Advancing Novel Approaches in Kinase Interface Networks

Objectives: Engineer TR-FRET dimer reporters using type-I AfBPs as donors/controls; establish Z’-validated windows across 3 kinases; Build orthogonal counterscreens to exclude ATP-pocket interference and optical artifacts; Define comparative dimerization fingerprints under ligands (type-I AfBP donors vs controls) to standardise cross-lab interpretation; Publish modular SOPs transferable to other kinases.

Expected results: Validated TR-FRET reporter set with per-kinase Z’ benchmarks, an assay-interference taxonomy (optical/aggregation/ATP-leak) and FAIR-deposited benchmark datasets; methods/protocol paper + dataset.

Secondments: NU: HDX-MS on ATP-locked vs apo kinases (M10–12; 3 months); EPFL: Assay-based peptide-hit triage (M28–32; 5 months).

Integration: Provides WP1 “common filter”; feeds WP3 hit triage and WP2 mechanistic signature mapping.

Interdisciplinarity: Combines chemical biology (AfBPs), assay engineering and biophysics (HDX) and phage-display screening

Objectives: Design and synthesise AfBP-anchored bisubstrate probes across 3 kinases with cleavable linkers and FRET pairs; establish substrate-displacement readouts that distinguish ATP-site binders from substrate-site competitors; benchmark transferability; implement AfBP-based counterselection; publish kit-style SOPs.

Expected results: A generalisable bisubstrate FRET format with construct-design rules, per-kinase benchmarking datasets and open, transferable SOPs; short methods paper + technical note demonstrating ATP-occlusion vs substrate-displacement discrimination; FAIR-deposited data.

Secondments: UoC: smFRET comparison of apo vs ATP-locked kinases (substrate-cleft geometry) (M10–12; 3 months); CNRS: Assay-based small-molecule hit triage for substrate binders (M28–32; 5 months).

Integration: Complements DC1 by providing the second WP1 “common filter”; supplies WP3 screening cascades and WP4 validation with substrate-site readouts.

Interdisciplinarity: Combines chemical biology, assay engineering, HTS screening and biophysics (smFRET, kinetic profiling).

Objectives: Generate site-specific labelled EGFR/BRAF/LTK constructs; quantify monomer↔dimer equilibria and conformational states under AfBP locking; derive FRET-signature fingerprints predictive of interface-inhibitor activity; propose interface mutants as mechanistic controls for WP1 and WP3.

Expected results: A smFRET atlas across three kinases, including state distributions, kinetic-transition parameters and mutant-based mechanistic controls; open analysis pipelines and FAIR-released datasets; a methodological paper integrating smFRET with ATP-site–locked conformational mapping.

Secondments: VIB: Cryo-EM correlation of FRET-defined states (M19–22; 4 months); AZ: MD simulations for atomistic validation of conformational transitions (M30–32; 3 months).

Integration: Provides mechanistic fingerprints for WP1 assay calibration and guides WP3 hit prioritisation; anchors structural interpretation across WP2 and supports WP4 functional analysis.

Interdisciplinarity: Integrates single-molecule biophysics, structural biology (cryoEM) and computational modelling

Objectives: Reconstitute EGFR and LTK (SMALPs) and soluble BRAF complexes; capture AfBP-locked conformations; map dimer and substrate interfaces; identify hotspot residues that report on interface engagement; cross-validate structural classes with smFRET states and feed geometric constraints back to WP1 assay design.

References: ³² Barniol-Xicota, M. et al. Commun. Biol. 4, 218 (2021); ³³ Barniol-Xicota, M. et al. J. Am. Chem. Soc. 140, 14557–14561 (2018); ³⁴ Merz, M. L. et al. Nat. Chem. Biol. 20, 624–633 (2024); ³⁵ Kong, X.-D. et al. Nat. Biomed. Eng. 4, 560–571 (2020); ³⁶ Kale, S. S. et al. Nat. Chem. 10, 715–723 (2018); ³⁷ Bosc, N. et al. ACS Chem. Biol. 15, 1566–1574 (2020).

Expected results: Medium-resolution cryo-EM maps and focused classifications of low-occupancy interface states; a hotspot/interfacial-geometry map for all three kinases; a methodological paper on SMALP-enabled cryo-EM for dynamic kinase assemblies plus FAIR-deposited structural classes and metadata.

