The objectives of FragNet are to (a) train a cohort of ESRs across FBLD methods and (b) develop individual skills in research into either new methods in FBLD or to apply FBLD to interrogate biological systems.
Download the full description of this project: ESR1: 3D Fragments with small aliphatic rings
3D Fragments with small aliphatic rings
Fragment screening libraries and high-throughput screening libraries both have an overwhelming prevalence of two-dimensional molecules containing planar aromatic rings. It is thought that the introduction of more 3D-character into a library may make it possible for screening compounds to interact with more complex targets such as PPIs or other as yet-unknown target classes. As the four-membered cyclobutyl motif is by far the most underrepresented of the small aliphatic ring structures, it offers an unexplored niche in the pursuit of 3D fragments.
The aim of this project is to develop methodology towards the design and synthesis of novel 3D fragments – with a particular focus on the cyclobutyl motif. This chemistry is then used to enumerate a chemically and spatially diverse library of 3D fragments containing four-membered rings.
Our first approach involves enaminone intermediates which can be furnished further prior to or following reduction to the corresponding amino alcohol. Our second approach involves the generation of enone intermediates followed by the addition of polar groups via nucleophilic addition and subsequent reduction. Both approaches begin with acyclic precursors and generate small highly water-soluble fragments – which, unlike the lipophilic hits that are more common after/during High-Throughput Screening, are ideal starting points for drug-discovery cascades.
Once a library of fragments has been synthesised, the molecular shape of its members will be analysed using principle moment of inertia plots (PMI) – a measure of 3D shape. We hope to show that we can supply a novel three-dimensional fragment library that gives the medicinal chemist access to uncharted chemical space, and make these compounds available for screening against an array of different targets.
The FragNet consortium offers a wealth of networking opportunities, which in the context of this project has produced two ongoing collaborations. The first, with academic partner RCNS Hungary, focuses on the design of electrophilic warheads for covalent approaches to drug discovery. The second, with industrial biotech partner ZoBio, Netherlands, is a more MedChem-based project that aims to develop a screening hit against HSP70 into a potent lead. In this project, ESR1 offers synthetic and design contributions, whilst fellow FragNet colleagues ESR5 and ESR11 perform biophysical/biochemical screening support and computational aid, respectively.
Publications and Dissemination:
• “Enone-based cyclobutyl fragment library design and synthesis, screening results and hit elaboration” – ULTIMATE Project, European Union’s Horizon 2020 research and innovation programme under grant agreement No. 777828. Ongoing work.
• “Enaminone-based cyclobutyl fragment library design and synthesis, screening results and hit elaboration.” Ongoing work. • “Potential of cyclobutenaminone scaffolds as electrophilic warheads for covalent-binding approaches to drug discovery.” Ongoing work.
• “Identification and optimisation of allosteric inhibitors targeting the molecular chaperone HSP70.” Ongoing work.
• Lectured to 2nd year BSc Pharmaceutical Sciences students and provided research content for synthesis practical courses.
• Poster presentation at the national CHAINS conference in Veldhoven, Netherlands (December 2018)
• Poster presentation at the Fragments 2019 conference in Cambridge, UK (March 2019)
Download the full description of this project: ESR2: Novel 3D fragments
The applicant will join a team working on the design, synthesis and assessment of novel 3D fragments. The focus of the project is on chemical synthesis with opportunities to explore aspects of cheminformatics (for analysing compounds), molecular modelling (how the compounds bind to proteins) and experimental fragment screening.
1. Design of 3-D fragment library.
2. Synthesis of selected fragments.
Most of the compounds in fragment libraries[1, 2] are commercially available small molecules which have been selected by medicinal chemists based on their experience on ease of synthesis and what they have seen before in existing drugs. This means that many fragments are flat heterocycles. This has worked well for many proteins that bind to and recognise metabolites such as ATP, but may not be ideal for other proteins, such as those that bind carbohydrates. There are also analyses which suggest that more 3D compounds have better properties as drugs. One of the major issues with such compounds is they contain multiple stereo-‐centres which makes synthesis to improve the compounds more challenging. Some of the synthetic chemistry developed at York provides a way to achieve this. This project builds on recent work in the O’Brien laboratory (aided by cheminformatics analysis by the Hubbard group) to design and synthesise novel 3D lead-‐like compounds. The compounds will be designed based on common features of drug molecules and some of our 3-‐D fragments are shown below. Principal moments of inertia (PMI) plots, which are a representation of 3-‐D space, will be used to evaluate the designed compounds and selection criteria will be developed to identify compounds. Selected compounds will then be synthesised.
