Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

A red light-triggered chemical tool for sequence-specific alkylation of G-quadruplex and I-motif DNA.

The importance of non-canonical DNA structures such as G-quadruplexes (G4) and intercalating-motifs (iMs) in the fine regulation of a variety of cellular processes has been recently demonstrated. As the crucial roles of these structures are being unravelled, it is becoming more and more important to develop tools that allow targeting these structures with the highest possible specificity. While targeting methodologies have been reported for G4s, this is not the case for iMs, as evidenced by the limited number of specific ligands able to bind the latter and the total absence of selective alkylating agents for their covalent targeting. Furthermore, strategies for the sequence-specific covalent targeting of G4s and iMs have not been reported thus far. Herein, we describe a simple methodology to achieve sequence-specific covalent targeting of G4 and iM DNA structures based on the combination of (i) a peptide nucleic acid (PNA) recognizing a specific sequence of interest, (ii) a pro-reactive moiety enabling a controlled alkylation reaction, and (iii) a G4 or iM ligand orienting the alkylating warhead to the reactive residues. This multi-component system allows for the targeting of specific G4 or iM sequences of interest in the presence of competing DNA sequences and under biologically relevant conditions.

Reactivity Modulation of Reactive OFF-ON Type G-Quadruplex Alkylating Agents.

Alkylating agents for nucleic acids have been widely used in cancer chemotherapy, as well as in chemical biology for strong inhibitors and tagging methods. We provide a series of reactive OFF-ON type alkylating agents which enable the reactivity modulation toward G-quadruplex (G4) DNA and RNA. Due to the protonation-accelerated process and equilibrium elimination method, the amine leaving groups show highly reactive and storable properties in an extensive investigation of vinyl quinazolinone (VQ) precursors with different leaving groups.

Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents.

Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.

Alkylating Agents, the Road Less Traversed, Changing Anticancer Therapy.

Cancer is considered one of the gruelling challenges and poses a grave health hazard across the globe. According to the International Agency for Research on Cancer (IARC), new cancer cases increased to 18.1 million in 2018, with 9.6 million deaths, bringing the global cancer rate to 23.6 million by 2030. In 1942, the discovery of nitrogen mustard as an alkylating agent was a tremendous breakthrough in cancer chemotherapy. It acts by binding to the DNA, and creating cross linkages between the two strands, leading to halt of DNA replication and eventual cell death. Nitrogen lone pairs of 'nitrogen mustard' produce an intermediate 'aziridinium ion' at the molecular level, which is very reactive towards DNA of tumour cells, resulting in multiple side effects with therapeutic consequences. Owing to its high reactivity and peripheral cytotoxicity, several improvements have been made with structural modifications for the past 75 years to enhance its efficacy and improve the direct transport of drugs to the tumour cells. Alkylating agents were among the first non-hormonal substances proven to be active against malignant cells and also the most valuable cytotoxic therapies available for the treatment of leukaemia and lymphoma patients. This review focus on the versatile use of alkylating agents and the Structure Activity Relationship (SAR) of each class of these compounds. This could provide an understanding for design and synthesis of new alkylating agents having enhanced target specificity and adequate bioavailability.

The functionality of plant mechanoproteins (forisomes) is dependent on the dual role of conserved cysteine residues.

Forisomes are giant polyprotein complexes that undergo reversible conformational rearrangements from a spindle-like to a plug-like state in response to Ca2+ or changes in pH. They act as valves in the plant vasculature, and reproduce this function in vitro to regulate flow in microfluidic capillaries controlled by electro-titration. Heterologous expression in yeast or plants allows the large-scale production of tailor-made artificial forisomes for technical applications. Here we investigated the unexpected disintegration of artificial forisomes in response to Ca2+ following the deletion of the M1 motif in the MtSEO-F1 protein or the replacement of all four conserved cysteine residues therein. This phenomenon could be mimicked in wild-type forisomes under reducing conditions by adding a thiol alkylating agent. We propose a model in which reversible changes in forisome structure depend on cysteine residues with ambiguous redox states, allowing the formation of intermolecular disulfide bridges (confirmed by mass spectrometry) as well as noncovalent thiol interactions to connect forisome substructures in the dispersed state. This is facilitated by the projection of the M1 motif from the MtSEO-F1 protein as part of an extended loop. Our findings support the rational engineering of disintegrating forisomes to control the release of peptides or enzymes in microfluidic systems.

Genotoxic C8-Arylamino-2'-deoxyadenosines Act as Latent Alkylating Agents to Induce DNA Interstrand Cross-Links.

