Periodic Reporting for period 2 - HiSCORE (Highly Informative Drug Screening by Overcoming NMR Restrictions)
Reporting period: 2022-11-01 to 2024-04-30
The need for drug screening with increasingly higher throughput is dictated both by the increasing number of drug targets becoming available through genomics and by the growing number of chemical molecules from combinatorial chemistry. Several high-throughput screening techniques can scan large libraries of compounds, but the ever-increasing throughput has not significantly increased promising drug candidates. HiSCORE presents a synergistic approach to high-throughput, high-information drug screening, leveraging the complementary skills of five laboratories supported by external drug screening experts:
(i) Research and design of innovative instrumentation for magnetic resonance that can provide small, hyperpolarized solid samples at intervals on the order of minutes, transfer and dissolve or liquefy these samples with minimal dilution, and acquire several high-resolution NMR spectra of the liquid samples in parallel, using contrast-enhancement methods in up to 1000 microfluidic detectors. The contrast between compounds binding to targets and those failing to bind can be enhanced by exploiting long-lived states.
(ii) Applications of this instrumentation for binding assays, measuring dissociation constants in the nano to micromolar range, and determining kinetic rates of association and dissociation for numerous complexes of drug compounds and protein targets.
(iii) Functional assays, particularly for systems with multiple enzyme steps and intermediate products, to determine the efficacy of potential inhibitors, fully exploiting the rich data from fluorine-19 NMR.
(iv) Metabolomic assays to observe compound metabolism in cell cultures, identifying potentially toxic side-products.
“HiSCORE” builds on the synergy of 4 institutions (beneficiairies): ENS (France), SKU (Netherlands), ETH (Switzerland), and KIT (Germany), the latter being represented by two different teams:
1. Geoffrey Bodenhausen ENS Paris
2. Benno Meier KIT Karlsruhe
3. Arno Kentgens SKU Nijmegen
4. Alvar Gossert ETH/Zürich
5. Jan Korvink KIT Karlsruhe
The groups contribute complementary expertise:
1 Geoffrey Bodenhausen and the pre-existing team at the Ecole Normale Supérieure in Paris (Abergel, Ferrage, Pelupessy, Baudin, Bouvignies, Birlirakis) develop NMR methodology, particularly by exciting long-lived states to extend the life-times of hyperpolarization, improving the determination of binding parameters, including kinetics, and developing libraries of compounds.
2 Benno Meier and his pre-existing team (Kourilova, Kouril): Alternatives to dissolution DNP: ballistic transfer of solid hyperpolarized “bullets” with minimal dilution, improved understanding and acceleration of the DNP process, parallelization.
3 Arno Kentgens and his pre-existing team (Tessari, Janssen, Aspers): Novel miniaturized NMR detectors, microliter samples, microfluidics, quantitation of nM compounds, rapid-melt DNP, supercritical CO2 as a solvent, para-hydrogen induced polarization (PHIP), iridium complexes, zero-quantum spectroscopy, Overhauser DNP.
4 Jan Korvink and his pre-existing team (Brandner, Mager, Lehmkuhl, Becker): miniaturization based on microelectromechanical systems (MEMS), complementary metal-oxide-semiconductors (CMOS), massively parallel detection, automatic data analysis, and machine learning.
In addition to the consortium comprising these 4 principal investigators, two external advisors contribute to the project:
1 Alvar Gossert and nearby pre-existing teams at ETH (Riek, Torres): Gossert worked for 10 years in drug development/screening labs of Novartis. Since 2017, he has worked at ETH Zürich. An amendment to add Alvar Gossert as a partner of the consortium was signed on April 24, 2023.
2 Claudio Dalvit worked 9 years in drug development/screening at Novartis in Basel, 9 years at Pharmacia & Upjohn in Milan, then at the Italian Institute of Technology (IIT) in Genova, and finally at the University of Neuchatel.
The project comprises several objectives that can be bundled into two groups:
(A) Development of novel instrumentation to produce hyperpolarized solid samples at intervals of 1 minute or less and to parallelize 4, 10, 100, …microfluidic micro-coils or strip-line detectors.
This meets two objectives: (i) Study different sample compositions, particularly varying concentration ratios, e.g. [L]:[P] = [ligand]:[protein] by titrations. (ii) Functional screening by varying [inhibitor]:[enzyme]. (iii) Complementary experiments (T1, T2, CPMG, CEST, TLLS, TLLC...) varying pulse intervals, relaxation delays, and observing nuclei such as 1H, 2H, 13C, 15N, 19F, 31P.