The skills required are an interest and aptitude for compound synthesis; the amount of time spent outside of the synthetic laboratory (on modelling or experimental screening) will depend on the interests of the successful applicant.
1. Doak et al. http://dx.doi.org/10.1071/CH13280, 2013.
2. Baurin et al. J Chem Inf Comput Sci, 2004. 44, 2157-66.
3. Lovering et al. J Med Chem, 2009, 52, 6752-6.
4. Luthy et al. Bioorg Med Chem, 2015, 23, 2680-94.
Host: RCNS, Hungary (PhD enrolment at Budapest University of Technology and Economics)
Academic supervisor: Prof. dr. György M. Keserű (RCNS)
Researcher: Aaron Keeley
Download the full description of this project: ESR3: Warhead Library of Covalent Fragment Binders
The starting points of FBLD studies are highly curated collections of small, chemically diverse and highly soluble fragments. To increase the diversity of the available fragment libraries, ESR3 will design and synthesise a reactive ‘warhead’ library and establish techniques for screening covalent binders against several FragNet protein targets, including kinases.
Designing and creating a compound library of fragment sized molecules with reactive warheads: “warhead library”. 2. Establishing techniques for efficient detection of covalent binders in a screening setup. 3. Screening of “warhead” library and virtual hits identified by ESR9 against various protein targets, e.g., Janus kinases. 4. Extending covalent binders into lead-‐like compounds.
By using synthetic organic chemistry, computational chemistry and (structure-‐based) drug design, a general fragment library for screening of covalent ligands will be created. The identification of covalent inhibitors for therapeutically relevant proteins, including Janus kinases, will be explored.
Preparative organic chemistry or theoretical organic chemistry or chemical biology knowledge and lab experience, analytical or bioanalytical background will be beneficial.
1. Jöst et al. J. Med. Chem. 2014, 57, 7590-7599
2. Mark et al. J. Med. Chem. 2014, 57, 10072-10079
3. Baskin et al. PLoS ONE 2014, 9(8), e105568.
Download the full description of this project: ESR4: Development of FBLD techniques for Intrinsically Disordered Proteins
FBLD technologies are continuously being improved to capture new opportunities. ESR4 will explore Intrinsically Disordered Proteins (IDPs) and Intrinsically Disordered Regions (IDRs). The significance of these proteins has recently become apparent. This project will begin to evaluate the possibility of using FBLD to develop small molecule ligands that bind to IDPs and IDRs, and modulate their folding and function.
1. Evaluate literature studies of ligands binding to intrinsically disordered proteins (IDPs).
2. Identify suitable IDP test system(s) with tractable expression, purification, stability and behaviour in aqueous solution.
3. Evaluate and develop fragment based screening (FBS) methods to identify fragments which bind to the IDP.
4. Validate and evolve initial fragments to show enhanced potency and characterise response of IDP to these ligands.
A number of IDP-‐ligand interactions have been identified in the literature, and assessment of these will provide both a key initial dataset and a valuable training in the biophysical techniques and approaches used to study protein-‐ligand interactions. Evaluation of the suitability of one or more IDPs for FBLD approaches will also provide a robust dataset, since any outcome will be of interest. Once the experimental methodology has been tested on the literature IDP systems, they will applied against one or more tractable IDP or IDR systems, identified either from the literature or through collaborations (for example, with research groups at the University of York who are investigating the structural biology of disease-‐related IDPs). The experimental methodology will then be extended to screen low affinity fragments for binding to this tractable IDP system, and to characterise the structural and kinetic basis for observed fragment: IDP interactions. Fragments which are determined as validated ligands for the IDP will then be explored through well-‐described FBLD evolution strategies, such as near neighbour analysis and template morphing, in order to enhance the affinity of the ligand: IDP interaction and if possible to characterise the response of the IDP to ligand binding
An MSc degree in Chemistry, Biochemistry, Biophysics or Molecular Life Sciences is required. Expertise in protein expression and/or protein biophysics is required, along with a keen interest in the protein folding and molecular interactions. The ability to work independently as part of a small team is essential, along with strong communication and interpersonal skills. Previous experience of NMR would be an advantage.