Secondments: UPF: SMALP membrane-reconstitution workflows (M19–22; 4 months); INS: In-cellulo validation (M30–32; 3 months)

Integration: Supplies structural determinants guiding WP1 assay geometry, calibrates WP2 smFRET interpretations and defines actionable interface hotspots for WP3 ligand optimisation.

Interdisciplinarity: Combines cryo-EM, membrane-protein biophysics (SMALPs) and integration with smFRET and cellular validation.

Objectives: Develop clickable/biotinylated SMALP nanodiscs for EGFR and LTK; quantify dimerization and conformational engagement using TR-FRET and smFRET; define how AfBP-locking reshapes membrane-embedded kinase dynamics; identify SMALP conditions that best approximate cellular phenotypes; provide a biophysical “translation layer’’ linking WP1/WP2 assays to WP4 cellular readouts.

Expected results: A SMALP-based native-membrane assay platform preserving functional kinase states; comparative TR-FRET/smFRET datasets benchmarked against soluble controls; protocols and datasets deposited as FAIR resources; a platform-style paper linking membrane-embedded conformations to cellular signatures.

Secondments: UoC: smFRET benchmarking of SMALP-incorporated kinases (M19–22; 4 months); KUL: Assay-harmonisation and cross-platform alignment with WP1 pipelines (M30–32; 3 months).

Integration: Provides membrane-native reference states for WP1 assay calibration, informs WP2 structural and dynamic interpretations and aligns WP3 discovery outputs with WP4 cellular behaviour.

Interdisciplinarity: Combines membrane biophysics (SMALPs), smFRET, assay integration and cross-WP methodological harmonisation.

Objectives: Generate and screen highly diverse combinatorial (bi)cyclic-peptide libraries against EGFR, BRAF and LTK regulatory surfaces under ATP-locked conditions; enrich selectively for non-ATP binders using WP1 filters; map minimal binding motifs via alanine scanning/truncations; derive early SAR and advance top series toward membrane-permeable, cell-compatible variants.

Expected results: At least two validated peptide chemotypes with interface specificity; a pre-lead data package and a publishable study demonstrating interface-targeting peptide scaffolds.

Secondments: UoC: MCR-enabled peptide diversification and conjugate design (M20–23; 4 months); SU: ML-based sequence prioritisation and SAR prediction workflows (M31–33; 3 months).

Integration: Primary peptide-hit generator for WP4 cellular validation; tightly coupled to WP2 (structural hotspots, smFRET fingerprints) and WP1 (dimer/substrate-site assay readouts) for iterative optimisation.

Interdisciplinarity: Combines combinatorial chemistry, peptide engineering, discovery and ML-assisted design.

Objectives: Screen focused PPI and fragment libraries under ATP-locking conditions; apply optical-interference counterscreens and ATP-site competition to confirm interface binding; integrate HTRF FRET probes; derive interface-anchored pharmacophores and iterate fragment follow-ups guided by WP2 hotspots and smFRET signatures; develop chemotypes with PPI-compatible profiles; optimize hit series using ChemoDOTS and structure-based growing to generate interface-selective hit-to-lead candidates.

Expected results: At least two small-molecule series with assay-confirmed interface engagement; a methodology paper on interface-directed triage and fragment-to-lead progression.

Secondments: GU: In-cellulo engagement and functional validation (M20–23; 4 months); LEI: AI-driven prioritisation and enrichment modelling (M31–33; 3 months).

Integration: Primary small-molecule discovery stream feeding WP4; tightly coupled to WP1 interference counterscreens and WP2 structural/mechanistic constraints.

Interdisciplinarity: Combines PPI-focused screening, biophysics-guided triage, in cellulo validation and AI-assisted optimisation.

Objectives: Design functionalised scaffolds using MCR chemistry; synthesise medium-sized, structurally diverse libraries biased for PPI/interface engagement; perform iterative SAR cycles (hit-to-lead), scaffold hopping and optimisation of physicochemical and conformational properties; profile all compounds in WP1 TR-FRET and bisubstrate assays under ATP-locking conditions.

Expected results: A rationally assembled small-molecule library, ≥1–2 validated chemotypes with interface-modulating activity and scalable lead compounds suitable for WP4 mechanistic and cellular evaluation.

Secondments: Servier: High-throughput analogue synthesis and hit-refinement support (M20–23; 4 months); GU: Cellular target-engagement and pathway-level validation (M31–33; 3 months).

