Drug–Protein Adducts: Chemistry, Mechanisms of Toxicity, and

Nov 29, 2016 - Dr. Jinping Gan is a Senior Principal Scientist in the Biotransformation group with the department of Pharmaceutical Candidate Optimiza...
2 downloads 9 Views 967KB Size
Subscriber access provided by University of Otago Library

Review

Drug-Protein Adducts—Chemistry, Mechanisms of Toxicity, and Methods of Characterization Jinping Gan, Haiying Zhang, and William Griffith Humphreys Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00274 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemical Research in Toxicology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Drug-Protein Adducts—Chemistry, Mechanisms of Toxicity, and Methods of Characterization Jinping Gan, Haiying Zhang and W. Griffith Humphreys* Bristol-Myers Squibb Pharmaceutical Company, Department of Biotransformation, Princeton, NJ 08540, USA KEYWORDS: Drug-protein adducts, covalent protein binding, drug-induced toxicity, proteomics, mass spectrometry, Major histocompatibility complex

ABSTRACT. The formation of drug-protein adducts is considered an important feature in the pharmacological and toxicological profiles of many drugs. Mechanistic insights into the role of specific protein adduct formation in pharmacology and toxicology remain scarce, partly due to the availability of tools to identify and characterize the specific protein adducts, and partly due to the scarcity of relevant in vitro and in vivo predictive models. This paper serves to provide a review on the current state of science on the chemistry, toxicology, and methods of detection and characterization of drug-protein adducts, and to offer some perspective on the future directions of research into the role of protein adducts in drug effects and toxicity.

ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction The formation of drug-protein adducts have been considered to be part of the pharmacological and toxicological profile of many drugs since the first description of drug bioactivation and reaction with biomolecules.1 In general, the process of drug bioactivation proceeds through oxidative transformation of the drug molecule to produce an electrophilic species followed by reaction with a nucleophilic moiety on protein. While there have been definitive mechanisms determined for the downstream effects of only a small set of protein adducts, this type of reaction is considered an important part of the overall process that leads to drug induced toxicity (DIT).2 The mechanisms through which protein adducts elicit toxicity is typically ascribed to either changing target protein function or provoking an antigen response.3 Acetaminophen induced toxicity has been studied for >40 years and there is still no universally accepted mechanism, with multiple protein adduct dependent and protein adduct independent pathways likely involved.4 Several drugs have been shown produce immune responses related to the formation of protein adducts and have all the hallmarks of immune-mediated response (little dose dependence, enhanced response upon re-challenge).5 Drugs in this class include halothane, tienilic acid, and abacavir. In the case of abacavir, the key step appears to be binding with a variant form of a specific major histocompatibility complex molecules (MHC) (HLAB*5701) to form a peptide-MHC complex that elicits a neoantigen response.6-7 This mechanism may be operative with abacavir itself, via direct binding to the peptide-MHC complex, as well as with a bioactivated version of abacavir, leading to a covalent drug-peptide/MHC complex.8-9 There is not a well characterized mechanistic understanding of toxicity pathways for the majority of drugs that form adducts and also demonstrate DIT. Proposed mechanisms for toxicity

ACS Paragon Plus Environment

Page 2 of 60

Page 3 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

include: 1) neoantigen formation, 2) depletion of cellular antioxidant reserve with concomitant oxidative stress and 3) inhibition of intracellular bile acid trafficking.10 Not all of these mechanisms are strictly dependent on the formation of protein adducts and it is likely that the full demonstration of DIT for many drugs is only fully captured through incorporation of protein adduct-dependent and -independent pathways. While the most common pathway for drugs to form covalent protein adducts is via oxidative biotransformation other important mechanisms to consider are: 1) drugs that have inherent chemical reactivity and 2) drugs that form conjugates that are reactive or are bioactivated through non-oxidative pathways. Formation of covalent adducts is directly involved in the mechanism of pharmacological activity for a small subset of drugs. These drugs form covalent bonds to sites on a pharmacologically important target protein. There are examples of drugs in this class that require bioactivation and those that are inherently reactive. While many of these drugs were discovered without full appreciation of their target and covalent mechanism (e.g. clopidogrel and omeprazole), the success of this class of drugs has sparked interest in designed target protein covalent modifiers. Modern efforts in targeted covalent modifiers have produced drugs that do not rely on bioactivation and attempt to maximize target binding vs non-target binding. One of the impediments to the study of the effects of protein adducts has been the lack of methods capable of characterization of sites of adduct formation when proteins were studied in complex mixtures.

Early studies were conducted with radiolabeled drug which allowed

visualization and some level of characterization with immunodetection of the labeled proteins. Proteomic approaches have been applied in recent years and have allowed not only detection of the labeled protein, but also the exact site of adduct formation. It is still difficult to translate this information into impact on protein function.

ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A second major problem in study of the impact of formation of protein adducts is the lack a good translational methods for predicting human toxicity based on toxicological findings determined in animals.11

The types of toxicity produced by protein adducts is still fairly

common in early drug development and a major cause of attrition. While there has been some recent advances in animal models that mimic what is seen in human, these models are still completely specific to certain drugs.12 It is interesting to note that some of these new models exploit alterations in immune tolerance pathways in conjunction with drug treatment to produce the desired toxicological endpoints.13-14 This review will briefly cover mechanisms of protein adduct formation, what is known about the toxicological consequences of adduct formation, recent advances in technologies to detect and characterize protein adducts and finally discuss what has been learned from adduct identification studies. 2. Formation of drug protein adducts Protein adducts can be derived either through chemical compounds that display inherent reactivity or through metabolic activation of a moiety of the compound to a reactive product. Another important distinction regarding drug-protein adducts is whether or not the adducted protein is a pharmacological target of the drug. This distinction is especially important when considering drugs that are designed to be covalent modifiers of target proteins. There have been numerous reviews written on the chemical and enzymatic properties of bioactivation reaction mechanisms. 15-17 Likewise, there have been several comprehensive reviews of the field of targeted covalent modifiers.18-20 This brief summary is not meant to be an exhaustive review but to serve as an introduction to a several important reaction types.

ACS Paragon Plus Environment

Page 4 of 60

Page 5 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

2.1 Oxidative transformations Acetaminophen is the most studied drug which is bioactivated to a reactive species, and also is a representative of a fairly common bioactivation pathway (Figure 1). The activation pathway was described in a number of publications throughout the 1970s as a P450-mediated oxidation to the electrophilic quinone imine, N-acetyl-p-aminobenzoquinone imine (NAPQI).1 The oxidation to a quinoid species occurs in a number of important drugs known to undergo bioactivation and several parent and metabolite pairs are illustrated in Figure 1. Another very important pathway for oxidative bioactivation is through epoxidation; several drugs with well described bioactivation through epoxidation pathways are shown in Figure 2. Epoxidation by P450s is a potential reaction for any double or triple bond, although P450 enzymes show a pronounced trend towards oxidation of electron rich rings, such as anilines. Also, furan, thiophene and related heterocycles are very susceptible to epoxidation. 2.2 Conjugate formation Acyl glucuronide formation is a very common pathway for bioactivation due to the high prevalence of carboxylic acid moieties in drugs as well as the efficiency with which the uridine glucuronosyl transferase enzymes (UGTs) conjugate these drugs. The exact chemical structure of the carboxylic acid substructure produces acyl glucuronides with widely different reactivity profiles and thus propensities to form protein adducts. The reactivity of the acyl glucuronide, along with the dose administered, has been demonstrated to have some correlation to risk of toxicity.21

Acyl glucuronides have been

postulated to be linked with DIT22 with zomiperac and tolmentin (Figure 3) being the two carboxylic acid containing drugs that have been most closely linked to the demonstration of toxicity through this type of bioactivation pathway. These drugs form relatively unstable acyl

5 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 60

glucuronides and are rapidly degraded in aqueous buffer or plasma. Drugs that form more stable conjugates have been found to be less susceptible to the production of toxicity.23-24 Other conjugation mechanisms besides acyl glucuronides can also lead to reactive metabolites. Carboxylate groups have been shown to form acyl coenzyme A and glutathione thioesters that may be important in their reactivity.25-27 Formation of acetate, sulfate or glucuronide conjugates of certain functional groups such as benzylic alcohols and hydroxylamines has been shown to lead to reactive species.28-30 2.3 Other Bioactivation Pathways Multiple additional pathways involving drug oxidation or conjugation that can lead to bioactivation have been reported. Besides P450 and UGT enzymes, other enzymes that have been demonstrated to be active in reactive metabolite formation are alcohol/aldehyde dehydrogenases and esterases.31 3. Protein Adduct Formation Leading to Binding to a Specific Pharmacological Target 3.1 Via Bioactivation Mechanism An interesting aspect of several of the bioactivation mechanisms illustrated above is that the activated species can sometimes lead to covalent interactions with a specific target leading to desired pharmacology. In the case of clopidogrel, the pathway to produce the active species that irreversibly binds to the P2Y12 receptor is shown in Figure 4.32 Other compounds that share the property of being bioactivated prior to binding irreversibly to their respective target protein are shown in Figure 5. Cobicistat33 and abiraterone34 are compounds in which the bioactivation pathway and the pharmacological activity are linked as the enzyme responsible for activation is also the pharmacological target; this property certainly helps to limit the amount of non-target protein adducts formed with these drugs. Cobicistat is a mechanism based inhibitor of P450 3A enzymes (and P450 2D6); the mechanism of enzyme inactivation is not completely understood. Abiraterone is a mechanism based inhibitor 6 ACS Paragon Plus Environment

Page 7 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

of steroid 17α-hydroxylase/C17,20-lyase (P450 17) and the mechanism of inactivation is hypothesized to proceed through an epoxide intermediate (site of activation shown on Figure 5) that binds to the P450 enzyme.35 This is distinct from clopidogrel, where the thiol metabolite is formed in the liver but binds to target in the blood. Despite this, clopidogrel has an excellent safety profile. However, predecessors in the same class that followed similar bioactivation pathways had considerable toxicity, which was likely related to reactive metabolite formation with binding to non-target proteins.36 3.2 Via Preactivation Mechanism There are several examples of compounds that contain inherently reactive moieties and are important, commonly used drugs. Two of the most important examples of these types of molecules are aspirin and the β-lactam antibiotics. Aspirin has multiple effects in vivo, but one of the properties central to the activity is the inhibition of cyclooxygenase (COX) enzymes. Aspirin irreversibly modifies COX-1 and COX-2 through acetylation of an active site serine, however, the activity of COX-1 is inhibited while the product profile of COX-2 is altered after covalent modification.37 Examples of β-lactams are shown in Figure 6. The β-lactams exert their pharmacological activity by binding directly, through the formation of a covalent link between an active site serine on the bacterial enzyme DDtranspeptidase and the β-lactam carbonyl, which inhibits formation of peptidoglycan cross-links in the bacterial cell wall. As a consequence, the decrease in cell wall cross-linking weakens the cell wall and ultimately leads to cell death. Another important class that is growing rapidly are designed covalent modifiers.18-20 These drug molecules contain two design elements that allow them to: 1) bind with high levels of specificity to a target active site and then 2) form a covalent link between the target protein and the drug molecule through the incorporation of a strategically placed electrophilic

7 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 60

moiety that after binding comes in proximity to an active site nucleophile. The design of these has been shown to lead to drugs with high levels of specificity to target and efficient irreversible inhibition. This strategy has been employed for the inhibition of enzyme function through interaction of the inhibitor with an active site catalytic amino acid for many years (e.g. fluorophosphates) and has been incorporated in a number of drug molecules; recent examples of approved drugs can be found in select inhibitors of hepatitis C virus (HCV) protease, dipeptidyl protease IV (DPP-IV), and proteosome function (Figure7).19,38 Another example of active site directed compounds is in the area of FAAH inhibitors, including many compounds in different stages of development.39 The newest class of covalent modifiers uses a non-catalytic site present in the binding pocket of the target protein as the site for covalent modification. The first and best characterized of these molecules is ibrutinib which targets the Bruton’s tyrosine kinase (BTK) protein, a tyrosine kinase important for B-cell development.40 The molecule employs an acrylamide functionality as the electrophilic moiety, often called the "warhead," that reacts with an active site cysteine. Acrylamide is the warhead moiety that has been used most often in published examples of covalent modifiers, but is by no means the only substructure that has been employed.18-19 Also, several drugs in late development or already on the market that employ this strategy are shown in Figure 8. Properties that are important in design of covalent modifiers are both the inherent specificity of the molecule as well as the reactivity of the warhead. The reactivity of the warhead is a balance between the efficient reaction with the target active site nucleophile and the reaction with all other off-target nucleophiles. Strategies for optimizing these properties have been published and are of much current interest in the pharmaceutical industry.41-42

8 ACS Paragon Plus Environment

Page 9 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

4. Toxicological Considerations Covalent modification of proteins has been hypothesized to be central to the toxicity exhibited by many drugs. Proposals for the mechanism of toxicological response are generally centered around either 1) alterations of biological function of the drug modified proteins, either producing exaggerated pharmacology of target proteins or unintended pharmacology of non-target proteins, or 2) stimulation of immunological response of the host immune system against modified proteins. There are multiple theories on the etiology of DIT, including the hapten hypothesis, danger hypothesis, the pharmacological interaction concept (p-i concept), and altered peptide pool hypothesis.5, 43-47 These theories are not necessarily exclusive and it is quite possible that multiple mechanisms are at play for DITs of a single drug.10, 46, 48 A recent review by Uetrecht and Naisbitt provides a very comprehensive summary of the clinical, biochemical, and toxicological models of DIT, especially idiosyncratic drug reactions that implicate immunological mechanisms.10

