Covalent Adduct Formation Research Articles

Proof of concept for poor inhibitor binding and efficient formation of covalent adducts of KRASG12C and ARS compounds.

The use of selective covalent inhibitors with low binding affinity and high reactivity with the target enzyme is a promising way to solve a long-standing problem of the "undruggable" RAS-like proteins. Specifically, compounds of the ARS family that prevent the activation of the GDP-bound G12C mutant of Kirsten RAS (KRAS) are in the focus of recent experimental research. We report the first computational characterization of the entire reaction mechanism of the covalent binding of ARS-853 to the KRASG12C·GDP complex. The application of molecular dynamics, molecular docking and quantum mechanics/molecular mechanics approaches allowed us to model the inhibitor binding to the protein and the chemical reaction of ARS-853 with Cys12 in the enzyme binding site. We estimated a full set of kinetic constants and carried out numerical kinetic analysis of the process. Thus, we were able to compare directly the physicochemical parameters of the reaction obtained in silico and the macroscopic parameters observed in experimental studies. From our computational results, we explain the observed unusual dependence of the rate constant of covalent complex formation, kobs, on the ARS concentration. The latter depends both on the non-covalent binding step with the equilibrium constant, Ki, and on the rate constant of covalent adduct formation, kinact. The calculated ratio kinact/Ki = 213 M-1 s-1 reproduces the corresponding experimental value of 250 ± 30 M-1 s-1 for the interaction of ARS-853 with KRASG12C. Electron density analysis in the reactive region demonstrates that covalent bond formation occurs efficiently according to the Michael addition mechanism, which assumes the activation of the C[double bond, length as m-dash]C bond of ARS-853 by a water molecule and Lys16 in the binding site of KRASG12C. We also refine the kinact and Ki constants of the ARS-107 compound, which shares common features with ARS-853, and show that the decrease in the kinact/Ki ratio in the case of ARS-107 is explained by changes in both Ki and kinact constants.

Reducing toxic reactive carbonyl species in e-cigarette emissions: testing a harm-reduction strategy based on dicarbonyl trapping.

Reducing the concentration of reactive carbonyl species (RCS) in e-cigarette emissions represents a major goal to control their potentially harmful effects. Here, we adopted a novel strategy of trapping carbonyls present in e-cigarette emissions by adding polyphenols in e-liquid formulations. Our work showed that the addition of gallic acid, hydroxytyrosol and epigallocatechin gallate reduced the levels of carbonyls formed in the aerosols of vaped e-cigarettes, including formaldehyde, methylglyoxal and glyoxal. Liquid chromatography mass spectrometry analysis highlighted the formation of covalent adducts between aromatic rings and dicarbonyls in both e-liquids and vaped samples, suggesting that dicarbonyls were formed in the e-liquids as degradation products of propylene glycol and glycerol before vaping. Short-term cytotoxic analysis on two lung cellular models showed that dicarbonyl-polyphenol adducts are not cytotoxic, even though carbonyl trapping did not improve cell viability. Our work sheds lights on the ability of polyphenols to trap RCS in high carbonyl e-cigarette emissions, suggesting their potential value in commercial e-liquid formulations.

In Vitro Biotransformation of the Nrf2 Activator Bardoxolone: Formation of an Epoxide Metabolite That Undergoes Two Novel Glutathione-Mediated Metabolic Pathways: Epoxide Reduction and Oxidative Elimination of Nitrile Moiety

Metabolism of bardoxolone methyl (BARD-Me), an oleanolic acid derivative, and its epoxide metabolite was studied in different in vitro systems. BARD-Me also undergoes glutathione (GSH)-adduct formation via direct nucleophilic attack at the β-carbon of the α,β-unsaturated ketone substituent on the A-ring. The presence of an electron-withdrawing nitrile residue on the α-carbon increases the α,β-unsaturated ketone's susceptibility to nucleophilic attack by thiols. This allows BARD-Me to generate reversible adducts with the thiol groups of cysteine residues in target proteins without the potential toxic liabilities of irreversible covalent adduct formation. However, BARD-Me epoxide can also react with thiols irreversibly. Therefore, the epoxide was synthesized and its metabolic fate studied in vitro. BARD-Me epoxide was found to undergo two novel metabolic biotransformations: epoxide reduction and oxidative elimination of nitrile moiety. Both metabolic pathways proceed via nucleophilic attack of the thiol group of GSH at each of the two carbon atoms of the epoxide as evidenced by the formation of two β-hydroxy sulfide regioisomers. Oxidative elimination of nitrile moiety proceeds via nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is β to the cyano group to give a cyanohydrin metabolite, which spontaneously decomposes to release cyanide and the corresponding ketone. Nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is α to the cyano group results in the formation of the GSH monoadduct that undergoes intermolecular reduction with another GSH molecule, followed by elimination of oxidized GSH (GS-SG) and the formation of an enolate intermediate. Upon protonation, the enolate intermediate gives rise to hydroxylated BARD-Me, which is readily converted back to BARD-Me through the elimination of water. The chemical reactivity of the epoxide metabolite and the liberation of cyanide are of significant toxicological interest for the potential utility of BARD-Me as a therapeutic agent.

Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer.

The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). Particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV-visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography, and mass spectrometry is used to establish the Ub changes triggered by UV-A irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes, that is, formation of a Ptr-Ub adduct, dimerization, and fragmentation of the protein.

Comparing and contrasting the coverage of publicly available structural alerts for protein binding

The molecular initiating event for many mechanisms of toxicological action comprise the reactive, covalent binding between an exogenous electrophile and an endogenous nucleophile. The target sites for electrophiles are typically peptides, proteins, enzymes or DNA. Of these, the formation of covalent adducts with proteins and DNA are perhaps the most established as they are most closely associated with skin sensitisation and genotoxicity endpoints. As such, being able to identify electrophilic features within a chemical structure provides a starting point to characterise its reactivity profile. There are a number of software tools that have been developed to help identify structural features indicative of electrophilic reactive potential to address various purposes, including: 1) to facilitate category formation for read-across of toxicity effects such as skin sensitisation potential, as well as 2) to profile substances to identify potential confounding factors to rationalise their activity in high-throughput screening (HTS) assays. Here, three such schemes that have been published in the literature as collections of SMARTS patterns and their associated chemical-biological reaction domains have been compared. The goals are 1) to better understand their scope and coverage, and 2) to assess their performance relative to a published skin sensitisation dataset where manual annotations to assign likely mechanistic domains based on expert judgement were already available. The 3 schemes were then applied to the Tox21 library and the consensus outcome was reported to highlight the proportion of chemicals likely to exhibit a reactivity response, specific to a mechanistic reaction domain, but non-specific with respect to target-tissue based activity. ToxPrint fingerprints were computed and activity enrichments computed to compare the structural features identified for the skin sensitisation dataset and Tox21 chemicals for each ‘consensus’ reaction domain. Enriched ToxPrints were also used to identify ToxCast assays potentially informative for reactivity.

Abstract CT055: Microdose induced oxaliplatin-DNA adducts as a predictive biomarker of response in colorectal cancer

Abstract Background Combination 5-FU and oxaliplatin (FOLFOX) is standard therapy for colorectal cancer (CRC) in neoadjuvant, adjuvant, and metastatic settings. However, oxaliplatin neuropathy is the main dose-limiting toxicity that often necessitates dose reduction or discontinuation before progression. Prediction of oxaliplatin sensitivity in tumor cells would be valuable for dose optimization to maximize anti-tumorigenic effect and limit sensory neuropathy. Oxaliplatin functions via the formation of covalent drug-DNA adducts which ultimately inhibit cell replication and promote apoptosis. Detection of these drug-DNA adducts could serve as a biomarker of oxaliplatin efficacy. We therefore hypothesized that drug-DNA adduct levels induced by sub-therapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are predictive of tumor responses. Methods The feasibility of this predictive diagnostic microdosing approach was assessed in CRC cell culture and a pilot clinical trial of six CRC patients. Four CRC cell lines were cultured with therapeutically relevant (100 µM) or diagnostic microdose (1 µM) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct levels with accelerator mass spectrometry, an ultrasensitive isotope analysis technique. Adduct formation was correlated with oxaliplatin cytotoxicity as measured by the MTT viability assay. In a pilot clinical trial (NCT02569723), six CRC patients received an intravenous diagnostic microdose containing [14C]oxaliplatin prior to treatment and a second [14C]oxaliplatin dose during FOLFOX chemotherapy. Adduct levels from peripheral blood mononuclear cells (PBMC), used as surrogates for tumor cells, were correlated to mean tumor volume change of evaluable target lesions. Results Drug-DNA adduct levels were linearly proportional between microdoses and therapeutically relevant concentrations in cell culture (R2 = 0.91, p < 0.0001) and patient samples (R2 = 0.63, p < 0.0001). Plasma pharmacokinetics of microdoses were consistent with those of therapeutic dosing. No microdose-associated toxicity was observed in this patient population. PBMC adduct levels significantly correlated with tumor shrinkage (microdose: R2 = 0.82, p = 0.033; therapeutic dose: R2=0.65, p = 0.099). Conclusion Diagnostic microdosing with oxaliplatin is feasible and is predictive of oxaliplatin sensitivity in CRC patients. Citation Format: Justin Chen, May Cho, Maike Zimmermann, Thomas Semrad, Chun-Yi Wu, Aiming Yu, Chong-Xian Pan, Paul T. Henderson, Edward J. Kim. Microdose induced oxaliplatin-DNA adducts as a predictive biomarker of response in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT055.

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Molecular insights into the carbon dioxide–carboxylate anion interactions and implications for carbon capture

In this work, we analyze the interaction of carbon dioxide with different carboxylate anion derivatives in the gas phase at the ab initio CCSD(T)//MP2 level using the aug-cc-pVTZ basis set. The systems considered here include the formate, acetate, propionate, bicarbonate, carbamate and glycinate anions. The study is relevant to get a better understanding of the interactions involved in novel carbon capture processes through either ionic liquids or amino acid salts. We describe the formation of covalent and non-covalent adducts and show that the formation energies are significantly larger than those previously reported for amines, which are used in conventional carbon capture processes. The nature of the interactions is analyzed using the natural bond orbitals methodology. The binding energy in the non-covalent processes does not depend much on the derivative, but covalent adducts display a rough correlation with nucleophilic/electrophilic indices provided distortion effects on the monomers are taken into account. In the case of glycinate, interactions with the amino and carboxylic moieties involve comparable energetics and the existence of several minima in the potential energy surface might be a factor contributing to the good CO2 capture capacity exhibited by this species in recent experimental studies.

