Enolate-Forming Compounds as a Novel Approach to Cytoprotection

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ENOLATE-FORMING COMPOUNDS AS A NOVEL APPROACH TO CYTOPROTECTION Richard Michael LoPachin, Brian C. Geohagen, Lars Ulrik Nordstrøm, and Terrence Gavin Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00300 • Publication Date (Web): 17 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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Chemical Research in Toxicology

ENOLATE-FORMING COMPOUNDS AS A NOVEL APPROACH TO CYTOPROTECTION Richard M. LoPachin1*, Brian C. Geohagen1, Lars Ulrik Nordstrøm2 and Terrence Gavin3 1

Department of Anesthesiology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2

Chemical Synthesis & Biology Core Facility, Albert Einstein College of Medicine, Bronx, NY 10461, USA 3

Department of Chemistry, Iona College, New Rochelle, NY 10801, USA

Running Title: Enolate-Forming Compounds are Cytoprotective. Key Words: phytopolyphenols, curcumin, phloretin, oxidative stress, acrolein, electrophile scavenging, metal ion chelation *Corresponding Author: Richard M. LoPachin, Ph.D. Department of Anesthesiology Montefiore Medical School 111 E. 210th St. Bronx, NY 10467 Fax: (718) 515-4903 Office: (718) 920-5054 E-mail: [email protected] 1

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TABLE OF CONTENTS GRAPHIC

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Abstract Evidence from laboratory studies and clinical trials suggest that plant-derived polyphenolic compounds such as curcumin, resveratrol or phloretin might be useful in the treatment of certain diseases (e.g., Alzheimer’s disease) and acute tissue injury states (e.g., spinal cord trauma). However, despite this potential, the corresponding chemical instability, toxic potential and low bioavailability of these compounds could limit their ultimate clinical relevance. We have shown that pharmacophores of curcumin (e.g., 2-acetylcyclopentanone; 2-ACP) and phloretin (e.g., 2’,4’,6’-trihydroxyacetophenone; THA) can provide cytoprotection in cell culture and animal models of oxidative stress injury. These pharmacophores are 1,3-dicarbonyl and polyphenol derivatives, the enol groups of which can ionize in biological solutions to form an enolate. This carbanionic moiety can chelate metal ions and, as a nucleophile, can scavenge toxic electrophiles (e.g., acrolein, 4-hydroxy-2-nonenal and N-acetyl-p-benzoquinone imine) involved in many pathogenic conditions. Aromatic derivatives such as THA can also trap free oxygen and nitrogen radicals and thereby provide another layer of cytoprotection.

The

multifunctional character of these enolate-forming compounds suggests an ability to block pathogenic processes (e.g., oxidative stress) at several steps. The purpose of this review is to discuss research supporting our theory that enolate-formation is a significant cytoprotective property that represents a platform for development of pharmacotherapeutic approaches to a variety of toxic and pathogenic conditions.

Our discussion will focus on mechanism and

structure-activity studies that defined enolate chemistry and corresponding relationship to cytoprotection.

Introduction 3



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Clinical trials supported by results from epidemiological and experimental laboratory studies have suggested that the severity and outcome of many subchronic conditions (e.g., Alzheimer’s disease, atherosclerosis) or acute tissue injury states (e.g., traumatic spinal cord injury, acetaminophen overdose) can be significantly improved by administration of plantderived polyphenolic compounds such as curcumin, resveratrol or phloretin.1,2,3 Substantial evidence implicates cellular oxidative stress as a common pathogenic process underlying these conditions.4-8 Accordingly, the ability of curcumin and other phytopolyphenols to behave as aromatic antioxidants and trap free radicals has been assumed to be an important cytoprotective attribute. Curcumin can also reduce the impact of oxidative stress through chelation of divalent metal ions9 and by scavenging unsaturated aldehydes.10,11 Although the molecular cytoprotective mechanism is unknown, additional evidence suggests that curcumin can activate protective cellular stress responses (e.g., Keap1/Nrf2 antioxidant pathway1) and can modify the toxicity of pathognomonic proteins involved in neurodegenerative diseases (e.g., amyloid- peptides12). Despite these cytoprotective features the ultimate clinical utility of curcumin and other phytopolyphenols is uncertain based on significant concerns regarding chemical instability, toxicity and low bioavailability.1,13,14,15 The potential clinical limitations of curcumin have prompted the search for analogues that exhibit adequate bioavailability and safety, while retaining multifunctional cytoprotection. In this regard, previous structure-activity studies revealed that the heptadienone bridge of curcumin was a critical component of cytoprotection.16-18 The central moiety of this bridge is a 1,3-dicarbonyl enol characterized by two keto carbonyl groups separated by a carbon atom (Fig. 1A). The dicarbonyl bridge of curcumin exists predominately as a keto-enol tautomer and, at physiological conditions, the  carbon can ionize (pKa = 7.8) to form an enolate nucleophile as 4


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the corresponding conjugate base (Fig. 1B).19,20 The chemistry of the dicarbonyl enolate has been well described over the past century11,21,22,23 and corroborative mechanistic research has implicated a role for this carbanion in curcumin cytoprotection.11,24 Specifically, although the 1,3-dicarbonyl pharmacophore of curcumin cannot trap free radicals, this moiety is a nucleophile that can form 1,4-Michael adducts with unsaturated aldehydes such as acrolein and 4-hydroxy-2nonenal (HNE) which are involved in oxidative stress.10,11,21

Subsequent reduction of the

cellular aldehyde load can provide cytoprotection by preventing secondary enzyme inactivation, mitochondrial dysfunction and glutathione (GSH) depletion.25-29