There are also multiple reviews covering the biological

consequences of covalent inhibitors, therefore these aspects will not be covered in details in this review.18-19 However, we will focus on some key questions of immunological consequences of covalently modified proteins by small molecule drugs, including the major determinants of tissue targets and types of immunological response, factors which determine individual risks beyond MHC restriction, and the predictive value of various in vitro and in vivo models in the evaluation of immunologic DIT. Several recent studies with select drugs that cause DITs have shed light on mechanistic aspects of the hapten hypothesis. This hypothesis states that the drug modifications of the protein lead to the formation of a neoantigen that is recognized as non-self by the immune system5 (Figure 9). For antigens to be recognized by the T lymphocytes (T cells), the antigen must first be

9 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 60

“processed” to an appropriate size, and then presented on the surface of antigen presenting cells (APCs) by MHC molecules.49-50 There are two classes of MHC molecules, Class I and II. The MHC class I molecules are expressed on most cell types, and they are typically responsible for presentation of cytoplasmic proteins (endogenous antigens) to be recognized by CD8+ T cells.5152

The processing and presentation of endogenous antigens is a multi-step process. First,

cytoplasmic proteins are targeted by ubiquitin conjugation before being degraded by the proteasome complex. The resulting peptides are then transported to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) family proteins; it is in the ER that the antigenic peptides of proper length (8-12 amino acid long) form complexes with MHC class I molecules before being transported to the cell membrane surface through the Golgi complex. In contrast, the MHC class II molecules are only expressed on the surface of professional APCs, including dendritic cells, macrophages, and B lymphocytes, and they are responsible for the presentation of extracellular antigens that are recognized by CD4+ cells. The processing of extracellular antigens is distinctively different than for MHC class I processing and features internalization by phagocytosis, endocytosis, or pinocytosis to form endosomal vacuoles which are then fused with lysosomes. It is in the lysosomes that proteolytic degradation occurs to generate peptides, which are associated with newly synthesized MHC II molecules.

The

peptides formed are typically of longer length than peptides bound to MHC class I molecules. MHC II molecules associated with antigenic peptides are then transported to the cell surface to present the antigen to CD4+ T cells.53 4.1 Tissue Targeting The liver, skin, and hematologic cells are the common targets of DITs. While some drugs such as halothane cause only liver injury,54 many drugs cause DITs in multiple organs.10 So what determines the target organ spectrum for a given drug? There is no

10 ACS Paragon Plus Environment

Page 11 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

simple answer to this question, but it may be possible to rationalize it through an ADME perspective. Absorption or route of exposure is a very important determining factor, as shown in the example of piperacillin versus flucloxacillin.55-56 Piperacillin and flucloxacillin are both beta lactams that readily react with lysine residues of albumin. Piperacillin is given at a very large dose (12 g per day) intravenously, and it causes skin DIT in 1 out of 3 patients. It was shown that it readily reacts with lysine residues in plasma albumin and stimulates drug-specific CD4+ T cell response without apparent HLA restriction. On the other hand, flucloxacillin is administered orally at a lower dose (1g per day), and its major target is liver at a much lower frequency (1 in 10,000). The liver injury is HLA-B*5701 restricted, and it stimulates a CD8+ T cell response. In studies with flucloxacillin, it was also shown in animals that there was multiple covalently modified proteins in the liver.57 The difference in route of administration is probably the major reason for the difference in the target tissues in this case.

It is possible, although not

experimentally determined, that orally absorbed flucloxacillin reaches high concentrations in the liver via first pass extraction where it readily reacts with newly synthesized albumin. Although flucloxacillin serum albumin adduct was detected in patients,58 systemic plasma concentrations may not reach the same levels as in liver and therefore levels of adducted albumin would also not be expected to be as high in the blood stream relative to liver tissue. In the case of intravenously administered piperacillin, the drug readily reacts with serum albumin, and the piperacillin-bound albumin presumably distributes to the skin to be processed and presented by resident APCs that trigger the skin reaction. For chemically inert drugs that require bioactivation to exert toxicity, the target tissue of toxicity depends on the localization of specific bioactivation enzymes and the reactivity of the resulting reactive metabolite and the ability of the organ to de-toxify the reactive species. Halothane is bioactivated by P450 2E1 in the liver,59-60 and the resulting reactive

11 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 60

metabolite is so reactive that it does not escape the active site of P450 2E1. Similarly tienilic acid is bioactivated by P450 2C9 to a very reactive intermediate that labels the P450 enzyme.61-62 For both drugs, the target organ of toxicity is liver and the onset of liver toxicity can be characterized by immune hepatitis with autoantibodies against their respective metabolizing cytochrome P450s. In the case of nevirapine, however, the initial metabolism occurs in the liver to generate a hydroxylated metabolite which is also inert. This metabolite circulates in the body and distributes to the skin where it is further metabolized to a sulfate metabolism by a sulfotransferase in the skin. This sulfate conjugate then reacts with proteins locally in the skin to cause ultimately cause skin rash.63 Therefore, the tissue targets depend on the localization of bioactivation enzymes, tissues where drug accumulates (especially in the case of directly reactive drugs), and the chemical stability of reactive metabolites. Another interesting question is what happens when antigens are presented by either the MHC class I or II molecules, and why do different drugs demonstrate different spectra of DIT. MHC class I molecules interact with the T cell receptors (TCR) on the surface of CD8+ cytotoxic T cells, and upon binding, CD8+ T cells release cytolytic factors. The release of different cytolytic factors seem to determine the nature and severity of drug reactions, but what determines the type of cytolytic factor release from CD8+ T cells is not fully understood. MHC class II molecules interact with TCR on the surface of CD4+ T cells, and depending upon which cytokine is present, the T cells differentiate into different T helper cells that each has its own spectrum of functions. The tissue microenvironment at the time of APC-T cell interaction therefore plays an important role in the manifestation of drug toxicity or the lack thereof. Our understanding in this area is still very limited and more basic research is needed.

12 ACS Paragon Plus Environment

Page 13 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

4.2 Determinants of Individual Response Other than MHC Restrictions Over the last decade there have been multiple reports of MHC genotypic associations with DITs, and some of those associations are quite exclusive, as is in the case of abacavir induced hypersensitivity reactions (HLA-B*5701)64-66 and carbamazepine induced Stevens-Johnson syndrome in the Han Chinese population (HLA-B*1502).67 Although these two drugs form reactive metabolites,8, 68-72 recent studies indicate that both drugs bind noncovalently to their respective MHC molecules to induce conformational changes in the MHC that affects the peptide bound conformation, which in turn changes the self-peptide pool and elicits a T cell response.44 For flucloxacillin induced liver DIT, although also highly associated with HLA-B*5701 genotype, it was shown that covalent modification of albumin is the antigenic culprit.56 Although some of these associations are quite exclusive, except in the case of abacavir where almost half of the patients with HLAB*5701 genotype developed hypersensitivity,65 the majority of patients with the associated allele don’t develop hypersensitivity for most other drugs. Therefore there must be other factors in addition to HLA genotype that define the susceptible population. This makes sense because antigen presentation and T cell response are multiple step process and there could be interindividual differences in any one or combinations of these steps including antigen generation, processing, transport in the APCs plus T cell receptor binding and costimulatory signaling. Beyond MHC restrictions, there have been reports of other genotypic associations with DITs, including drug metabolizing enzymes and transporters.73-74 There are also other risk factors such as viral infection, age, and gender that may define susceptibility to some drugs.75-76 4.3 In Vitro and In Vivo Models. Although there are many bioanalytical tools to detect and quantify bioactivation of drugs leading to downstream products of reactive metabolites, establishment of in vitro or in vivo models that accurately predict human toxicity has been

13 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 60

extremely challenging. Human relevant hepatic in vitro models are used as predictive tools for a number of ADME-Tox related assays, but the correct models and endpoints for truly predictive toxicology have not been identified. Similarly, it has been challenging to find relevant in vivo models as it is often the case that typical animal models do not develop the same toxicity as observed in human for many drugs causing DIT. Significant efforts have been made in the attempt to find susceptibility factors that change sensitivity to DIT by perturbing the immune response of standard models or by scanning for genetic factors that influence response by looking at inter-strain differences. The following section captures some of these studies, for additional details on progress towards in vitro and in vivo models there have been several recent reviews on the topic.77-79 Recent work in the area of in vitro models for DIT have examined 1) advanced cell cultures or co-cultures 2) interruption of trafficking of endogenous compounds, such as bile acids, 3) activation of the inflammasome response 4) performance multi-parameter prediction. Multiple liver co-culture models have been introduced with hepatocyte and at least one non-parenchymal cell type including macrophages.80

One interesting results from such studies was the

demonstrated that trovafloxacin kills hepatocytes effectively when the hepatocyte/macrophage co-culture was challenged with lipopolysaccharide, indicating the involvement of nonparenchymal cells in the manifestation of trovafloxacin hepatotoxicity.81-83 Several published reports have described the interaction of hepatotoxic compounds with biliary transporters with the hypothesis that interruption of transport of endogenous compounds may lead to toxic levels of these compounds within the hepatocyte. These studies have focused on inhibition of bile salt export protein (BSEP), multidrug resistance-associated proteins (MRPs) and multidrug resistance protein 3 (MDR3).84-86 There has also been a cellular model reported that is responsive and able

14 ACS Paragon Plus Environment

Page 15 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

to detect inflammasome activation, which would presumably be sensitive to drugs triggering a danger signal.87 The utility of this model is yet to be demonstrated with more drugs, but if demonstrated, it could provide another important assay endpoint. There have been multiple reports from the pharmaceutical industry describing multi-factorial algorithms, which included hepatocyte cytotoxicity, reactive metabolite formation, inhibition of key transporters including BSEP and MRP2, mitochondrial toxicity, among others, which seem to increase the predictive power as compared with single assay endpoints.17, 88 Another advance in in vitro mechanistic studies of immune mediated DIT is the in vitro T-cell priming assay.89-90

Typical T-cell priming assay uses immature dendritic cells as antigen

presenting cells. Upon incubations with drugs of interest with or without a carrier protein such as albumin, the resulting haptenized antigen is present to naive T-cells. T-cell proliferation and/or interferon γ production are then measured after 1 to 2 weeks of priming. It is hoped that human PBMC banks with large sample sizes and matching genomic data could provide an interesting tool set to evaluate immune-mediated DIT potential of new molecular entities when the drug, its metabolites and/or its protein adduct are incubated with the PBMC tissue array to identify T cell responsive clones.89-92 Studies have demonstrated that administration of ranitidine to lipopolysaccharide-sensitized mice induced liver toxicity, which is consistent with the “danger hypothesis”, i.e., that the activation of innate immune response and release of pro-inflammatory cytokines are potentially important in the development of DIT.93-94 However, the lipopolysaccharide challenge model seems to only work with a limited number of drugs, therefore, it is not suitable as a universal animal model.

15 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 60

Shenton et al. recapitulated nevirapine-induced skin rash in Brown Norway rats,95-96 after scanning several species/strains for susceptibility to nevirapine toxicity. Although the Brown Norway rat model appears to be specific as a sensitive species/strain for nevirapine toxicity, it does illustrate the importance of genetic factors in determining appropriate models. The search for genetic diversity leading to susceptibility to DIT is the basis of mouse diversity model method of investigation, where a panel of outbred mouse strains is administered a toxic agent and then the genetics of the susceptible strain(s) is evaluated to attempt to find the genes responsible.97-98 There has been recent efforts to elicit DIT through modulation of immune checkpoint inhibitors in animal models as a way to overcome the immune tolerance that is displayed in liver.14 Amodiaquine induced liver toxicity was shown to be augmented in programmed death receptor-1 (PD-1) knockout mice also treated with a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody. The toxicity in mice was very similar to that seen in humans and is consistent with the blockage of immune tolerance of the liver.14 More recently, similar data was shown with isoniazid and nevirapine in PD-1 knockout mice.13 There studies cited here demonstrate some of the challenges in the search for animal models for DIT and illustrate why there has been relatively little progress in the area.

