Novel Bruton’s Tyrosine Kinase (BTK) Substrates for Time-Resolved Luminescence Assays

Widstrom, Naomi E.; Perez, Minervo; Pratt, Erica D.; Heier, Jason L.; Blankenhorn, John F.; Breidenbach, Lindsay; Peterson, Hannah; Parker, Laurie L.

doi:10.1021/acschembio.2c00106

Cited by 4 publications

(17 citation statements)

References 27 publications

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“…Following phosphopeptide enrichment of the resulting material, samples were analyzed on an Orbitrap Velos mass spectrometer and sequences identified by PEAKS Studio X Pro. The identified phosphopeptides were used as input into our KINATEST-ID V2 pipeline . In brief, KINATEST-ID V2 creates a Fisher Odds-based PSSM for each amino acid at positions surrounding a central phosphorylation site.…”

Section: Resultsmentioning

confidence: 99%

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Novel Substrate Prediction for the TAM Family of RTKs Using Phosphoproteomics and Structure-Based Modeling

Widstrom,

Andrianov,

Heier

et al. 2023

ACS Chem. Biol.

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The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date, there are no FDA-approved small molecule inhibitors for the TAM kinases. Inhibitor design and screening rely on tools to study the kinase activity. Our goal was to address this gap by designing a set of synthetic peptide substrates for each of the TAM family members: Tyro3, Axl, and Mer. We used an in vitro phosphoproteomics workflow to determine the substrate profile of each TAM kinase and input the identified substrates into our data processing pipeline, KINATEST-ID, producing a position-specific scoring matrix for each target kinase and generating a list of candidate synthetic peptide substrates. We synthesized and characterized a set of those substrate candidates, systematically measuring their initial phosphorylation rate with each TAM kinase by LC-MS. We also used the multimer modeling function of AlphaFold2 (AF2) to predict peptide−kinase interactions at the active site for each of the novel candidate peptide sequences against each of the TAM family kinases and observed that, remarkably, every sequence for which it predicted a putative catalytically competent interaction was also demonstrated biochemically to be a substrate for one or more of the TAM kinases. This work shows that kinase substrate design can be achieved using a combination of preference motifs and structural modeling, and it provides the first demonstration of peptide−protein interaction modeling with AF2 for predicting the likelihood of constructive catalytic interactions.

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Section: Resultsmentioning

confidence: 99%

“…Italic Y indicates phosphorylation site. Underlined scores indicate the score is above threshold for target kinase (see ref for details). B denotes biotinylated lysine.…”

Section: Resultsmentioning

confidence: 99%

Novel Substrate Prediction for the TAM Family of RTKs Using Phosphoproteomics and Structure-Based Modeling

Widstrom,

Andrianov,

Heier

et al. 2023

ACS Chem. Biol.

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“…28 Previously, we have successfully used an adapted in vitro phosphoproteomics workflow to expand the number of known substrates, identify the preferences and design synthetic peptide substrates for target kinase that lacked sufficient numbers of reported substrates. 25, 29 To determine the substrates for the TAM family kinases, cell lysate was reduced and alkylated, digested with trypsin, and treated with lambda phosphatase to dephosphorylate endogenous phosphosites. This was used as a natural peptide library, reacted in kinase reaction buffer (as described in the Experimental Section) for 2 hrs in triplicate with recombinantly prepared enzyme comprising the intracellular portion of each: Tyro3 (455-end), Axl (473-end), and Mer (528-end).…”

Section: Resultsmentioning

confidence: 99%

“…Data was processed as previously described. 25 In brief, peptides were identified using PEAKS Studio Xpro (Bioinformatics Solutions Inc.) and exported protein-peptides.csv lists were used for input into the PEAKS ModExtractor 35 , which combines all the input lists into a concatenated output file, containing a modification-centered list of all peptides. Tables S1-3 show a summary of the total number of unique peptides and phosphorylated peptides with A-score ≥ 30 identified for each kinase and replicate.…”

Section: Methodsmentioning

confidence: 99%

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Novel substrate prediction for the TAM family of RTKs using phosphoproteomics and structure-based modeling

Widstrom,

Andrianov,

Heier

et al. 2023

Preprint

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The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date there are no FDA-approved small molecule inhibitors for the TAM kinases. Inhibitor design and screening rely on tools to study kinase activity. Our goal was to address this gap by designing a set of synthetic peptide substrates for each of the TAM family members: Tyro3, Axl and Mer. We used an in vitro phosphoproteomics workflow to determine the substrate profile of each TAM kinase and input the identified substrates into our data processing pipeline, KINATEST-ID, producing a position-specific scoring matrix for each target kinase and generating a list of candidate synthetic peptide substrates. We synthesized and characterized a set of those substrate candidates, systematically measuring their initial phosphorylation rate with each TAM kinase by LC-MS. We also used the multimer modeling function of AlphaFold2 to predict peptide-kinase interactions at the active site for each of the novel candidate peptide sequences against each of the TAM family kinases, and observed that remarkably, every sequence for which it predicted a putatively catalytically competent interaction was also demonstrated biochemically to be a substrate for one or more of the TAM kinases. This work shows that kinase substrate design can be achieved using a combination of preference motifs and structural modeling, and it provides the first demonstration of peptide-protein interaction modeling for predicting likelihood of constructive catalytic interactions.

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Luminescent lanthanide metallopeptides for biomolecule sensing and cellular imaging

Sánchez-Fernández,

Obregon-Gomez,

Sarmiento

et al. 2024

Chem. Commun.

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Novel Bruton’s Tyrosine Kinase (BTK) Substrates for Time-Resolved Luminescence Assays

Cited by 4 publications

References 27 publications

Novel Substrate Prediction for the TAM Family of RTKs Using Phosphoproteomics and Structure-Based Modeling

Novel Substrate Prediction for the TAM Family of RTKs Using Phosphoproteomics and Structure-Based Modeling

Novel substrate prediction for the TAM family of RTKs using phosphoproteomics and structure-based modeling

Luminescent lanthanide metallopeptides for biomolecule sensing and cellular imaging

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