The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins

Abstract

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.4 and 37 °C results in the formation of putative S-tetrahydrothiophenium adducts at their catalytic Cys residues, followed by β-elimination to yield dehydroalanine. Both proteins contain a second Cys, in their catalytic C-X-X-C motif, which reacts with the dehydroalanine, the initial Cys adduct with busulfan, or the S-tetrahydrothiophenium, to form novel intramolecular cross-links. The reactivity of the dehydroalanine (DHA) formed is further demonstrated by adduction with glutathione to yield a lanthionine and by a novel reaction with the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which yields a phosphine adduct via Michael addition to the DHA. Formation of a second quaternary organophosphonium salt via nucleophilic substitution with TCEP on the initial busulfan-protein adduct or on the THT(+)-Redoxin species is also observed. These results reveal a rich potential for reactions of busulfan with proteins in vitro, and likely in vivo. It is striking that several of the chemically altered protein products retain none of the atoms of busulfan, in contrast to typical drug-protein adducts or traditional protein modification reagents. In particular, the ability of a clinically used drug to convert Cys to dehydrolanine in intact proteins, and its subsequent reaction with biological thiols, is unprecedented.

Figure 1

Deconvoluted ESI-MS spectra of (a) Grx-1 or (b) Thrx-1 incubated with busulfan for 24 h at pH 7.4 and 37 °C in the presence of 250 μM TCEP and after reduction with 10 mM DTT for 1 h at RT. The native redoxin, DHA-Redoxin, and putative THT⁺-Redoxin are clearly present. In panel b, the asterisk indicates Met1-cleaved Thrx-1 species. The insets show the time course of formation or depletion of the species present. The relative amount (percent of total protein) of each species was calculated from the fractional area of the most intense charge state (i.e., [M + 11H]¹¹⁺ or [M + 10H]¹¹⁺) in the MS spectra. The isobaric species DHA-Redoxin and Cyclo-Redoxin were integrated as if they were a single species. A similar approach was applied to THT⁺-Redoxin and CL-Redoxin.

Figure 2

Deconvoluted ESI-MS spectrum of Grx-1 incubated with busulfan for 24 h at pH 7.4 and 37 °C in the presence of 5 mM TCEP. Two new species with a mass shift of 250 Da were identified as TCEP adducts, [M − 33 + 250]⁺ or [M + 55 + 250]⁺.

Figure 3

Products of iodoacetamide alkylation of Grx-1 samples previously incubated with busulfan (24 h at pH 7.4 and 37 °C) in the presence of (a) 250 μM or (b) 5 mM TCEP. Grx-1/busulfan products formed in the presence of 250 μM TCEP (a) had three cysteines available for S-carbamidomethylation, whereas the TCEP adducts formed in the presence of 5 mM TCEP (b) conserved four cysteines amenable for IAM alkylation. These results suggested a cyclic nature of the Grx-1/busulfan products.

Figure 4

Deconvoluted mass spectrum of a busulfan-incubated Grx-1 sample (24 h at pH 7.4 and 37 °C in the presence of 250 μM TCEP) treated with GSH (24 h at pH 7.4 and 37 °C the presence of 250 μM DTT). A mass shift of 307 Da indicates formation of a DHA-Grx-1/glutathione adduct.

Figure 5

High-mass accuracy MS/MS spectra of the Grx-1 tryptic peptides (a) Cyclo-peptide and (b) CL-peptide obtained from fragmentation of precursor ions with masses of 745.91 Da (i.e., [M + 2H]²⁺) and 789.93 Da (i.e., [M + 2H]²⁺), respectively. Selected fragment ions are highlighted. Atoms colored green belong to the side chains of cysteine residues, whereas those colored blue are from busulfan. The symbol “CAM” in the peptide sequence indicates that the specific Cys residue was S-carbamidomethylated with iodoacetamide.

Figure 6

MS/MS spectra of the Grx-1 tryptic peptides (a) THT⁺-peptide and (b) DHA-peptide obtained from fragmentation of precursor ions with masses of 409.72 Da (i.e., [M + 3H]⁴⁺) and 516.62 Da (i.e., [M + 3H]³⁺), respectively. Atoms colored green belong to side chains of cysteine residues, whereas those colored blue are from busulfan. Asterisks denote fragments characteristic of the DHA-peptide formed by gas-phase decomposition of the THT⁺-peptide.

Figure 7

MS/MS spectra of the Grx-1 tryptic peptides (a) Phospho-DHA-peptide and (b) Phospho-THT⁺-peptide obtained from fragmentation of precursor ions with masses of 450.23 Da (i.e., [M + 3H]⁴⁺) and 472.24 Da (i.e., [M + 3H]⁴⁺), respectively. Atoms colored green belong to side chains of cysteine residues, whereas those colored blue are from busulfan or TCEP.

Figure 8

MS/MS spectrum of the Grx-1 tryptic peptide GS-DHA-peptide obtained from fragmentation of precursor ions with a mass of 464.49 Da (i.e., [M + 4H]⁴⁺). In addition to ions resulting from fragmentation of the peptide backbone, a characteristic neutral loss of pyroglutamic acid (pGlu, monoisotopic mass of 129.04 Da) is observed for many fragments (colored red). Atoms colored green belong to side chains of cysteine residues, whereas those colored blue are from busulfan or GSH.

Figure 9

Electrostatic potential surface and cartoon representation of Grx-1 (adapted from Protein Data Bank entry 1JHB, model 1) and Thrx-1 (adapted from Protein Data Bank entry 1ERT). The close-ups show the structural details of the catalytic Cys residues (residues 23 and 26 and residues 32 and 35 for Grx-1 and Thrx-1, respectively). Images were generated using PyMol Molecular Graphics System, version 1.2r1 (Schrödinger, LLC). Arrows indicate the catalytic Cys residues that specifically react with busulfan. Other Cys residues do not react.

Scheme 1

Proposed Reaction Mechanism for Products Formed from Reaction of Busulfan with Redoxin Thiols