DNA interstrand cross-links (ICLs) are extremely deleterious and structurally diverse, driving the evolution of ICL repair pathways. Discovering ICL-inducing agents is, thus, crucial for the characterization of ICL repair pathways and Fanconi anemia, a genetic disease caused by mutations in ICL repair genes. Although several studies point to oxidative stress as a cause of ICLs, oxidative stress-induced cross-linking events remain poorly characterized. Also, polycyclic aromatic amines, potent environmental carcinogens, have been implicated in producing ICLs, but their identities and sequences are unknown. To close this knowledge gap, we tested whether ICLs arise by the oxidation of 8-arylamino-2'-deoxyadenosine (ArNHdA) lesions, adducts produced by arylamino carcinogens. Herein, we report that ArNHdA acts as a latent cross-linking agent to generate ICLs under oxidative conditions. The formation of an ICL from 8-aminoadenine, but not from 8-aminoguanine, highlights the specificity of 8-aminopurine-mediated ICL production. Under the influence of the reactive oxygen species (ROS) nitrosoperoxycarbonate, ArNHdA (Ar = biphenyl, fluorenyl) lesions were selectively oxidized to generate ICLs. The cross-linking reaction may occur between the C2-ArNHdA and N2-dG, presumably via oxidation of ArNHdA into a reactive diiminoadenine intermediate followed by the nucleophilic attack of the N2-dG on the diiminoadenine. Overall, ArNHdA-mediated ICLs represent rare examples of ROS-induced ICLs and polycyclic aromatic amine-mediated ICLs. These results reveal novel cross-linking chemistry and the genotoxic effects of arylamino carcinogens and support the hypothesis that C8-modified adenines with low redox potential can cause ICLs in oxidative stress.

Toxic blister agents: Chemistry, mode of their action and effective treatment strategies.

Since their use during the First World War, Blister agents have posed a major threat to the individuals and have caused around two million casualties. Major incidents occurred not only due to their use as chemical warfare agents but also because of occupational hazards. Therefore, a clear understanding of these agents and their mode of action is essential to develop effective decontamination and therapeutic strategies. The blister agents have been categorised on the basis of their chemistry and the biological interactions that entail post contamination. These compounds have been known to majorly cause blisters/bullae along with alkylation of the contaminated DNA. However, due to the high toxicity and restricted use, very little research has been conducted and a lot remains to be clearly understood about these compounds. Various decontamination solutions and detection technologies have been developed, which have proven to be effective for their timely mitigation. But a major hurdle seems to be the lack of proper understanding of the toxicological mechanism of action of these compounds. Current review is about the detailed and updated information on physical, chemical and biological aspects of various blister agents. It also illustrates the mechanism of their action, toxicological effects, detection technologies and possible decontamination strategies.

Halomethyl-Triazoles for Rapid, Site-Selective Protein Modification.

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.

Simple and sensitive quantification of glutathione hydropersulfide alkylated using iodoacetamide by high-performance liquid chromatography with post-column derivatization.

A novel analytical method was developed for the quantification of glutathione hydropersulfide (G-SSH) in biological samples by high-performance liquid chromatography (HPLC) with post-column derivatization. G-SSH was treated with iodoacetamide as an alkylating agent for 5 min at 37 °C, and the resultant acetamide-labeled G-SSH (G-SS-acetamide) was subjected to HPLC. After separation on a reversed-phase column, G-SS-acetamide was quantified by detection using a post-column reaction with orthophthalaldehyde under alkaline conditions. The standard G-SS-acetamide was synthesized through the S-S exchange reaction between oxidized glutathione and 2-mercaptoacetamide. It should be noted that some types of alkylating agents, including N-ethylmaleimide and monobromobimane, cleave the polysulfide chains of polysulfides that consist of glutathione, resulting in the production of alkylated G-SSHs. We confirmed that iodoacetamide did not enhance the cleavage of acetamide-labeled glutathione trihydropersulfide (G-SSS-acetamide). The lowest quantification limit was estimated to be 25 nM for G-SS-acetamide. This method can be useful for studying the dynamics of sulfane sulfur in glutathione-containing matrices.

The Transient Covalent Bond in Abiotic Nonequilibrium Systems.

Biochemical systems accomplish many critical functions with by operating out-of-equilibrium using the energy of chemical fuels. The formation of a transient covalent bond is a simple but very effective tool in designing analogous reaction networks. This Minireview focuses on the fuel chemistries that have been used to generate transient bonds in recent demonstrations of abiotic nonequilibrium systems (i.e., systems that do not make use of biological components). Fuel reactions are divided into two fundamental classifications depending on whether the fuel contributes structural elements to the activated state, a distinction that dictates how they can be used. Reported systems are further categorized by overall fuel reaction (e.g., hydrolysis of alkylating agents, carbodiimide hydration) and illustrate how similar chemistry can be used to effect a wide range of nonequilibrium behavior, ranging from self-assembly to the operation of molecular machines.