First, by

definition, DITs occur in very low frequency, therefore the sample size in a typical animal toxicity testing is too small to pick up a low frequency event; secondly, the ADME profile including bioactivation may be very different in the animals than in human, thus not producing the same antigen at the same tissue location; thirdly, for MHC restricted DITs, animals simply may not have the same MHC molecules/TCR pairs to trigger an immune response; finally the

16 ACS Paragon Plus Environment

Page 17 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

immune tolerance to drug derived antigens may be very different than humans so that it provokes a more severe response. 5. Identification of Protein Targets for Covalent Reactive Metabolite Modifications Protein target identification is required to allow the comprehensive understanding of the consequences of drug protein adduct formation. Ideally, such investigations should reveal the specifics of a) what proteins are targeted, and b) which residues on the protein become modified and c) the exact chemical structure of the conjugate. As well, for the methods to really allow complete understanding of the impact of adduct formation, they must allow for quantitative information on the extent and nature of the covalent interactions. As pointed out above, methods are necessary to track drug protein adducts in multiple tissues and may also need to focus on the whole modified proteins as well as modified peptides formed from those proteins. In reality, such tasks are very challenging due to the extremely low abundance of drug-protein adducts. This is because reactive metabolites are typically a fraction of a drug’s total metabolism to begin with, and the levels are further reduced by reaction with small molecule nucleophiles such as GSH, leaving only a small portion for covalent binding to proteins.99 Quite often, the drug-bound portion of a target protein only accounts for a tiny percentage of its unmodified form, and protein targets subject to reactive metabolite modifications are only a small fraction of a proteome under investigation. Therefore, the task of identifying drug-protein adducts has been described in the literature as an exercise of hunting a “needle in a haystack”.100 Typically the combination of several analytical procedures is necessary, including at least one separation technique to resolve the protein/peptide mixtures, a detection technique to recognize the drug-adducted proteins, and an identification technique to determine the identity of the drugadducted proteins. Historically, the detection procedure and the identification procedure are two 17 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 60

distinct procedures, leading to the possibility that the adduct signals detected and the proteins identified do not match. Not surprisingly, the specificity and sensitivity of the detection techniques have been the key factors for the success of such exercises. Initial efforts towards global detection of drug-protein adducts in a proteome came in the 1980’s with Western blot-based immunochemical detection. For example, antibodies against trifluoroacetylated proteins were raised and used in Western blot analyses in combination with 1D SDS-PAGE separation.101-103 Numerous protein targets were detected as having formed adducts with the halothane metabolite trifluroacetyl chloride or other trifluoroacetylating chemicals.104-106 Similar approaches have been used for detection of protein targets of other electrophilic chemicals.107-119 The applicability of an immunochemical detection method is subject to the availability, quality, and specificity of antibodies to protein adducts. As the detection procedure only reveals immunoreactivity bands after SDS-PAGE separation, this approach suffers from the likelihood of co-migrating proteins confounding analysis and ambiguity about the proteins identified. Further, because of the protein identification technology available at the time (Edman sequencing or MS-based finger-printing), the sequence information of the proteins was limited, further adding to uncertainty of the protein identities. Since late 1990’s, the advancement of proteomics analysis with the incorporation of 2D gelelectrophoresis separation and advanced mass spectrometric protein identification technologies has significantly improved the efficiency and reliability of global reactive metabolite protein target identification efforts. A benchmark study was completed with these new technologies and identified 23 acetaminophen-bound mouse liver proteins from mouse liver in one 2D-gel experiment.120 The detection technology for these types of studies remained immunochemistrybased Western blotting, or auto-radiography for radio-labeled drug. In this method, 2D gel-

18 ACS Paragon Plus Environment

Page 19 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

electrophoresis provided improved resolution for protein separation, which was achieved by a first dimension of isoelectric focusing separation based on pI of proteins, followed by a second, orthogonal dimension SDS-PAGE separation. The impact of the improved resolution was significant in that it reduced, but did not eliminate, the chance of the detected proteins being mixtures of adducted targets with co-migrated proteins. After visualization of potential protein adducts on the 2D gel, areas of the gel could be excised for in-gel protease digestion, and the resultant peptide samples subject to mass spectrometry for protein target identification. Advanced mass spectrometric identification techniques also began to be applied to adduct detection, with application of both matrix assisted laser desorption/ionization (MALDI) MS and LC-ESI-MS.

Both techniques had sufficient sensitivity for peptide samples and provided

MS/MS data reflecting target protein sequence information, which significantly improved the efficiency and reliability of protein identification via protein database search and match.121-123 The 2D gel-electrophoresis and tandem mass spectrometry-based proteomics approach has been widely used for detecting and characterizing reactive metabolite protein targets. For example, protein targets have been identified for acetaminophen,120 naphthalene,124-127 monocrotaline pyrrole,128-129 bromobenzene,130-131 tienilic acid,132 and thioacetamide133 with autoradiography detection, and for tienilic acid,134 4-hydroxynonenal,135-137 2,6-di-tert-butyl-4methylphenol,138-139 and menthofuran140 with Western blot-based immunochemical detection. Figure 10 exhibits an exemplary autoradiogram showing detected spots of acetaminophenprotein adducts on a 2D-gel.120 It should be pointed out that although the 2D gel and MS-based proteomics approach provides significantly improved separation resolution and typically sufficient target protein information via peptide identification, the approach often cannot directly detect the adduct-bearing peptides.

19 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 60

Strictly speaking, in such cases there is still some level of ambiguity between the spots detected on the gel and the protein identified, due to the possibility of proteins co-migrating with the detected targets. Also, structural information on which residues of the target proteins become modified is typically not determined. For example, although there have been reports of more than thirty non-redundant acetaminophen target proteins, the specific site of adduction is not known for any of them, and this is the general case for most reactive metabolite adducts of target proteins.69 With further advances of LC/MS and MS/MS technologies and the improved capability of protein identification based on database search, the shotgun proteomics strategy emerged and has become a standard proteomic method to directly analyze digested peptides of a protein mixture for discerning changes in a proteome.141-142 This was accomplished based on chromatographic separation of peptide mixtures obtained from a biological sample of interest after protein digestion and relies on producing a comprehensive MS/MS dataset of the peptides for database search and identification of proteins. Attempts have been made to adopt this strategy for protein target identification for reactive metabolites. The hope is to detect metabolite-adducted peptides in the MS/MS dataset and thus provide unambiguous identification of the target proteins along with exact site of adduct formation.

However, although the strategy is very effective for

characterizing changes of protein profiles in a proteome upon a stimulus or treatment, there has been limited success in the identification of peptides adducted with reactive metabolites of typical drugs.143 The method has been successful in cases where high levels of protein adducts are formed.144

The lack of success with typical protein adduct containing samples is not

surprising given the extremely low abundance nature of adducted protein targets in a proteome. The low level adducted peptides in digested samples of a shotgun proteomics study present

20 ACS Paragon Plus Environment

Page 21 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

significant detection challenges because with the duty cycle limitation of current LC/MS & MS/MS instrumentation; it is simply not possible to generate quality MS/MS spectra for all precursor ions in an LC/MS run. Despite these challenges there have been several reports that have led to successful identification of acetaminophen adduct proteins using either a 2Dimensional LC-MS/MS in rat liver microsome samples145 or data independent acquisition MS methodology in 3 dimensional human liver microtissues.146 In both cases, there were limited numbers of target protein adducts identified and it remains to be seen how much more can be identified with the advances in mass spectrometry technology alone. There have been several other recent efforts to directly detect adduct-bearing peptides. One such effort used isotope patterns and mass spectrometry to attempt to identify ions of potential adduct-bearing peptides. A key to the technique was the use of an equimolar mixture of 14Clabeled and non-labeled furan-containing 2-amino-pyrimidine drugs, along with other procedures including preparative SDS-PAGE, in gel digestion and strong cation exchange chromatography enrichment.147 A perhaps more prevailing strategy has been the combination of proteomic methods with an affinity capture procedure which allowed direct and specific pre-enrichment of adducted proteins/peptides to overcome the low abundance issue, and thus provided sufficient sensitivity for detecting adducted peptides. Affinity capture can be achieved by employing a biotin-labeled drug,148-150 and after protease digestion of the protein mixtures, the resultant peptides are subject to affinity purification to remove bulk, unmodified peptides to enrich the adducted peptides (Figure 11). The subsequent LC/MS/MS analysis allows direct detection of the adducted peptides, leading to unambiguous identification of the protein targets. This is a practical and unambiguous approach in that the targets identified is directly based on the adduct signals detected. However, there is a potential that a biotin-labeled drug (i.e., a bulky modified

21 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 60

version of the drug) may not display the same binding profile as the original drug in context of their protein targets.100 To alleviate such a concern, another affinity capture strategy has been developed using smaller azido- or alkynyl groups to label a drug. In these methods the affinity tag (biotin) is added via click chemistry and not added until later in the study, at the sample workup stage and post protein reaction.151 The azido or alkynyl groups on a drug are relatively inert in typical cell or tissue environments and add only a modest amount of steric bulk and thus have a good probability of not altering the protein target profile of a drug. Nevertheless, it is prudent in practice to run some validation studies to ensure that the “clickable” drug and its native form exhibit the same characteristics in their toxicity and/or activity profiles. Figure 12 exhibits structures of three alkynyl labeled versions of endogenous reactive species or designed covalent inhibitors that have been utilized in click chemistry based protein adduct studies.41, 152153

Figure 13 demonstrates the click chemistry based workflows that have been used to

characterize the reaction products of endogenous reactive lipid oxidation products with protein targets. Post-reaction biotin labeling chemistry has also been reported for affinity capture of certain drug-protein adducts without the need of prior labeling of the drug. For example, if the adduct structure contains an aldehyde moiety it may allow for direct tagging with a biotinylated hydroxyl amine for affinity capture.154 However, most drug protein conjugates will not provide a unique chemical handle for direct modification, so the application is limited. Other methods for adduct detection has been developed that take advantage of high resolution mass spectrometry data sets. The methods use high resolution MS1 data sets obtained directly from LC/MS runs as a global detection mechanism to discern and highlight drug-related species in a complex biological sample without prior knowledge of their masses or structures.155 One such example is high resolution LC/MS-based background subtraction, which leverages the high

22 ACS Paragon Plus Environment

Page 23 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

mass precision feature of LC/MS datasets, and sets a variable retention time window to remove all “matched” ions (based on mass to charge ratio (m/z)) in a test sample that are also present in a control sample, thus revealing ions unique to the test sample. There are multiple methodologies for background subtraction available commercially; the success of the protocol used in the referenced study hinges on the use of the variable retention time window, the criteria set for m/z match and the threshold of peak intensity, all leading to the decision of whether a peak in the study sample is unique is present in the control. Recently, a proof-of-principle study was conducted applying this mode of detection for adducted peptides formed after acetaminophen bioactivation in liver microsomal samples followed by digestion.156 As illustrated in Figure 14, instead of traditional proteomics relying on data-dependent MS/MS, this study first used MS1 data collected from paired LC/MS analysis of 13C and unlabeled acetaminophen-treated samples for background subtraction and detection of putative adducted peptide ions. This step circumvents the distinct possibility of low abundant adduct ions going undetected in datadependent MS/MS. After background subtraction-based detection of putative adducted peptide ions, focused MS/MS analyses were conducted to determine the peptide sequences and adduct site information and, hence, the identification of protein targets. Both affinity capture-based and background subtraction-based proteomic approaches provide unambiguous identification of protein targets along with structural information on binding sites. The background subtraction-based approach does not require alteration of a drug’s structure, hence free of any validity concerns. However, the detection sensitivity of such an approach is dictated by the sensitivity of currently available mass spectrometry technology, the quality of the background subtraction algorithm as well as possible ion suppression effects due to the multitude of peptide ions in a shotgun proteomics sample. It is envisioned that the combination with

23 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 60

preparative SDS-PAGE or other protein fractionation technique can potentially provide preconcentration of target proteins and reduction of ion suppression in subsequent background subtraction-based adduct peptide detection. In addition, with the advancement of sheathless coupling of capillary electrophoresis to mass spectrometry,157 it is possible that CE/MS may provide a better detection/identification technology than LC/MS for further improvement of the detection sensitivity of adducted peptides. 6. Profiling of Clinical Samples for Reactive Metabolite-mediated Protein Conjugates The identification of reactive metabolite protein targets and determination of the nature and structure of the protein adducts paves the way for developing specific assays for profiling reactive metabolite-mediated protein adducts from non-clinical or clinical samples. Much of the research in this area to date has focused on monitoring clinical samples. The purposes of such assays can be for revealing the kinetics and disposition of protein adducts in patients, for understanding the relationship between the dose, damage, and toxicity effects, or ultimately for using the features of the protein adducts as potential biomarkers to monitor adverse effects or disease processes. The assays typically are quantitative and specific, targeting either features of the protein adduct structures or some surrogate markers associated with the protein adducts. For example, assays for detecting and quantifying 3-p-cysteinyl acetaminophen, a protein degradation product of acetaminophen protein adducts, in clinical samples have been developed and optimized over the years in attempt to assess the causal association between acetaminophen bioactivation and hepatic injury.158-165 A second example is the assays that have been developed for detecting antibodies against diclofenac metabolite-mediated liver protein adducts in sera to establish the relationship between protein damage and hepatotoxicity effects in patients.166-167 In addition, targeted profiling of drug-bound adducts with human serum albumin have been

24 ACS Paragon Plus Environment

Page 25 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

developed based on in vitro studies168 or projection from prior knowledge.169 These assays have been used to monitor exposure to various chemicals or drugs. Modern advances in mass spectrometry have also allowed for the detection of various protein adducts in clinical samples from patients treated with multiple drugs implicated in immunologically-based drug induced toxicities.

For example, the research groups lead by

Naisbitt and Park have detected and characterized protein adducts from patients treated with isoniazid,170 abacavir,9 nevirapine,171 and beta-lactam antibiotics.172-174 The combination of in vitro mechanistic studies and in vivo characterization in patients hold promise to be a powerful tool in the unraveling of complex pathology of immune-mediated drug induced toxicities.

