Library · Biology & lab methods
What Kinase Inhibition Assays got wrong, from 14 theses
Summary written by a model from the records below: The records describe methodological challenges across kinase activity assays, kinome screening pipelines, substrate identification workflows, and computational binding models. Investigators frequently faced assay artifacts, recombinant enzyme inactivation, unconfirmed screening hits, and computational predictions that failed experimental validation. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 9 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
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In vitro kinase activity assays suffer from non-specific artifacts, lack of physiological context, or high signal variance
Assays such as TR-FRET and cellular thermal shifts exhibited tag-dependent dimerization artifacts or irregular thermal stabilization profiles. Other in vitro platforms suffered from high variance, lack of kinase dependence, low throughput, or the absence of necessary cellular cofactors and substrate tertiary structure.
Tried and failed
cellular thermal shift assay applied to kinase-inhibitor target engagement. Outcome: unstable. Reason: concentration-dependent irregular stabilization and destabilization at elevated temperatures
Target identification of novel necroptosis inhibitors · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected relying exclusively on commercial ULight-H3 TR-FRET kinase activity assay due to its artifactual dependency on tag-mediated VRK1 dimerization.
Targeting VRK1: Selective Inhibitors, Chemical Probes, and Biochemical Insights · Harvard · PhD
Considered and rejected
Considered and rejected: Rejected RNA pol II CTD immunoblot kinase assay for CDK8 module inhibition kinetics because it was low throughput, non-quantitative, and consumed large quantities of purified complex.
Identifying CDK8/19 as a Novel Regulator of EWS-FLI1 Function in Ewing Sarcoma · UT Southwestern · PhD
Tried and failed
reconstituted in vitro kinase activity assay applied to small molecule paradox activation modeling. Outcome: no signal. Reason: simplified monomeric kinase system lacked necessary cellular cofactors or full-length scaffold constraints required for paradoxical activation
Biochemical, Biophysical, and Computational Characterization of RAF Dimer Inhibition and Paradoxical Activation by Diverse RAF Inhibitors · Harvard · PhD
Tried and failed
in vitro kinase validation using peptide fragments applied to mass spectrometry identified kinase substrates. Outcome: no signal. Reason: isolated peptide fragments lacked required tertiary structure or context for phosphorylation by the kinase
Identification of brain-specific serine/threonine-protein kinase 1 as a protein kinase c epsilon substrate and its role in alcohol-related behaviors · UT Austin · PhD
Kinome screening and high-throughput profiling fail to validate candidate hits or produce false positives
Screening techniques like yeast surface display and multi-kinase inhibitor screens generated candidate hits that failed to replicate in validation assays. Furthermore, single-dose kinome profiling risked assay over-saturation and false-positive candidate identification, while bead-conjugated pull-downs raised structural alteration concerns.
Tried and failed
yeast surface display kinase activity screening applied to drug-resistant kinase mutant identification. Outcome: did not generalise. Reason: enrichment in yeast display screen did not replicate in orthogonal biochemical and cellular validation assays
High-throughput screen reveals novel resistance mechanisms of Bruton’s Tyrosine Kinase to targeted therapeutics · UT Austin · PhD
Tried and failed
multi-kinase inhibitor drug screening validation applied to drug-resistant cancer cell lines. Outcome: no signal. Reason: candidate inhibitors failed to replicate increased inhibition during dose-response validation
Identifying Vulnerabilities and Novel Drug Candidates in Platinum-Resistant Ovarian Cancer. · Harvard · Master's
Considered and rejected
Considered and rejected: Single-dose (50 µM TTM) kinome profiling was rejected as an endpoint due to potential assay over-saturation and false-positive candidate kinase identification, prompting multi-dose profiling (10, 25, 50 µM)
Copper Metabolism Directly Influences Kinase Functionality To Modulate Signal Transduction In Cancer · Penn · PhD
Kinase substrate identification and mapping assays fail due to biological redundancy and low phosphopeptide yields
Systematic kinase deletion screens and knockout phosphoproteomic workflows failed to detect novel substrates or resolve kinases due to functional redundancy. Additionally, biochemical mapping assays failed to confirm target phosphorylation, and knockout backgrounds resulted in reduced phosphopeptide yields compared to wild-type controls.
