Thiol-Dependent Recovery of Catalytic Activity from Oxidized Protein Tyrosine Phosphatases

Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme’s catalytic cysteine thiolate group. Importantly, low-molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively inactivated PTPs (PTP1B and SHP-2) by various low-molecular weight thiols and the enzyme thioredoxin. All monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. In general, molecules bearing low-p<i>K</i>_a thiol groups were particularly effective. The biological thiol glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M^–1 s^–1, while the dithiol dithiothreitol (DTT) displayed an apparent second-order rate constant of 0.325 ± 0.007 M^–1 s^–1. The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 μM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10^–3 s^–1. The rates at which these agents regenerated oxidized PTP1B followed the order Trx > DTT > GSHand comparable values observed at 2 μM Trx, 4 mM DTT, and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1–20 mM. The common biochemical reducing agent tris­(2-carboxyethyl)­phosphine regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M^–1 s^–1. We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low-molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events

Electron and Spin-Density Analysis of Tirapazamine Reduction Chemistry

Tirapazamine (TPZ, <b>1</b>, 3-amino-1,2,4-benzotriazine 1,4-<i>N</i>,<i>N</i>-dioxide), the radical anion <b>2</b> formed by one-electron reduction of <b>1</b>, and neutral radicals <b>3</b> and <b>4</b> formed by protonation of <b>2</b> at O­(N4) or O­(N1), respectively, and their N–OH homolyses <b>3</b> → <b>5</b> + ·OH and <b>4</b> → <b>6</b> + ·OH have been studied with configuration interaction theory, perturbation theory, and density functional theory. A comprehensive comparative analysis is presented of structures and electronic structures and with focus on the development of an understanding of the spin-density distributions of the radical species. The skeletons of radicals <b>3</b> and <b>4</b> are distinctly nonplanar, several stereoisomeric structures are discussed, and there exists an intrinsic preference for <b>3</b> over <b>4</b>. The <i>N</i>-oxides <b>1</b>, <b>5</b>, and <b>6</b> have closed-shell singlet ground states and low-lying, singlet biradical (<b>SP-1</b>, <b>SP-6</b>) or biradicaloid (<b>SP-5</b>) excited states. The doublet radicals <b>2</b>, <b>3</b>, and <b>4</b> are heavily spin-polarized. Most of the spin density of the doublet radicals <b>2</b>, <b>3</b>, and <b>4</b> is located in one (N,O)-region, and in particular, <b>3</b> and <b>4</b> are not C3-centered radicals. Significant amounts of spin density occur in both rings in the singlet biradical­(oid) excited states of <b>1</b>, <b>5</b>, and <b>6</b>. The dipole moment of the N2–C3­(X) bond is large, and the nature of X provides a powerful handle to modulate the N2–C3 bond polarity with opposite effects on the two NO regions. Our studies show very low proton affinities of radical anion <b>2</b> and suggest that the p<i>K</i>_a of radical [<b>2</b>+H] might be lower than 6. Implications are discussed regarding the formation of hydroxyl from <b>3</b> and/or <b>4</b>, regarding the ability of <b>5</b> and <b>6</b> to react with carbon-centered radicals in a manner that ultimately leads to oxygen transfer, and regarding the interpretation of the EPR spectra of reduced TPZ species and of their spin-trap adducts

Electron and Spin-Density Analysis of Tirapazamine Reduction Chemistry

Tirapazamine (TPZ, <b>1</b>, 3-amino-1,2,4-benzotriazine 1,4-<i>N</i>,<i>N</i>-dioxide), the radical anion <b>2</b> formed by one-electron reduction of <b>1</b>, and neutral radicals <b>3</b> and <b>4</b> formed by protonation of <b>2</b> at O­(N4) or O­(N1), respectively, and their N–OH homolyses <b>3</b> → <b>5</b> + ·OH and <b>4</b> → <b>6</b> + ·OH have been studied with configuration interaction theory, perturbation theory, and density functional theory. A comprehensive comparative analysis is presented of structures and electronic structures and with focus on the development of an understanding of the spin-density distributions of the radical species. The skeletons of radicals <b>3</b> and <b>4</b> are distinctly nonplanar, several stereoisomeric structures are discussed, and there exists an intrinsic preference for <b>3</b> over <b>4</b>. The <i>N</i>-oxides <b>1</b>, <b>5</b>, and <b>6</b> have closed-shell singlet ground states and low-lying, singlet biradical (<b>SP-1</b>, <b>SP-6</b>) or biradicaloid (<b>SP-5</b>) excited states. The doublet radicals <b>2</b>, <b>3</b>, and <b>4</b> are heavily spin-polarized. Most of the spin density of the doublet radicals <b>2</b>, <b>3</b>, and <b>4</b> is located in one (N,O)-region, and in particular, <b>3</b> and <b>4</b> are not C3-centered radicals. Significant amounts of spin density occur in both rings in the singlet biradical­(oid) excited states of <b>1</b>, <b>5</b>, and <b>6</b>. The dipole moment of the N2–C3­(X) bond is large, and the nature of X provides a powerful handle to modulate the N2–C3 bond polarity with opposite effects on the two NO regions. Our studies show very low proton affinities of radical anion <b>2</b> and suggest that the p<i>K</i>_a of radical [<b>2</b>+H] might be lower than 6. Implications are discussed regarding the formation of hydroxyl from <b>3</b> and/or <b>4</b>, regarding the ability of <b>5</b> and <b>6</b> to react with carbon-centered radicals in a manner that ultimately leads to oxygen transfer, and regarding the interpretation of the EPR spectra of reduced TPZ species and of their spin-trap adducts

