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Targeted SuFEx-mediated covalent modification of a tyrosine residue in the catalytic pocket of tyrosyl-DNA phosphodiesterase 1

ABSTRACT: Developing effective inhibitors of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) is complicated by the shallow catalytic pocket and the difficulties of overcoming binding interactions of substrate. Recently, we discovered a quinolone-binding hot spot in TDP1s active site proximal to the evolutionary conserved Y204 and F259 residues that position DNA. Sulfur (VI) fluoride exchange (SuFEx) is a biocompatible click chemistry reaction that enables acylation of protein residues, including tyrosine. Selective protein modifications can provide valuable insights into the biological roles of proteins and inform ligand design. As we report herein, we used SuFEx chemistries to prepare several covalent TDP1-bound binders showing site-specific covalent bonds with Y204. Our work presents the first application of SuFEx chemistries to TDP1-binding ligands. It validates the ability to covalently modify specific TDP1 residues by designed targeting. Adding this capability to the chemical biology toolbox of resources for studying TDP1 could have far reaching consequences.

INSTRUMENT(S): Orbitrap Eclipse, Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Terrence Burke

PROVIDER: MSV000095388 | MassIVE | Fri Jul 19 09:12:00 BST 2024

REPOSITORIES: MassIVE

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Project description:Matthew F. Hockin, Kevin M. Cawthern, Michael Kalafatis & Kenneth G. Mann. A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition. Biochemistry 38, 21 (1999). The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.

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Targeted SuFEx-mediated covalent modification of a tyrosine residue in the catalytic pocket of tyrosyl-DNA phosphodiesterase 1

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