1. Tompa et al. 2015 Curr. Opin. Struct. Biol. 35, 49–59.
2. Follis et al. 2008 Chem. Biol. 15, 1149–55.
3. Krishnan et al. 2014 Nat. Chem. Biol. 10, 558-66.
Download the full description of this project: ESR5: Biophysics Based FBLD
FBLD technologies are continuously being improved to capture new opportunities. This project will investigate emerging antimicrobial targets with state-‐of-‐the-‐art biophysical screening technologies.
Use NMR and SPR to screen a fragment library for validated hits against the target protein. 2. Develop NMR structural biology approaches to enable structure based drug design to elaborate hits to potent lead-‐like molecules. 3. Collaborate with the medicinal chemistry group of Prof. Iwan de Esch to design, synthesize and test elaborated hits.
This project will seek to develop inhibitors of critical bacterial and/or viral enzymes. In order to do so we will first concentrate on expressing the target in E. coli in a form that is suitable for biophysical and structural biological work. The recombinant protein will be used to screen for ligands specific for the target using ZoBio’s proprietary, NMR-‐based TINS technology and SPR. The structure of validated hits from this effort bound to the target will be elucidated using protein observed NMR methods. Collaboration with other Fragnet members will bring the possibility to use X-‐ray crystallography as well. The structural information will be used to design compounds with better potency and ligand efficiency in collaboration with the medicinal chemistry group of Prof. Iwan de Esch. We expect to develop novel compounds that have biological activity in anti-‐bacterial or anti-‐viral assays.
A strong bachelors background in chemistry and physical chemistry is important. The successful applicant will have demonstrated some ability to recombinantly express and purify proteins. Any previous experience with NMR, either theoretical or practical, would be a help.
1. van Linden et al. Eur. J. Med. Chem. 2012, 47, 493-500.
2. Vanwetswinkel et al. Chem. Biol. 2005, 12, 207-216.
3. Shah et al. J. Med. Chem. 2012, 55, 23, 10786-10790.
Download the full description of this project: ESR6: FBLD experimental methods
FBLD technologies are continuously being improved to capture new opportunities. This project will study the use of biosensor-‐based technologies to study ligand-‐protein binding events.
Develop biosensor-‐based assays for epigenetic target proteins interacting with histones. 2. Screen proprietary FragNet fragment libraries against selected target proteins. 3. Characterize fragment hits using same biosensor-‐based methods and orthogonal assays. 4. Use experimental data in computer-‐assisted drug design. 5. Optimise fragment hits.
New biosensor instruments and methods will be used for development of highly sensitive and informative assays suitable for epigenetic targets that interact with and modify histone proteins. The methods will address the challenges associated with detection of weakly interacting small molecules (fragments) and will be focused on distinguishing ligands with a functional effect from binders that simply interact with the protein. The assays will be designed for direct or indirect detection of fragments that can directly block interactions with the protein substrate/binding partner or that have enough binding energy to induce the required conformational changes for allosteric inhibition of protein-‐protein interactions. Biophysical methods will be developed for identifying ligand binding sites, i.e., binding to the protein-‐protein interaction surface (corresponding to the active site for non-‐enzyme targets) or an allosteric site. Computational studies of hits will be performed as a complement to experimental studies, with a focus on identifying potential binding sites, binding modes and interaction features of weakly interacting ligands. The design of any new ligands can be supported by computer-‐aided drug design studies and synthesis will be performed in collaboration with other ESRs.
Required diploma: MSc degree in Biochemistry, Biophysics or related Molecular Life Science degree. Required expertise: Experience in biochemical and/or biophysical characterization of proteins. The candidate has a strong background in biochemistry or biophysics, and has experience in variety of methods for producing proteins and characterizing their structural and functional properties. Recommended expertise: Use of advanced biophysical instruments and development of new biochemical and biophysical assays. An interest in computer-‐aided drug design and mathematical modelling and statistical analysis of biochemical data would be an advantage. The candidate needs to be able to discuss and develop methods in collaboration with other ESRs.
1. Winquist et al. Biochemistry, 2013, 52, 613-626.
2. Gossas et al. Med. Chem. Commun, 2013, 4, 432 – 442
3. Seeger et al . Journal of Molecular recognition 2012, 25, 495–503.
4. Geitmann et al. J. Med. Chem. 2011, 54, 699-708.
5. Elinder et al. J. of Biomolecular Screening, 2011, 16, 15-25.
Download the full description of this project: ESR7: Understanding PDE binding kinetics
This project will investigate of binding kinetic while growing hit fragments, building on interesting data that has already been generated for Trypanosoma brucei Tbr-PDE ligands. The project will combine design, synthesis and molecular dynamics studies to unravel the molecular features of structure-kinetics relationships.