7. Overall Impact of Drug Protein Adduct Research to Date and Future Directions Although the field is still far from a complete understanding of how protein adducts produce downstream effects, there are a number of new developments that will likely drive research developments in the coming years. These include both improved understanding of how protein adducts can provoke toxicological response, especially through interaction with the immune system, and in the methodology necessary to characterize protein adducts. Improvements in methodology of adduct detection via proteomic type approaches have begun to help unravel the biological consequences of adduct formation for drugs such as acetaminophen. Numerous studies have been conducted to attempt to determine the key protein targets of NAPQI. Along with older studies conducted with radioactive drug, more recent studies utilizing LC-MS-based proteomic methods with detection of modified peptides have a allowed a more thorough understanding of the inventory of modified proteins.175-176 Although there is a preference for reaction with mitochondrial proteins that correlates with a key

25 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 60

functional effect of APAP, it is still unclear how the full array of protein adducts formed after acetaminophen exposure alter function. There has been limited demonstration of the functional impact of adduct formation with one protein target, microsomal glutathione S-transferase, shown to increase its function after acetaminophen administration in rats.177 Theoretically it is possible to explore the functional consequences of target proteins by either direct biochemical readouts of adducted proteins or indirect pharmacodynamic readouts of downstream effects once key protein targets are identified. Other methods for inferring the functional consequences of modified proteins may be through pathway analysis. Recent studies have extensively cataloged the protein target of quinone, dubbed the “quinonome”, to attempt to delineate functional effects by tracking all proteins in particular pathways.178 While there are still many gaps in the understanding of how protein adducts effect cell function, there has been more impact in the delineation of the mechanism by which protein adducts can mediate immune response. The detection of albumin adducts of a wide array of drugs in patient samples is encouraging. In some cases the formation of these adducts has been directly linked to T-cell activation response.179 This approach could potentially lead to a method to examine albumin adducts in clinical setting for drugs under development as part of a risk assessment strategy if potential bioactivation is suspected. There is also opportunity for studies in relevant experimental animals to explore the toxicological consequences of adduct formation with or without immunomodulation. At the heart of all these potential applications is the advances in modern mass spectrometry technology. The need to better understand and characterize the risk of drug protein adducts remains an acute need for drug discovery and development. There have been significant advances in the understanding of what drives formation of these adducts and most companies use a combination

26 ACS Paragon Plus Environment

Page 27 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

of structure alert as well as active screening to limit formation of high levels of adducts.15, 17, 180 However, while bioactivation to reactive species can be reduced, it will never be completely eliminated from new drugs as long as they contain traditional organic subunits. The new focus on designed covalent inhibitors will certainly require continued risk assessment.18 Additional understanding of the basic biology of drug protein adduct formation will be the primary driver for improved risk assessment, with key developments likely coming from progress towards translational models to predict how formation of drug protein adducts might impact immune response and overall protein function. These new models may come from maturation of existing models to examine T-cell activation and could also take advantage of advances in complex 3D cell systems.80

New developments in animal models along with

improved animal and human biomarkers will also play a major role in risk assessment.11, 14, 181 New assay methodology is making it possible to thoroughly characterize drug protein adducts, even in complex biological samples.178 Advances in proteomic methods, along with new enrichment strategies such as click chemistry based approaches,41 are now allowing individual proteins and pathways to be identified from cell and tissue samples. These studies should help in identifying what type of adducts are driving effects. Also, studies of how mixtures of chemicals forming adducts over extended exposure windows may help to determine functional consequences.182-183 Overall understanding of the biological effects of formation of drug protein adducts through advances in translational models and improvements in characterization should allow significant improvements in the ability to risk assessment activities. The current practice of minimization and avoidance of the property will likely be replaced by a more defined set of criteria for evaluation of how and when drug protein adducts are likely to provoke toxicological response.

27 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 60

28 ACS Paragon Plus Environment

Page 29 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

AUTHOR INFORMATION Corresponding Author *W. Griffith Humphreys, Ph.D. Address: Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA; Email: [email protected]; phone: (609) 252-3636

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ABBREVIATIONS ADME, absorption, distribution, metabolism, excretion; APAP, acetaminophen; APC, antigen presenting cell; CE/MS, capillary electrophoresis/mass spectrometry; COX, cyclooxygenase; DIT, drug induced toxicity; HLA, human leukocyte antigen; MHC, major histocompatability complex; NAPQI, N-acetyl-p-benzoquinone imine; pI, isoelectric point; SDS-PAGE, sodium dodecyl

sulfate-polyacrylamide

electrophoresis;

TCR,

T-cell

receptor;

UGT,

uridine

glucuronosyl transferase

29 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 60

REFERENCES 1. Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Davis, D. C.; Gillette, J. R.; Brodie, B. B., Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J Pharmacol Exp Ther 1973, 187 (1), 185-94. 2. Tailor, A.; Waddington, J. C.; Meng, X.; Park, B. K., Mass Spectrometric and Functional Aspects of Drug-Protein Conjugation. Chem Res Toxicol 2016. 3. Cho, T. E.; Uetrecht, J., How Reactive Metabolites Induce an Immune Response that Sometimes Leads to an Idiosyncratic Drug Reaction. Chem Res Toxicol 2016. 4. Jaeschke, H.; Xie, Y.; McGill, M. R., Acetaminophen-induced Liver Injury: from Animal Models to Humans. J Clin Transl Hepatol 2014, 2 (3), 153-61. 5. Faulkner, L.; Meng, X.; Park, B. K.; Naisbitt, D. J., The importance of hapten-protein complex formation in the development of drug allergy. Curr Opin Allergy Clin Immunol 2014, 14 (4), 293-300. 6. Norcross, M. A.; Luo, S.; Lu, L.; Boyne, M. T.; Gomarteli, M.; Rennels, A. D.; Woodcock, J.; Margulies, D. H.; McMurtrey, C.; Vernon, S.; Hildebrand, W. H.; Buchli, R., Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity. AIDS 2012, 26 (11), F21-9. 7. Ostrov, D. A.; Grant, B. J.; Pompeu, Y. A.; Sidney, J.; Harndahl, M.; Southwood, S.; Oseroff, C.; Lu, S.; Jakoncic, J.; de Oliveira, C. A.; Yang, L.; Mei, H.; Shi, L.; Shabanowitz, J.; English, A. M.; Wriston, A.; Lucas, A.; Phillips, E.; Mallal, S.; Grey, H. M.; Sette, A.; Hunt, D. F.; Buus, S.; Peters, B., Drug hypersensitivity caused by alteration of the MHC-presented selfpeptide repertoire. Proc Natl Acad Sci U S A 2012, 109 (25), 9959-64. 8. Grilo, N. M.; Charneira, C.; Pereira, S. A.; Monteiro, E. C.; Marques, M. M.; Antunes, A. M., Bioactivation to an aldehyde metabolite--possible role in the onset of toxicity induced by the anti-HIV drug abacavir. Toxicol Lett 2014, 224 (3), 416-23. 9. Meng, X.; Lawrenson, A. S.; Berry, N. G.; Maggs, J. L.; French, N. S.; Back, D. J.; Khoo, S. H.; Naisbitt, D. J.; Park, B. K., Abacavir forms novel cross-linking abacavir protein adducts in patients. Chem Res Toxicol 2014, 27 (4), 524-35. 10. Uetrecht, J.; Naisbitt, D. J., Idiosyncratic adverse drug reactions: current concepts. Pharmacol Rev 2013, 65 (2), 779-808. 11. Ng, W.; Lobach, A. R.; Zhu, X.; Chen, X.; Liu, F.; Metushi, I. G.; Sharma, A.; Li, J.; Cai, P.; Ip, J.; Novalen, M.; Popovic, M.; Zhang, X.; Tanino, T.; Nakagawa, T.; Li, Y.; Uetrecht, J., Animal models of idiosyncratic drug reactions. Adv Pharmacol 2012, 63, 81-135. 12. Sayeh, E.; Uetrecht, J. P., Factors that modify penicillamine-induced autoimmunity in Brown Norway rats: failure of the Th1/Th2 paradigm. Toxicology 2001, 163 (2-3), 195-211. 13. Mak, A.; Uetrecht, J., The Combination of Anti-CTLA-4 and PD1-/- Mice Unmasks the Potential of Isoniazid and Nevirapine To Cause Liver Injury. Chem Res Toxicol 2015, 28 (12), 2287-91. 14. Metushi, I. G.; Hayes, M. A.; Uetrecht, J., Treatment of PD-1(-/-) mice with amodiaquine and anti-CTLA4 leads to liver injury similar to idiosyncratic liver injury in patients. Hepatology 2015, 61 (4), 1332-42. 15. Doss, G. A.; Baillie, T. A., Addressing metabolic activation as an integral component of drug design. Drug Metab Rev 2006, 38 (4), 641-9.

30 ACS Paragon Plus Environment

Page 31 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

16. Kalgutkar, A. S.; Dalvie, D., Predicting toxicities of reactive metabolite-positive drug candidates. Annu Rev Pharmacol Toxicol 2015, 55, 35-54. 17. Stepan, A. F.; Walker, D. P.; Bauman, J.; Price, D. A.; Baillie, T. A.; Kalgutkar, A. S.; Aleo, M. D., Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States. Chem Res Toxicol 2011, 24 (9), 1345410. 18. Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A., The resurgence of covalent drugs. Nat Rev Drug Discov 2011, 10 (4), 307-17. 19. Kalgutkar, A. S.; Dalvie, D. K., Drug discovery for a new generation of covalent drugs. Expert Opin Drug Discov 2012, 7 (7), 561-81. 20. Baillie, T. A., Targeted Covalent Inhibitors for Drug Design. Angew Chem Int Ed Engl 2016, 55 (43), 13408-13421. 21. Sawamura, R.; Okudaira, N.; Watanabe, K.; Murai, T.; Kobayashi, Y.; Tachibana, M.; Ohnuki, T.; Masuda, K.; Honma, H.; Kurihara, A.; Okazaki, O., Predictability of idiosyncratic drug toxicity risk for carboxylic acid-containing drugs based on the chemical stability of acyl glucuronide. Drug metabolism and disposition: the biological fate of chemicals 2010, 38 (10), 1857-64. 22. Boelsterli, U. A., Acyl glucuronides: mechanistic role in drug toxicity? Curr Drug Metab 2011, 12 (3), 213-4. 23. Jinno, N.; Ohashi, S.; Tagashira, M.; Kohira, T.; Yamada, S., A simple method to evaluate reactivity of acylglucuronides optimized for early stage drug discovery. Biol Pharm Bull 2013, 36 (9), 1509-13. 24. Iwamura, A.; Ito, M.; Mitsui, H.; Hasegawa, J.; Kosaka, K.; Kino, I.; Tsuda, M.; Nakajima, M.; Yokoi, T.; Kume, T., Toxicological evaluation of acyl glucuronides utilizing halflives, peptide adducts, and immunostimulation assays. Toxicol In Vitro 2015, 30 (1 Pt B), 241-9. 25. Skonberg, C.; Olsen, J.; Madsen, K. G.; Hansen, S. H.; Grillo, M. P., Metabolic activation of carboxylic acids. Expert Opin Drug Metab Toxicol 2008, 4 (4), 425-38. 26. Grillo, M. P., Drug-S-acyl-glutathione thioesters: synthesis, bioanalytical properties, chemical reactivity, biological formation and degradation. Curr Drug Metab 2011, 12 (3), 22944. 27. Darnell, M.; Breitholtz, K.; Isin, E. M.; Jurva, U.; Weidolf, L., Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S-Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes. Chem Res Toxicol 2015, 28 (5), 886-96. 28. Surh, Y. J., Bioactivation of benzylic and allylic alcohols via sulfo-conjugation. Chem Biol Interact 1998, 109 (1-3), 221-35. 29. Guengerich, F. P., N-hydroxyarylamines. Drug Metab Rev 2002, 34 (3), 607-23. 30. Hoie, A. H.; Monien, B. H.; Glatt, H.; Hjertholm, H.; Husoy, T., DNA adducts induced by food mutagen PhIP in a mouse model expressing human sulfotransferases 1A1 and 1A2. Toxicol Lett 2016, 248, 34-8. 31. Gan, J.; Ma, S.; Zhang, D., Non-cytochrome P450-mediated bioactivation and its toxicological relevance. Drug Metab Rev 2016, 48 (4), 473-501. 32. Dansette, P. M.; Levent, D.; Hessani, A.; Mansuy, D., Bioactivation of clopidogrel and prasugrel: factors determining the stereochemistry of the thiol metabolite double bond. Chem Res Toxicol 2015, 28 (6), 1338-45. 31 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 60