High-Precision Sulfur Metabolomics Innovated by a New Specific Probe for Trapping Reactive Sulfur Species.

Aims: Persulfides and other reactive sulfur species are endogenously produced in large amounts in vivo and participate in multiple cellular functions underlying physiological and pathological conditions. In the current study, we aimed to develop an ideal alkylating agent for use in sulfur metabolomics, particularly targeting persulfides and other reactive sulfur species, with minimal artifactual decomposition. Results: We synthesized a tyrosine-based iodoacetamide derivative, N-iodoacetyl l-tyrosine methyl ester (TME-IAM), which reacts with the thiol residue of cysteine identically to that of ß-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM), a commercially available reagent. Our previous study revealed that although various electrophilic alkylating agents readily decomposed polysulfides, HPE-IAM exceptionally stabilized the polysulfides by inhibiting their alkaline hydrolysis. The newly synthesized TME-IAM stabilizes oxidized glutathione tetrasulfide more efficiently than other alkylating agents, including HPE-IAM, iodoacetamide, and monobromobimane. In fact, our quantitative sulfur-related metabolome analysis showed that TME-IAM is a more efficient trapping agent for endogenous persulfides/polysulfides containing a larger number of sulfur atoms in mouse liver and brain tissues compared with HPE-IAM. Innovation and Conclusions: We developed a novel iodoacetamide derivative, which is the most ideal reagent developed to date for detecting endogenous persulfides/polysulfides formed in biological samples, such as cultured cells, tissues, and plasma. This new probe may be useful for investigating the unique chemical properties of reactive persulfides, thereby enabling identification of novel reactive sulfur metabolites that remain unidentified because of their instability, and thus can be applied in high-precision sulfur metabolomics in redox biology and medicine. We did not perform any clinical experiments in this study. Antioxid. Redox Signal. 34, 1407-1419.

Tunable Properties of Nature-Inspired N,N'-Alkylated Riboflavin Semiconductors.

A series of novel soluble nature-inspired flavin derivatives substituted with short butyl and bulky ethyl-adamantyl alkyl groups was prepared via simple and straightforward synthetic approach with moderate to good yields. The comprehensive characterization of the materials, to assess their application potential, has demonstrated that the modification of the conjugated flavin core enables delicate tuning of the absorption and emission properties, optical bandgap, frontier molecular orbital energies, melting points, and thermal stability. Moreover, the thin films prepared thereof exhibit smooth and homogeneous morphology with generally high stability over time.

Palladium-Catalyzed Cross-Coupling of Gem-Bromofluoroalkenes with Alkylboronic Acids for the Synthesis of Alkylated Monofluoroalkenes.

Monofluoroalkenes are versatile fluorinated synthons in organic synthesis, medicinal chemistry and materials science. In light of the importance of alkyl-substituted monofluoroalkenes efficient synthesis of these moieties still represents a synthetic challenge. Herein, we described a mild and efficient methodology to obtain monofluoroalkenes through a stereospecific palladium-catalyzed alkylation of gem-bromofluoroalkenes with primary and strained secondary alkylboronic acids under mild conditions. This novel strategy gives access to a wide range of functionalized tri- and tetrasubstituted monofluoroalkenes in high yield, with good functional group tolerance, independently from the gem-bromofluoroalkenes geometry.

Pre-clinical studies of EC2629, a highly potent folate- receptor-targeted DNA crosslinking agent.

Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors. EC2629 treatment of nude mice bearing FR-positive KB human xenografts led to cures in 100% of the test animals with very low dose levels (300 nmol/kg) following a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing). Complete responses were also observed against FR-positive paclitaxel (KB-PR) and cisplatin (KB-CR) resistant models. When evaluated against FR-positive patient derived xenograft (PDX) models of ovarian (ST070), endometrial (ST040) and triple negative breast cancers (ST502, ST738), EC2629 showed significantly greater anti-tumor activity compared to their corresponding standard of care treatments. Taken together, these studies thus demonstrated that EC2629, with its distinct DNA reacting mechanism, may be useful in treating FR-positive tumors, including those that are classified as drug resistant.

Polycyclic aromatic hydrocarbons: bioaccumulation in dragonfly nymphs (Anisoptera), and determination of alkylated forms in sediment for an improved environmental assessment.