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage, DNA Cleavage Inhibition, and Formation of Ligand-DNA Adducts.

Ligands interacting with abasic (AP) sites in DNA may generate roadblocks in base-excision DNA repair (BER) due to indirect inhibition of DNA repair enzymes (e.g., APE1) and/or formation of toxic byproducts, resulting from ligand-induced strand cleavage or covalent cross-links. Herein, a series of 12 putative AP-site ligands, sharing the common naphthalenophane scaffold, but endowed with a variety of substituents, have been prepared and systematically studied. The results demonstrate that most naphthalenophanes bind to AP sites in DNA and inhibit the APE1-induced hydrolysis of the latter in vitro. Remarkably, their APE1 inhibitory activity, as characterized by IC50 and KI values, can be directly related to their affinity and selectivity to AP sites, as assessed by means of fluorescence melting experiments. On the other hand, the molecular design of naphthalenophanes has a crucial influence on their intrinsic AP-site cleavage activity (i.e., ligand-catalyzed β- and β,δ-elimination reactions at the AP site), as illustrated by the compounds either having an exceptionally high AP-site cleavage activity (e.g., 2,7-BisNP-S, 125-fold more efficacious than spermine) or being totally devoid of this activity (four compounds). Finally, the unprecedented formation of a stable covalent DNA adduct upon reaction of one ligand (2,7-BisNP-NH) with its own product of the AP-site cleavage is revealed.

Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens.

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.

Vibrational spectroscopy of flavoproteins.

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum.

Formation of large oligomers of DOPAL-modified α-synuclein is modulated by the oxidation of methionine residues located at C-terminal domain

The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients.

Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.

Exploring the role of methionine residues on the oligomerization and neurotoxic properties of DOPAL-modified α-synuclein

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.

Covalent binding of food-derived blue pigment phycocyanobilin to bovine β-lactoglobulin under physiological conditions

In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine β-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (ka = 0.065 min−1), of moderate affinity (Ka = 4 × 104 M−1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (ka = 0.101 min−1). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB.

Development of a Profiler for Facile Chemical Reactivity Using the Open-Source Konstanz Information Miner

Abstract It is prudent to assess the chemical reactivity of compounds during early stages of product development, given that formation of covalent adducts between electrophilic compounds and biological nucleophiles serves as the molecular initiating event in a range of adverse outcome pathways (AOPs). In vitro assays such as the direct peptide reactivity assay (DPRA) have been developed to assess facile reactivity. However, these approaches still require test material and laboratory resources, are less conducive to rapid high-throughput screening than computational approaches, and have been developed with a primary focus on a single AOP (e.g., skin sensitization). Given the known set of specific mechanisms that drive facile chemical reactivity between electrophilic compounds and biological nucleophiles, we developed an in-house profiler to identify reactive moieties using an open-source Konstanz Information Miner (KNIME) workflow. This covered the chemical space for five known classes of chemical facile r...

Profiling Acute Oral and Inhalation Toxicity Data Using a Computational Workflow to Screen for Facile Chemical Reactivity

Abstract We are developing computational approaches for mechanisms of acute toxicity. Facile chemical reactivity involves formation of covalent adducts between electrophiles and nucleophiles and can be molecular initiating events for adverse outcome pathways. Using an open-source Konstanz Information Miner workflow, we developed a profiler to identify reactive moieties that covers five chemically facile-reactive classes: (1) Michael acceptors, (2) Schiff base formation, (3) acylation, (4) nucleophilic aromatic substitution, and (5) bimolecular nucleophilic substitution. We hypothesized that nonspecific chemical reactivity may be a more prominent molecular initiating event for high acute toxicity for inhalation compared to oral exposure routes because of ensuing respiratory irritation with pulmonary edema resulting in lethality. We tested this hypothesis by stratifying rodent acute oral and 4-hour inhalation toxicity databases (DBs) according to the globally harmonized system (GHS) classification and our p...

Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins

BackgroundRegulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.HypothesisDifferences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.ApproachThe rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.ResultsDeuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.ConclusionThese results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.Support or Funding InformationNIH T32 GM092715NSF 1507588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches

Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug-induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk.Etravirine (ETR) was the first second-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV-infected patients who developed resistance to the first-generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post-marketing reports of severe ETR-induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent.In the current study, high resolution mass spectrometry-based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co-factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV-infected patients on ETR therapy.We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-L-cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR-based regimens.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs <em>In Vitro</em> and Their Determination by <sup>32</sup>P-postlabeling

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs <em>In Vitro</em> and Their Determination by <sup>32</sup>P-postlabeling