Irreversible Complex Research Articles

Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases.

The speciation of lead in seawater by pseudopolarography using a vibrating silver amalgam microwire electrode

A novel electrode based on a vibrating silver amalgam microwire was used for pseudopolarography of lead (Pb) in seawater. Pseudopolarography is a method to study the stability of metal species by examination of their reduction potential. The reduction potential of kinetically irreversible species is related to the thermodynamic complex stability constant, whilst for labile species it is related to the conditional stability constant. The new electrode enables the speciation of Pb at picomolar level in uncontaminated seawater. The potential shift of electrochemically irreversible lead complexes was calibrated against the complex stability of several ligands in pH8 seawater. The method was compared against the mercury drop electrode at various concentrations of lead and various deposition times, and the response was in general agreement with the theory. The method is tested on samples from oceanic (Atlantic), coastal (Irish Sea) and estuarine (Mersey) origins at Pb concentrations between 30pM and 2nM. The pseudopolarograms were found to resolve the speciation of lead in terms of labile and irreversibly bound Pb species. The lead in the samples was largely in the labile component of the pseudopolarograms, indicating that it was reversibly complexed with values for log α′Pb2+L of 2 for the oceanic sample, and of 4 for the coastal sample. A smaller fraction was resolved as stable, irreversibly bound species with values for the thermodynamic stability constant of log KPb2+L=15–16. The speciation method was able to resolve metal species occurring at levels of only a fraction of that of the metal. Unlike copper, where organic complexation is strong and electrochemically irreversible, and able to significantly decrease its bioavailability, the Pb mostly occurs in the labile complexed fraction and is therefore readily available for geochemical scavenging or biological uptake reactions.

Mechanism of nitric oxide synthase dimerization inhibition by novel pyrimidine imidazoles

Over-production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been etiologically linked to several degenerative diseases. Thus its therapeutic control has become clinically imperative. As dimerization of NOS is essential for its activity, several dimerization inhibitors including pyrimidine imidazoles are being evaluated for therapeutic intervention of NOS activity. However, the mechanism of their action is still unclear. Here, we examined the mechanism of inhibition by two novel pyrimidine imidazole derivatives (PIDs) having low toxicity, high affinity and strong selectivity for iNOS using stopped-flow kinetic studies, gel-filtration and spectrophotometric analysis. The binding of our PIDs to iNOS produced an irreversible monomer-inhibitor complex with intact heme, contrary to reported behavior of bulky analogues. Treatment with tetrahydrobiopterin (H 4 B) and l -arginine ( l -Arg) failed to recover any dimer from such monomers, demonstrating the irreversible nature of such inhibition. To elucidate the precise mechanism of action of the PIDs, we engaged two monomeric mutants of the iNOS oxygenase domain (iNOSoxy) whose dimerization capabilities were either recoverable (K82AiNOSoxy) or non-recoverable (D92AiNOSoxy) with H 4 B. We observed that PID bound the H 4 B-pretreated dimeric K82A mutant in a biphasic manner triggering monomerization but with fast monophasic kinetics to the monomeric form of both mutants, disallowing dimer recovery by H 4 B and l -Arg. This indicated that PIDs could not only interact with the iNOS monomer to prevent dimerization but also with the dimer to elicit irreversible monomerization. Overall, this study establishes the strong potential of PIDs as candidates for therapeutic control of diseases related to NO overproduction by iNOS.

Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation

We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein-protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA(W37V), which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplA(W37V) labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein-protein interactions can be imaged (FRB-FKBP, Fos-Jun, and neuroligin-PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution.

Pivotal Role for α1-Antichymotrypsin in Skin Repair

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.

Delayed Adverse Effects of Neonatal Exposure to Diethylstilbestrol and Their Dose Dependency in Female Rats

