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ANNUAL AEGIS NEWSLETTER 2019
The 5rd AEGIS Training School took place in Krakow, Poland, from April 1-5, 2019, at the picturesque old buildings of Jagiellonian University. Focus was layed on modern methods for structure determinations including NMR, Xray diffraction and Cryo Electron Microscopy. The event was organized by Prof. Grzegorz Dubin.
Meeting Excursion of the ESR students to the famous Wieliczka salt mine of next to Karkow
The topic of the school were methods of structural biology including a lecture of Prof. Sattler on NMR in drug discovery, talks on synchrotron radiation and measurements at such
facilities, the use of free electron lasers and high resolution cryo-electron microscopy in structural biology. In addition visits of the synchrotron facility Solaris at Krakow and the crystallization laboratory of the group of Prof. Dubin were included. The school also comprised a shot course in methods for structure solving.
The final meeting of the AEGIS International Training Network was organized as a conference summarizing the results of the AEGIS consortium and putting them into the context of the fast developing field of fragment-based lead discovery. The meeting was organized together with the EU ITN TASPPI. It was run under the title “New Frontiers in Structure-based Drug Design” in the marvelous city of Florence, September 23 -25, 2019.
We succeeded to attract an impressive list of internationally well-known speakers that covered many of the aspects being in focus of the AEGIS and TASPPI consortia but which also gave perspectives to novel approaches developed in the field of structure-based drug design. The talks of the invited speakers were complemented by contributions from students and PIs of both schools.
Overall, the meeting was attended by 120 participant who came from all over the world. An extensive poster session was hold and students from the two schools succeeded to win poster prices.
Prof. Sattler and Prof. Ottmann Poster Prize Awardees in Florence
Speakers of AEGIS and TASPPI Geqing Wang, Madita Wolter, Engi Hassaan
The program of the meeting covered the following different aspects:
Structure- and Fragment-based Drug Discovery
Intrinsically Disordered and Amyloidogenic Proteins
Chemical Biology, Targeting & New Concepts
Peptides, Natural Products and Non-small Molecules
Structural biology, biophysics & methods
Computational Drug Discovery
Finally the meeting was concluded by a workshop offered by BioSolveIT who develops software tools for drug discovery, particularly for docking, virtual screening and exploring chemical spaces.
Lecture Hall at the Convitto della Calza, Florence
Selected Scientific Achievements
Publications:
1. M. Konstantinidou, J. Li, B. Zhang, Z. Wang, S. Shaabani, F. ter Brake, K. Essa, A. Dömling, PROTAC – a game changing technology, Expert Opinion Drug Discov. 2019, DOI: 10.1080/17460441.2019.1659242
2. Q. Wang, K.C. Mgimpatsang, M. Konstantinidou, S. V. Shishkina, A. Dömling, 1,3,4-Oxadiazoles by Ugi-tetrazole and Huisgen reactions, Org. Lett. 2019,DOI: 10.1021/acs.orglett.9b02614
3. Q. Wang, A. Osipyan, M. Konstantinidou, R. Butera, K. C. Mgimpatsang, S. V. Shishkina, A. Dömling, Pd-Catalyzed de novo assembly of diversely substituted indole-fused polyheterocycles, J.Org. Chem. 2019, DOI: 10.1021/acs.joc.9b01258
4. S. Kurhade, M. Konstantinidou, F.Sutanto, K. Kurpiewska, J. Kalinowska –Tłuscik, A. Dömling, Sequential multicomponent synthesis of 2-(imidazo[1,5-α]pyridin-1-yl)-1,3,4-oxadiazoles, Europ. J. Org. Chem. 2019, 10, 2029-2034,DOI: 10.1002/ejoc.201801880
5. Neochoritis, C. G.; Shaabani, S.; Ahmadianmoghaddam, M.; Zarganes-Tzitzikas, T.; Gao, L.; Novotná, M.; Mitríková, T.; Romero, A. R.; Irianti, M. I.; Xu, R.; et al. Rapid Approach to Complex Boronic Acids. Sci. Adv. 2019, 5 (7), eaaw4607. https://doi.org/10.1126/sciadv.aaw4607.
6. Al-Qahtani, A. D.; Bashraheel, S. S.; Rashidi, F. B.; O’Connor, C. D.; Romero, A. R.; Domling, A.; Goda, S. K. Production of “Biobetter” Variants of Glucarpidase with Enhanced Enzyme Activity. Biomed. Pharmacother. Biomedecine Pharmacother. 2019, 112, 108725. https://doi.org/10.1016/j.biopha.2019.108725.
