Opportunities and challenges for the development of covalent chemical immunomodulators

Compounds that react irreversibly with cysteines have reemerged as potent and selective tools for altering protein function, serving as chemical probes and even clinically approved drugs. The exquisite sensitivity of human immune cell signaling pathways to oxidative stress indicates the likely, yet still underexploited, general utility of covalent probes for selective chemical immunomodulation. Here, we provide an overview of immunomodulatory cysteines, including identification of electrophilic compounds available to label these residues. We focus our discussion on three protein classes essential for cell signaling, which span the ‘druggability’ spectrum from amenable to chemical probes (kinases), somewhat druggable (proteases), to inaccessible (phosphatases). Using existing inhibitors as a guide, we identify general strategies to guide the development of covalent probes for selected undruggable classes of proteins and propose the application of such compounds to alter immune cell functions.     

Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles

Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here, we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately two- to three-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity.     

Lysine richness in human snurps - possible sites for electrophilic attacks

Gene-expression strategies are remodeled following exposure to stress. The reactive oxidants and electrophiles generated after stress actually affects the structural and functional properties of different cellular proteins. It is also seen that lysine rich motifs of proteins play crucial role in electrophilic attack and modification. Therefore, this study revealing lysine richness in 5 main human snrups (Small Nuclear Ribonucleoproteins) indicates a possible mechanism of gene regulation under stress. This possibility is highly supported by the findings that surface residues of the molecules were full of lysine rich clusters. Lysine richness is also found to be a highly conserved pattern across the various domains of life indicative of stress adaptation in the prebiotic to biotic world transition. Moreover the modeled structures showed good all atom contacts and minimal outliers.     

Dicarbonyliridium(I) complexes of pyridine ester ligands and their reactivity towards various electrophiles

The reaction of dimeric precursor [Ir(CO)₂Cl]₂ with two molar equivalent of the pyridine-ester ligands (L) like methyl picolinate (a), ethyl picolinate (b), methyl nicotinate (c), ethyl nicotinate (d), methyl isonicotinate (e) and ethyl isonicotinate (f) affords the tetra coordinated neutral complexes of the type [Ir(CO)₂ClL] (1a-f). The single crystal X-ray structure of 1d reveals that the Ir atom occupies the centre of an approximately square planar geometry with two CO groups cis- to each other. Intermolecular C-H?O and Ir?C interactions greatly stabilize the supramolecular structure of 1d in the solid state. The oxidative addition (OA) reactions of 1a-f with different electrophiles such as CH₃I, C₂H₅I and I₂ undergo decarbonylation of one CO group to generate the oxidized products of the type [Ir(CO)RClIL] where R = -CH₃ (2a-f); -C₂H₅ (3a-f) and [Ir(CO)ClI₂L] (4a-f). Kinetic study of the reaction of 1c-f with CH₃I indicates a first order reaction which follow the order 1d > 1c > 1f > 1e. All the synthesized complexes were characterized by elemental analyses, IR, and multinuclear NMR spectroscopy.     

The redox‐sensing regulator YodB senses quinones and diamide via a thiol‐disulfide switch in Bacillus subtilis

The MarR/DUF24‐type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox‐sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6‐Ala mutant that the conserved Cys6 apparently contributes to the DNA‐binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox‐sensing in response to diamide and 2‐methylhydroquinone (MHQ) in vivo. DNA‐binding experiments indicate that YodB is regulated by a reversible thiol‐modification in response to diamide and MHQ in vitro. Redox‐regulation of YodB involves Cys6‐Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C‐terminal Cys101' or Cys108' residues. This study reveals a thiol‐disulfide switch model of redox‐regulation for the YodB repressor to sense electrophilic compounds in vivo.     