(B) Development of novel chemistry: develop binding assays to measure dissociation constants in the range nM < KD < µM, determine kinetic rates kon and koff for ligand-protein interactions, develop functional assays to determine IC50 of enzyme cascade inhibitors, and explore metabolomic assays in cell cultures.
(i) Research and design of innovative instrumentation for magnetic resonance that can provide small, hyperpolarized solid samples at intervals on the order of minutes, transfer and dissolve or liquefy these samples with minimal dilution, and acquire several high-resolution NMR spectra of the liquid samples in parallel, using contrast-enhancement methods in up to 1000 microfluidic detectors. The contrast between compounds binding to targets and those failing to bind can be enhanced by exploiting long-lived states.
(ii) Applications of this instrumentation for binding assays, measuring dissociation constants in the nano to micromolar range, and determining kinetic rates of association and dissociation for numerous complexes of drug compounds and protein targets.
(iii) Functional assays, particularly for systems with multiple enzyme steps and intermediate products, to determine the efficacy of potential inhibitors, fully exploiting the rich data from fluorine-19 NMR.
(iv) Metabolomic assays to observe compound metabolism in cell cultures, identifying potentially toxic side-products.
“HiSCORE” builds on the synergy of 4 institutions (beneficiairies): ENS (France), SKU (Netherlands), ETH (Switzerland), and KIT (Germany), the latter being represented by two different teams:
1. Geoffrey Bodenhausen ENS Paris
2. Benno Meier KIT Karlsruhe
3. Arno Kentgens SKU Nijmegen
4. Alvar Gossert ETH/Zürich
5. Jan Korvink KIT Karlsruhe
The groups contribute complementary expertise:
1 Geoffrey Bodenhausen and the pre-existing team at the Ecole Normale Supérieure in Paris (Abergel, Ferrage, Pelupessy, Baudin, Bouvignies, Birlirakis) develop NMR methodology, particularly by exciting long-lived states to extend the life-times of hyperpolarization, improving the determination of binding parameters, including kinetics, and developing libraries of compounds.
2 Benno Meier and his pre-existing team (Kourilova, Kouril): Alternatives to dissolution DNP: ballistic transfer of solid hyperpolarized “bullets” with minimal dilution, improved understanding and acceleration of the DNP process, parallelization.
3 Arno Kentgens and his pre-existing team (Tessari, Janssen, Aspers): Novel miniaturized NMR detectors, microliter samples, microfluidics, quantitation of nM compounds, rapid-melt DNP, supercritical CO2 as a solvent, para-hydrogen induced polarization (PHIP), iridium complexes, zero-quantum spectroscopy, Overhauser DNP.
4 Jan Korvink and his pre-existing team (Brandner, Mager, Lehmkuhl, Becker): miniaturization based on microelectromechanical systems (MEMS), complementary metal-oxide-semiconductors (CMOS), massively parallel detection, automatic data analysis, and machine learning.
In addition to the consortium comprising these 4 principal investigators, two external advisors contribute to the project:
1 Alvar Gossert and nearby pre-existing teams at ETH (Riek, Torres): Gossert worked for 10 years in drug development/screening labs of Novartis. Since 2017, he has worked at ETH Zürich. An amendment to add Alvar Gossert as a partner of the consortium was signed on April 24, 2023.
2 Claudio Dalvit worked 9 years in drug development/screening at Novartis in Basel, 9 years at Pharmacia & Upjohn in Milan, then at the Italian Institute of Technology (IIT) in Genova, and finally at the University of Neuchatel.
The project comprises several objectives that can be bundled into two groups:
(A) Development of novel instrumentation to produce hyperpolarized solid samples at intervals of 1 minute or less and to parallelize 4, 10, 100, …microfluidic micro-coils or strip-line detectors.
This meets two objectives: (i) Study different sample compositions, particularly varying concentration ratios, e.g. [L]:[P] = [ligand]:[protein] by titrations. (ii) Functional screening by varying [inhibitor]:[enzyme]. (iii) Complementary experiments (T1, T2, CPMG, CEST, TLLS, TLLC...) varying pulse intervals, relaxation delays, and observing nuclei such as 1H, 2H, 13C, 15N, 19F, 31P.