The -diketone bridge of

curcumin is also a bidentate chelator of metal ions (e.g., iron [Fe(II)], copper [Cu(II)])21,30 that can participate in the Fenton reaction9,11,21

Inhibition of this metal-catalyzed reaction can

decrease the generation of free-radicals, such as hydroxyl radicals, that play a significant role in oxidative stress.13,31 The seemingly important cytoprotective role of the 1,3-dicarbonyl pharmacophore of curcumin suggested that acetylacetone (AcAc), 2-acetylcyclopentanone (2-ACP) and other simple dicarbonyl compounds (Fig. 2) might also be cytoprotective. Indeed, like the central bridge of curcumin, -dicarbonyl compounds exist as keto-enol tautomers that can form nucleophilic enolates following ionization.23,24 Furthermore, there is direct evidence that these chemicals chelate metal ions and scavenge unsaturated aldehyde electrophiles.11,21,23 However, unlike curcumin and other phytopolyphenols, the 1,3-dicarbonyls are chemically stable, relatively water-soluble chemicals with lower toxicity and large volumes of distribution.32 These compounds might therefore be candidates for pharmacological development. The goal of this review is to present evidence in support of our theory that enolate-forming compounds represent a viable platform for the development of pharmacotherapeutic approaches to pathogenic 5



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conditions (e.g., atherosclerosis, drug-induced toxicity, ischemia-reperfusion injury) that have a common etiology of oxidative stress. Our discussion will focus on mechanistic studies and analyses of structure-activity relationships (SAR) that defined enolate chemistry and corresponding relevance to cytoprotection. We will begin by introducing quantum mechanical concepts that govern the covalent interactions of electrophiles and nucleophiles and are therefore important to our discussions of enolate cytoprotection.

HSAB Concepts. As we will discuss, electrophiles (electron deficient species) play an important role in many pathogenic processes by disabling biological macromolecules through the formation of covalent adducts with functionally critical nucleophilic (electron rich species) sites. Therefore, 2-ACP and other structural analogues provide cytoprotection by acting as surrogate nucleophile targets for these electrophiles. However, electrophiles do not react arbitrarily with nucleophiles, and instead, these interactions are relatively selective as predicted by the Hard and Soft, Acids and Bases (HSAB) theory of Pearson (for detailed discussions see22,29,33). Thus, based on relative polarizability (electron mobility), electrophiles and nucleophiles are classified as being either soft (polarizable) or hard (non-polarizable). In accordance with HSAB principles, toxic electrophiles will react preferentially with nucleophilic biological targets of comparable softness or hardness. The designation of “hard” or “soft” is quantifiable based on corresponding inherent electronic characteristics that can be computed from the energies of the respective frontier molecular orbitals; i.e., the Highest Occupied Molecular Orbital (EHOMO) and the Lowest Unoccupied Molecular Orbital (ELUMO). These energies have been used to develop parameters that define the electrophilicity () and nucleophilicity (-) of chemical species. For example, 6


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Table 1 shows that quinones such as N-acetyl-p-benzoquinoneimine (NAPQI), the electrophilic metabolite of acetaminophen, are in general exceptionally soft, highly electrophilic chemicals (larger  and  values). Acrolein and other -unsaturated carbonyl derivatives listed in Table 1, exhibit a significantly lower range of softness and electrophilicity (see also LoPachin et al.28,34,35). As soft electrophiles, these toxicants will rapidly form covalent adducts with soft nucleophilic thiolate groups (see detailed discussions in11,36). The relative nucleophilicity (-) of 2-ACP and other putative cytoprotectants can also be calculated. Data presented in Table 2 show that, as a carbon-based nucleophile, the enolate of 2-ACP is comparable to the respective nucleophilic thiolate forms of NAC and GSH. As discussed in the next sections, we used HSAB concepts as guiding principles in our studies of enolate-based cytoprotection in experimental models of oxidative stress cytotoxicity.

Oxidative stress: enolate-based cytoprotection in cell culture models. Oxidative stress-induced cell injury is mediated by sequential involvement of electrophiles such as reactive oxygen and nitrogen species (ROS, RNS; e.g., hydroxyl radicals), transition metal ions and toxic unsaturated aldehyde by-products of membrane lipid peroxidation (e.g., acrolein, HNE; reviewed in4,26,29). As indicated in the previous section, the ability of 1,3dicarbonyl compounds to scavenge -unsaturated aldehydes and chelate metal ions is well documented in the chemical literature.11,21,23 Therefore, we surmised that these enolate-forming compounds could block oxidative stress at several steps along the cascade.

To test this

hypothesis, we determined the structure-activity relationships (SAR) for a series of 1,3dicarbonyl derivatives (Figs. 2 and 3) in cell culture models of oxidative stress.11 7



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1,3-Dicarbonyl prevention of oxidative stress in cultured

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MN9D cells.