Neonatal exposure to estrogenic chemicals causes irreversible complex damage to the hypothalamus-pituitary-gonadal axis and reproductive system in females. Some lesions are noted after maturation as delayed adverse effects. We investigated the characteristics and dose dependence of delayed effects using female rats neonatally exposed to diethylstilbestrol (DES). Female Donryu rats were subcutaneously injected with a single dose of DES of 0 (control), 0.15, 1.5, 15, 150, or 1,500 µg/kg bw after birth. All except the lowest dose had estrogenic activity in a uterotrophic assay. All rats at 1500 µg/kg and some at 150 µg/kg showed abnormal morphologies in the genital tract, indicating they were androgenized before maturation. Although no morphological abnormalities were noted at 15 µg/kg or lower, onset of persistent estrus was significantly accelerated in the 1.5, 15, and 150 µg/kg groups with dose dependency, and the latest onset was from seventeen to twenty-one weeks of age at 1.5 µg/kg. The neonatal exposure to DES increased uterine adenocarcinoma development only at 150 µg/kg, although uterine anomalies were detected at 1,500 µg/kg. These results indicate that neonatal exposure to DES, which exerts estrogenic activity in vivo, induces delayed adverse effects in female rats in a dose-dependent manner. Early onset of persistent estrus appears to be the most sensitive parameter.

Heterotropic Effect of β-lactam Antibiotics on Antioxidant Property of Haptoglobin (2-2)-Hemoglobin Complex.

Haptoglobin (Hp) is a mammalian serum glycoprotein showing a genetic polymorphism with three types, 1-1, 2-2 and 1-2. Hp appears to conserve the recycling of heme-iron by forming an essentially irreversible but non-covalent complex with hemoglobin which is released into the plasma by erythrocyte lysis. As an important consequence, Haptoglobin-Hemoglobin complex (Hp-Hb) shows considerable antioxidant property. In this study, antioxidant activity of Hp (2-2)-Hb complex on hydrogen peroxide has been studied and analyzed in the absence and presence of two beta-lactam antibiotics in-vitro. For this purpose, non-Michaelis behavior of peroxidase activity of Hp (2-2)-Hb complex was analyzed using Eadie-Hofstee, Clearance and Hill plots, in the absence and presence of pharmaceutical dose of ampicillin and coamoxiclav. The results have shown that peroxidase activity of Hp (2-2)-Hb complex is modulated via homotropic effect of hydrogen peroxide as an allostric substrate. On the other hand antioxidant property of Hp (2-2)-Hb complex increased via heterotropic effect of both antibiotics on the peroxidase activity of the complex. Both drugs also have mild effect on quality of homotropic property of the peroxidase activity of Hp (2-2)-Hb complex. Therefore, it can be concluded from our study that both beta-lactam antibiotics can increase peroxidase activity of Hp (2-2)-Hb complex via heterotropic effect. Thus, the two antibiotics (especially ampicillin) may help those individuals with Hp (2-2) phenotype to improve the Hp-Hb complex efficiency of removing hydrogen peroxide from serum under oxidative stress. This can be important in the individuals with phenotype Hp 2-2 who have less antioxidant activity relative to other phenotypes and are susceptible to cardiovascular disorders, as has been reported by other researchers.

Tracking losses of brevetoxins on exposure to phytoplankton competitors: Mechanistic insights

The increasing frequency of devastating blooms of the harmful dinoflagellate Karenia brevis has motivated investigations into understanding bloom dynamics and the potential for mitigation. Previous findings indicate that waterborne concentrations of the most abundant brevetoxin (brevetoxin B or PbTx-2) associated with these blooms decrease in the presence of other phytoplankton species. The current study explores the mechanism of brevetoxin removal from seawater upon exposure to phytoplankton competitors. Live phytoplankton removed waterborne brevetoxins more rapidly than lysates, but phytoplankton did not need to be in a state of active metabolism. Biomolecules, probably proteins, exuded from phytoplankton appeared to be responsible for the loss of brevetoxins, either by irreversible complexation or by degradation. Selective removal of PbTx-2 and -1, but not PbTx-3, -9 or BTX-B5, by cultured phytoplankton revealed that brevetoxin removal is dependent upon the presence of an α,β-unsaturated aldehyde functionality. The mechanism of biotransformation appears to be common among phytoplankton, since members of various taxonomic groups including diatoms, dinoflagellates, and a cryptophyte each caused 75–90% decrease in PbTx-2 concentration, as did a generic protein (bovine serum albumin) added to seawater at high concentration. These findings support the concept of potentially using competitor phytoplankton species or compounds derived from phytoplankton as biocontrol agents for waterborne toxins associated with red tide.

