
Student research projects
The Cryle Lab offers a variety of Honours, Masters and PhD projects related to neuroscience and natural drug screening for students interested in joining our group. There are also a number of short term research opportunities available.
Please visit Supervisor Connect to explore the projects currently available in our Lab.