Hydrogen

peroxide (H2O2) is a stable membrane permeable compound that is used as an experimental model of oxidative stress. The resulting cell injury is mediated by the sequential generation of intracellular electron deficient species such as hydroxyl radicals, transition metal ions and toxic unsaturated aldehyde by-products of membrane lipid peroxidation (e.g., acrolein, HNE; reviewed in4,26,29). MN9D cells are a murine dopaminergic hybridoma cell line derived from mesencephalic neurons and neuroblastoma cells. In our initial studies11, MN9D cells were exposed to graded H2O2 concentrations (200-800 µM; LC50 = 254 µM) and the relative abilities of dicarbonyl derivatives (100-750 µM) to prevent cell death were determined (Fig. 3A). 2-ACP provided nearly complete protection in this injury model, whereas AcAc and 1,1,1-trifluoro-2,4pentanedione (TFPD) were less effective (Fig. 3A). N-Acetyl cysteine (NAC) provided weak cytoprotection, whereas 1,3-cyclopentanedione (CPD) was ineffective. 2,5-Hexanedione (HD), a -diketone that does not form an enolate in physiological conditions, was used as a negative control and was shown to be completely ineffective. Our studies indicated that the rank-order of -dicarbonyl protection in MN9D cell cultures exposed to H2O2 was: 2-ACP >> AcAc, TFPD, NAC >> CPD, HD. Because the pathophysiology of oxidative stress involves toxic unsaturated aldehyde electrophiles (see above), we also determined the ability of the 1,3-dicarbonyl series to provide cytoprotection in acrolein-exposed MN9D cells (Fig. 3B). Pre-incubation (1 hr) of cells with 2ACP, CPD or NAC (750 µM) completely prevented cell lethality induced by subsequent exposure to graded concentrations of acrolein (25-200 µM). AcAc and TFPD significantly increased the acrolein LC50 (72 µM) by approximately 9 fold as indicated by corresponding rightward shifts in the acrolein concentration-response curves. In contrast, HD was ineffective; 8


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i.e., the corresponding LC50 was comparable to that of acrolein alone. The rank-order of protection in MN9D cultures exposed to acrolein was 2-ACP, NAC, CPD >> AcAc, TFPD >> HD. It is noteworthy that 2-ACP was highly protective in both the H2O2 (Fig. 3A) and acrolein (Fig. 3B) models. Furthermore, although CPD and NAC were significantly less protective in H2O2–exposed cells (Fig. 3A), these compounds were highly effective against acrolein-induced cytotoxicity (Fig. 3B). As a kinetic index of enolate electrophile scavenging, we determined the relative in chemico abilities of enolate-forming compounds to slow the rate of acrolein-induced sulfhydryl loss. Fig. 3C shows that acrolein rapidly depleted NAC sulfhydryl concentrations (78.4 nmol/s). Consistent with corresponding nucleophilicity and pKa values, 2-ACP completely prevented acrolein-mediated thiol loss. MA and DMD provided significant protection (-3.2 and 2.4 nmol/s; respectively), whereas AcAc, CPD and TFPD were only modestly effective; i.e., 24.9, -47.7 and -52.1 nmol/s, respectively. HD is a -diketone that cannot form an enolate and was, therefore, completely ineffective (-76.7 nmo/s). These data indicate that enolate formation is a prerequisite for thiol protection in this model and that the magnitude of protection is governed by nucleophilicity and pKa (see detailed discussion in LoPachin et al.11). These initial findings suggested that the 1,3-dicarbonyl compounds, in particular 2-ACP, can reduce cytotoxicity in cell culture models of oxidative stress. Cytoprotective mechanisms of enolate-forming compounds in cell culture models of oxidative stress. Oxidative stress is mediated by ROS/RNS production, transition metal ions and toxic unsaturated aldehyde by-products of membrane lipid peroxidation; e.g., acrolein, HNE. The aldehydes are soft electrophiles that mediate cytotoxicity by depleting cellular GSH and inhibiting enzyme/protein function through their ability to undergo conjugate (Michael) addition reactions with soft nucleophilic cysteine residues.26-29 Metal ions (e.g., Cu2+, Fe2+) catalyze the 9



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free-radical generating Fenton reaction and thereby also play an important pathophysiological role in oxidative stress.31 SAR analyses indicated that 1,3-dicarbonyl cytoprotection was not mediated by free-radical trapping.11 Instead, ionization of the  carbon yields a soft anionic enolate (Fig. 1B) that can act as a surrogate target for soft electrophiles such as the unsaturated aldehydes involved in oxidative stress. In fact, the ability of 1,3-dicarbonyl compounds to form Michael adducts with -unsaturated aldehydes is well established in the chemical literature.21,23

Consistent with this, we found that the rank order of cytoprotection for a -

dicarbonyl series was related to the respective abilities of these compounds to scavenge electrophiles such as acrolein (Fig. 3). The rate of adduct formation between a 1,3-dicarbonyl and a given electrophile is a determinant of cytoprotective potency and is related to differences in: (1) the acidity (pKa) of the dicarbonyl, which governs the concentration of the reacting enolate nucleophile and (2) the inherent nucleophilicity of the enolate, which influences the second order rate constant (k) for the adduct reaction. The nucleophilicity of an enolate anion can be described by the nucleophilic index (-; Table 2), an HSAB parameter derived from quantum mechanical calculations (see above). Relative differences in the respective - and pKa values among individual compounds in the 1,3-dicarbonyl series can provide an explanation for corresponding differences in adduct reaction rates and, hence, cytoprotective potencies. For example, 2-ACP is relatively acidic (pKa = 7.8) and the enolate - value (204; Table 2) is significant and, accordingly, this dicarbonyl provided substantial cytoprotection in the cell culture models tested in our research (Figs. 3A-B). In contrast, AcAc and CPD are less acidic dicarbonyls (pKa = 8.9) that are not well ionized (~2%) at cellular conditions.

