PXD054878

PXD054878 is an original dataset announced via ProteomeXchange.

Title	An SH3-binding allosteric modulator stabilizes the global conformation of the AML-associated Src-family kinase, Hck
Description	While ATP-site inhibitors for protein-tyrosine kinases are often effective drugs, their clinical utility can be limited by off-target activity and acquired resistance mutations due to the conserved nature of the ATP-binding site. However, combining ATP-site and allosteric kinase inhibitors can overcome these shortcomings in a double-drugging framework. Here we explored the allosteric effects of two pyrimidine diamines, PDA1 and PDA2, on the conformational dynamics and activity of the Src-family tyrosine kinase Hck, a promising drug target for acute myeloid leukemia. Using 1H/15N-HSQC NMR, we mapped the binding site for both analogs to the PPII-binding surface of the SH3 domain. Despite the shared binding site, PDA1 and PDA2 had opposing effects on near-full-length Hck dynamics by hydrogen-deuterium exchange mass spectrometry, with PDA1 stabilizing and PDA2 disrupting the overall kinase conformation. Kinase activity assays were consistent with these observations, with PDA2 enhancing kinase activity while PDA1 was without effect. Molecular dynamics simulations predicted selective bridging of the kinase domain N-lobe and SH3 domain by PDA1, a mechanism of allosteric stabilization supported by site-directed mutagenesis of N-lobe contact sites. Cellular thermal shift assays confirmed SH3 do-main-dependent interaction of PDA1 with wild-type Hck in myeloid leukemia cells and with a kinase domain gatekeeper mutant (T338M). These results identify PDA1 as a starting point for Src-family kinase allosteric inhibitor development that may work in concert with ATP-site inhibitors to suppress the evolution of resistance.
HostingRepository	PRIDE
AnnounceDate	2025-05-07
AnnouncementXML	Submission_2025-05-07_00:21:59.074.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Thomas Wales
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Synapt MS

Revision	Datetime	Status	ChangeLog Entry
0	2024-08-14 02:58:17	ID requested
⏵ 1	2025-05-07 00:21:59	announced

10.1016/j.jbc.2024.108088;

Selzer AM, Gerlach G, Gonzalez-Areizaga G, Wales TE, Cui SY, Iyer P, Engen JR, Camacho C, Ishima R, Smithgall TE, An SH3-binding allosteric modulator stabilizes the global conformation of the AML-associated Src-family kinase, Hck. J Biol Chem, 301(1):108088(2025) [pubmed]

submitter keyword: HCK

HDX-MS

hydrogen/deuterium exchange mass spectrometry

allosteric modulator

global conformation

Thomas Wales
contact affiliation	Northeastern University
contact email	t.wales@northeastern.edu
lab head
Thomas Wales
contact affiliation	Northeastern University
contact email	t.wales@northeastern.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD054878
     1. Label: PRIDE project
     2. Name: An SH3-binding allosteric modulator stabilizes the global conformation of the AML-associated Src-family kinase, Hck