On the Reaction Mechanism of Tirapazamine Reduction Chemistry: Unimolecular N–OH Homolysis, Stepwise Dehydration, or Triazene Ring-Opening

The initial steps of the activation of tirapazamine (TPZ, <b>1</b>, 3-amino-1,2,4-benzotriazine 1,4-<i>N</i>,<i>N</i>-dioxide) under hypoxic conditions consist of the one-electron reduction of <b>1</b> to radical anion <b>2</b> and the protonation of <b>2</b> at O­(N4) or O­(N1) to form neutral radicals <b>3</b> and <b>4</b>, respectively. There are some questions, however, as to whether radicals <b>3</b> and/or <b>4</b> will then undergo N–OH homolyses <b>3</b> → <b>5</b> + ·OH and <b>4</b> → <b>6</b> + ·OH or, alternatively, whether <b>3</b> and/or <b>4</b> may react by dehydration and form aminyl radicals via <b>3</b> → <b>11</b> + H₂O and <b>4</b> → <b>12</b> + H₂O or phenyl radicals via <b>3</b> → <b>17</b> + H₂O. These outcomes might depend on the chemistry <i>after the homolysis</i> of <b>3</b> and/or <b>4</b>, that is, dehydration may be the result of a two-step sequence that involves N–OH homolysis and formation of ·OH aggregates of <b>5</b> and <b>6</b> followed by H-abstraction within the ·OH aggregates to form hydrates of aminyls <b>11</b> and <b>12</b> or of phenyl <b>17</b>. We studied these processes with configuration interaction theory, perturbation theory, and density functional theory. All stationary structures of OH aggregates of <b>5</b> and <b>6</b>, of H₂O aggregates of <b>11</b>, <b>12</b>, and <b>17</b>, and of the transition state structures for H-abstraction were located and characterized by vibrational analysis and with methods of electron and spin-density analysis. The doublet radical <b>17</b> is a normal spin-polarized radical, whereas the doublet radicals <b>11</b> and <b>12</b> feature quartet instabilities. The computed reaction energies and activation barriers allow for dehydration in principle, but the productivity of all of these channels should be low for kinetic and dynamic reasons. With a view to plausible scenarios for the generation of latent aryl radical species <i>without</i> dehydration, we scanned the potential energy surfaces of <b>2</b>–<b>4</b> as a function of the (O)­N1–Y (Y = C5a, N2) and (O)­N4–Z (Z = C4a, C3) bond lengths. The elongation of any one of these bonds by 0.5 Å requires less than 25 kcal/mol, and this finding strongly suggests the possibility of bimolecular reactions of the spin-trap molecules with <b>2</b>–<b>4</b> concomitant with triazene ring-opening

Diethylaminobenzaldehyde Is a Covalent, Irreversible Inactivator of ALDH7A1

There is growing interest in aldehyde dehydrogenases (ALDHs) because of their overexpression in cancer stem cells and the ability to mediate resistance to cancer drugs. Here, we report the first crystal structure of an aldehyde dehydrogenase complexed with the inhibitor 4-diethylaminobenzaldehyde (DEAB). Contrary to the widely held belief that DEAB is a reversible inhibitor of ALDHs, we show that DEAB irreversibly inactivates ALDH7A1 via formation of a stable, covalent acyl-enzyme species

Covalent Adduct Formation between the Antihypertensive Drug Hydralazine and Abasic Sites in Double- and Single-Stranded DNA

Hydralazine (<b>4</b>) is an antihypertensive agent that displays both mutagenic and epigenetic properties. Here, gel electrophoretic, mass spectroscopic, and chemical kinetics methods were used to provide evidence that medicinally relevant concentrations of <b>4</b> rapidly form covalent adducts with abasic sites in double- and single-stranded DNA under physiological conditions. These findings raise the intriguing possibility that the genotoxic properties of this clinically used drug arise via reactions with an endogenous DNA lesion rather than with the canonical structure of DNA

Toward Hypoxia-Selective DNA-Alkylating Agents Built by Grafting Nitrogen Mustards onto the Bioreductively Activated, Hypoxia-Selective DNA-Oxidizing Agent 3‑Amino-1,2,4-benzotriazine 1,4-Dioxide (Tirapazamine)