33. Deeks, E. D., Cobicistat: a review of its use as a pharmacokinetic enhancer of atazanavir and darunavir in patients with HIV-1 infection. Drugs 2014, 74 (2), 195-206. 34. Yin, L.; Hu, Q., CYP17 inhibitors--abiraterone, C17,20-lyase inhibitors and multitargeting agents. Nat Rev Urol 2014, 11 (1), 32-42. 35. Jarman, M.; Barrie, S. E.; Llera, J. M., The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien3beta-ol) and related steroidal inhibitors. J Med Chem 1998, 41 (27), 5375-81. 36. Andres, E.; Maloisel, F., Idiosyncratic drug-induced agranulocytosis or acute neutropenia. Curr Opin Hematol 2008, 15 (1), 15-21. 37. Lucido, M. J.; Orlando, B. J.; Vecchio, A. J.; Malkowski, M. G., Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry 2016, 55 (8), 1226-38. 38. Potashman, M. H.; Duggan, M. E., Covalent modifiers: an orthogonal approach to drug design. J Med Chem 2009, 52 (5), 1231-46. 39. Keith, J. M.; Jones, W. M.; Tichenor, M.; Liu, J.; Seierstad, M.; Palmer, J. A.; Webb, M.; Karbarz, M.; Scott, B. P.; Wilson, S. J.; Luo, L.; Wennerholm, M. L.; Chang, L.; Rizzolio, M.; Rynberg, R.; Chaplan, S. R.; Breitenbucher, J. G., Preclinical Characterization of the FAAH Inhibitor JNJ-42165279. ACS Med Chem Lett 2015, 6 (12), 1204-8. 40. Gayko, U.; Fung, M.; Clow, F.; Sun, S.; Faust, E.; Price, S.; James, D.; Doyle, M.; Bari, S.; Zhuang, S. H., Development of the Bruton's tyrosine kinase inhibitor ibrutinib for B cell malignancies. Ann N Y Acad Sci 2015, 1358, 82-94. 41. Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F., A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nat Chem Biol 2014, 10 (9), 760-7. 42. Cheng, H.; Nair, S. K.; Murray, B. W.; Almaden, C.; Bailey, S.; Baxi, S.; Behenna, D.; Cho-Schultz, S.; Dalvie, D.; Dinh, D. M.; Edwards, M. P.; Feng, J. L.; Ferre, R. A.; Gajiwala, K. S.; Hemkens, M. D.; Jackson-Fisher, A.; Jalaie, M.; Johnson, T. O.; Kania, R. S.; Kephart, S.; Lafontaine, J.; Lunney, B.; Liu, K. K.; Liu, Z.; Matthews, J.; Nagata, A.; Niessen, S.; Ornelas, M. A.; Orr, S. T.; Pairish, M.; Planken, S.; Ren, S.; Richter, D.; Ryan, K.; Sach, N.; Shen, H.; Smeal, T.; Solowiej, J.; Sutton, S.; Tran, K.; Tseng, E.; Vernier, W.; Walls, M.; Wang, S.; Weinrich, S. L.; Xin, S.; Xu, H.; Yin, M. J.; Zientek, M.; Zhou, R.; Kath, J. C., Discovery of 1{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyr imidin-4yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants. J Med Chem 2016, 59 (5), 2005-24. 43. Gerber, B. O.; Pichler, W. J., Noncovalent interactions of drugs with immune receptors may mediate drug-induced hypersensitivity reactions. AAPS J 2006, 8 (1), E160-5. 44. Illing, P. T.; Vivian, J. P.; Dudek, N. L.; Kostenko, L.; Chen, Z.; Bharadwaj, M.; Miles, J. J.; Kjer-Nielsen, L.; Gras, S.; Williamson, N. A.; Burrows, S. R.; Purcell, A. W.; Rossjohn, J.; McCluskey, J., Immune self-reactivity triggered by drug-modified HLA-peptide repertoire. Nature 2012, 486 (7404), 554-8. 45. Pichler, W. J.; Beeler, A.; Keller, M.; Lerch, M.; Posadas, S.; Schmid, D.; Spanou, Z.; Zawodniak, A.; Gerber, B., Pharmacological interaction of drugs with immune receptors: the p-i concept. Allergol Int 2006, 55 (1), 17-25.

32 ACS Paragon Plus Environment

Page 33 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

46. Pichler, W. J.; Naisbitt, D. J.; Park, B. K., Immune pathomechanism of drug hypersensitivity reactions. J Allergy Clin Immunol 2011, 127 (3 Suppl), S74-81. 47. Pirmohamed, M.; Naisbitt, D. J.; Gordon, F.; Park, B. K., The danger hypothesis-potential role in idiosyncratic drug reactions. Toxicology 2002, 181-182, 55-63. 48. Tailor, A.; Faulkner, L.; Naisbitt, D. J.; Park, B. K., The chemical, genetic and immunological basis of idiosyncratic drug-induced liver injury. Hum Exp Toxicol 2015, 34 (12), 1310-7. 49. Kalish, R. S., Antigen processing: the gateway to the immune response. J Am Acad Dermatol 1995, 32 (4), 640-52. 50. Neefjes, J.; Jongsma, M. L. M.; Paul, P.; Bakke, O., Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 2011, 11 (12), 823-836. 51. Lazaro, S.; Gamarra, D.; Del Val, M., Proteolytic enzymes involved in MHC class I antigen processing: A guerrilla army that partners with the proteasome. Mol Immunol 2015, 68 (2 Pt A), 72-6. 52. York, I. A.; Goldberg, A. L.; Mo, X. Y.; Rock, K. L., Proteolysis and class I major histocompatibility complex antigen presentation. Immunol Rev 1999, 172, 49-66. 53. Roche, P. A.; Furuta, K., The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 2015, 15 (4), 203-16. 54. Kharasch, E. D., Adverse Drug Reactions With Halogenated Anesthetics. Clinical Pharmacology & Therapeutics 2008, 84 (1), 158-162. 55. El-Ghaiesh, S.; Monshi, M. M.; Whitaker, P.; Jenkins, R.; Meng, X.; Farrell, J.; Elsheikh, A.; Peckham, D.; French, N.; Pirmohamed, M.; Park, B. K.; Naisbitt, D. J., Characterization of the antigen specificity of T-cell clones from piperacillin-hypersensitive patients with cystic fibrosis. J Pharmacol Exp Ther 2012, 341 (3), 597-610. 56. Monshi, M. M.; Faulkner, L.; Gibson, A.; Jenkins, R. E.; Farrell, J.; Earnshaw, C. J.; Alfirevic, A.; Cederbrant, K.; Daly, A. K.; French, N.; Pirmohamed, M.; Park, B. K.; Naisbitt, D. J., Human leukocyte antigen (HLA)-B*57:01-restricted activation of drug-specific T cells provides the immunological basis for flucloxacillin-induced liver injury. Hepatology 2013, 57 (2), 727-39. 57. Carey, M. A.; van Pelt, F. N., Immunochemical detection of flucloxacillin adduct formation in livers of treated rats. Toxicology 2005, 216 (1), 41-8. 58. Jenkins, R. E.; Meng, X.; Elliott, V. L.; Kitteringham, N. R.; Pirmohamed, M.; Park, B. K., Characterisation of flucloxacillin and 5-hydroxymethyl flucloxacillin haptenated HSA in vitro and in vivo. Proteomics. Clinical applications 2009, 3 (6), 720-9. 59. Bourdi, M.; Chen, W.; Peter, R. M.; Martin, J. L.; Buters, J. T.; Nelson, S. D.; Pohl, L. R., Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis. Chem Res Toxicol 1996, 9 (7), 1159-66. 60. Eliasson, E.; Kenna, J. G., Cytochrome P450 2E1 is a cell surface autoantigen in halothane hepatitis. Molecular pharmacology 1996, 50 (3), 573-82. 61. Neuberger, J.; Williams, R., Immune mechanisms in tienilic acid associated hepatotoxicity. Gut 1989, 30 (4), 515-519. 62. Lecoeur, S.; Andre, C.; Beaune, P. H., Tienilic acid-induced autoimmune hepatitis: antiliver and-kidney microsomal type 2 autoantibodies recognize a three-site conformational epitope on cytochrome P4502C9. Molecular pharmacology 1996, 50 (2), 326-33.

33 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 60

63. Sharma, A. M.; Novalen, M.; Tanino, T.; Uetrecht, J. P., 12-OH-nevirapine sulfate, formed in the skin, is responsible for nevirapine-induced skin rash. Chem Res Toxicol 2013, 26 (5), 817-27. 64. Mallal, S.; Nolan, D.; Witt, C.; Masel, G.; Martin, A. M.; Moore, C.; Sayer, D.; Castley, A.; Mamotte, C.; Maxwell, D.; James, I.; Christiansen, F. T., Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir. Lancet (London, England) 2002, 359 (9308), 727-32. 65. Mallal, S.; Phillips, E.; Carosi, G.; Molina, J. M.; Workman, C.; Tomazic, J.; JagelGuedes, E.; Rugina, S.; Kozyrev, O.; Cid, J. F.; Hay, P.; Nolan, D.; Hughes, S.; Hughes, A.; Ryan, S.; Fitch, N.; Thorborn, D.; Benbow, A., HLA-B*5701 screening for hypersensitivity to abacavir. The New England journal of medicine 2008, 358 (6), 568-79. 66. Martin, M. A.; Kroetz, D. L., Abacavir Pharmacogenetics – From Initial Reports to Standard of Care. Pharmacotherapy 2013, 33 (7), 765-775. 67. Chung, W. H.; Hung, S. I.; Hong, H. S.; Hsih, M. S.; Yang, L. C.; Ho, H. C.; Wu, J. Y.; Chen, Y. T., Medical genetics: a marker for Stevens-Johnson syndrome. Nature 2004, 428 (6982), 486. 68. Bell, C. C.; Santoyo Castelazo, A.; Yang, E. L.; Maggs, J. L.; Jenkins, R. E.; Tugwood, J.; O'Neill, P. M.; Naisbitt, D. J.; Park, B. K., Oxidative bioactivation of abacavir in subcellular fractions of human antigen presenting cells. Chem Res Toxicol 2013, 26 (7), 1064-72. 69. Charneira, C.; Grilo, N. M.; Pereira, S. A.; Godinho, A. L.; Monteiro, E. C.; Marques, M. M.; Antunes, A. M., N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo. British journal of pharmacology 2012, 167 (6), 1353-61. 70. Bu, H. Z.; Kang, P.; Deese, A. J.; Zhao, P.; Pool, W. F., Human in vitro glutathionyl and protein adducts of carbamazepine-10,11-epoxide, a stable and pharmacologically active metabolite of carbamazepine. Drug metabolism and disposition: the biological fate of chemicals 2005, 33 (12), 1920-4. 71. Lu, W.; Uetrecht, J. P., Peroxidase-mediated bioactivation of hydroxylated metabolites of carbamazepine and phenytoin. Drug metabolism and disposition: the biological fate of chemicals 2008, 36 (8), 1624-36. 72. Pirmohamed, M.; Kitteringham, N. R.; Guenthner, T. M.; Breckenridge, A. M.; Park, B. K., An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro. Biochemical pharmacology 1992, 43 (8), 1675-82. 73. Daly, A. K.; Aithal, G. P.; Leathart, J. B.; Swainsbury, R. A.; Dang, T. S.; Day, C. P., Genetic susceptibility to diclofenac-induced hepatotoxicity: contribution of UGT2B7, CYP2C8, and ABCC2 genotypes. Gastroenterology 2007, 132 (1), 272-81. 74. Huang, Y. S.; Su, W. J.; Huang, Y. H.; Chen, C. Y.; Chang, F. Y.; Lin, H. C.; Lee, S. D., Genetic polymorphisms of manganese superoxide dismutase, NAD(P)H:quinone oxidoreductase, glutathione S-transferase M1 and T1, and the susceptibility to drug-induced liver injury. J Hepatol 2007, 47 (1), 128-34. 75. Chalasani, N.; Bjornsson, E., Risk factors for idiosyncratic drug-induced liver injury. Gastroenterology 2010, 138 (7), 2246-59. 76. Bjornsson, E. S., Epidemiology and risk factors for idiosyncratic drug-induced liver injury. Seminars in liver disease 2014, 34 (2), 115-22. 77. Sison-Young, R. L.; Lauschke, V. M.; Johann, E.; Alexandre, E.; Antherieu, S.; Aerts, H.; Gerets, H. H.; Labbe, G.; Hoet, D.; Dorau, M.; Schofield, C. A.; Lovatt, C. A.; Holder, J. C.; 34 ACS Paragon Plus Environment

Page 35 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Stahl, S. H.; Richert, L.; Kitteringham, N. R.; Jones, R. P.; Elmasry, M.; Weaver, R. J.; Hewitt, P. G.; Ingelman-Sundberg, M.; Goldring, C. E.; Park, B. K., A multicenter assessment of singlecell models aligned to standard measures of cell health for prediction of acute hepatotoxicity. Arch Toxicol 2016. 78. Lauschke, V. M.; Hendriks, D. F.; Bell, C. C.; Andersson, T. B.; Ingelman-Sundberg, M., Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the Hepatotoxicity of Drugs and Drug Candidates. Chem Res Toxicol 2016. 79. Maes, M.; Vinken, M.; Jaeschke, H., Experimental models of hepatotoxicity related to acute liver failure. Toxicol Appl Pharmacol 2016, 290, 86-97. 80. Godoy, P.; Hewitt, N. J.; Albrecht, U.; Andersen, M. E.; Ansari, N.; Bhattacharya, S.; Bode, J. G.; Bolleyn, J.; Borner, C.; Böttger, J.; Braeuning, A.; Budinsky, R. A.; Burkhardt, B.; Cameron, N. R.; Camussi, G.; Cho, C.-S.; Choi, Y.-J.; Craig Rowlands, J.; Dahmen, U.; Damm, G.; Dirsch, O.; Donato, M. T.; Dong, J.; Dooley, S.; Drasdo, D.; Eakins, R.; Ferreira, K. S.; Fonsato, V.; Fraczek, J.; Gebhardt, R.; Gibson, A.; Glanemann, M.; Goldring, C. E. P.; GómezLechón, M. J.; Groothuis, G. M. M.; Gustavsson, L.; Guyot, C.; Hallifax, D.; Hammad, S.; Hayward, A.; Häussinger, D.; Hellerbrand, C.; Hewitt, P.; Hoehme, S.; Holzhütter, H.-G.; Houston, J. B.; Hrach, J.; Ito, K.; Jaeschke, H.; Keitel, V.; Kelm, J. M.; Kevin Park, B.; Kordes, C.; Kullak-Ublick, G. A.; LeCluyse, E. L.; Lu, P.; Luebke-Wheeler, J.; Lutz, A.; Maltman, D. J.; Matz-Soja, M.; McMullen, P.; Merfort, I.; Messner, S.; Meyer, C.; Mwinyi, J.; Naisbitt, D. J.; Nussler, A. K.; Olinga, P.; Pampaloni, F.; Pi, J.; Pluta, L.; Przyborski, S. A.; Ramachandran, A.; Rogiers, V.; Rowe, C.; Schelcher, C.; Schmich, K.; Schwarz, M.; Singh, B.; Stelzer, E. H. K.; Stieger, B.; Stöber, R.; Sugiyama, Y.; Tetta, C.; Thasler, W. E.; Vanhaecke, T.; Vinken, M.; Weiss, T. S.; Widera, A.; Woods, C. G.; Xu, J. J.; Yarborough, K. M.; Hengstler, J. G., Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Archives of Toxicology 2013, 87 (8), 1315-1530. 81. Rose, K. A.; Holman, N. S.; Green, A. M.; Andersen, M. E.; LeCluyse, E. L., Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity. Journal of pharmaceutical sciences 2016, 105 (2), 950-64. 82. Vernetti, L. A.; Senutovitch, N.; Boltz, R.; DeBiasio, R.; Shun, T. Y.; Gough, A.; Taylor, D. L., A human liver microphysiology platform for investigating physiology, drug safety, and disease models. Experimental biology and medicine (Maywood, N.J.) 2016, 241 (1), 101-14. 83. Shaw, P. J.; Ganey, P. E.; Roth, R. A., Idiosyncratic drug-induced liver injury and the role of inflammatory stress with an emphasis on an animal model of trovafloxacin hepatotoxicity. Toxicological sciences : an official journal of the Society of Toxicology 2010, 118 (1), 7-18. 84. Morgan, R. E.; Trauner, M.; van Staden, C. J.; Lee, P. H.; Ramachandran, B.; Eschenberg, M.; Afshari, C. A.; Qualls, C. W., Jr.; Lightfoot-Dunn, R.; Hamadeh, H. K., Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicological sciences : an official journal of the Society of Toxicology 2010, 118 (2), 485-500. 85. Morgan, R. E.; van Staden, C. J.; Chen, Y.; Kalyanaraman, N.; Kalanzi, J.; Dunn, R. T., 2nd; Afshari, C. A.; Hamadeh, H. K., A multifactorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development. Toxicological sciences : an official journal of the Society of Toxicology 2013, 136 (1), 216-41.