Thus, although moderate nucleophiles (- = 160 and 185,

respectively), these dicarbonyls exhibited lower cytoprotective capacity than 2-ACP. TFPD is 10


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almost completely ionized at physiologic pH (99.1% anion), but the lower nucleophilicity (- = 108) results in relatively poor cytoprotective properties. In contrast to 1,3-dicarbonyl compounds, the negative control HD is a 1,4-dicarbonyl and was therefore not protective since it cannot form an enolate in physiological conditions. The preceding discussion indicates that enolate-based aldehyde scavenging is a mechanism of 1,3-dicarbonyl cytoprotection. However, the ability to chelate metal ions is also a well-established chemical trait of the 1,3-dicarbonyls.21,23 This trait has significant cytoprotective potential, since metal ions (e.g., Cu2+, Fe2+) catalyze the free-radical generating Fenton reaction.31 Our analyses11 confirmed the metal ion chelating properties of 2-ACP, AcAc and TFPD and showed that these 1,3-dicarbonyls provided substantial cytoprotection in the oxidative stress models. In contrast, CPD, which cannot chelate metal ions due to a rigid structure that precludes bidentate coordination, was not protective (LoPachin et al., 2011). Our research also indicated that NAC was a relatively ineffective metal ion chelator, which could be directly related to the inconsistent cytoprotection observed.11 Considered together, our findings indicate that 2-ACP was the most effective cytoprotectant among the 1,3-dicarbonyl compounds tested.

The enolate of 2-ACP is a

significant nucleophile (Table 2) that acts by at least two mechanisms: 1) sequestration of toxic unsaturated aldehydes, which prevents subsequent protein inhibition and GSH depletion and, 2) metal ion chelation, which reduces the free radical load by inhibiting the metal-catalyzed Fenton reaction. In contrast, NAC provided full protection in the acrolein model (Fig. 3B), but was relatively ineffective at preventing H2O2–induced cytotoxicity (Fig. 3A). This differential efficacy is consistent with an inability of NAC to cross cell membranes in physiological conditions37. Specifically, acrolein was added to the culture media where it was accessible to 11



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NAC scavenging. In contrast, NAC is negatively charged at cellular pH (7.4) and therefore membrane impermeable. Since H2O2 is permeable, NAC cannot prevent subsequent intracellular bioactivation and cytotoxicity. Furthermore, although NAC has modest antioxidant abilities, it does not chelate metal ions and has a very high pKa (9.6). This latter attribute means that NAC exists primarily in the thiol state, which is a significantly weaker nucleophile (- = 98 x 10-3ev) than the anionic thiolate state (- = 266 x 10-3ev).33 Therefore, the relatively low concentration of the reactive thiolate nucleophile could limit electrophile scavenging.

Enolate cytoprotection in animal models of oxidative stress. Enolate-forming compounds modify warm ischemia/reperfusion injury. To evaluate cytoprotection in an animal model of oxidative stress, we determined the ability of 2-ACP to modify warm ischemia/reperfusion injury (IRI) in rat liver.36 The pathophysiology of liver IRI involves oxidative stress, mitochondrial dysfunction, Kuppfer cell activation and inflammation. These processes are initiated as a consequence of ischemia and are subsequently exacerbated when cellular reoxygenation occurs during the reperfusion phase.38 In our experimental model, IRI was induced by clamping the portal vasculature for 45 min (ischemia phase) followed by recirculation for 180 min (reperfusion phase). This sequence was associated with substantial derangement of plasma liver enzyme activities, histopathological indices and hepatocyte markers of oxidative stress (Fig. 4).

2-ACP (1.60-2.40mmol/kg), administered by intraperitoneal (i.p.)

injection 10 min prior to either ischemia (clamping the portal circulation; Fig. 4) or reperfusion (unclamping the portal circulation; data not shown), provided dose-dependent cytoprotection as indicated by normalization of the IRI-altered liver biochemical (Fig. 4A) and histological (Fig. 4B) parameters. In contrast, an equimolar dose of NAC (2.40 mmol/kg; i.p.) was not hepatoprotective when administered prior to reperfusion (data not shown). 12


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Recent evidence suggests that reversal of mitochondrial succinate dehydrogenase (SDH) during ischemia promotes accumulation of the substrate succinate. During reoxygenation, SDH oxidation of excess succinate drives reactive oxygen species (ROS) production through reverse electron transport involving complex I.39 Malonic acid is a 1,3-dicarbonyl succinate analogue and therefore a competitive inhibitor of mitochondrial SDH. Although malonate has been shown to provide cytoprotection in several IRI models39, our research indicated that 2-ACP did not act through substrate inhibition.36 Instead, the chemical nature of 2-ACP (see preceding discussion) indicates a mechanism of cytoprotection involving enolate-mediated scavenging of unsaturated aldehydes and metal ion chelation. Decreasing the aldehyde load has significant cytoprotective implications since acrolein, HNE and other ,-unsaturated aldehydes appear to be critically involved in the pathophysiological processes of IRI.40-42 The ability of 1,3-dicarbonyl compounds to form metal ion complexes also has cytoprotective potential, given the pathogenic role of iron and copper ions in IRI.31,43 In contrast to 2-ACP, intraperitoneal administration of equimolar NAC did not affect the development of IRI, which is consistent with our finding that i.p. NAC also did not prevent acetaminophen (APAP) hepatotoxicity (see ahead). Enolate-based prevention of hepatotoxicity associated with acetaminophen overdose. To evaluate enolate-based cytoprotection in a more complex animal model of oxidative stress, we determined the relative ability of 2-ACP to prevent liver damage in a mouse model of acetaminophen (APAP, Tylenol™) overdose.44 APAP is a popular analgesic drug and, although considered safe at therapeutic doses, overdose is associated with severe hepatotoxicity, acute liver failure and death.45 APAP is one of the most investigated drugs available and, accordingly, the mechanism of APAP-induced hepatic damage is reasonably well known (see ahead). This level of mechanistic understanding suggested that APAP intoxication of mice was a relevant 13