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a heterocyclic di-<i>N</i>-oxide that undergoes enzymatic deoxygenation selectively in the oxygen-poor (hypoxic) cells found in solid tumors to generate a mono-<i>N</i>-oxide metabolite. This work explored the idea that the electronic changes resulting from the metabolic deoxygenation of tirapazamine analogues might be exploited to activate a DNA-alkylating species selectively in hypoxic tissue. Toward this end, tirapazamine analogues bearing nitrogen mustard units were prepared. In the case of the tirapazamine analogue <b>18a</b> bearing a nitrogen mustard unit at the 6-position, it was found that removal of the 4-oxide from the parent di-<i>N</i>-oxide to generate the mono-<i>N</i>-oxide analogue <b>17a</b> did indeed cause a substantial increase in reactivity of the mustard unit, as measured by hydrolysis rates and DNA-alkylation yields. Hammett sigma values were measured to quantitatively assess the magnitude of the electronic changes induced by metabolic deoxygenation of the 3-amino-1,2,4-benzotriazine 1,4-dioxide heterocycle. The results provide evidence that the 1,2,4-benzotiazine 1,4-dioxide unit can serve as an oxygen-sensing prodrug platform for the selective unmasking of bioactive agents in hypoxic cells

Characterization of Interstrand DNA–DNA Cross-Links Derived from Abasic Sites Using Bacteriophage ϕ29 DNA Polymerase

Interstrand cross-links in cellular DNA are highly deleterious lesions that block transcription and replication. We recently characterized two new structural types of interstrand cross-links derived from the reaction of abasic (Ap) sites with either guanine or adenine residues in duplex DNA. Interestingly, these Ap-derived cross-links are forged by chemically reversible processes, in which the two strands of the duplex are joined by hemiaminal, imine, or aminoglycoside linkages. Therefore, understanding the stability of Ap-derived cross-links may be critical in defining the potential biological consequences of these lesions. Here we employed bacteriophage φ29 DNA polymerase, which can couple DNA synthesis and strand displacement, as a model system to examine whether dA-Ap cross-links can withstand DNA-processing enzymes. We first demonstrated that a chemically stable interstrand cross-link generated by hydride reduction of the dG-Ap cross-link completely blocked primer extension by φ29 DNA polymerase at the last unmodified nucleobase preceding cross-link. We then showed that the nominally reversible dA-Ap cross-link behaved, for all practical purposes, like an irreversible, covalent DNA–DNA cross-link. The dA-Ap cross-link completely blocked progress of the φ29 DNA polymerase at the last unmodified base before the cross-link. This suggests that Ap-derived cross-links have the power to block various DNA-processing enzymes in the cell. In addition, our results reveal φ29 DNA polymerase as a tool for detecting the presence and mapping the location of interstrand cross-links (and possibly other lesions) embedded within regions of duplex DNA

Near-Silence of Isothiocyanate Carbon in ¹³C NMR Spectra: A Case Study of Allyl Isothiocyanate

¹H and ¹³C NMR spectra of allyl isothiocyanate (AITC) were measured, and the exchange dynamics were studied to explain the near-silence of the ITC carbon in ¹³C NMR spectra. The dihedral angles α = ∠(C1–C2–C3–N4) and β = ∠(C2–C3–N4–C5) describe the <i>conformational dynamics</i> (conformation change), and the bond angles γ = ∠(C3–N4–C5) and ε = ∠(N4–C5–S6) dominate the <i>molecular dynamics</i> (conformer flexibility). The conformation space of AITC contains three minima, <i>C</i>_<i>s</i>-M1 and enantiomers M2 and M2′; the exchange between conformers is very fast, and conformational effects on ¹³C chemical shifts are small (ν_M1 – ν_M2 < 3 ppm). Isotropic chemical shifts, ICS­(γ), were determined for sp, sp^<i>x</i>, and sp² N-hybridization, and the γ dependencies of δ­(N4) and δ­(C5) are very large (10–33 ppm). Atom-centered density matrix propagation trajectories show that every conformer can access a large region of the potential energy surface AITC­(γ,ε,...) with 120° < γ < 180° and 155° < ε < 180°. Because the extreme broadening of the ¹³C NMR signal of the ITC carbon is caused by the structural flexibility of every conformer of AITC, the analysis provides a general explanation for the near-silence of the ITC carbon in ¹³C NMR spectra of organic isothiocyanates

Allylation and Alkylation of Biologically Relevant Nucleophiles by Diallyl Sulfides

Allyl sulfides are bioactive phytochemicals found in garlic, onion, and other members of the genus <i>Allium</i>. Here we showed that diallyl disulfide and diallyl trisulfide can transfer allyl side chains to low molecular weight thiols. Diallyl monosulfide is inert with respect to this allyl transfer reaction. On the other hand, diallyl sulfone, a known metabolite of diallyl monosulfide, alkylates both amines and thiols under physiologically relevant conditions via isomerization to an electrophilic vinyl sulfone