1. Use SPR biosensors to measure the differences in binding kinetics of ligands for human hPDE4 and parasite TbrPDE proteins.
2. Perform Random Acceleration Molecular Dynamics (RAMD) studies to determine ligand access and egress mechanisms and factors determining the kinetics of ligand binding to PDEs.
3. Guide fragment hit growing to develop optimised compounds with well-‐defined kinetic selectivity profiles.
Trypanosoma brucei (Tbr) is the causative parasite of human African trypanosomiasis (HAT), also known asAfrican sleeping sickness, a disease that has been grossly neglected as it is only a problem in the poorest areas of Africa. Tbr-‐PDE enzymes are validated drug targets to treat this illness. These Fragnet studies will lead to a better understanding of kinetic binding properties of Tbr-‐ PDE ligands. This molecular understanding can be used to achieve kinetic selectivity and thereby create safe and efficient drugs for this neglected disease.
AApplicants must have a background in molecular modelling (including molecular dynamics studies). Experience with organic synthesis or SPR biosensor instruments would be an advantage. The project can be fine-‐tuned according to the background and interests of the successful candidate.
1. Jansen et al. J. Med. Chem. 2013, 56, 2087-2096.
2. Orrling et al. Discov Today. 2012, 55, 8745-8756.
Host: RCNS, Hungary (PhD enrolment at Budapest University of Technology and Economics)
Academic supervisor: Prof. dr. György M. Keserű (RCNS)
Researcher: Andrea Scarpino
Download the full description of this project: ESR8: Virtual Screening of Fragment Libraries of Covalent Binders
Design of computational protocols for targeted covalent inhibitors
Covalent drug discovery has recently received increased interest thanks to the definition of guidelines for the design of safe and potent targeted covalent inhibitors . As in conventional discovery programs, considerable attention is devoted to optimizing the selectivity of a compound to the protein target, while simultaneously tailoring the chemical reactivity of the electrophile (also known as the “warhead”) to the specific nucleophilicity of the targeted residue .
A key role in the design and characterization of compounds acting via a covalent mechanism of action is played by computer-aided drug design (CADD) approaches. Quantum mechanics (QM) simulations can be used to inspect the energy profile of covalent fragments equipped with different warhead groups, thus providing insight into their relative reactivity . Due to the correlation between calculated and experimental reactivity descriptors within specific warhead classes, QM simulations can be used as predictive tools for prioritizing the selection of covalent fragment candidates for further optimization.
In parallel, molecular docking is used to predict the binding mode adopted by compounds in a pocket, and to rank ligands in a set by means of scoring functions devised to capture their relative binding affinity. Although a number of covalent docking methods are available to model the structural changes occurring with covalent ligands, the scoring functions they use do not consider the contribution of covalent bond formation to binding energy, thus neglecting the gap between warheads with different reactivity .
Andrea Scarpino’s research focuses on the design of computational protocols featuring conceptually distinct covalent docking algorithms. By evaluating the ability of these algorithms to predict experimental binding modes of several covalent complexes available in the Protein Data Bank, he highlighted the key factors influencing their docking performance and provided guidelines to improve the prediction based on the system being studied .
He is also exploring alternative docking and scoring solutions for covalent binders. By customizing an open-source docking protocol (AutoDock4) to account for differences in warheads’ reactivity, he is developing methods to enable virtual screening of fragment libraries that include mixed chemistries.
As well as protocol development, Andrea performs virtual screenings of commercial libraries to identify covalent compounds that act on targets of pharmacological relevance (e.g., proteases, kinases and GTPases) [6,7]. Overall, by providing the computational support necessary to tackle the challenges raised by covalent drug discovery, he contributes to the projects of the Medicinal Chemistry Research Group at RCNS, Budapest.
• Singh, J. et al., Nature Rev. Drug Discov. 2011, 10, 307–317.
• Lonsdale, R. et al., J. Chem. Inf. Mod. 2017, 57, 3124-3137.
• Flanagan, M. E. et al., J. Med. Chem. 2014, 57, 10072-10079.
• De Cesco, S. et al., Eur. J. Med. Chem. 2017, 138, 96-114.