Tried and failed
systematic kinase deletion screening applied to identifying substrate phosphorylation site kinase. Outcome: no signal. Reason: redundant or non-canonical kinases likely mediate phosphorylation, preventing complete abolishment in single deletion strains
Functional roles of N-terminal intrinsic disorder and phosphorylation in the yeast heterotrimeric G protein gamma subunit, Ste18 · Georgia Tech · PhD
Tried and failed
comparative phosphoproteomics across kinase knockout lines applied to identifying novel kinase substrate peptides. Outcome: no signal. Reason: failed to detect consensus motif-containing candidate substrates beyond known targets
Functional and mechanistic roles of Ezrin-Radixin-Moesin protein family in regulating apical cell polarity and cell division in epithelial cells · Cornell · PhD
Tried and failed
in vitro biochemical phosphorylation and kinase mapping assays applied to specific target protein phosphorylation site. Outcome: no signal. Reason: could not biochemically confirm phosphorylation in vitro or identify the responsible upstream kinase
INVESTIGATION OF MOTOR PROTEINS IN HOMOLOGOUS RECOMBINATION · Cornell · PhD
Tried and failed
using knockout background for substrate labeling enrichment applied to kinase substrate identification in tissue lysates. Outcome: worse than baseline. Reason: knockout tissue produced lower phosphopeptide yields and fewer detected peptides than wild-type
Identification of brain-specific serine/threonine-protein kinase 1 as a protein kinase c epsilon substrate and its role in alcohol-related behaviors · UT Austin · PhD
Computational kinase docking and predictive scoring fail experimental confirmation or lose to simpler baselines
Three-dimensional convolutional neural network docking models predicted inhibitor binding that could not be confirmed in experimental assays. Moreover, complex data augmentation methods were outperformed by unaugmented baselines, and multi-domain kinases had to be excluded because of violated ATP-site binding assumptions.
Tried and failed
3D CNN scoring of molecular docking poses applied to protein kinase ligand binding prediction. Outcome: did not generalise. Reason: experimental assays failed to confirm predicted inhibitor binding to homology-modeled targets
Leveraging diverse data modalities to study kinase inhibitor polypharmacology · Harvard · PhD
Considered and rejected
Considered and rejected: Decided against using allosteric kinase inhibitors and multi-domain kinases in the KinCo docking dataset due to lack of domain-level binding annotations and violation of ATP-site binding assumptions.
Leveraging diverse data modalities to study kinase inhibitor polypharmacology · Harvard · PhD
Lost to a baseline
On ChEMBL kinase target 10752, unaugmented OriginalFS achieved PR-AUC of 0.289, beating augmented TransFS (0.272) and RedockedFS (0.270)
Deep neural networks for pose validation, affinity prediction, and input attribution · Oxford · PhD
Recombinant kinase purification protocols lead to contaminating activities or loss of catalytic function
Attempts to isolate recombinant protein kinases for in vitro assays were hindered by contaminating background kinase activity. Additionally, extended multi-step chromatographic purification caused kinase cascade enzymes to misfold and lose their catalytic activity.
Tried and failed
recombinant kinase purification for in vitro assays applied to large protein kinase preparation. Reason: could not isolate protein without contaminating background kinase activity
MAPPING AND SPATIAL MONITORING OF THE DNA DAMAGE SIGNALING NETWORK USING MASS SPECTROMETRY · Cornell · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Determine if SteE interacts with and directs phosphorylation on other host kinase substrates using kinase assays or interactome screening. Blocker: Requires wet-lab biochemical assays, protein expression, and kinase interaction screening.
Structural and Functional characterisation of SteE, a Salmonella type III secretion system effector · Imperial · PhD
Left open
Map the kinase landscape across tumor-infiltrating immune populations and evaluate cell-specific responses to kinase inhibitors across cancer types. Blocker: Requires wet-lab experimental phosphoproteomics, FACS sorting, cell co-cultures, and mass spectrometry across multiple cancer types.
Signaling at the Tumor-Immune Interface in Glioblastoma · MIT · PhD
Left open
Perform in vitro kinase assays with candidate kinases (Ksp1, Ime2, Vhs1, Ptk2, Rim15) to identify the kinase responsible for Ste18Nt S7 phosphorylation. Blocker: Requires a wet-lab biochemistry facility and purified candidate kinases to perform in vitro kinase assays.
