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SITE-SPECIFIC ACTIVITY-BASED PROTEIN PROFILING USING PHOSPHONATE HANDLES

ABSTRACT: Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses IMAC-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe (ABP), enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID–ABPP (Activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated Afatinib derivative (PF-06672131). As PhosID–ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor (EGFR), which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID–ABPP is highly complementary to biotin-based enrichment strategies in activity-based protein profiling studies, with PhosID–ABPP providing the advantage of direct ABP-interaction site identification.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin Cancer Cell

SUBMITTER: Wouter van Bergen

LAB HEAD: A.J.R. Heck

PROVIDER: PXD036569 | Pride | 2023-01-10

REPOSITORIES: Pride

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Project description:Clinically significant radiation-induced lung injury (RILI) is associated with significant morbidity and mortality and a common toxicity in patients administered thoracic radiotherapy. While the molecular etiology of RILI is poorly understood, we previously characterized a murine model of RILI in which alterations in lung endothelial barrier integrity surfaced as a potentially important pathobiologic event. In these studies, inhibition of HMG-CoA reductase activity (simvastatin) reduced murine RILI-associated lung inflammation and vascular leak and attenuated radiation-induced dysregulation of sphingolipid metabolic pathway genes identified by genome-wide lung gene expression profiling. In the present study, we test the hypothesis that sphingolipid signaling components serve as important modulators of RILI pathobiology and novel therapeutic targets. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to cumulative sphingosine-1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage (BAL) fluid and lung tissue following 25 Gy exposure (6 weeks). Moreover, genetically-engineered mice with either targeted deletion of SphK1 (SphK1-/-), or with reduced expression of selective members of the S1P receptor family (S1PR1+/-, S1PR2-/-, S1PR3-/-,), exhibited marked susceptibility to RILI-mediated lung inflammation. Finally, we assessed the efficacy of three potent vascular barrier-protective S1P analogues FTY720 (FTY), fTysiponate (fTyS) and SEW-2871 (SEW) in attenuating indices of RILI. The phosphonate analogue, fTyS, and to a lesser degree SEW, exhibited significant attenuation of RILI and RILI-induced gene dysregulation compared to control RILI-challenged mice (6 weeks). In contrast, FTY failed to significantly alter physiologic or genomic changes compared to RILI-challenged controls. Together, these results support the targeting of sphingolipid components as a novel and effective therapeutic strategy in RILI. Four mice were treated with PBS as a control. Three mice were treated with (S)-FTY-phosphonate (0.1mg/kg) as a drug control. Three mice were treated with SEW-2871 (0.1mg/kg) as a drug control. Three mice were treated with FTY720 (0.1mg/kg) as a drug control. Three mice were treated with administered radiation (25 Gy) alone. Three mice were treated with both administered radiation (25 Gy) and (S)-FTY-phosphonate (0.1mg/kg). Three mice were treated with both administered radiation (25 Gy) and SEW-2871 (0.1mg/kg). Three mice were treated with both administered radiation (25 Gy) and FTY720 (0.1mg/kg).

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SITE-SPECIFIC ACTIVITY-BASED PROTEIN PROFILING USING PHOSPHONATE HANDLES

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