• Scarpino, A. et al., J. Chem. Inf. Model. 2018, 58, 1441-1458.
• Rachman, M. et al., ChemMedChem 2019, 14, 1011.
• Scarpino, A. et al., Molecules 2019, 24, 2590.
Download the full description of this project: ESR9: Fragment evolution platform - chemical navigation
An Automated Computational Platform for Fragment to Lead Optimization
To automate fragment to lead optimization, this project uses a computational platform, FrEvolution. The developed fragment evolution platform can be seen as a very unique virtual screening (VS) application as it is performed iteratively. In every iteration, similar analogues of a fragment that are slightly larger are screened. Based on several orthogonal structure-based approaches, the most likely to bind analogues are retrieved. From these, the chemical space is determined for the next round of VS, by again finding similar analogues that are slightly larger. The three orthogonal structure-based approaches are docking with rDock to determine a plausible binding mode where the fragment core is maintained, MMGBSA with Schrodinger to account for solvation effects, and the in-house developed method, Dynamic Undocking, to assess the robustness of key hydrogen bonds. All calculations are performed in parallel, for optimum efficiency.
The platform was used within the FragNet consortium, the Barril Lab and externally, as a tool to progress on-going projects. Furthermore, development of the platform led to the following projects:
Comparing Virtual Fragment Growing Strategies (in collaboration with XChem) - The platform was validated on the NUDT21 protein by comparing it with a standard VS. Based on experimental (SPR) data, the iterative VS approach gave higher hit rates, more ligand efficient compounds and more diversity.
Efficiently Exploring Accessible Chemical Space - As the platform has been optimized to be efficient, it can screen up to hundreds of millions of compounds. The effects of using a larger chemical space with the platform were studied for another fragment found to bind NUDT21. Docking scores suggested that using a larger chemical space, better compounds can be found. The compounds will be purchased and tested by SPR.
Fragment Growing from Multiple Binding Modes - This project aims to rescue fragment hits that are found to bind, but for which no binding mode could be obtained by crystallography, by using the platform to automatically evolve fragments from viable predicted binding modes. Four case studies for HSP90, BRD4-BD1 and DYRK1A kinase of fragments were used and experimental (DSF and NMR) results showed binding for evolved compounds from each predicted binding mode.
In addition to the main project, the following side projects were conducted:
1. At the Research Center for Natural Sciences in Budapest.
• Scaffold Hopping with a Novel Hinge Binding Fragment Discovered through Virtual Screening
• DUckCov: a Dynamic Undocking-based Virtual Screening Protocol for Covalent Binders
2. At the Uppsala University:
• Binding pocket assessment and fragment growing with FrEvolution for SMYD3 methyltransferase.
Download the full description of this project: ESR10: Fragment evolution platform – molecular simulations
Hydrogen Bonds as determinants of structural stability: implications for ligand design
The aim of the project is to develop new computational approaches for structure-based drug design. These tools will also be used to investigate new properties of macromolecular complexes and their role in molecular recognition.
Our methods rely on a fundamental property of protein-ligand complexes that has been neglected in drug design so far: structural stability, which describes the resistance of the system to structural changes. Thanks to sharp distance and angular dependencies1, stability can be provided by hydrogen bonds. Certain hydrogen bonds present strong opposition to small structural distortions and can act as kinetic traps2, thus influencing the whole dissociation process. This concept has been implemented in the molecular-dynamics based method, Dynamic Undocking (DUck)3, which allows to assess the structural stability of the complex by calculating the work used in the process of breaking the hydrogen bond (WQB).
In the first part of the project we performed a first large-scale assessment of robustness of hydrogen bonds on a set of 77 protein-ligand complexes (341 hydrogen bonds)4 sourced from the Iridium data set5. We have shown that hydrogen bond-driven structural stability is very common. Stable bonds can be found in 75% of complexes and tend to group in fragment-sized structural anchors. Additional calculations have shown that we can modulate the stability of the bond by modifying the structure of ligand. Manipulating the local environment around the bond has important implications for structural stability, and is a useful drug design principle.
In the second part of the project we used the same data set to evaluate the usefulness of structural stability in binding mode prediction. Post-docking pose evaluation with DUck was performed on a set of binding modes generated with rDock6. The results show that DUck is equally good as rDock at selecting poses. Additionally, the performance of DUck surpasses the docking software in predicting the binding mode of the structural anchor. That has been confirmed on the set of protein-fragment complexes, gathered in SERAPhiC data set7. DUck is also more resistant to conformational changes in the receptor, which was confirmed in cross-docking experiment.