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animal model for evaluating the hepatoprotective properties of enolate-forming 1,3-dicarbonyl compounds and their derivatives.46,47 In addition to 2-ACP, we also included assessment of 2’,4’,6’-trihydroxyacetophenone (THA) and 1,3,5,-trihydroxybenzene (phloroglucinol; Fig. 5). These are aromatic pharmacophores of the phytopolyphenol, phloretin, which demonstrated cytoprotectant properties in animal models48 and earlier in vitro SAR analyses.49 Whereas phloretin and corresponding derivatives are not obvious 1,3-dicarbonyl analogues, ionization by proton loss at enol sites leads to the formation of nucleophilic enolates that are electronically similar to those formed by 2-ACP. In our previous animal studies44,36, APAP overdose (500 mg/kg; oral) in mice was 90% lethal within 72 hrs of administration (Fig. 6).

Severe hepatocyte injury was revealed by

histopathological indices of necrotic cell death that were temporally correlated with significantly elevated plasma liver enzyme activities (e.g., alanine aminotransferase) and changes in biomarkers of oxidative stress (e.g., thiol depletion, HNE liver content). 2-ACP administered by intraperitoneal (i.p.) injection 20 mins before or after oral APAP intoxication provided dosedependent (0.80 – 2.40 mmol/kg) protection against lethality determined over a 7 day observation period. Fig. 6A shows the protective effects of 2-ACP at the 2.4 mmol/kg dose. The increased survival rate was correlated to normalization of the measured liver toxicity parameters. In contrast, 2-ACP administered by oral injection was relatively ineffective at preventing APAPinduced lethality. Fig. 6B shows the effects of 2-ACP at 4.8 mmol/kg dose. Zhang et al.44 also determined the ability of the thiol-based nucleophile, NAC, to prevent APAP-induced lethality. Results indicated that i.p. injection of NAC (Fig. 6A; 2.40mmol/kg) was ineffective, whereas oral administration provided hepatoprotection (2.40 mmol/kg; Fig. 6B).

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Animal research48 showed that the phloretin pharmacophores, THA and phloroglucinol (PG; Fig. 5), produced significant hepatoprotection in the APAP toxicity model (Fig. 6). THA was also effective regardless of administration route; i.e., i.p. or oral (Figs. 6A and B). On a comparative basis, THA was more potent than PG; e.g., i.p. THA completely prevented APAPinduced lethality at 0.8 mmol/kg, whereas PG provided only 70% protection at this dose (Fig. 6). The hepatoprotective profile of PG was similar to that of 2-ACP; i.e., PG produced routedependent hepatoprotection (i.p. only) over an equivalent dose range (0.40 – 2.40 mmol/kg; Fig. 6). THA was effective regardless of route and over much smaller dose ranges; i.e., i.p. = 0.200.80 mmol/kg; oral = 0.80 – 2.40 mmol/kg (Fig. 6). Together, the results of these studies indicated that the enolate-forming compounds, 2-ACP, THA and PG, can provide dosedependent hepatoprotection in an animal model of APAP poisoning. Our results showed that NAC, the clinical antidote for APAP hepatotoxicity (Mucomyst™) was also hepatoprotective in this model.

However, unlike NAC and the other compounds tested, THA provided effective

protection at lower doses and independent of administration route. A noteworthy aspect of these animal studies was the observation that 2-ACP and PG were effective by i.p. injection only, whereas NAC hepatoprotection occurred only following oral administration (Figs. 6A-B). Compromised enolate hepatoprotection associated with oral administration was likely due to the susceptibility of -diketones to acid-catalyzed reactions (e.g., aldol condensation) that can occur in the low pH environment of the stomach.44 The finding that NAC hepatoprotection was dependent upon oral administration is consistent with the fact that at pH 7.4, this thiol nucleophile is negatively charged and only 0.001% of NAC is present in the neutral membrane permeable form. The neutral form of NAC predominates at pH < 3.3, which allows passive diffusion from the highly acidic (pH 1.5 – 3.3) stomach to the 15



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intestinal circulation37.

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Evidence suggests that NAC undergoes bioactivation via intestinal

pathways50 to sulfite (SO32-), a significant nucleophile that can scavenge NAPQI, acrolein and other electrophiles involved in APAP hepatotoxicity51

NAC-derived sulfite can also reduce

disulfide bonds; e.g., GSSG, protein-GSH mixed disulfide bonds, which can yield free GSH50 This could account for the increased GSH content in liver following NAC administration (e.g., see52,53) and subsequent restoration of the cellular redox environment, which would improve mitochondrial function of APAP intoxicated hepatocytes (see ahead). Pharmacological characterization of enolate-forming compound in APAP-exposed isolated hepatocytes.

The findings in our animal model were supported by results from

corroborative experiments in APAP-exposed isolated mouse hepatocytes that demonstrated direct hepatoprotection by enolate-forming compounds during APAP intoxication.48 The level of hepatocyte injury was determined by measurements of cell viability, elevated liver enzyme activities (e.g., alanine aminotransferase) in media and mitochondrial membrane depolarization

(Ψm). Extensive pharmacological characterization of the putative cytoprotectants showed that THA and to a lesser extent, PG, were substantially more potent than either 2-ACP or NAC. Geohagen et al.48 also found that THA and PG were more effective at rescuing isolated mouse hepatocytes from ongoing APAP intoxication (2 hr exposure). This could have significant clinical implications for cell preservation and recovery from certain diseases and traumatic tissue injuries. The relationship of enolate chemistry to the prevention of APAP hepatotoxicity. In this section we will discuss the relationship of enolate chemistry to possible mechanisms of hepatoprotection in APAP overdose. The cytoprotection afforded by 2-ACP, THA and other 16


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enolate-forming compounds might be simply related to inhibition of oxidative metabolism and subsequent reduced formation of the NAPQI metabolite.53 However, Zhang et al.44 found that transient APAP-induced changes in liver injury biomarkers (e.g., unsaturated aldehyde and GSH contents) were initially evident in 2-ACP pretreated mice. This indicated that APAP metabolism and NAPQI generation were not impaired by the dicarbonyl compounds. Alternatively, it is possible that hepatoprotection involved changes in APAP tissue disposition that slowed metabolism and therefore NAPQI generation (e.g., see55).

