26 research outputs found
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><sub>a</sub> 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<sup>–1</sup> s<sup>–1</sup>, while the
dithiol dithiothreitol (DTT) displayed an apparent second-order rate
constant of 0.325 ± 0.007 M<sup>–1</sup> s<sup>–1</sup>. 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<sup>–3</sup> s<sup>–1</sup>. 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 trisÂ(2-carboxyethyl)Âphosphine
regenerates enzymatic activity from oxidized PTP1B somewhat faster
than the thiol-based reagents, with a rate constant of 1.5 ±
0.5 M<sup>–1</sup> s<sup>–1</sup>. 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 OÂ(N4) or OÂ(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 biradicalÂ(oid) excited states of <b>1</b>, <b>5</b>, and <b>6</b>. The dipole moment of
the N2–C3Â(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><sub>a</sub> 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 OÂ(N4) or OÂ(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 biradicalÂ(oid) excited states of <b>1</b>, <b>5</b>, and <b>6</b>. The dipole moment of
the N2–C3Â(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><sub>a</sub> 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 OÂ(N4) or OÂ(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> + H<sub>2</sub>O and <b>4</b> → <b>12</b> + H<sub>2</sub>O or phenyl radicals via <b>3</b> → <b>17</b> + H<sub>2</sub>O. 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 H<sub>2</sub>O 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)ÂN1–Y (Y = C5a, N2) and (O)ÂN4–Z
(Z = C4a, C3) bond lengths. The elongation of any one of these bonds
by 0.5 Ã… 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 <sup>13</sup>C NMR Spectra: A Case Study of Allyl Isothiocyanate
<sup>1</sup>H and <sup>13</sup>C NMR spectra of allyl isothiocyanate
(AITC) were measured, and the exchange dynamics were studied to explain
the near-silence of the ITC carbon in <sup>13</sup>C 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><sub><i>s</i></sub>-M1 and enantiomers M2 and M2′; the exchange between
conformers is very fast, and conformational effects on <sup>13</sup>C chemical shifts are small (ν<sub>M1</sub> – ν<sub>M2</sub> < 3 ppm). Isotropic chemical shifts, ICSÂ(γ), were
determined for sp, sp<sup><i>x</i></sup>, and sp<sup>2</sup> N-hybridization, and the γ dependencies of δÂ(N4) and
δÂ(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 AITCÂ(γ,ε,...)
with 120° < γ < 180° and 155° < ε
< 180°. Because the extreme broadening of the <sup>13</sup>C 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 <sup>13</sup>C 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