35 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 60

86. He, K.; Cai, L.; Shi, Q.; Liu, H.; Woolf, T. F., Inhibition of MDR3 Activity in Human Hepatocytes by Drugs Associated with Liver Injury. Chem Res Toxicol 2015, 28 (10), 1987-90. 87. Weston, J. K.; Uetrecht, J., Activation of inflammasomes by agents causing idiosyncratic skin reactions: a possible biomarker. Chem Res Toxicol 2014, 27 (6), 949-51. 88. Thompson, R. A.; Isin, E. M.; Li, Y.; Weaver, R.; Weidolf, L.; Wilson, I.; Claesson, A.; Page, K.; Dolgos, H.; Kenna, J. G., Risk assessment and mitigation strategies for reactive metabolites in drug discovery and development. Chem Biol Interact 2011, 192 (1-2), 65-71. 89. Martin, S. F.; Esser, P. R.; Schmucker, S.; Dietz, L.; Naisbitt, D. J.; Park, B. K.; Vocanson, M.; Nicolas, J. F.; Keller, M.; Pichler, W. J.; Peiser, M.; Luch, A.; Wanner, R.; Maggi, E.; Cavani, A.; Rustemeyer, T.; Richter, A.; Thierse, H. J.; Sallusto, F., T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays. Cell Mol Life Sci 2010, 67 (24), 4171-84. 90. Ogese, M. O.; Ahmed, S.; Alfirevic, A.; Betts, C. J.; Dickinson, A.; Faulkner, L.; French, N. S.; Gibson, A.; Hirschfield, G. M.; Kammuller, M. E.; Meng, X.; Martin, S. F.; Musette, P.; Norris, A.; Pirmohamed, M.; Park, B. K.; Purcell, A. W.; Spraggs, C. F.; Whritenour, J.; Naisbitt, D. J., New approaches to investigate drug-induced hypersensitivity. Chem Res Toxicol 2016. 91. Gibson, A.; Kim, S. H.; Faulkner, L.; Evely, J.; Pirmohamed, M.; Park, K. B.; Naisbitt, D. J., In Vitro Priming of Naive T-cells with p-Phenylenediamine and Bandrowski's Base. Chem Res Toxicol 2015, 28 (10), 2069-77. 92. Faulkner, L.; Martinsson, K.; Santoyo-Castelazo, A.; Cederbrant, K.; Schuppe-Koistinen, I.; Powell, H.; Tugwood, J.; Naisbitt, D. J.; Park, B. K., The development of in vitro culture methods to characterize primary T-cell responses to drugs. Toxicological sciences : an official journal of the Society of Toxicology 2012, 127 (1), 150-8. 93. Luyendyk, J. P.; Maddox, J. F.; Cosma, G. N.; Ganey, P. E.; Cockerell, G. L.; Roth, R. A., Ranitidine treatment during a modest inflammatory response precipitates idiosyncrasy-like liver injury in rats. J Pharmacol Exp Ther 2003, 307 (1), 9-16. 94. Roth, R. A.; Ganey, P. E., Animal models of idiosyncratic drug-induced liver injury-current status. Crit Rev Toxicol 2011, 41 (9), 723-39. 95. Shenton, J. M.; Popovic, M.; Chen, J.; Masson, M. J.; Uetrecht, J. P., Evidence of an immune-mediated mechanism for an idiosyncratic nevirapine-induced reaction in the female Brown Norway rat. Chem Res Toxicol 2005, 18 (12), 1799-813. 96. Shenton, J. M.; Teranishi, M.; Abu-Asab, M. S.; Yager, J. A.; Uetrecht, J. P., Characterization of a potential animal model of an idiosyncratic drug reaction: nevirapineinduced skin rash in the rat. Chem Res Toxicol 2003, 16 (9), 1078-89. 97. Harrill, A. H.; Ross, P. K.; Gatti, D. M.; Threadgill, D. W.; Rusyn, I., Population-based discovery of toxicogenomics biomarkers for hepatotoxicity using a laboratory strain diversity panel. Toxicological sciences : an official journal of the Society of Toxicology 2009, 110 (1), 235-43. 98. Harrill, A. H.; Watkins, P. B.; Su, S.; Ross, P. K.; Harbourt, D. E.; Stylianou, I. M.; Boorman, G. A.; Russo, M. W.; Sackler, R. S.; Harris, S. C.; Smith, P. C.; Tennant, R.; Bogue, M.; Paigen, K.; Harris, C.; Contractor, T.; Wiltshire, T.; Rusyn, I.; Threadgill, D. W., Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminopheninduced liver injury in humans. Genome Res 2009, 19 (9), 1507-15. 99. Zhou, S., Separation and detection methods for covalent drug-protein adducts. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2003, 797 (1-2), 63-90. 36 ACS Paragon Plus Environment

Page 37 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

100. Liebler, D. C., Protein Damage by Reactive Electrophiles: Targets and Consequences. Chem. Res. Toxicol. 2008, 21 (1), 117-128. 101. Satoh, H.; Fukuda, Y.; Anderson, D. K.; Ferrans, V. J.; Gillette, J. R.; Pohl, L. R., Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes. J. Pharmacol. Exp. Ther. 1985, 233 (3), 857-62. 102. Satoh, H.; Gillette, J. R.; Davies, H. W.; Schulick, R. D.; Pohl, L. R., Immunochemical evidence of trifluoroacetylated cytochrome P-450 in the liver of halothane-treated rats. Mol. Pharmacol. 1985, 28 (5), 468-74. 103. Satoh, H.; Martin, B. M.; Schulick, A. H.; Christ, D. D.; Kenna, J. G.; Pohl, L. R., Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase. Proc. Natl. Acad. Sci. U. S. A. 1989, 86 (1), 322-6. 104. Butler, L. E.; Thomassen, D.; Martin, J. L.; Martin, B. M.; Kenna, J. G.; Pohl, L. R., The calcium-binding protein calreticulin is covalently modified in rat liver by a reactive metabolite of the inhalation anesthetic halothane. Chem. Res. Toxicol. 1992, 5 (3), 406-10. 105. Hayden, P. J.; Ichimura, T.; McCann, D. J.; Pohl, L. R.; Stevens, J. L., Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts. J. Biol. Chem. 1991, 266 (28), 18415-18. 106. Pohl, L. R., An immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolites. Chem. Res. Toxicol. 1993, 6 (6), 786-93. 107. Bartolone, J. B.; Sparks, K.; Cohen, S. D.; Khairallah, E. A., Immunochemical detection of acetaminophen-bound liver proteins. Biochem. Pharmacol. 1987, 36 (8), 1193-6. 108. Bulera, S. J.; Birge, R. B.; Cohen, S. D.; Khairallah, E. A., Identification of the mouse liver 44-kDa acetaminophen-binding protein as a subunit of glutamine synthetase. Toxicol Appl Pharmacol 1995, 134 (2), 313-20. 109. Cohen, S. D.; Pumford, N. R.; Khairallah, E. A.; Boekelheide, K.; Pohl, L. R.; Amouzadeh, H. R.; Hinson, J. A., Selective protein covalent binding and target organ toxicity. Toxicol Appl Pharmacol 1997, 143 (1), 1-12. 110. Halmes, N. C.; Hinson, J. A.; Martin, B. M.; Pumford, N. R., Glutamate dehydrogenase covalently binds to a reactive metabolite of acetaminophen. Chem Res Toxicol 1996, 9 (2), 5416. 111. Hargus, S. J.; Amouzedeh, H. R.; Pumford, N. R.; Myers, T. G.; McCoy, S. C.; Pohl, L. R., Metabolic Activation and Immunochemical Localization of Liver Protein Adducts of the Nonsteroidal Anti-inflammatory Drug Diclofenac. Chem. Res. Toxicol. 1994, 7 (4), 575-82. 112. Hargus, S. J.; Martin, B. M.; George, J. W.; Pohl, L. R., Covalent Modification of Rat Liver Dipeptidyl Peptidase IV (CD26) by the Nonsteroidal Anti-Inflammatory Drug Diclofenac. Chem. Res. Toxicol. 1995, 8 (8), 993-6. 113. Hoivik, D. J.; Manautou, J. E.; Tveit, A.; Mankowski, D. C.; Khairallah, E. A.; Cohen, S. D., Evidence suggesting the 58-kDa acetaminophen binding protein is a preferential target for acetaminophen electrophile. Fundam Appl Toxicol 1996, 32 (1), 79-86. 114. Kleiner, H. E.; Jones, T. W.; Monks, T. J.; Lau, S. S., Immunochemical Analysis of Quinol-Thioether-Derived Covalent Protein Adducts in Rodent Species Sensitive and Resistant to Quinol-Thioether-Mediated Nephrotoxicity. Chem. Res. Toxicol. 1998, 11 (11), 1291-1300.

37 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 60

115. Kleiner, H. E.; Rivera, M. I.; Pumford, N. R.; Monks, T. J.; Lau, S. S., Immunochemical Detection of Quinol-Thioether-Derived Protein Adducts. Chem. Res. Toxicol. 1998, 11 (11), 1283-1290. 116. Kretz-Rommel, A.; Boelsterli, U. A., Selective protein adducts to membrane proteins in cultured rat hepatocytes exposed to diclofenac: radiochemical and immunochemical analysis. Mol. Pharmacol. 1994, 45 (2), 237-44. 117. Pumford, N. R.; Halmes, N. C.; Hinson, J. A., Covalent binding of xenobiotics to specific proteins in the liver. Drug Metab. Rev. 1997, 29 (1 & 2), 39-57. 118. Pumford, N. R.; Hinson, J. A.; Benson, R. W.; Roberts, D. W., Immunoblot analysis of protein containing 3-(cystein-S-yl)acetaminophen adducts in serum and subcellular liver fractions from acetaminophen-treated mice. Toxicol. Appl. Pharmacol. 1990, 104 (3), 521-32. 119. Wade, L. T.; Kenna, J. G.; Caldwell, J., Immunochemical Identification of Mouse Hepatic Protein Adducts Derived from the Nonsteroidal Anti-Inflammatory Drugs Diclofenac, Sulindac, and Ibuprofen. Chem. Res. Toxicol. 1997, 10 (5), 546-555. 120. Qiu, Y.; Benet, L. Z.; Burlingame, A. L., Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. J Biol Chem 1998, 273 (28), 17940-53. 121. Katayama, H.; Nagasu, T.; Oda, Y., Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2001, 15 (16), 1416-1421. 122. Soskic, V.; Godovac-Zimmermann, J., Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents. Proteomics 2001, 1 (11), 1364-1367. 123. Chambers, G.; Lawrie, L.; Cash, P.; Murray, G. I., Proteomics: a new approach to the study of disease. J Pathol 2000, 192 (3), 280-8. 124. Isbell, M. A.; Morin, D.; Boland, B.; Buckpitt, A.; Salemi, M.; Presley, J., Identification of proteins adducted by reactive naphthalene metabolites in vitro. Proteomics 2005, 5 (16), 41974204. 125. Lame, M. W.; Jones, A. D.; Wilson, D. W.; Segall, H. J., Protein targets of 1,4benzoquinone and 1,4-naphthoquinone in human bronchial epithelial cells. Proteomics 2003, 3 (4), 479-95. 126. Lin, C. Y.; Boland, B. C.; Lee, Y. J.; Salemi, M. R.; Morin, D.; Miller, L. A.; Plopper, C. G.; Buckpitt, A. R., Identification of proteins adducted by reactive metabolites of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques. Proteomics 2006, 6 (3), 972982. 127. Lin, C. Y.; Isbell, M. A.; Morin, D.; Boland, B. C.; Salemi, M. R.; Jewell, W. T.; Weir, A. J.; Fanucchi, M. V.; Baker, G. L.; Plopper, C. G.; Buckpitt, A. R., Characterization of a Structurally Intact in Situ Lung Model and Comparison of Naphthalene Protein Adducts Generated in This Model vs. Lung Microsomes. Chem. Res. Toxicol. 2005, 18 (5), 802-813. 128. Lame, M. W.; Jones, A. D.; Wilson, D. W.; Dunston, S. K.; Segall, H. J., Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells. J. Biol. Chem. 2000, 275 (37), 29091-29099. 129. Lame, M. W.; Jones, A. D.; Wilson, D. W.; Segall, H. J., Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells. Proteomics 2005, 5 (17), 4398-4413.