SerpinB1 in cystic fibrosis airway fluids: quantity, molecular form and mechanism of elastase inhibition

Neutrophil serine proteases (NSPs), especially elastase, are major agents of lung destruction in cystic fibrosis (CF) patients. This study investigated SerpinB1, a highly efficient inhibitor of NSPs, in CF lung disease. Bronchoalveolar lavage fluid (BALF) from 31 children with CF and 24 control children was examined for amount and molecular species of SerpinB1, and its mechanism of action was studied. CF BALF had more SerpinB1 than control BALF (geometric mean 3.9 (95% CI 2.60-5.62) versus 1.37 (1.20-1.55) μg·mL⁻¹; p<0.001). BALF levels of SerpinB1 were higher for infected versus uninfected CF subjects (5.5 versus 2.7 μg·mL⁻¹; p<0.04) and substantially higher for elastase-positive versus -negative CF subjects (8.41 versus 1.89 μg·mL⁻¹; p<0.001). Most SerpinB1 in CF BALF had been cleaved. Adding recombinant SerpinB1 to CF BALF stoichiometrically inhibited endogenous elastase, indicating that the inhibitor functions in the CF microenvironment. In vitro simulations comparing SerpinB1 and α₁-antitrypsin (SerpinA1) showed that both rapidly form irreversible inhibitory covalent complexes with elastase and that these differed in survival time. The SerpinB1-elastase complex survived only briefly due to fragmentation of bound elastase, releasing cleaved SerpinB1, the molecular form in CF BALF. The findings define an innate role for SerpinB1 in CF airways.

Binary polypeptide system for permanent and oriented protein immobilization

BackgroundMany techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling.ResultsHere we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution.ConclusionsIPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

A computational workflow for the design of irreversible inhibitors of protein kinases

Design of irreversible inhibitors is an emerging and relatively less explored strategy for the design of protein kinase inhibitors. In this paper, we present a computational workflow that was specifically conceived to assist such design. The workflow takes the form of a multi-step procedure that includes: the creation of a database of already known reversible inhibitors of protein kinases, the selection of the most promising scaffolds that bind one or more desired kinase templates, the modification of the scaffolds by introduction of chemically reactive groups (suitable cysteine traps) and the final evaluation of the reversible and irreversible protein-ligand complexes with molecular dynamics simulations and binding free energy predictions. Most of these steps were automated. In order to prove that this is viable, the workflow was tested on a database of known inhibitors of ERK2, a protein kinase possessing a cysteine in the ATP site. The modeled ERK2-ligand complexes and the values of the estimated binding free energies of the putative ligands provide useful indicators of their aptitude to bind reversibly and irreversibly to the protein kinase. Moreover, the computational data are used to rank the ligands according to their computed binding free energies and their ability to bind specific protein residues in the reversible and irreversible complexes, thereby providing a useful decision-making tool for each step of the design. In this work we present the overall procedure and the first proof of concept results.

4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesulfonate) (NSC 88915) and related novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo when topoisomerase I (Top1) processes DNA. For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes. Tdp1 inhibitors have been regarded as potential therapeutics in combination with Top1 inhibitors, such as the camptothecin derivatives, topotecan, and irinotecan, which are used to treat human cancers. Using a novel high-throughput screening assay, we have identified the C21-substituted progesterone derivative, NSC 88915 (1), as a potential Tdp1 inhibitor. Secondary screening and cross-reactivity studies with related DNA processing enzymes confirmed that compound 1 possesses specific Tdp1 inhibitory activity. Deconstruction of compound 1 into discrete functional groups reveals that both components are required for inhibition of Tdp1 activity. Moreover, the synthesis of analogues of compound 1 has provided insight into the structural requirements for the inhibition of Tdp1. Surface plasmon resonance shows that compound 1 binds to Tdp1, whereas an inactive analogue fails to interact with the enzyme. On the basis of molecular docking and mechanistic studies, we propose that these compounds are competitive inhibitors, which mimics the oligonucleotide-peptide Tdp1 substrate. These steroid derivatives represent a novel chemotype and provide a new scaffold for developing small molecule inhibitors of Tdp1.

Prostate-specific antigen kallikrein complexes and acute myocardial infarction

Recently, attention has focused on prostate-specific antigen kallikrein and the cardiovascular system. The finding of diminished PSA during acute myocardial infarction and the correlation of variation of PSA with coronary lesions and occurrence of major adverse cardiac events have opened a possible new intriguing scenario. As with many stories in biology, the tale of PSA can be more complex than was first appreciated. In the bloodstream, the proteolytic activity of PSA is inhibited by the formation of irreversible complexes with serum protease inhibitors and other acute-phase proteins. We noticed that the formation of irreversible PSA complexes (reflected by the increased bound PSA) has significant correlation with high-sensitivity C-reactive protein (hsCRP) and that seems to play a crucial role in the adverse event prevention.