Functional roles and underlying mechanism of site-specific N-terminal phosphorylation in a heterotrimeric G protein gamma subunit · Georgia Tech · PhD
Left open
Identify the proline-directed kinase responsible for phosphorylating the 112 ATR/RAD1-dependent S/T-P-X-K motifs in mouse spermatocytes. Blocker: Requires wet-lab biochemical or genetic validation (e.g., in vitro kinase assays, kinase perturbation in spermatocytes)
THE ATR-TOPBP1 SIGNALING AXIS IN MAMMALIAN MEIOSIS AND SOMATIC DNA REPAIR · Cornell · PhD
Left open
Perform kinome profiling and evaluate off-target specificity for the lead reversible inhibitor YKL-04-125. Blocker: Requires wet-lab kinase screening assay panels and physical samples of YKL-04-125.
Biochemical characterization of EGFR exon 20 insertion variants and their inhibitor sensitivities · Harvard · PhD
Left open
Test SPARK for in vitro kinase activity using recombinant protein and complementation with a kinase-dead allele. Blocker: Requires a molecular biology wet lab, recombinant protein expression, and in vitro kinase assays.
High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress · MIT · PhD
Left open
Perform phosphoproteomic analysis to measure phosphorylation states of interactors downstream of oncogenic kinase-containing fusion proteins. Blocker: Requires wet-lab experimental phosphoproteomics assays and cell culture.
PROTEIN-PROTEIN INTERACTION NETWORK PERTURBATIONS CAUSED BY ONCOGENIC FUSION EVENTS · Cornell · PhD
Left open
Synthesize and evaluate flavonoid derivatives including flavones, isoflavones, flavanones, and isoflavanones as potential FMS kinase inhibitors. Blocker: Requires wet lab facilities for organic synthesis and kinase inhibition assays
Development of structurally novel FMS (CSF-1R) inhibitors for cancer · Imperial · PhD
Left open
Obtain pure recombinant Tel1 kinase and perform in vitro kinase assays to confirm direct phosphorylation of the D/E-S/T motif. Blocker: Requires a wet biology lab for protein purification and in vitro kinase assays.
MAPPING AND SPATIAL MONITORING OF THE DNA DAMAGE SIGNALING NETWORK USING MASS SPECTROMETRY · Cornell · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Will Comstock · Cornell · PhD · 2025
MAPPING AND SPATIAL MONITORING OF THE DNA DAMAGE SIGNALING NETWORK USING MASS SPECTROMETRY - Changchang Liu · Harvard · PhD · 2021
Leveraging diverse data modalities to study kinase inhibitor polypharmacology - Emma Kathryn Crowley-Dolen · Harvard · PhD · 2026
Targeting VRK1: Selective Inhibitors, Chemical Probes, and Biochemical Insights - Jingyi Hu · Cornell · PhD · 2025
INVESTIGATION OF MOTOR PROTEINS IN HOMOLOGOUS RECOMBINATION - Alicia D. D'Souza · MIT · PhD · 2025
Signaling at the Tumor-Immune Interface in Glioblastoma - Emre Tkacik · Harvard · PhD · 2025
Biochemical, Biophysical, and Computational Characterization of RAF Dimer Inhibition and Paradoxical Activation by Diverse RAF Inhibitors - Xinya Su · Georgia Tech · PhD · 2024
Functional roles of N-terminal intrinsic disorder and phosphorylation in the yeast heterotrimeric G protein gamma subunit, Ste18 - Chotima Seripracharat · Imperial · PhD · 2024
Target identification of novel necroptosis inhibitors - Chiara Alden Victor · Harvard · Master's · 2024
Identifying Vulnerabilities and Novel Drug Candidates in Platinum-Resistant Ovarian Cancer. - Karim Omar · Cornell · PhD · 2024
PROTEIN-PROTEIN INTERACTION NETWORK PERTURBATIONS CAUSED BY ONCOGENIC FUSION EVENTS - Hanchen Zhao · Harvard · PhD · 2023
Biochemical characterization of EGFR exon 20 insertion variants and their inhibitor sensitivities - James Henry McGinnis III · UT Southwestern · PhD · 2023
Identifying CDK8/19 as a Novel Regulator of EWS-FLI1 Function in Ewing Sarcoma
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