38 ACS Paragon Plus Environment

Page 39 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

130. Koen, Y. M.; Gogichaeva, N. V.; Alterman, M. A.; Hanzlik, R. P., A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo. Chem. Res. Toxicol. 2007, 20 (3), 511-519. 131. Koen, Y. M.; Hanzlik, R. P., Identification of Seven Proteins in the Endoplasmic Reticulum as Targets for Reactive Metabolites of Bromobenzene. Chem. Res. Toxicol. 2002, 15 (5), 699-706. 132. Koen, Y. M.; Sarma, D.; Williams, T. D.; Galeva, N. A.; Obach, R. S.; Hanzlik, R. P., Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes. Chem. Res. Toxicol. 2012, 25 (5), 1145-1154. 133. Koen, Y. M.; Sarma, D.; Hajovsky, H.; Galeva, N. A.; Williams, T. D.; Staudinger, J. L.; Hanzlik, R. P., Protein Targets of Thioacetamide Metabolites in Rat Hepatocytes. Chem. Res. Toxicol. 2013, 26 (4), 564-574. 134. Methogo, R. M.; Dansette, P. M.; Klarskov, K., Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach. Int. J. Mass Spectrom. 2007, 268 (2-3), 284-295. 135. Carbone, D. L.; Doorn, J. A.; Kiebler, Z.; Ickes, B. R.; Petersen, D. R., Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease. J. Pharmacol. Exp. Ther. 2005, 315 (1), 8-15. 136. Carbone, D. L.; Doorn, J. A.; Kiebler, Z.; Sampey, B. P.; Petersen, D. R., Inhibition of Hsp72-Mediated Protein Refolding by 4-Hydroxy-2-nonenal. Chem. Res. Toxicol. 2004, 17 (11), 1459-1467. 137. Kapphahn, R. J.; Giwa, B. M.; Berg, K. M.; Roehrich, H.; Feng, X.; Olsen, T. W.; Ferrington, D. A., Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets. Exp. Eye Res. 2006, 83 (1), 165-175. 138. Meier, B. W.; Gomez, J. D.; Kirichenko, O. V.; Thompson, J. A., Mechanistic Basis for Inflammation and Tumor Promotion in Lungs of 2,6-Di-tert-butyl-4-methylphenol-Treated Mice: Electrophilic Metabolites Alkylate and Inactivate Antioxidant Enzymes. Chem. Res. Toxicol. 2007, 20 (2), 199-207. 139. Meier, B. W.; Gomez, J. D.; Zhou, A.; Thompson, J. A., Immunochemical and Proteomic Analysis of Covalent Adducts Formed by Quinone Methide Tumor Promoters in Mouse Lung Epithelial Cell Lines. Chem. Res. Toxicol. 2005, 18 (10), 1575-1585. 140. Khojasteh, S. C.; Hartley, D. P.; Ford, K. A.; Uppal, H.; Oishi, S.; Nelson, S. D., Characterization of rat liver proteins adducted by reactive metabolites of menthofuran. Chem. Res. Toxicol. 2012, 25 (11), 2301-2309. 141. McDonald, W. H.; Yates, J. R., III, Shotgun proteomics: integrating technologies to answer biological questions. Curr. Opin. Mol. Ther. 2003, 5 (3), 302-309. 142. Wu, C. C.; MacCoss, M. J., Shotgun proteomics: Tools for the analysis of complex biological systems. Curr. Opin. Mol. Ther. 2002, 4 (3), 242-250. 143. Stamper, B. D.; Mohar, I.; Kavanagh, T. J.; Nelson, S. D., Proteomic analysis of acetaminophen-induced changes in mitochondrial protein expression using spectral counting. Chem Res Toxicol 2011, 24 (4), 549-58. 144. Yang, Y.; Xiao, Q.; Humphreys, W. G.; Dongre, A.; Shu, Y.-Z., Identification of Human Liver Microsomal Proteins Adducted by a Reactive Metabolite Using Shotgun Proteomics. Chem. Res. Toxicol. 2014, 27 (9), 1537-1546. 145. Golizeh, M.; LeBlanc, A.; Sleno, L., Identification of Acetaminophen Adducts of Rat Liver Microsomal Proteins using 2D-LC-MS/MS. Chem Res Toxicol 2015, 28 (11), 2142-50. 39 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 60

146. Bruderer, R.; Bernhardt, O. M.; Gandhi, T.; Miladinovic, S. M.; Cheng, L. Y.; Messner, S.; Ehrenberger, T.; Zanotelli, V.; Butscheid, Y.; Escher, C.; Vitek, O.; Rinner, O.; Reiter, L., Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Mol Cell Proteomics 2015, 14 (5), 1400-10. 147. Tzouros, M.; Pahler, A., A Targeted Proteomics Approach to the Identification of Peptides Modified by Reactive Metabolites. Chem. Res. Toxicol. 2009, 22 (5), 853-862. 148. Dennehy, M. K.; Richards, K. A. M.; Wernke, G. R.; Shyr, Y.; Liebler, D. C., Cytosolic and Nuclear Protein Targets of Thiol-Reactive Electrophiles. Chem. Res. Toxicol. 2006, 19 (1), 20-29. 149. Liu, J.; Li, Q.; Yang, X.; Van Breemen, R. B.; Bolton, J. L.; Thatcher, G. R. J., Analysis of Protein Covalent Modification by Xenobiotics Using a Covert Oxidatively Activated Tag: Raloxifene Proof-of-Principle Study. Chem. Res. Toxicol. 2005, 18 (9), 1485-1496. 150. Marcone, S.; Fitzgerald, D. J., Proteomic identification of the candidate target proteins of 15-deoxy-delta12,14-prostaglandin J2S. Proteomics 2013, 13 (14), 2135-2139. 151. Vila, A.; Tallman, K. A.; Jacobs, A. T.; Liebler, D. C.; Porter, N. A.; Marnett, L. J., Identification of Protein Targets of 4-Hydroxynonenal Using Click Chemistry for ex Vivo Biotinylation of Azido and Alkynyl Derivatives. Chem. Res. Toxicol. 2008, 21 (2), 432-444. 152. Codreanu, S. G.; Liebler, D. C., Novel approaches to identify protein adducts produced by lipid peroxidation. Free Radic Res 2015, 49 (7), 881-7. 153. Federspiel, J. D.; Codreanu, S. G.; Goyal, S.; Albertolle, M. E.; Lowe, E.; Teague, J.; Wong, H.; Guengerich, F. P.; Liebler, D. C., Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells. Mol Cell Proteomics 2016, 15 (10), 3233-3242. 154. Tzeng, S.-C.; Maier, C. S., Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE). Front Chem 2016, 4, 2. 155. Zhu, M.; Zhang, H.; Humphreys, W. G., Drug Metabolite Profiling and Identification by High-resolution Mass Spectrometry. J. Biol. Chem. 2011, 286 (29), 25419-25425. 156. Zhang, H.; Gan, J.; Shu, Y.-Z.; Humphreys, W. G., High-Resolution Mass SpectrometryBased Background Subtraction for Identifying Protein Modifications in a Complex Biological System: Detection of Acetaminophen-Bound Microsomal Proteins Including Argininosuccinate Synthetase. Chem. Res. Toxicol. 2015, 28 (4), 775-781. 157. Chapman, J. D.; Busnel, J.-M., Identifying previously undetected proteins: capillary electrophoresis, combined with electrospray ionization, can help researchers identify new proteins. Drug Discovery Dev. 2011, 14 (7), 18-20. 158. Davern, T. J., II; James, L. P.; Hinson, J. A.; Polson, J.; Larson, A. M.; Fontana, R. J.; Lalani, E.; Munoz, S.; Shakil, A. O.; Lee, W. M., Measurement of serum acetaminophen-protein adducts in patients with acute liver failure. Gastroenterology 2006, 130 (3), 687-694. 159. James, L. P.; Alonso, E. M.; Hynan, L. S.; Hinson, J. A.; Davern, T. J.; Lee, W. M.; Squires, R. H., Detection of acetaminophen protein adducts in children with acute liver failure of indeterminate cause. Pediatrics 2006, 118 (3), e676-81. 160. James, L. P.; Farrar, H. C.; Sullivan, J. E.; Givens, T. G.; Kearns, G. L.; Wasserman, G. S.; Walson, P. D.; Hinson, J. A.; Pumford, N. R., Measurement of acetaminophen-protein adducts in children and adolescents with acetaminophen overdoses. J. Clin. Pharmacol. 2001, 41 (8), 846-851. 40 ACS Paragon Plus Environment

Page 41 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

161. Muldrew, K. L.; James, L. P.; Coop, L.; McCullough, S. S.; Hendrickson, H. P.; Hinson, J. A.; Mayeux, P. R., Determination of acetaminophen-protein adducts in mouse liver and serum and human serum after hepatotoxic doses of acetaminophen by using high-performance liquid chromatography with electrochemical detection. Drug Metab. Dispos. 2002, 30 (4), 446-451. 162. Bond, G. R., Acetaminophen protein adducts: A review. Clin. Toxicol. 2009, 47 (1), 2-7. 163. Cook, S. F.; King, A. D.; Chang, Y.; Murray, G. J.; Norris, H.-R. K.; Dart, R. C.; Green, J. L.; Curry, S. C.; Rollins, D. E.; Wilkins, D. G., Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatographyelectrospray ionization-tandem mass spectrometry: Clinical and animal model applications. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2015, 985, 131-141. 164. James, L. P.; Letzig, L.; Simpson, P. M.; Capparelli, E.; Roberts, D. W.; Hinson, J. A.; Davern, T. J.; Lee, W. M., Pharmacokinetics of acetaminophen-protein adducts in adults with acetaminophen overdose and acute liver failure. Drug Metab. Dispos. 2009, 37 (8), 1779-1784. 165. Xie, Y.; McGill, M. R.; Cook, S. F.; Sharpe, M. R.; Winefield, R. D.; Wilkins, D. G.; Rollins, D. E.; Jaeschke, H., Time course of acetaminophen-protein adducts and acetaminophen metabolites in circulation of overdose patients and in HepaRG cells. Xenobiotica 2015, 45 (10), 921-929. 166. Aithal, G. P.; Ramsay, L.; Daly, A. K.; Sonchit, N.; Leathart, J. B. S.; Alexander, G.; Kenna, J. G.; Caldwell, J.; Day, C. P., Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity. Hepatology (Hoboken, NJ, U. S.) 2004, 39 (5), 1430-1440. 167. Bougie, D.; Johnson, S. T.; Weitekamp, L. A.; Aster, R. H., Sensitivity to a metabolite of diclofenac as a cause of acute immune hemolytic anemia. Blood 1997, 90 (1), 407-413. 168. Meng, X.; Howarth, A.; Earnshaw, C. J.; Jenkins, R. E.; French, N. S.; Back, D. J.; Naisbitt, D. J.; Park, B. K., Detection of Drug Bioactivation in Vivo: Mechanism of NevirapineAlbumin Conjugate Formation in Patients. Chem. Res. Toxicol. 2013, 26 (4), 575-583. 169. Hammond, T. G.; Meng, X.; Jenkins, R. E.; Maggs, J. L.; Castelazo, A. S.; Regan, S. L.; Bennett, S. N. L.; Earnshaw, C. J.; Aithal, G. P.; Pande, I.; Kenna, J. G.; Stachulski, A. V.; Park, B. K.; Williams, D. P., Mass spectrometric characterization of circulating covalent protein adducts derived from a drug acyl glucuronide metabolite: multiple albumin adductions in diclofenac patients. J. Pharmacol. Exp. Ther. 2014, 350 (2), 387-402, 16 pp. 170. Meng, X.; Maggs, J. L.; Usui, T.; Whitaker, P.; French, N. S.; Naisbitt, D. J.; Park, B. K., Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin. Chem Res Toxicol 2015, 28 (1), 51-8. 171. Meng, X.; Howarth, A.; Earnshaw, C. J.; Jenkins, R. E.; French, N. S.; Back, D. J.; Naisbitt, D. J.; Park, B. K., Detection of drug bioactivation in vivo: mechanism of nevirapinealbumin conjugate formation in patients. Chem Res Toxicol 2013, 26 (4), 575-83. 172. Jenkins, R. E.; Yaseen, F. S.; Monshi, M. M.; Whitaker, P.; Meng, X.; Farrell, J.; Hamlett, J.; Sanderson, J. P.; El-Ghaiesh, S.; Peckham, D.; Pirmohamed, M.; Park, B. K.; Naisbitt, D. J., beta-Lactam antibiotics form distinct haptenic structures on albumin and activate drug-specific T-lymphocyte responses in multiallergic patients with cystic fibrosis. Chem Res Toxicol 2013, 26 (6), 963-75. 173. Whitaker, P.; Meng, X.; Lavergne, S. N.; El-Ghaiesh, S.; Monshi, M.; Earnshaw, C.; Peckham, D.; Gooi, J.; Conway, S.; Pirmohamed, M.; Jenkins, R. E.; Naisbitt, D. J.; Park, B. K., Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis. J Immunol 2011, 187 (1), 200-11. 41 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 60