However, the observation that

THA/PG provided cytoprotection in an isolated cell system argues against this possibility.48 It is also unlikely that hepatoprotection is mediated by activation of cellular stress responses (e.g., Keap1/Nrf2 pathway), as has been suggested for curcumin and other phytopolyphenols.3,56 In this case, the corresponding molecular targets at which a nucleophilic compound such as 2-ACP might interact are unclear. Instead, an understanding of enolate chemistry provides a rational basis for defining molecular mechanisms of cytoprotection.

Differences in respective

cytoprotective potency can then be understood within the context of individual physicochemical traits. Results from our initial cell culture studies (see above) have suggested that enolate-based scavenging of reactive electrophiles and metal ion chelation are common mechanisms of hepatoprotection. NAPQI can produce injury to hepatocytes through both oxidant- and electrophile-based mechanisms.57,58 Thus, NAPQI can oxidize cysteine sulfhydryl groups on cellular glutathione (GSH) and proteins (S-hydroxylation).59-61 NAPQI is also a soft electrophile that will form Michael adducts with soft nucleophilic sulfhydryl groups on GSH and proteins.58,62-65 The oxidant and electrophile mechanisms of NAPQI lead to cellular GSH depletion, protein inactivation and secondary oxidative stress (Fig. 7).66,67 Mitochondrial function is especially 17



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sensitive to NAPQI68 since depletion of the GSH pool69 and adduction of corresponding proteins causes selective disruption of this organelle.65,70,71 Accordingly, early NAPQI-mediated damage to mitochondria is thought to play a critical role in liver cell demise.67,72 This toxic cascade (Fig. 7) can be disrupted by the respective enolates of 2-ACP, THA and PG that function as surrogate nucleophile targets for NAPQI.44,48 In direct support of this mechanism, recent in chemico studies48 showed that these soft nucleophiles can scavenge NAPQI, which increases the inhibitory concentration of this electrophile needed to produce 50% loss of GSH (IC50; Table 3). However, results from our studies in animal and isolated hepatocyte models indicated that 2-ACP and NAC were substantially less effective protectants than THA or PG. Nonetheless, HSAB calculations of the respective nucleophilic indices (-; Table 3) showed that the 2-ACP enolate was more reactive with NAPQI than either the PG or THA enolates. This is inconsistent with the rank order of GSH protection demonstrated in our in chemico model where 2-ACP was substantially less effective than THA (Table 3). This discrepancy can be explained by the redox properties of THA. Thus, similar to GSH57,58, THA can decrease the effective NAPQI concentration via reduction to APAP (Fig. 7). In contrast, when acrolein was the reacting electrophile (Table 3), the respective abilities of 2-ACP and THA to prevent GSH loss were consistent with the corresponding values for nucleophilicity, provided the data were interpreted in accordance with the corresponding pKa-based anion concentrations (see below). Our studies have also revealed pronounced differences in the cytoprotective potency of THA and PG. This differential effectiveness can be explained by underlying physicochemical differences. Specifically, although PG is a better nucleophile, the pKa of THA is closer to cytological pH and, therefore, 33% of this analogue will be in the electrophile-scavenging enolate state (Table 2). In contrast, with a pKa of 8.5 only 7% of PG will exist in the anionic 18


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enolate state at physiological pH (Table 2). Based on this difference in acidity, THA will readily scavenge electrophiles, whereas the lower PG enolate concentration at physiological pH (7%) will decrease the respective adduct reaction rates with NAPQI and other electrophiles thereby reducing cytoprotection. In addition to scavenging NAPQI, the secondary oxidative stress instigated by this metabolite appears to involve acrolein, HNE and other unsaturated aldehydes generated during membrane lipid peroxidation (Table 1)25,73,74. These aldehydes are also soft electrophiles that cause cytotoxicity via a mechanism similar to that of NAPQI.26-29

Thus, 2-ACP and the

aromatic enols can scavenge unsaturated aldehydes that mediate NAPQI-initiated secondary oxidative stress (Fig. 7). Metal ions, in particular Fe(II) and Cu(II), appear to be critically important to the pathogenesis of APAP-induced hepatotoxicity presumably through their abilities to catalyze the free radical generating Fenton reaction and to promote intramitochondrial ROS.75-77 Indeed, previous studies have indicated that metal ion chelation (e.g., dipyridyl) is hepatoprotective in experimental APAP overdose.75-78 In this regard, the ability of 1,3-dicarbonyl compounds to chelate metal ions has been well documented (see preceding discussion) and we have shown that chelation is a vital component of 2-ACP cytoprotection in oxidative stress (Fig. 7). Furthermore, in contrast to the more rigid PG analogue, the flexible structure of THA permits bidentate complexation with iron and copper metal ions.79,80 In previous in chemico studies11, NAC was found to be a weak metal chelator that was ineffective in cell culture models of oxidative stress. Nonetheless, in subsequent experimental animal models of APAP overdose44, oral NAC provided significant hepatoprotection. In this case, NAC likely undergoes bioactivation to 19