The project branched into a few subprojects, which were performed by other members of the group. One of them is attempting to find a relationship between structural stability and the binding free energy on the set of carefully selected activity cliffs. The other uses a large collection of DUck simulation data and machine learning approaches to construct a predictor of structural stability of macromolecular complexes.
• C. Bissantz, et al., J. Med. Chem. 2010, 53, 5061-5084.
• P. Schmidtke, et al., J. Am. Chem. Soc., 2011, 133, 18903-18910.
• S. Ruiz-Carmona, et al., Nature Chemistry, 2017, 9, 201.
• M. Majewski, et al., bioRxiv, 2018, 454165.
• G. L. Warren, et al., Drug Discovery Today, 2012, 17, 1270-1281.
• S. Ruiz-Carmona, et al. PLoS computational biology, 2014, 10.4, e1003571.
• A. D. Favia, et al., J. Chem. Inf. Model., 2011, 51.11, 2882-2896.
Download the full description of this project: ESR11: Fragment-based approaches to identify novel PPI inhibitors
FBLD technologies are continuously being improved to capture new opportunities. This project will interrogate emerging antimicrobial targets by fragment hit identification and fragment growing, linking and merging approaches.
Design and synthesise compounds for antimicrobial protein targets, starting from existing hits of fragment library screening. 2. Perform ITC and SPR screening and develop ligand binding models. 3. Develop accurate ligand-‐protein binding models using X-‐ray, 15N-‐NMR data and CADD data (in collaboration with ESR5).
In a joined effort with Zobio, VU University Amsterdam is exploring a couple of antimicrobial targets using FBLD approaches. In this project we will design and synthesize optimized hit fragments and drug-‐like compounds. Computer-‐aided drug design will be combined with the synthesis of series of compounds that interrogate the protein targets. Using the biochemical and biophysical screening data that will be generated (amongst others by ESR5), structure-‐activity relationships, structure-‐kinetics relationships and structure-‐thermodynamics relationships will be explored and used to optimize the hit fragments.
Applicants must have a background in medicinal chemistry and have ample experience in computer-‐aided drug design and the synthesis and characterisation of novel ligands. We are looking for an enthusiastic team player that is eager to collaborate with others.
1. Edink et al. J.Am. Chem. Soc. 2011, 133, 5363-5371.
2. De Kloe J. Med. Chem. 2010, 53, 7192-7201.
Download the full description of this project: ESR12: Covalent fragments to activate industrial enzymes
Covalent fragments to activate industrial enzymes
Fragment-based ligand discovery (FBLD) has been widely used to identify selective small organic molecules that inhibit protein activity, either inhibiting the catalytic action or disrupting a protein-protein interaction. However, there has been very little reported on the use of FBLD to identify activators of enzyme activity.
The phenomenon of activation is well established for receptors. There are relatively few examples of naturally occurring small molecules that activate enzymes.The York laboratory has recently demonstrated that small fragments can increase the activity of the glycoside hydrolase, BtGH84  which were subsequently engineered to bind covalently to the enzyme and give further increased activity .
In this project we are using fragment methods to identify fragments that activate industrial glycoside hydrolases, a class of enzymes used industrially in cellulose degradation. Low activity has been considered as one of the main limiting steps in the process.
Figure 1. 4MU-GlcNAc cleavage assay Michaelis -Menten plotfor TrBgl2 in the absence and presence of the initial hit.
A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule activator of the fungus glycoside hydrolase (TrBgl2) (Figure 1). An analogue by catalogue approach was used to identify improved compounds which result in approximately up to 2-fold increase in maximum activation and behave as nonessential activators increasing the Vmax(Vmax= maximum rate of reaction) and decreasing the KMvalue. The activators showed no activation of the related bacterial glycoside hydrolase CcBglA demonstrating that the effect is specific.
Interestingly, a fragment analogue of the initial hit appears to inhibit both TrBgl2 and CcBglA through a mixed-model mechanism.
The activators appear to stabilize the TrBgl2-substrate complex -productive conformation- leading to enhanced activity of TrBgl2 while the inhibitor appear to destabilize the TrBgl2-substrate complex -non-productive conformation- leading to inhibition.
The actual mechanism of activation is being investigated and will suggest where to covalently tether the activator to constitutively activate the enzyme.