Integration: Provides binder feed for WP4; tightly linked to WP1 counterscreens and WP2 mechanistic constraints.

Interdisciplinarity: Spans synthetic/medicinal chemistry (MCR scaffolds), chemical biology (assay-guided SAR) and AI-supported design, with translational validation in cell biology

Objectives: Build docking, shape-phonon and ML models that integrate WP2 cryo-EM/smFRET states with WP1 assay outcomes; train classifiers to penalise ATP-like geometries and prioritise interface-compatible scaffolds; generate prospective design hypotheses and SAR expansions for DC6–DC8 discovery streams.

Expected results: Open, validated modelling pipelines and benchmark datasets; a methods paper demonstrating improved enrichment for shallow kinase interfaces; a prospective case study showing hit-rate uplift in WP3 screens.

Secondments: EPFL: Experimental grounding in peptide interface binders (M20–23; 4 months); KUL: Testing DC9-prioritised designs in the bisubstrate assay (M31–33; 3 months).

Integration: Provides the central computational design loop linking WP2 structural signatures, WP1 assay labels and WP3 screening/optimisation workflows.

Interdisciplinarity: Combines cheminformatics & machine learning, phage-display screening and assay-bases hit triage.

Objectives: Develop DL architectures integrating text-mined, cheminformatic and biophysical descriptors to predict interface binding, allosteric signatures and resistance liabilities; jointly learn across peptide and small-molecule modalities; generate hybrid peptidomimetic design hypotheses; deliver consortium-wide prioritisation dashboards for hit triage and analogue selection.

Expected results: A resistance-aware AI workflow for non-ATP interface ligands, accompanied by public models/datasets and interpretable feature maps; prospective design case studies demonstrating improved optimisation trajectories in WP3 campaigns.

Secondments: VIB: Structural-class extraction and conformational-feature mapping from cryo-EM datasets (M20–23; 4 months); Skylight: SHG profiling of ligand-induced conformational changes (M31–33; 3 months).

Integration: Provides resistance- and allosterism-aware prioritisation engines for WP3 and closes the computational–mechanistic feedback loop with WP2.

Interdisciplinarity: Bridges AI/ML, cheminformatics, structural biology (cryo-EM constraints) and chemical-probe/screening environments.

Objectives: Establish NanoBRET and CETSA cellular lines for EGFR/BRAF; quantify intracellular engagement of top peptide and small-molecule interface hits; map pathway rewiring under interface modulation and benchmark against ATP-site inhibitors; establish resistance-evolution baselines; define cell-level go/no-go criteria to guide WP3 optimisation.

Expected results: Quantitative NanoBRET/CETSA engagement datasets; pathway-profiling resource and comparative analysis versus ATP-site drugs; validated decision-gates for chemical-series advancement; a resistance-trajectory dataset to benchmark the adaptive resilience of interface modulators.

Secondments: AuTh: Assay-based hits refinement (M20–23; 4 months); LEI: AI-assisted resistance-modelling inputs (M31–33; 3 months).

Integration: Provides the translational anchor for the consortium; feeds engagement and signalling constraints back to WP3 design streams and WP1 assay tuning.

Interdisciplinarity: Combines chemical biology, quantitative cell signalling, resistance biology and AI-guided interpretation.

Objectives: Establish LTK cellular systems and define whether signalling depends on dimerization/interface integrity; generate interface-disrupting mutants and quantify their effects on activation and pathway output; map substrate motifs via quantitative phosphoproteomics; validate AfBP engagement; measure how interface-targeting ligands reshape signalling networks; provide a generalizable blueprint for extending interface-centric analysis to other dark-kinome RTKs.

Expected results: First mechanistic map of LTK; public phosphoproteomic and signalling dataset; a cellular engagement + pathway paper defining interface dependency and substrate specificity.

Secondments: KUL (Prof Derua): Phosphoproteomics optimisation and workflow refinement (M20–23; 4 months); AZ: MD simulations to interpret structural/dynamic determinants of LTK regulation (M31–33; 3 months).

Integration: Establishes the mechanistic basis of an understudied RTK; informs WP1/DC2 bisubstrate-probe design and provides structural/functional constraints for WP3 optimisation and WP4 comparative benchmarking.

Interdisciplinarity: Combines cell signalling, phosphoproteomics, chemical biology and computational biophysics.