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nucleophilic sulfite that scavenges NAPQI, acrolein and other electrophiles involved in APAP hepatotoxicity51. In addition, NAC-derived sulfite can reduce disulfide bonds which yields GSH50 that can restore the cellular redox environment and thereby improve hepatocyte mitochondrial energy production (e.g., see53-55,81). Our data indicate that 2-ACP and other 1,3-dicarbonyls are incapable of free radical trapping, whereas NAC has only modest trapping abilities (summarized in Fig. 7). However, as aromatic phenols THA and PG can behave as antioxidants and trap free radicals generated during oxidative stress.49,82-85 Thus, 2-ACP can provide dose-dependent hepatoprotection in the APAP mouse model via enolate-based scavenging of soft electrophiles (e.g., NAPQI, unsaturated aldehyde by-products of lipid peroxidation) and metal ion chelation. PG was also capable of providing hepatoprotection over a dose-range similar to that of 2-ACP. However, the rigid structure of PG precludes metal ion coordination and with a relatively basic pKa only a small percentage is in the anionic state and therefore available for electrophile scavenging. As an aromatic compound, the hepatoprotection provided by PG was likely a product of ROS/RNS trapping. In contrast to 2-ACP and PG, THA has multifunctional properties (i.e., reduction, electrophile scavenging, metal ion chelation and ROS/RNS trapping) that can inhibit the NAPQI cascade at multiple steps. The pleiotropic actions of THA (Fig. 7) can account for the superior hepatoprotective performance of this compound in our APAP overdose models.

Summary Our research has shown that enolate-forming compounds offer significant protection in rodent models of hepatotoxicity. The well-described chemistry of these compounds21,23, in conjunction with our in chemico and cell culture studies11,48, indicate that cytoprotection is due to 20


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enolate-based electrophile scavenging and metal ion chelation. The 1,3-dicarbonyl compounds are structural derivatives of the curcumin heptadienone bridge and therefore the cytoprotective potential of this phytopolyphenol likely involves enolate formation. Our studies also identified a highly potent aromatic enol derivative of phloretin, THA, which represents a cytoprotective chimeric of enolate mechanisms and free radical trapping.

In general, these findings are

consistent with our premise that the enolate moiety has significant cytoprotective potential and could be used as a structural platform for the development of potent multifunctional pharmacotherapeutic compounds. In this regard, it is noteworthy that the clinical relevance of phytopolyphenols (e.g., curcumin, phloretin) as general cytoprotectants is uncertain given their known toxicity, poor bioavailability and chemical instability.14,15,86

In contrast, the enolate-

forming compounds tested in our studies are chemically stable, relatively non-toxic and water soluble compounds with large volumes of distribution.11,32,43 That enolate-forming compounds can be developed for pharmaceutical use is evidenced by the fact that PG is marketed as the antispasmodic, Spasfon® (Cephalon, France). As alluded to previously, acrolein, HNE and other unsaturated aldehyde electrophiles are critically involved in disease states and tissue injuries that have oxidative stress as a common molecular etiology; e.g., Alzheimer’s disease, ischemia/reperfusion injury or traumatic spinal cord injury.87-89 Furthermore, most drug-induced toxicities are caused by compounds that are themselves electrophiles (e.g., cisplatin, clopidogrel) or that are metabolized to electrophilic intermediates (e.g., acetaminophen, cyclophosphamide).90 Finally, many environmental pollutants (e.g., acrolein, methyl mercury, organophosphate insecticides) are electrophiles that cause toxicity through acute or chronic exposures.29,91,92 The ability of nucleophilic enolate-forming compounds to scavenge electrophiles might therefore

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have particular relevance as a cytoprotective trait, since electrophiles clearly play a substantial role in chemically acquired toxicities and pathogenic processes.

AUTHOR INFORMATION Corresponding Author E-Mail: [email protected] Funding The research discussed in this Review was supported by NIH grants from the National Institutes of Environmental Health Sciences RO1 ES03830-29; RO1 ESO7912-13. ABBREVIATIONS HSAB, hard and soft, acids and bases; LUMO – lowest unoccupied molecular orbital; HOMO – highest occupied molecular orbital; ELUMO, LUMO energy; EHOMO, HOMO energy; LD50, 2ACP, 2-acetylcyclopentanone; AcAc, acetylacetone; CPD, 1,3-cyclopentanedione; TFPD, 1,1,1trifluoro-2,4-pentanedione; APAP,

acetaminophen; NAPQI, N-acetyl-p-benzoquinone imine;

lethal oral dose for 50% of the population; IC50, inhibitory concentration for 50% activity; HNE, 4-hydroxy-2-nonenal; NAC, N-acetylcysteine; H2O2, hydrogen peroxide; Nrf2/Keap1 – nuclear factor erythroid 2-related factor 2/kelch-like erythroid cell-derived protein with CNS homologyassociated protein 1; eV, electron volt; GSH, glutathione; SAR, structure-activity relationship; GSSG, oxidized glutathione; Ψm;

mitochondrial membrane polarization; THA, 2’,4’,6’-

trihydroxyacetophenone; PG, 1,3,5,-trihydroxybenzene; HD, 2,5-hexanedione; IRI, ischemiareperfusion injury; ROS, reactive oxygen species; RNS, reactive nitrogen species; H2O2, hydrogen peroxide .