7. Nakladal, D.; Buikema, H.; Romero, A. R.; Lambooy, S. P. H.; Bouma, J.; Krenning, G.; Vogelaar, P.; van der Graaf, A. C.; Groves, M. R.; Kyselovic, J.; et al. The (R)-Enantiomer of the 6-Chromanol Derivate SUL-121 Improves Renal Graft Perfusion via Antagonism of the Α1-Adrenoceptor. Sci. Rep. 2019, 9 (1), 13. https://doi.org/10.1038/s41598-018-36788-0.
8. Batista, F. A.; Bosch, S. S.; Butzloff, S.; Lunev, S.; Meissner, K. A.; Linzke, M.; Romero, A. R.; Wang, C.; Müller, I. B.; Dömling, A. S. S.; et al. Oligomeric Protein Interference Validates Druggability of Aspartate Interconversion in Plasmodium Falciparum. Microbiology Open 2019, e779. https://doi.org/10.1002/mbo3.779.
9. Kuusk A, Neves JF, Bravo Rodriguez K, Gunnarsson A, Ruiz-Blanco YB, Ehrmann M, Chen H, Landrieu I, Sanchez-Garcia E, Boyd H, Ottmann C, Doveston RG. Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3σ. ACS Chem. Biol. 2019 Nov 19. https://doi.org/10.1021/acschembio.9b00893.
10. Byrne R, Schneider G. In Silico Target Prediction for Small Molecules. Methods. Mol Biol. 2019;1888:273-309. https://doi.org/10.1007/978-1-4939-8891-4_16
11. Hassaan E, Eriksson PO, Geschwindner S, Heine A, Klebe G. Fragments as Novel Starting Points for tRNA-Guanine Transglycosylase Inhibitors Found by Alternative Screening Strategies ChemMedChem 2019, https://doi.org/10.1002/cmdc.201900604
12. Dawidowski M, Kalel VC, Napolitano V, Fino R, Schorpp , Emmanouilidis L, Lenhart D,
Ostertag M, Kaiser M, Kolonko M, Tippler B, Schliebs W, Dubin G, Mäser P, Tetko IV, Hadian K, Plettenburg O, Erdmann R, Sattler M, Popowicz GM. Structure-Activity Relationship in Pyrazolo[4,3-c]pyridines, First Inhibitors of PEX14-PEX5 Protein-Protein Interaction with Trypanocidal Activity. J Med Chem. 2020 Jan 6. https://doi.org/10.1021/acs.jmedchem.9b01876.
13. Xin Yang, Yifei Wang, Ryan Byrne, Gisbert Schneider, Shengyong Yang. Concepts of Artificial Intelligence for Computer-Assisted Drug Discovery. Chem. Rev. 2019, 119, 18, 10520-10594, https://doi.org/10.1021/acs.chemrev.8b00728
Talks: Engi Hassaan Busted! Recognizing False Positives and False Negatives: Learnings from Comparative Analysis of Fragment Binding Using X-Ray Crystallography and NMR. Drug Discovery Chemistry in San Diego 2019.
Giulia Opassi Targeting Chagas Disease Using Fragment-based Lead Discovery: Validation and Screening of FPPS Enzyme. XXVI National Meeting in Med. Chemistry, Milano, July 16.- 19, 2019
Markella Konstantinidou Applications of multi-component reaction chemistry in heterocyclic synthesis, ACS Spring National Meeting, 31/3-1/4/2019, Orlando, USA
Markella Konstantinidou Discovery of proteolysis targeting chimeras for leucine-rich repeat kinase 2 (LRRK2) “New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Laura Ortega Targeting α4/α5 nicotinic acetylcholine receptors to tackle addiction. Jacques-Monod Conference Ligand-gated ion channels from atomic structure to synaptic transmission, May 2019 in Roscoff, France
Valeria Napolitano Inhibition of glycosomal protein import: the sweet death of Trypanosoma New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
João Encarnação Bioengineering subunit B from Shiga Toxin 1 for intracellular drug delivery. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Charlotte Softley Placing fragments with lanthanide tags using paramagnetic NMR. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Flash Talks & Posters Francesca Magari Exhaustive X-Ray Crystallographic Screening of a Hit-Enriched 96 Fragment Library Against Diverse Targets. Drug Discovery Chemistry in San Diego 2019
Valeria Napolitano Structure-based design and synthesis of PEX5/PEX14 PPI inhibitors blocking glycosomal protein import: a way to kill Trypanosoma. EMBO Workshop “Current advances in protein translocation across membranes” Girona, Spain, 2019
Charlotte Softley Use of paramagnetic NMR with lanthanide binding tags to locate fragment binding sites. 7th RSC-BMCS Fragment-based Drug Discov. meeting, March 24-26, 2019, Cambridge
Markella Konstantinidou Inhibitors for Asp-proteases: anchor-based virtual screening, innovative chemistry and protein crystallography, ACS Spring National Meeting, 31/3-1/4/2019, Orlando, USA
Roberto Fino Introducing the CSP Analyzer: a Novel Machine Learning-based Application For Automatic Analysis of Bidimensional NMR Data in Fragment-Based NMR Screening. Gordon
Research Conference on Computational Aspects of Biomolecular NMR, Les Diablerets (CH), 9-14 June 2019
Atilio Reyes Romero In Vitro Fragment-Based Drug Discovery of PfMDH Allosteric Inhibitor. New Frontiers in Structure-Based Drug Discovery, 23-25 September 2019, Italy, Florence
