Fig. 3: Intact protein LC-MS analysis applied to covalent drug screening. | Nature Communications

Fig. 3: Intact protein LC-MS analysis applied to covalent drug screening.

From: Mass spectrometry methods and mathematical PK/PD model for decision tree-guided covalent drug development

Fig. 3

Covalent drug candidates—disulfiram (green, column 1), cisplatin (light blue, column 2), ebselen (purple, column 3), and S-XL6 (pink, column 4) were chosen for screening as they target SOD1G93A, a fALS variant of SOD1 protein involved in ALS. (First row) The proposed mechanism of binding and the predicted mass shift for each covalent drug using intact protein LC-MS analysis. (Second row) Control sample for target protein SOD1G93A. Representative raw and deconvoluted spectra (inset) for each covalent drug. (Third row) Assessment of target engagement of covalent drug using purified SOD1G93A. a Disulfiram shows the predicted molecular mass at 16006 Da ( + 148 Da shift). *However, it also forms a SOD1G93A dimer via a non-native disulfide bond with an unknown mechanism, which can result in misfolding and aggregation41,80. b Cisplatin shows the predicted molecular mass at 16311 Da ( + 452 Da i.e., 226 × 2). c Ebselen shows a molecular mass of 16133 Da ( + 274 Da shift), which confirms its proposed MoA. d S-XL6 cross-links SOD1G93A to form a dimer at 31834 Da [15976 Da shift from (15858*2 + 136 – 18 = 118 Da)], that also confirms the proposed MoA45. (Fourth row). Specifically, the mechanism of S-XL6 involves (step 1) reversible thiolate disulfide interchange between a protein cysteine and S-XL6, which due to Brönstead characteristics results in ring-opening to expose a terminal sulfenic acid, which (step 2) forms a second disulfide (and crosslink) with a second cysteine via condensation. Evaluation of in vitro target engagement in target tissue homogenate (i.e., transgenic ALSG93A mouse brain homogenate). Both ebselen and S-XL6 show the predicted mass shifts at 16133 Da and 31834 Da, respectively. (Fifth row) In vivo target engagement (SC RoA for all candidates). Only S-XL6 in vivo dosing was feasible and resulted in successful detection of cross-linked dimer at 31834 Da in brain (1-hr post-dose via SC). A red “no” symbol indicates a “no-go” decision.

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