Protective effects of brain-derived neurotrophic factor against neurotoxicity of 3-nitropropionic acid in rat cortical neurons

Brain-derived neurotrophic factor (BDNF) deficiency has been implicated in pathogenesis of Huntington's disease (HD). 3-Nitropropionic acid (3-NP), an irreversible mitochondrial complex II inhibitor, has been commonly used as a pharmacological model recapitulating HD phenotypes in rodents and nonhuman primates. Herein we test whether BDNF may exert neuroprotective effects against mitochondrial dysfunction caused by 3-NP in primary culture of fetal rat cortical neurons. Preconditioning of neuronal cells with BDNF (100 ng/ml for 8 h) attenuated 3-NP toxicity (2.5 mM for additional 24 h) based on Hoechst and propidium iodide (PI) staining. BDNF effects can be inhibited by the nitric oxide synthase (NOS) inhibitor l-nitroarginine methylester ( l-NAME, 100 μM), the cGMP-dependent protein kinase (PKG) inhibitor KT5823 (2 μM), the thioredoxin reductase inhibitor 1-chloro-2,4-dinitrobenzene (DNCB, 5 μM), and a membrane-permeable Bcl-2 inhibitor (12.5 μM). 8-Br-cGMP is a cGMP analogue capable of activating PKG independent of NO. Exogenous application of 8-Br-cGMP (3–30 μM) and purified thioredoxin (3–5 μM) partially mimicked BDNF effects in conferring 3-NP resistance to cortical cells. These results, together with our previous report showing NO donor S-nitrosoglutathione (GSNO)-mediated neuroprotective effects against 3-NP toxicity, suggest that BDNF may protect neurons from mitochondrial dysfunction at least partly via activation of the signaling cascades involving NOS/NO, PKG, thioredoxin and Bcl-2.

Reducing the allergenic capacity of peanut extracts and liquid peanut butter by phenolic compounds

Phenolic compounds are known to form soluble and insoluble complexes with proteins. The objective of this study was to determine if phenolics such as caffeic, chlorogenic and ferulic acids form insoluble and irreversible complexes with major peanut allergens, and if such complexation reduces immunoglobulin E (IgE) binding. After adding each of the phenolics to peanut extracts and liquid peanut butter, the soluble materials were analysed by SDS-PAGE and inhibition ELISA. Results showed that addition of the phenolics precipitated most of the major peanut allergens, Ara h 1 and Ara h 2, and that complexation was irreversible. IgE binding was reduced approximately 10- to 16-fold. We concluded that reducing IgE binding by phenolics is feasible. The research, if proven by clinical studies, could lead to the development of less allergenic liquid peanut-based products.

A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity.

Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds

Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta (Abeta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Abeta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Abeta analog identified rare IgVs that hydrolyzed Abeta mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t'). The IgVs hydrolyzed Abeta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Abeta hydrolysis, suggesting catalysis by the unpaired VL domain.Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.

The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Crotonaldehyde is a representative α,β-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA–Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA–Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA–Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a recently discovered enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo following the DNA processing activity of topoisomerase I (Top1). For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes, which can be generated by either exogenous or endogenous factors. Tdp1 has been regarded as a potential therapeutic co-target of Top1 in that it seemingly counteracts the effects of Top1 inhibitors, such as camptothecin and its clinically used derivatives. Thus, by reducing the repair of Top1-DNA lesions, Tdp1 inhibitors have the potential to augment the anticancer activity of Top1 inhibitors provided there is a presence of genetic abnormalities related to DNA checkpoint and repair pathways. Human Tdp1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolates and 3'-abasic sites indicating it may function as a general 3'-DNA phosphodiesterase and repair enzyme. The importance of Tdp1 in humans is highlighted by the observation that a recessive mutation in the human TDP1 gene is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1). This review provides a summary of the biochemical and cellular processes performed by Tdp1 as well as the rationale behind the development of Tdp1 inhibitors for anticancer therapy.

Towards Covalent Vaccination: IMPROVED POLYCLONAL HIV NEUTRALIZING ANTIBODY RESPONSE INDUCED BY AN ELECTROPHILIC gp120 V3 PEPTIDE ANALOG

Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) approximately 50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.