Valeria Napolitano Glycosomal protein import: a new target against Trypanosomiasis. FEBS Congress 2019: From Molecules to Living Systems" (Krakow (Poland), 6-11 July 2019
Ryan Byrne Filling in the gaps: geometric complementarity and its role in ligand-protein interaction prediction. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Maxime Denis Design of new paramagnetic tags for NMR Spectroscopy: A new strategy to yield fast, selective and irreversible tagging of protein. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Roberto Fino Introducing the CSP Analyzer: a Novel Machine Learning-based Application For Automatic Analysis of Bidimensional NMR Data in Fragment-Based NMR Screening. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Engi Hassaan Do All Roads Really Lead to Rome? Learnings from Comparative Analysis of Fragment Binding using SPR, NMR, and X-Ray Crystallography. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Giulia Opassi Validation of tcFPPS as a fragment-based lead discovery target. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Ave Kuusk Stabilization of 14-3-3/p53 protein-protein interaction. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Joy Petrick "Novel active site and allosteric site binders identified for farnesyl pyrophosphate synthase of Trypanosoma cruzi". New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Atilio Reyes Romero Protein Interference Assay (PIA) Enables the Validation in vivo of New Drug Target Against Malaria and the Discovery in Vitro of the First Class of Fragments Acting at the Oligomeric Interface. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Patrick Walter "From target characterisation to novel inhibitor discovery – A biophysical way". New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Fancesca Magari “The Power of Fragments: FBLD approach to investigate proteins structure”. New Frontiers in Structure-based Drug Design” Florence, Sept. 23-25, 2019.
Markella Konstantinidou Recent advances in heterocyclic synthesis via multi-component reaction schemes, EFMC International Symposium on Advances in Synthetic and Medicinal Chemistry (EFMC-ASMC), 1-5/9/2019, Athens, Greece
Selected AEGIS ESR activities
As 2019 was the last year of the AEGIS EU ITN five of our ESRs succeeded already to finish and defense their PhD thesis. In short, the following pages present the highlights of their theses:
Dr. Engi Hassaan, ESR 4 PhD from the Phillips University in Marburg, Germany (PUM)
Drug Design group of Prof. Dr. Gerhard Klebe.
Thesis Title: “Do All Roads Really Lead to Rome? Learnings from Comparative
Analysis using SPR, NMR, & X-Ray Crystallography to Optimize
Fragment Screening in Drug Discovery.”
Thesis Highlights
Fragment Screening Hit Draws Attention to a Novel
Transient Pocket Adjacent to the Recognition Site of
the tRNA-Modifying Enzyme TGT
This led to the design and synthesis of a series of
model compounds to target the opening of the
pocket. A cysteine residue Cys158, only present in the
bacterial TGT isoforms, appeared as a gate keeper to
the transient sub-pocket. It becomes accessible upon
pocket opening and may be covalently blocked by
covalent inhibitors.
First crystal structure of the wild type PEX14 of T.
brucei in complex with a compound
Fragment screening on the mutated PEX14 revealed
only one fragment hit due to pockets blocked by
neighboring crystal mates. The fragment hit was
developed into a lead compound and soaked into the
wild type PEX14, which has free binding pockets and
led to a crystal structure at a resolution of 1.83 Å.
Comparative analysis of fragment binding on endothiapepsin
using STD NMR and X-ray crystallography revealed several
phenomena that may cause false negatives in fragment
screening
Flexible Region
Closed pocket Opened pocket
Binding of F002 alone (PDB: 4YCY)
Ritonavir Relocated
F002
F002
PEG
Binding of F002 with Ritonavir
Dispersed electron density
Only the fragment fits in
Light Water (H2O) Heavy Water (D2O)
Fragment & Protein
STD Protein
STD Protein & Fragment
Relocation of catalytic dyad
fragments to new binding sites in
the presence of a potent active-
site inhibitors. Fragments
relocate to PEG binding position.
Displacement of the potent
active-site inhibitors by
fragments, likely caused by
reduced affinity of inhibitor
due to crystal packing.
P
STD NMR can yield false negatives in H2O as seen with some
fragments. This is owing to H2O being a magnetization sink,
whereas D2O does not interfere with transfer of magnetization.