- Darby et al. Angewandte Chemie, 2014, 53, 13419-13423
- Darby et al. Chemical Science, 2017 , 11, 7772-7779
Host: University of York, UK
Academic supervisors: Prof. dr. Peter O'Brien and Prof. dr. Rod Hubbard (University of York)
Researcher: Bas Lamoree
Download the full description of this project: ESR13: Fragment-based assessment of new antibiotic targets
Fragment-based assessment of new antibiotic targets
We are using the fragment-based approach to discover new antibiotics. Specifically, we are investigating new ways of targeting bacteria, by looking at a set of their DNA-replicating proteins: the bacterial replisome.
The bacterial replisome is a molecular machine made up of at least twelve essential components, including a DNA polymerase, an RNA polymerase, a nuclease, and three different ATPases, as well as other non-catalytic but structurally important proteins. These proteins form three main subcomplexes: the polymerase, the clamp loader, and the helicase. Structural details are known for these subcomplexes, but not for the active whole replisome complex. Instead, it is better characterized by the many different dynamic interactions between proteins during various stages of the catalytic cycle.
As the machine only works when all these components are present, inactivating one of them would lead to slow-down or arrest of DNA replication. Such inhibition of DNA replication can therefore happen in many ways. We have reconstituted the functional bacterial replisome in vitro and screened a fragment library against it. We found that fragments can target several known sites, while some fragments appear to act by unknown mechanisms. This demonstrates a novel way of working with fragments in a complex system.
The current project focuses on 1) using structural and functional experiments to optimize fragment screening hits against a protein-protein interaction in the helicase subcomplex, and 2) using covalent modification of the replisome to directly identify the sites of action of fragments with unknown mechanism.
Download the full description of this project: ESR14: Targeting allosteric pockets with FBLD
New inhibitors for Trypanosoma brucei farnesyl pyrophosphate synthase by fragment-based drug discovery
African sleeping sickness, caused by the parasite Trypanosoma brucei (T. brucei), is a neglected disease with an endemic occurrence in 36-sub-Saharan African countries As the current options for drug treatment have low efficacy and also strong side effects, new drugs with a better safety and efficacy profile are urgently needed. This project therefore took a fragment-based drug-discovery approach to identify new inhibitors for the T. brucei farnesyl pyrophosphate synthase (FPPS).
Nitrogen-containing bisphosphonates are the current treatment for bone diseases and have been shown to block the growth of the T. brucei parasites by inhibiting FPPS, an enzyme involved in sterol biosynthesis. However, due to their particular pharmacokinetic properties they are not well suited for non-bone indications. In previous efforts at Novartis, an allosteric pocket of human FPPS was identified by fragment-based drug discovery, allowing modulation of human FPPS by non-bisphosphonate inhibitors. As a similar pocket also exists in T. brucei FPPS, we drew on a fragment-based drug discovery approach to identify inhibitors of a new chemotype for T. brucei FPPS.
This work involved expressing and purifying T. brucei FPPS protein and applying it for an X-ray and NMR fragment screen. Fragments were screened by 1D-NMR and further validated and characterized in protein-observed 2D-NMR and the exact binding mode was determined in crystallization experiments. An X-ray fragment screen was performed in collaboration with the EMBL Grenoble using the automatic crystal harvesting pipeline. A 500 fragment library was screened and weak fragment binders were identified with the PanDDA (Pan-Dataset Density Analysis) software.
Fragment hits identified and characterized by NMR and X-ray techniques provided the starting points for medicinal chemistry, the overall goal being to optimize initial fragment hits into tool compounds with a high binding affinity that inhibit the FPPS enzyme function and parasitic growth.
• Jahnke, W., et al. (2010). "Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery." Nat Chem Biol 6(9): 660-666.
• Marzinzik, A. L., et al. (2015). "Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding." ChemMedChem 10(11): 1884-1891.