Road runoff carries a mixture of contaminants that threatens the quality of natural water bodies and the health of aquatic organisms. The use of sedimentation ponds is a nature-based solution for the treatment of road runoff. This study assessed the concentration of polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in sediment from seven highway sedimentation ponds and three natural urban ponds. In addition, the study explored the bioaccumulation of PAHs in dragonfly nymphs (Anisoptera). Finally, biota-sediment accumulation factors (BSAFs) were estimated. The results revealed a significant difference in the concentrations of 16 priority PAHs in sediment, with overall higher levels in sedimentation ponds (2,911 µg/kg on average) compared to natural urban ponds (606 µg/kg on average). PAH levels increased substantially once alkylated homologues were considered, with alkylated comprising between 42 and 87% of the total PAH in sediment samples. These results demonstrate the importance of alkylated forms in the environmental assessment of PAHs. The bioaccumulation assessment indicates that dragonfly nymphs bioaccumulate PAHs to a certain degree. It is not clear, however, whether they metabolize PAHs. BSAF results ranged from approx. 0.006 to 10 and indicate that BSAFs can be a powerful tool to determine the functionality of sedimentation ponds.

Biological Basis for Threshold Responses to Methylating Agents.

The cellular outcomes of chemical exposure are as much about the cellular response to the chemical as it is an effect of the chemical. We are growing in our understanding of the genotoxic interaction between chemistry and biology. For example, recent data has revealed the biological basis for mutation induction curves for a methylating chemical, which has been shown to be dependent on the repair capacity of the cells. However, this is just one end point in the toxicity pathway from chemical exposure to cell death. Much remains to be known in order for us to predict how cells will respond to a certain dose. Methylating agents, a subset of alkylating agents, are of particular interest, because of the variety of adverse genetic end points that can result, not only at increasing doses, but also over time. For instance, methylating agents are mutagenic, their potency, for this end point, is determined by the cellular repair capacity of an enzyme called methylguanine DNA-methyltransferase (MGMT) and its ability to repair the induceed methyl adducts. However, methyl adducts can become clastogenic. Erroneous biological processing will convert mutagenic adducts to clastogenic events in the form of double strand breaks (DSBs). How the cell responds to DSBs is via a cascade of protein kinases, which is called the DNA damage response (DDR), which will determine if the damage is repaired effectively, via homologous recombination, or with errors, via nonhomologous end joining, or whether the cell dies via apoptosis or enters senescence. The fate of cells may be determined by the extent of damage and the resulting strength of DDR signaling. Therefore, thresholds of damage may exist that determine cell fate. Such thresholds would be dependent on each of the repair and response mechanisms that these methyl adducts stimulate. The molecular mechanism of how methyl adducts kill cells is still to be fully resolved. If we are able to quantify each of these thresholds of damage for a given cell, then we can ascertain, of the many adducts that are induced, what proportion of them are mutagenic, what proportion are clastogenic, and how many of these clastogenic events are toxic. This review examines the possibility of dose and damage thresholds for methylating agents, from the perspective of the underlying evolutionary mechanisms that may be accountable.

Metabolic-Hydroxy and Carboxy Functionalization of Alkyl Moieties in Drug Molecules: Prediction of Structure Influence and Pharmacologic Activity.

Alkyl moieties-open chain or cyclic, linear, or branched-are common in drug molecules. The hydrophobicity of alkyl moieties in drug molecules is modified by metabolic hydroxy functionalization via free-radical intermediates to give primary, secondary, or tertiary alcohols depending on the class of the substrate carbon. The hydroxymethyl groups resulting from the functionalization of methyl groups are mostly oxidized further to carboxyl groups to give carboxy metabolites. As observed from the surveyed cases in this review, hydroxy functionalization leads to loss, attenuation, or retention of pharmacologic activity with respect to the parent drug. On the other hand, carboxy functionalization leads to a loss of activity with the exception of only a few cases in which activity is retained. The exceptions are those groups in which the carboxy functionalization occurs at a position distant from a well-defined primary pharmacophore. Some hydroxy metabolites, which are equiactive with their parent drugs, have been developed into ester prodrugs while carboxy metabolites, which are equiactive to their parent drugs, have been developed into drugs as per se. In this review, we present and discuss the above state of affairs for a variety of drug classes, using selected drug members to show the effect on pharmacologic activity as well as dependence of the metabolic change on drug molecular structure. The review provides a basis for informed predictions of (i) structural features required for metabolic hydroxy and carboxy functionalization of alkyl moieties in existing or planned small drug molecules, and (ii) pharmacologic activity of the metabolites resulting from hydroxy and/or carboxy functionalization of alkyl moieties.

An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG).

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG.

Collision-Induced Dissociation Studies of Protonated Ions of Alkylated Thymidine and 2'-Deoxyguanosine.

Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O4-alkylthymidine (O4-alkyldT), O2-alkyldT, O6-alkyl-2'-deoxyguanosine (O6-alkyldG), and N2-alkyldG through MS3 analysis. The MS3 of O2- and O4-MedT exhibit different fragmentation patterns from each other and from other O2- and O4-alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O2-alkyldT, O4-alkyldT, and O6-alkyldG adducts carrying larger alkyl groups, whereas O6-MedG mainly undergoes elimination of ammonia. The breakdown of N2-alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.