(B) Development of novel chemistry: develop binding assays to measure dissociation constants in the range nM < KD < µM, determine kinetic rates kon and koff for ligand-protein interactions, develop functional assays to determine IC50 of enzyme cascade inhibitors, and explore metabolomic assays in cell cultures.
1. Team of Geoffrey Bodenhausen:
Evaluating and ordering new instrumentation. Purchasing a 500 MHz wide-bore liquid-state spectrometer with 5 and 10 mm probes for 1H, 13C, and 19F NMR, with a console capable of running 4 parallel experiments. Purchasing a 60 MHz ‘table-top’ liquid-state spectrometer. Developing MRI methods at 800 MHz using concentric samples. Purchasing a robot for handling samples. Developing a prototype liquid-state NMR probe with four parallel samples. Purchasing a 9.4 T polarizer with cryogenics, probes, microwave source, etc. Installing a console provided by Jan Korvink.
Developing new experimental methods: Excitation and observation of long-lived states (LLSs) and long-lived coherences (LLCs). Effects of paramagnetic relaxation agents and of protein-ligand binding on the lifetimes of LLLs and LLCs.
2. Team of Benno Meier
Evaluating and ordering new instrumentation: Purchasing a 400 MHz wide-bore liquid-state spectrometer. Purchasing a 9.4 T polarizer with cryogenics, probes, microwave source, etc. Acquiring a power supply for a 100 W microwave klystron provided by Arno Kentgens. Acquiring a flow cryostat.
Developing new experimental methods: enhancing proton polarization by transfer from carbon-13 polarization (reverse INEPT).
Studying relaxation in hyperpolarized solids at low temperature and low field.
Developing an automated DNP system for unsupervised screening experiments.
3. Team of Arno Kentgens
Evaluating and ordering new instrumentation: microwave source.
Developing new experimental methods: hyperpolarization by Parahydrogen Induced Polarization (PHIP); transferring hyperpolarization to target molecules in the solid state using a “rapid freeze” probe.
4. Team of Jan Korvink
Evaluating and ordering new instrumentation: NMR of miniaturized samples.
Developing new experimental methods: MRI of parallel samples.
5. Team of Alvar Gossert
Preparing and evaluating several protein/ligand systems.
Developing suitable pulse sequences for hyperpolarization.
Developing methods to evaluate protein/ligand affinity for the 4 teams.
Evaluating and ordering new instrumentation. Purchasing a 500 MHz wide-bore liquid-state spectrometer with 5 and 10 mm probes for 1H, 13C, and 19F NMR, with a console capable of running 4 parallel experiments. Purchasing a 60 MHz ‘table-top’ liquid-state spectrometer. Developing MRI methods at 800 MHz using concentric samples. Purchasing a robot for handling samples. Developing a prototype liquid-state NMR probe with four parallel samples. Purchasing a 9.4 T polarizer with cryogenics, probes, microwave source, etc. Installing a console provided by Jan Korvink.
Developing new experimental methods: Excitation and observation of long-lived states (LLSs) and long-lived coherences (LLCs). Effects of paramagnetic relaxation agents and of protein-ligand binding on the lifetimes of LLLs and LLCs.
2. Team of Benno Meier
Evaluating and ordering new instrumentation: Purchasing a 400 MHz wide-bore liquid-state spectrometer. Purchasing a 9.4 T polarizer with cryogenics, probes, microwave source, etc. Acquiring a power supply for a 100 W microwave klystron provided by Arno Kentgens. Acquiring a flow cryostat.
Developing new experimental methods: enhancing proton polarization by transfer from carbon-13 polarization (reverse INEPT).
Studying relaxation in hyperpolarized solids at low temperature and low field.
Developing an automated DNP system for unsupervised screening experiments.
3. Team of Arno Kentgens
Evaluating and ordering new instrumentation: microwave source.
Developing new experimental methods: hyperpolarization by Parahydrogen Induced Polarization (PHIP); transferring hyperpolarization to target molecules in the solid state using a “rapid freeze” probe.
4. Team of Jan Korvink
Evaluating and ordering new instrumentation: NMR of miniaturized samples.
Developing new experimental methods: MRI of parallel samples.
5. Team of Alvar Gossert
Preparing and evaluating several protein/ligand systems.
Developing suitable pulse sequences for hyperpolarization.
Developing methods to evaluate protein/ligand affinity for the 4 teams.
Many unanticipated discoveries that had not been foreseen at the time of writing the proposal are currently being evaluated. It appears premature to give an overview of these aspects.