Dr. Joy Petrick, ESR 12 PhD from the group of Dr. Wolfgang Jahnke, Novartis Institute for Biomedical Research, Basel, Switzerland. Thesis Title
“Targeting farnesyl pyrophosphate synthase of Trypanosoma cruzi by fragment-based lead discovery”
Thesis highlights
A crystallization system was installed for T. cruzi FPPS (TcFPPS) that was suited for FBS.
FBS by NMR identified 109 validated fragment hits and showed selectivity between the enzyme of different sources (T. cruzi, T. brucei and human FPPS).
Follow up of the NMR hits by X-ray crystallography and using this method as initial screening method identified fragment hits in sites spread over the entire protein
Key finding 10 fragment binders were identified in the so-called allosteric site. Fragments were found binding either in an open- or closed-state of this pocket, which is caused by conformational changes of the side chain of residue Phe50.
Patrick Walter, PhD
PhD from the Institut Pasteur, Unit of Chemistry
and Biocatalysis, under the supervision of Dr.
Hélène Munier-Lehmann.
Title of the thesis:
“Identification and characterization of allosteric
sites of UMP kinase”
The uridine monophosphate kinase from M. tuberculosis is a potential new anti-infective target.
My aim was to structurally and biophysically characterize this protein, as well as screen a fragment
library for new potential inhibitor discovery.
For the first time, we solved the
structure of mycobacterial UMP
kinase in complex with its natural
negative effector UTP via X-ray
crystallography (Fig. 1A) and cryo-
EM (Fig. 1B). The determination of
the previously unknown UTP-
binding site gives a new starting
point for novel allosteric inhibitor
design.
Information on the enzyme kinetics have been investigated by
activity assays (Fig. 2) and SPR experiments. Latter technique,
complemented with ITC was used to determine the affinity
parameters for the natural substrate UMP, the positive effector
GTP and the negative effector UTP. These information will be
valuable for lead validation during the fragment-2-lead
optimization process.
A thermal shift assay protocol has been established (Fig.
3) and been used to screen a small-compound library.
Two hits have been validated by ligand-observed STD-
NMR, Waterlogsy and NOESY experiments. The
fragment-2-lead optimization process can now be
entered.
Fig. 1: A) Crystal structure of UMP kinase, B) cryo-EM electron density map of UMP kinase. Both structures in complex with the negative effector UTP and the catalysis product UDP.
Fig. 2: Enzyme activity of UMP kinase in the absence (red) and the presence of GTP (blue) or UTP (green) (Labesse et al. 2011).
Fig. 3: Melting curves of the UMP kinase apo enzyme (blue), in the presence of GTP (green) and UTP (brown).
A B
a5-nAChRsa5-Nicotinic Acetylcholine Receptors
PfLDHPvSUB1
Dr. Laura Ortega Varga, ESR 8PhD from Sorbonne University
Thesis Directors: Prof. Michael Nilges and Dr. Arnaud BlondelStructural Bioinformatics Unit | Institut Pasteur | Paris
Thesis Title: “Innovative inhibition strategy against functional structural transitions of essential pathogenic factors: Computational applications to Malarial and Neurotransmitter targets”
S1’
S1
FlexX score (kJ/mol)
Hyde score (kJ/mol)
Hyde LE (kcal/mol)
Ac-Gly-Ala-Asp-Asp-Nma -40.41 -11.0 -0.09
S4
S2Scissile bond
S2’
Design and optimization of reversible covalent inhibitors to block parasite egress/invasion stage.
Malarial Subtilisin-like serine protease 1
Docking studies were used to pock S1-S4 subsiteswith natural and non-natural amino acids and toassess the binding of covalent peptidomimetics.
The designed inhibitors have shown enzymaticinhibitory activity in the sub-micromolar range anddetectable egress/invasion inhibition in cells. Co-crystallographic structures have been obtained.
Enumeration and screening in silicoof a combinatorial virtual libraryaiming to cover the whole cofactor-substrate binding site.
Malarial Lactate dehydrogenase
Fifty molecules have been selectedfor synthesis and ex vivo testing.
Design of inhibitory cofactor analogsto halt parasite metabolism.
In collaboration with J-C. Barale, P. Alzari (InstitutPasteur, Paris) & J-F. Hernandez (IBMM, Montpellier)
In collaboration with A. Tahghighi(Institut Pasteur, Iran) & A. Lawrence(Institut Pasteur, Cameron)
a5-nAChRs were targeted to address addiction using an AChBPchimera mimicking the a5/a4 binding interface, which structure wassolved in complex with the first known a5 ligands. Two dockingmodels were also built to better cover the allosteric mechanism.
In silico and STD-NMR screening returned hit molecules confirmed asbinders. Physiological effect can then be evaluated on oocytesexpressing these subunits.
In collaboration with P-J. Corringer, A. Nemecz, N. Wolff (Institut Pasteur), D. Joseph (Uni. Paris-Sud) & I. Krimm (INS, Lyon)
Identification of effectors targeting complex allosteric mechanism.