Redox regulation of antioxidants,autophagy, and the response to stress: Implications for electrophile therapeutics

Redox networks in the cell integrate signaling pathways that control metabolism, energetics, cell survival, and death. The physiological second messengers that modulate these pathways include nitric oxide, hydrogen peroxide, and electrophiles. Electrophiles are produced in the cell via both enzymatic and nonenzymatic lipid peroxidation and are also relatively abundant constituents of the diet. These compounds bind covalently to families of cysteine-containing, redox-sensing proteins that constitute the electrophile-responsive proteome, the subproteomes of which are found in localized intracellular domains. These include those proteins controlling responses to oxidative stress in the cytosol—notably the Keap1-Nrf2 pathway, the autophagy-lysosomal pathway, and proteins in other compartments including mitochondria and endoplasmic reticulum. The signaling pathways through which electrophiles function have unique characteristics that could be exploited for novel therapeutic interventions; however, development of such therapeutic strategies has been challenging due to a lack of basic understanding of the mechanisms controlling this form of redox signaling. In this review, we discuss current knowledge of the basic mechanisms of thiol-electrophile signaling and its potential impact on the translation of this important field of redox biology to the clinic. Emerging understanding of thiol-electrophile interactions and redox signaling suggests replacement of the oxidative stress hypothesis with a new redox biology paradigm, which provides an exciting and influential framework for guiding translational research.     

Nitroalkenes confer acute cardioprotection via adenine nucleotide translocase 1

Electrophilic nitrated lipids (nitroalkenes) are emerging as an important class of protective cardiovascular signaling molecules. Although species such as nitro-linoleate (LNO(2)) and nitro-oleate can confer acute protection against cardiac ischemic injury, their mechanism of action is unclear. Mild uncoupling of mitochondria is known to be cardioprotective, and adenine nucleotide translocase 1 (ANT1) is a key mediator of mitochondrial uncoupling. ANT1 also contains redox-sensitive cysteines that may be targets for modification by nitroalkenes. Therefore, in this study we tested the hypothesis that nitroalkenes directly modify ANT1 and that nitroalkene-mediated cardioprotection requires ANT1. Using biotin-tagged LNO(2) infused into intact perfused hearts, we obtained mass spectrometric (MALDI-TOF-TOF) evidence for direct modification (nitroalkylation) of ANT1 on cysteine 57. Furthermore, in a cell model of ischemia-reperfusion injury, siRNA knockdown of ANT1 inhibited the cardioprotective effect of LNO(2). Although the molecular mechanism linking ANT1-Cys(57) nitroalkylation and uncoupling is not yet known, these data suggest that ANT1-mediated uncoupling may be a mechanism for nitroalkene-induced cardioprotection.     

Drug Metabolism for the Perplexed Medicinal Chemist

Two related and significant issues may elicit perplexity in medicinal chemists and are discussed here. First, a broad presentation of the pharmacological and toxicological consequences of drug metabolism should justify the significance of drug metabolism and serve as an incentive to further study. When comparing the pharmacological activities of a drug and its metabolite(s), a continuum is found which ranges from soft drugs (no active metabolites) to prodrugs (inactive per se, as illustrated here with clopidogrel and prasugrel). Innumerable intermediate cases document drugs whose activity is shared by one or more metabolites, as exemplified with tamoxifen. The toxicological consequences of metabolism at the molecular, macromolecular, and macroscopic levels are manyfold. A brief overview is offered together with a summary of the reactions of toxification and detoxification of the antiepileptic valproic acid. The second issue discussed in the review is a comparison of the relative significance of cytochromes P450 and other oxidoreductases (EC 1), hydrolases (EC 3), and transferases (EC 2) in drug metabolism, based on a ‘guesstimate’ of the number of drug metabolites that are known to be produced by them. The conclusion is that oxidoreductases are the main enzymes responsible for the formation of toxic or active metabolites, whereas transferases play the major role in producing inactive and nontoxic metabolites.     

Reactions of 1-amino-n′-ferrocenemethylcarboxylate with electrophiles: A combined synthetic, electrochemical, and theoretical study

The reactivity of 1-amino-n′-ferrocenemethylcarboxylate, 1,n′-H₂N-Fc-COOCH₃ (2) toward alkylating agents is reported and MeNH-Fc-COOMe (3), EtNH-Fc-COOMe (4), EtSCH₂CH₂NH-Fc-COOMe (5), (EtO)₂P(O)NH-Fc-COOMe (6) are synthesized by reacting 2 with MeI, EtI, EtSCH₂CH₂Cl and (CN)(EtO)₂P(O), respectively. Cyclic voltammetry of these compounds shows different half-wave potential characteristics compared to aminoferrocene and is dependent on the nature of the substituents, which is rationalized by molecular orbital calculations.