174. Meng, X.; Jenkins, R. E.; Berry, N. G.; Maggs, J. L.; Farrell, J.; Lane, C. S.; Stachulski, A. V.; French, N. S.; Naisbitt, D. J.; Pirmohamed, M.; Park, B. K., Direct evidence for the formation of diastereoisomeric benzylpenicilloyl haptens from benzylpenicillin and benzylpenicillenic acid in patients. J Pharmacol Exp Ther 2011, 338 (3), 841-9. 175. Birge, R. B.; Bartolone, J. B.; Tyson, C. A.; Emeigh Hart, S. G.; Cohen, S. D.; Khairallah, E. A., Selective binding of acetaminophen (APAP) to liver proteins in mice and men. Adv Exp Med Biol 1991, 283, 685-8. 176. McGill, M. R.; Williams, C. D.; Xie, Y.; Ramachandran, A.; Jaeschke, H., Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity. Toxicol Appl Pharmacol 2012, 264 (3), 387-94. 177. Yonamine, M.; Aniya, Y.; Yokomakura, T.; Koyama, T.; Nagamine, T.; Nakanishi, H., Acetaminophen-derived activation of liver microsomal glutathione S-transferase of rats. Jpn J Pharmacol 1996, 72 (2), 175-81. 178. Pierce, E. N.; Piyankarage, S. C.; Dunlap, T.; Litosh, V.; Siklos, M. I.; Wang, Y. T.; Thatcher, G. R., Prodrugs Bioactivated to Quinones Target NF-kappaB and Multiple Protein Networks: Identification of the Quinonome. Chem Res Toxicol 2016, 29 (7), 1151-9. 179. Meng, X.; Earnshaw, C. J.; Tailor, A.; Jenkins, R. E.; Waddington, J. C.; Whitaker, P.; French, N. S.; Naisbitt, D. J.; Park, B. K., Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients. Chem Res Toxicol 2016, 29 (10), 1762-1772. 180. Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.; Callegari, E.; Henne, K. R.; Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.; Nakai, Y.; O'Donnell, J. P.; Boer, J.; Harriman, S. P., A comprehensive listing of bioactivation pathways of organic functional groups. Curr Drug Metab 2005, 6 (3), 161-225. 181. Uetrecht, J.; Kaplowitz, N., Inhibition of immune tolerance unmasks drug-induced allergic hepatitis. Hepatology 2015, 62 (2), 346-8. 182. Balbo, S.; Turesky, R. J.; Villalta, P. W., DNA adductomics. Chem Res Toxicol 2014, 27 (3), 356-66. 183. Rappaport, S. M.; Li, H.; Grigoryan, H.; Funk, W. E.; Williams, E. R., Adductomics: characterizing exposures to reactive electrophiles. Toxicol Lett 2012, 213 (1), 83-90.

42 ACS Paragon Plus Environment

Page 43 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

AUTHOR BIOGRAPHIES Dr. Jinping Gan is a Senior Principal Scientist in the Biotransformation group with the department of Pharmaceutical Candidate Optimization at Bristol-Myers Squibb Company. He has a long standing interest in the biochemical basis for tissue and species-specific pharmacological and toxicological effects of xenobiotics.

Dr. Haiying Zhang is a Principal Scientist in the Biotransformation group with the department of Pharmaceutical Candidate Optimization at Bristol-Myers Squibb Company. His research interests include the development of HRMS-based technologies, such as the mass defect filter, to drug metabolite identification and the application of biotransformation knowledge to the lead optimization process.

Dr. Griff Humphreys is a Senior Research Fellow in the Biotransformation group with the department of Pharmaceutical Candidate Optimization at Bristol-Myers Squibb Company. His interests include the consequences of reactive metabolite formation, development of new analytical methodologies, reaction phenotyping of CYP and UGT catalyzed biotransformations, predictive metabolism and toxicology models, in vitro-in vivo correlations, and strategies for candidate optimization. He has served in various roles within the ACS-Division of Chemical Toxicology and ISSX as well as serving on the Editorial Boards for Chemical Research Toxicology and Drug Metabolism and Disposition.

43 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 60

FIGURE LEGENDS

Figure 1. Examples of drugs bioactivated through the formation of quinoid intermediates. Figure 2. Examples of drugs or xenobiotics bioactivated through epoxide intermediates. Arrows denote site of epoxidation. Figure 3. Examples of drugs bioactivated through the formation of acyl glucuronides Figure 4. Mechanism of clopidogrel bioactivation. Figure 5. Examples of drugs that bind select target proteins that are involved in their bioactivation. Site of bioactivation of abiraterone circled, site of bioactivation of cobicistat not completely defined. Figure 6. Examples of drugs with inherent reactivity, β-lactams or lactones. Site of reactivity circled. Figure 7. Examples of drugs with inherent reactivity that covalently modify select target proteins. Site of reactivity circled. Figure 8. Examples of drugs with inherent reactivity designed to covalently modify active sight residues of select target proteins. Site of reactivity circled. Figure 9. Schematic diagram of a simplified model for immune mediated drug reaction through reactive metabolite formation and covalent protein binding.

Protein adducts are processed

through either endogenous or exogenous pathway and presented to the surface of APC, then recognized by T cells through the TCR binding of MHC molecules. Not shown are alternative pathways of immune system activation including the p-i concept and altered peptide pool by direct MHC binding. Figure 10. Autoradiogram illustrating acetaminophen-protein targets detected on a twodimensional preparative gel. Protein sample was from a homogenate of whole liver from a phenobarbital-induced B6C3F1 mouse after treatment with a toxic dose of 14C-labeled acetaminophen. Two milligram of total protein was loaded. Protein samples were focused (x axis, cathode on the right) and then separated by SDS-PAGE (y axis, dye front at the bottom). Reprinted with permission from Prof. A.L. Burlingame, Department of Pharmaceutical Chemistry, University of California San Francisco.120

44 ACS Paragon Plus Environment

Page 45 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 11. Schematic representation of affinity capture strategy for protein adduct analysis. Reproduced from Dennehy, M. K., Richards, K. A. M., Wernke, G. R., Shyr, Y., Liebler, D. C. Cytosolic and Nuclear Protein Targets of Thiol-Reactive Electrophiles. Chem. Res. Toxicol. 19, 20-29.148 Copyright 2006 American Chemical Society.

Figure 12. Examples of click chemistry based probes used to characterize the protein targets of drug or xenobiotics containing inherently reactive functional groups.

Figure 13. Examples of click chemistry based probes used to characterize the protein targets of drug or xenobiotics containing inherently reactive functional groups. Scheme presented for characterization of lipid oxidation products.

Figure 14. Illustration of high-resolution mass spectrometry-based background subtraction approach for detecting acetaminophen-bound peptides in peptide mixtures of a shotgun proteomics study.156

45 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 60

Figure 1. Examples of drugs bioactivated through the formation of quinoid intermediates. OH

O

X = O, NR, CR2 XH

XH

O

X

O

HN

N

OH

O

O

Acetaminophen

Cl

Cl

Cl

OH N

OH

H N

HO

Cl

O

Cl

O

Cl

Cl

Diclofenac

H N Cl

N(Et)2 HO

O OH

Cl

OH N

OH

Cl

O

N(Et)2 O

NH

Cl

OH

H N

O

O

N

N

Cl

N

Amodiaquine

46 ACS Paragon Plus Environment

Page 47 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 2. Examples of drugs and toxicants or xenobiotics bioactivated through epoxide intermediates. Arrows denote site of epoxidation. Cl H3CO

Cl

O

N

N

R

O

H 2N

NH2

Carbamazepine O

Precocene

N

N

NH2

Lamotrigine OH

O

O

O

S N H

N

S

O O

Aflatoxin B

O

O

OCH3

Sudoxicam

47 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 60

Figure 3. Examples of drugs bioactivated through the formation of acyl glucuronides. Direct reaction with nucleophiles O

O

OH

OGluc

Amadori rearrangement, then reaction with nucleophiles O

O

N

N

O

O

Cl Zomepirac

OH

OH

Tolmentin

48 ACS Paragon Plus Environment

Page 49 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 4. Mechanism of clopidogrel bioactivation.

CYP

N S

COOCH3

COOCH3

COOCH3

CYP

N S

Cl

N O

O

S

Cl

Cl

O

Clopidogrel

HOOC

HOOC

HOOC HS

N

N

N Cl SH

P2Y12 Platelet

COOCH3

COOCH3

COOCH3

RSS

HOS

Cl

Cl

Transit from hepatocyte X SSR P2Y12 to bloodstream Platelet

R = activated clopidogrel

49 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 60

Figure 5. Examples of drugs that bind select target proteins that are involved in their bioactivation. Site of bioactivation of cobicistat not completely defined, site of bioactivation of abiraterone circled. O N N O N S

N

N H

H N O

O N H

O

H

S H N

Cobicistat

H

HO

Abiraterone

50 ACS Paragon Plus Environment

Page 51 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 6. Examples of drugs with inherent reactivity, β-lactams or lactones. Site of reactivity circled. NH2 H N HO

O

O

S N

N CO2H

O

OH nC6H13

O

O

Amoxicillin

O

H

H

C10H21 O O

H O

CO2H

Clavulanic acid

H N

Orlistat

51 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 60

Figure 7. Examples of drugs with inherent reactivity that covalently modify select target proteins. Site of reactivity circled. H

H

O

NH2

H N H N

H N

O

N O

HO

O

N

N H

O

CN

O

Boceprevir

Vildagliptin

O

O

O

N H

O

H N O

N H

H N

N O

O

Ph Ph

Carflizomib

52 ACS Paragon Plus Environment

Page 53 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 8. Examples of drugs with inherent reactivity designed to covalently modify active sight residues of select target proteins. Site of reactivity circled. Pharmacological target for these inhibitors is list in parentheses.

F O Cl NH N N

H 2N

N

O

N

N N

O

N O

N

Afatinib (HER-2, EGFR)

Ibrutinib (BTK)

N

H N

O N Cl NH NC N

O H N O

Neratinib (HER-2, EGFR)

N

N

N O

NH

N

N H

N

O

Osimertinib (EGFR)

53 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 60

Figure 9. Schematic diagram of a simplified model for immune mediated drug reaction through reactive metabolite formation and covalent protein binding.

Protein adducts are processed

through either endogenous or exogenous pathway and presented to the surface of APC, then recognized by T cells through the TCR binding of MHC molecules. Not shown are alternative pathways of immune system activation including the p-i concept and altered peptide pool by direct MHC binding.

54 ACS Paragon Plus Environment

Page 55 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 10. Autoradiogram illustrating acetaminophen-protein targets detected on a twodimensional preparative gel. Protein sample was from a homogenate of whole liver from a phenobarbital-induced B6C3F1 mouse after treatment with a toxic dose of 14C-labeled acetaminophen. Two milligram of total protein was loaded. Protein samples were focused (x axis, cathode on the right) and then separated by SDS-PAGE (y axis, dye front at the bottom). Reprinted with permission from Prof. A.L. Burlingame, Department of Pharmaceutical Chemistry, University of California San Francisco.120

55 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 60

Figure 11. Schematic representation of affinity capture strategy for protein adduct analysis. Reproduced from Dennehy, M. K., Richards, K. A. M., Wernke, G. R., Shyr, Y., Liebler, D. C. Cytosolic and Nuclear Protein Targets of Thiol-Reactive Electrophiles. Chem. Res. Toxicol. 19, 20-29.148 Copyright 2006 American Chemical Society.

56 ACS Paragon Plus Environment

Page 57 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 12. Examples of click chemistry based probes used to characterize the protein targets of drug or xenobiotics containing inherently reactive functional groups.

O OH

O

4-hydroxy nonenal derivative NH2 O

O

O

N H

N

O

H N O

H N

N H

N

N O

N N O

O

N

Ph

Carflizomib derivative - "OP829"

Ibrutinib derivative

57 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 58 of 60

Figure 13. Examples of click chemistry based probes used to characterize the protein targets of drug or xenobiotics containing inherently reactive functional groups. Scheme presented for characterization of lipid oxidation products.

+

HS

S

+

N3

Biotin

O

O

OH

OH

click chemistry 1. biotin capture 2. protein/peptide characterization

Biotin

S

N N N

O OH

58 ACS Paragon Plus Environment

Page 59 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Figure 14. Illustration of high-resolution mass spectrometry-based background subtraction approach for detecting acetaminophen-bound peptides in peptide mixtures of a shotgun proteomics study.156

59 ACS Paragon Plus Environment

Chemical Research in Toxicology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

+ Xenobiotic

Technologies in Development

Biological Response Direct and Indirect

Protein Adducts

Analytical techniques to characterize modified proteins

Page 60 of 60

+

Methodology to assess: 1) modified protein function and 2) innate/acquired immune response

ACS Paragon Plus Environment

Toxicity

Translational models to predict protein adduct mediated toxicity