• Muenzker L., et al. Identification of an allosteric binding pocket on Trypanosoma brucei FPPS and implications for Human African Sleeping sickness, manuscript in preparation
• Muenzker L., et al. Allosteric and active site non-bisphosphonate binders identified by fragment-based discovery, manuscript in preparation Poster: • Muenzker L., Petrick J., Klebe G., Marzinzik A., Jahnke W., Targeting Trypanosoma brucei FPPS by fragment-based drug discovery, 9th International Conference on Structural Biology, 18th-20th September 2017, Zurich, Switzerland
• Muenzker L., Petrick J., Klebe G., Marzinzik A., Jahnke W., Fragment-based discovery of novel active and allosteric site binders of T. brucei farnesyl pyrophosphate synthase, 3rd Integrative Structural Biology School, 16th – 21st July 2018, Institut Pasteur, Paris, France Presentation:
• Muenzker L., Fragment-based discovery of novel active and allosteric site binders of T. brucei farnesyl pyrophosphate synthase, Fragment-based Lead Discovery Conference, 7th – 10th October 2018, San Diego, USA
Host: VU University Amsterdam, The Netherlands
Academic supervisors: Prof. Peter van der Sijde, dr. Iina Hellsten & dr. Jacqueline van Muijlwijk (VU University Amsterdam)
Researcher: Angelo Kenneth Romasanta
Download the full description of this project: ESR15: Science, Business & Innovation in the pharmaceutical sciences
Catching the Waves of a New Scientific Field: Creating Opportunities in Fragment-based Drug Discovery
Creating opportunities from scientific advances is a complex challenge in the pharmaceutical sciences. On one hand, researchers and firms need to differentiate to gain competitive advantage against other players in the field. At the same time, they still need to conform in order to gain access to various resources and build legitimacy. The main question then in this research is: how do firms manage the tension between differentiation and conformity to create opportunities in a new scientific field.
Managing this tension between being different yet remaining familiar is known as optimal distinctiveness. In a multidisciplinary study involving innovation management and the pharmaceutical sciences, we conduct multilevel empirical research to explore how individuals and organizations pursued optimal distinctiveness in fragment-based drug discovery.
In our first study presented at the R&D Management conference, we build a picture of how optimal distinctiveness is pursued in the pharmaceutical industry. We use bibliometric methods to survey 1,800 articles by innovation scholars who looked at the pharmaceutical industry from various perspectives. Drawing out from their insights, we then distilled various areas where individuals and firms compete and cooperate for optimal distinctiveness.
In our second study, we used 4,000 publications to analyze the emergence of fragment-based drug discovery and its development over time. We show how academia and industry were both important to the scientific development and commercialization of the field. We also show how a crucial role in knowledge development lay in various researchers’ integration of scientific ideas. This study has been published in Drug Discovery Today.
Our third study shows how firms pursued optimal distinctiveness. In a qualitative case study, we peeked inside the black box of how two big pharmaceutical companies adopted FBDD. Working from the process perspective of absorptive capacity, we explored how firms pursued legitimacy and distinctiveness as they built capabilities in FBDD. We show that it is important to manage not only the external pursuits of optimal distinctiveness but also the parallel internal dynamics within firms.
In our fourth study, we look at how researchers are gaining skills in new fields. Since it is not straightforward to learn skills in an emerging scientific field through traditional routes, we look at the EU’s Innovative Training Networks (ITN) initiative. In this study, we discuss how ITNs can respond to training required in developing fields by allowing researchers to general skills required to practice drug discovery while at the same time distinguishing themselves through the specialized techniques such as FBDD.
In our fifth study, which we are currently conducting, we look at how small firms pursued differentiation and legitimacy when FBDD was still emerging. Through interviews with new ventures created to take advantage of FBDD, we are exploring the balance necessary to be as different as legitimately possible.
Publications and conference presentations
• Romasanta, Angelo KS, IJP de Esch and Peter van der Sijde. “Conforming to Differentiate: The Pursuit of Optimal Distinctiveness in R&D.” Under Review
• Romasanta, Angelo KS, IJP de Esch and Peter van der Sijde. “Riding the wave of a new scientific field: How pioneers create opportunities amidst hype and hope” Accepted at Babson College Entrepreneurship Research Conference (2019).
• Romasanta, Angelo KS and Peter van der Sijde. “Innovation in the pharmaceutical industry: Mapping the research landscape.” R&D Management Conference (Milan IT, 2018).
• Romasanta, Angelo KS, et al. "When fragments link: a bibliometric perspective on the development of fragment-based drug discovery." Drug Discovery Today (2018).
• Romasanta, Angelo KS, et al. "Patents and publications landscape in fragment-based drug discovery." High Tech Small Firms Conference (Amsterdam NL, 2017).
• Romasanta, Angelo KS, et al. "The patent landscape of fragment-based drug discovery." Fragments 2017: 6th RSC-BMCS Fragment-based Drug Discovery Meeting (Vienna AT, 2017).