Human cells are exposed to the electrophilic α,β-unsaturated aldehyde acrolein from a variety of sources. The reaction of acrolein with functionally critical protein thiol residues can yield ...important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well-characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B (k inact = 0.02 ± 0.005 s−1 and K I = 2.3 ± 0.6 × 10−4 M). The enzyme activity does not return upon gel filtration of the inactivated enzyme, and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together, these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon–carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally related α,β-unsaturated aldehydes.
Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H2O2-mediated ...cellular signal transduction processes, but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer bicarbonate/CO2 potentiates the ability of H2O2 to inactivate PTPs. The results of biochemical experiments and high-resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H2O2 with HCO3 –/CO2.
Bortezomib N-(2,3-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid is a potent first-in-class dipeptidyl boronic acid proteasome inhibitor that was approved in May 2003 in the United States ...for the treatment of patients with relapsed multiple myeloma where the disease is refractory to conventional lines of therapy. Bortezomib binds the proteasome via the boronic acid moiety, and therefore, the presence of this moiety is necessary to achieve proteasome inhibition. Metabolites in plasma obtained from patients receiving a single intravenous dose of bortezomib were identified and characterized by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Metabolite standards that were synthesized and characterized by LC/MS/MS and high field nuclear magnetic resonance spectroscopy (NMR) were used to confirm metabolite structures. The principal biotransformation pathway observed was oxidative deboronation, most notably to a pair of diastereomeric carbinolamide metabolites. Further metabolism of the leucine and phenylalanine moieties produced tertiary hydroxylated metabolites and a metabolite hydroxylated at the benzylic position, respectively. Conversion of the carbinolamides to the corresponding amide and carboxylic acid was also observed. Human liver microsomes adequately modeled the in vivo metabolism of bortezomib, as the principal circulating metabolites were observed in vitro. Using cDNA-expressed cytochrome P450 isoenzymes, it was determined that several isoforms contributed to the metabolism of bortezomib, including CYP3A4, CYP2C19, CYP1A2, CYP2D6, and CYP2C9. The development of bortezomib has provided an opportunity to describe the metabolism of a novel boronic acid pharmacophore.
Reversible phosphorylation of tyrosine residues serves as a biochemical “switch” that alters the functional properties of many proteins involved in cellular signal transduction processes. Protein ...tyrosine phosphatases (PTPs) catalyze the removal of phosphoryl groups from tyrosine residues in target proteins, thereby playing a central role in the regulation of diverse cellular processes including glucose metabolism, cell cycle control, and immune responses. Accordingly, small molecules capable of inactivating PTPs may find use as therapeutic agents and tools for the study of diverse signal transduction pathways. In the work reported here, peroxymonophosphate (2-O3POOH) was reported to be an exceptional inactivator of PTP1B, an archetypal member of the PTP enzyme family (K I = 6.6 × 10-7 M and k inact = 0.043 s-1). In this regard, peroxymonophosphate is over 7000 times more potent than hydrogen peroxide, the endogenous regulator of PTP1B. Inactivation of PTP1B by peroxymonophosphate is active site directed and, like that by hydrogen peroxide, is readily reversed by treatment with dithiothreitol (5 mM). Together the findings suggest that peroxymonophosphate oxidizes the active site cysteine residue of PTP1B to the sulfenic acid oxidation state. Importantly, peroxymonophosphate (100 nM) yields substantial inactivation of PTP1B even in the presence of physiologically relevant concentrations of the biological thiol glutathione (1 mM). Collectively, these properties may make peroxymonophosphate a useful tool for probing the role of cysteine-dependent PTPs in various signal transduction pathways. Finally, it is interesting to note that peroxymonophosphate may be biosynthetically accessible via the reaction of endogenous hydrogen peroxide with phosphoryl donors. Peroxymonophosphate possesses key properties expected for the endogenous signaling molecule involved in the redox regulation of PTP activity.
It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-derived regulator of cellular protein tyrosine phosphatase activity under physiological or ...pathophysiological conditions. To facilitate further consideration of the potential role of peroxymonophosphate in biological systems we present studies related to the preparation, characterization, stability, and fluorometric detection of this agent.
Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell signaling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a ...manner that mirrors the endogenous signaling agent hydrogen peroxide.
Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell signaling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a manner that mirrors the endogenous signaling agent hydrogen peroxide.
Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of ...salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time- and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated.
It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-derived regulator of cellular protein tyrosine phosphatase activity under physiological or ...pathophysiological conditions. To facilitate further consideration of the potential role of peroxymonophosphate in biological systems we present studies related to the preparation, characterization, stability, and fluorometric detection of this agent.
Bortezomib (1) is a potent first-in-class dipeptidyl boronic acid proteasome inhibitor employed in the treatment of patients with relapsed multiple myeloma where the disease is refractory to ...conventional lines of therapy. The potency of 1 is owed primarily to the presence of the boronic acid moiety, one which is suited to establish a tetrahedral intermediate with the active site N-terminal threonine residue of the proteasome. Hence, deboronation of 1 represents a deactivation pathway for this chemotherapeutic agent. Deboronation of 1 affords a near equal mixture of diastereomeric carbinolamide metabolites (M1/M2) and represents the principal metabolic pathway observed in humans. In vitro results from human liver microsomes and human cDNA-expressed cytochrome P450 enzymes (P450) indicate a role for P450 in the deboronation of 1. Use of 18O-labeled oxygen under controlled atmospheres confirmed an oxidative mechanism in the P450-mediated deboronation of 1, as 18O was found incorporated in both M1 and M2. Chemically generated reactive oxygen species (ROS), such as those generated as byproducts during P450 catalysis, were also found to deboronate 1 resulting in the formation of M1 and M2. Known to undergo efficient redox cycling, P450 2E1 was found to catalyze the deboronation of 1 predominantly to the carbinolamide metabolites M1 and M2, as well as to a pair of peroxycarbinolamides, 2 and 3. The presence of superoxide dismutase (SOD) and catalase prevented the deboronation of 1, thus, supporting the involvement of ROS in the P450 2E1-catalyzed deboronation reaction. The presence of SOD and catalase also protected 1 against P450 3A4-catalyzed deboronation, albeit to a lesser extent. The remaining deboronation activity observed in the P450 3A4 reaction may suggest the involvement of the more conventional activated enzyme-oxidants previously described for P450. Our present findings indicate that the oxidase activity of P450 (i.e., formation of ROS) represents a mechanism of deboronation.
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