Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.

Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

Posttranslational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the recently identified family of nuclear protein kinases that act as corepressors for homeodomain transcription factors. Here, we show that HIPK2 is regulated by a ubiquitin-like protein, SUMO-1. We demonstrate that HIPK2 localizes to nuclear speckles (dots) by means of a speckle-retention signal. This speckle-retention signal contains a domain that interacts with a mouse ubiquitin-like protein conjugating (E2) enzyme, mUBC9. In cultured cells, HIPK2 is covalently modified by SUMO-1, and the SUMO-1 modification of HIPK2 correlates with its localization to nuclear speckles (dots). Thus, our results provide firm evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots).

Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats. (C) 2001 Elsevier Science Inc. All rights reserved.

Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouse

Background and Aims: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. Methods and Results: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. Conclusion: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG. (C) 2002 Blackwell Science Asia Pty Ltd.

Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the CNS and PNS that is activated by the endogenous agonist serotonin (5-hydroxytryptamine, 5-HT). 5-HT3R is the only serotonin receptor belonging to the Cys-loop superfamily of neurotransmitter receptors. Different structural biology approaches can be applied, such as crystallization and x-ray analysis. Nonetheless, characterizing the exact ligand binding site(s) of these dynamic receptors is still challenging. The use of photo-crosslinking probes is an alternative validated approach allowing identification of regions in the protein that are important for the binding of small molecules. We designed our probes based on the core structure of the 5-HT3R antagonist granisetron, a FDA approved drug used for the treatment of chemotherapy-induced nausea and vomiting. We synthesized a small library of photo-crosslinking probes by conjugating diazirines and benzophenones via various linkers to granisetron. We were able to obtain several compounds with diverse linker lengths and different photo-crosslinking moieties that show nanomolar binding affinity for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Several experiments showed unambiguously that we are able to photo-crosslink our probes with the receptor site-specifically. The functionalised protein was analysed by Western blot and MS-analysis. This yielded the exact covalent modification site, corroborating current ligand binding models derived from mutagenesis and docking studies.

UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.

Covalent protein adduction by drugs of abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Covalent protein adduction of nitrogen mustards and related compounds

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

Covalent Protein Adduction by Drugs of Abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Hypervalent organochalcogenanes as inhibitors of protein tyrosine phosphatases

A series of organochalcogenanes was synthesized and evaluated as protein tyrosine phosphatases (PTPs) inhibitors. The results indicate that organochalcogenanes inactivate the PTPs in a time- and concentration-dependent fashion, most likely through covalent modification of the active site sulfur-moiety by the chalcogen atom. Consequently, organochalcogenanes represent a new class of mechanism-based probes to modulate the PTP-mediated cellular processes.

Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid

Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that catalyses the acetylation of arylamine and hydrazine drugs as well as several known carcinogens, and so its levels in the body may have toxicological importance with regard to drug toxicity and cancer risk. Recently, we showed that p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultured cells, but the exact mechanism by which PABA acts remains unclear. In the present study, we investigated the possibility that PABA-induced down-regulation involves its metabolism to N-OH-PABA, since N-OH-AAF functions as an irreversible inhibitor of hamster and rat NAT1. We show here that N-OH-PABA irreversibly inactivates human NAT1 both in cultured cells and cell cytosols in a time- and concentration-dependent manner. Maximal inactivation in cultured cells occurred within 4 hr of treatment, with a concentration of 30 muM reducing activity by 60 +/- 7%. Dialysis studies showed that inactivation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement of the cofactor-binding site. In agreement with these data, kinetic studies revealed a 4-fold increase in cofactor K-m, but no change in substrate K-m for N-OH-PABA-treated cytosols compared to control. We conclude that N-OH-PABA decreases NAT1 activity by a direct interaction with the enzyme and appears to be a result of covalent modification at the cofactor-binding site. This is in contrast to our findings for PABA, which appears to reduce NAT1 activity by down-regulating the enzyme, leading to a decrease in NAT1 protein content. BIOCHEM PHARMACOL 60;12: 1829-1836, 2000. (C) 2000 Elsevier Science Inc.

Inhibition of proliferation of HT-29 colon adenocarcinorna cells by carboxylate NSAIDs and their acyl glucuronides

Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma. HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1-3000 muM with HT-29 cells for 24 hr, with [H-3]-thymidine added for an additional 2 hr incubation. IC50 values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC50 values about 200-500 muM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present. (C) 2001 Elsevier Science Inc. All rights reserved.

Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.

Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.

Epigenetics in sepsis: targeting histone deacetylases.

Severe sepsis and septic shock are lethal complications of infection, characterised by dysregulated inflammatory and immune responses. Our understanding of the pathogenesis of sepsis has improved markedly in recent years, but unfortunately has not been translated into efficient treatment strategies. Epigenetic mechanisms such as covalent modification of histones by acetylation are master regulators of gene expression under physiological and pathological conditions, and strongly impact on inflammatory and host defence responses. Histone acetylation is controlled by histone acetyltransferases and histone deacetylases (HDACs), which affect gene expression also by targeting non-histone transcriptional regulators. Numerous HDAC inhibitors (HDACi) are being tested in clinical trials, primarily for the treatment of cancer. We performed the first comprehensive study of the impact of HDACi on innate immune responses in vitro and in vivo. We showed that HDACi act essentially as negative regulators of the expression of critical immune receptors and antimicrobial pathways in innate immune cells. In agreement, HDACi impaired phagocytosis and killing of bacteria by macrophages, and increased susceptibility to non-severe bacterial and fungal infections. Strikingly, proof-of-principle studies demonstrated that HDACi protect from lethal toxic shock and septic shock. Overall, our observations argue for a close monitoring of the immunological and infection status of patients treated with HDACi, especially immunocompromised cancer patients. They also support the concept of pharmacological inhibitors of HDACs as promising drugs to treat inflammatory diseases, including sepsis.

Fulereno[C60]: química e aplicações

Fullerene chemistry has become a very active research field in the two last decades, largely because of the exceptional properties of the C60 molecule and the variety of fullerene derivatives that appear to be possible. In this review, a general analysis of fullerene C60 reactivity is performed. The principal methods for the covalent modification of this fascinating carbon cage are presented. The prospects of using fullerene derivatives as medicinal drugs and photoactive materials in light converting devices are demonstrated.