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74. Brown, J.M., Kuhlman, C., Terneus, M.V., Labenski, M.T., Lamyaithong, A.B., Ball, J.G., Lau,

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(2014)

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of

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Author Biographies

Lars Ulrik Nordstrøm received a BS (2003) and MS (2005) in carbohydrate chemistry from Aarhus University working with Dr. Mikael Bols. In 2009, Lars received a Ph.D. in Synthetic Chemistry from the Technical University of Denmark under the supervision of Robert Madsen. He was a Postdoctoral Fellow in the laboratory of Dr. David Gin at Memorial Sloan-Kettering Cancer Center. He then joined the Chemical Synthesis Core Facility at Albert Einstein College of Medicine in 2012 where he is currently an Assistant Research Professor. His research interests focus on synthesis of small molecules for the use in biological research and drug development. 35



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Brian Geohagen received a Bachelor of Science in Chemistry from Iona College (New Rochelle, NY) in 2003. In 2005, Mr. Geohagen joined the laboratory of Richard M. LoPachin, Ph.D. as a Research Associate in the Anesthesia Research Labs at Montefiore Medical Center (Bronx, NY). Over the past decade he has contributed significantly to the pharmacological understanding and development of enolate-forming compounds as cytoprotectants.

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Terrence Gavin received his Ph.D. in Chemistry from the State University of New York at Stony Brook in 1982. Early in his career, Dr. Gavin worked as an Associate Scientist at CIBA-Giegy (Ardsley, NY) and later joined the faculty of the Department of Chemistry at Iona College (New Rochelle, NY). Terry rose in rank and eventually became a full Professor (2003) and Chair (1999) of the Iona Chemistry Department. We announce with great sadness that on August 22 2016, Terry Gavin passed away as a result of esophageal cancer. It was Dr. Gavin’s remarkable understanding of enol chemistry that led us to the discovery that the nucleophilic enolate state was cytoprotective and therefore represented a platform for drug development.

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Richard M. LoPachin received a Ph.D. in Pharmacology from the University of WisconsinMadison (UWM) in 1981. Subsequently (1981-1984), he received Post-Doctoral training in Neurotoxicology in the laboratory of Richard E. Peterson (UWM) and thereafter joined the Pharmacology faculty at the University of Houston (1984 – 1988) as an Assistant Professor. In 1988, Dr. LoPachin moved to the Department of Anesthesiology at the State University of New York-Stony Brook where he was promoted to the rank of Associate Professor. He is currently a Professor of Anesthesiology at the Albert Einstein/Montefiore Medical Center. Dr. LoPachin’s research interests include drug development, computational toxicology and the role of environmental toxicants in disease progression.

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Table 1. HSAB Parameters for Selected Electrophiles Participating in IRI and APAP Hepatotoxicity Softness () and electrophilicity () for the compounds were calculated as described in the Materials and Methods section. Abbreviations: NAPQI - N-acetyl-p-benzoquinoneimine; HNE – 4-hydroxy-2-nonenal Electrophile

Structure

Softness

Electrophilicity

(x 103 ev-1)

(, ev)

NAPQI

499

6.83

Acrolein

371

3.82

377

3.80

384

3.39

HNE

Malondialdehyde

39



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Table 2. HSAB and Ionization Parameters of Thiolate and Enolate Anions Softness () and nucleophilicity (-) for the selected chemicals were calculated as described in Vydyanathan et al. (2015). Abbreviations: 2-ACP – 2-acetylcyclopentanone; AcAc – acetylacetone; TFPD - 1,1,1,-trifluoro-2,4-pentanedione; CPD – 1,3-cyclopentanedione; PG – phloroglucinol (1,3,5-trihydroxybenzene); THA – 2’,4’,6’-trihydroxyacetophenone; NAC – Nacetylcysteine.

Anion

Nucleophilicity with Acrolein

pKa

% Anion (pH 7.4)

 x10-3 ev)

2-ACP

204

7.8

28.0

AcAc

160

8.9

2.0

TFPD

108

4.7

99.1

CPD

185

8.9

2.0

PG

133

8.5

7.40

THA

114

7.7

33.0

NAC

316

9.5

0.80

Phloretin

105

7.3

55.0

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Table 3. Enolate-Forming Compounds: Relationship of Nucleophilicity to the IC50 for Protection of GSH Sulfhydryl Groups. Correspondence of IC50 values (µM) for in chemico depletion of GSH sulfhydryl groups by NAPQI or acrolein with the respective nucleophilicities (-) of the enolate forming compounds (see Geohagen et al., 2015 for details). Abbreviations: 2-ACP – 2-acetylcyclopentanone; PG – phloroglucinol (1,3,5-trihydroxybenzene) and THA – 2’,4’,6’-trihydroxyacetophenone.

NAPQI (IC50; µM)

Nucleophilicity with  NAPQI x10-3

Acrolein (IC50; µM)

ev)

Nucleophilicity with  Acrolein  x10-3 ev)

Alone

10

-

21

-

PG

20

366

27

133

2-ACP

25

485

62

204

THA

73

325

34

114

Figure Legends 41



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Figure 1. (A). Color-coded line structures are presented for curcumin and 2-ACP. The circled curcumin moiety designates the 1,3-dicarbonyl pharmacophore (red), which was the bases for the evaluation of 2-ACP (red) and other structurally related compound as possible cytoprotectants. B. Schematic diagram illustrating enolate formation for a 1,3-dicarbonyl compound. Figure 2. Line structures for the 1,3-dicarbonyl compounds used in the cell culture and animal models described in the present review. Abbreviations: AcAc – acetylacetone;; TFPD - 1,1,1,trifluoro-2,4-pentanedione; DEM – diethylmalonate; CPD – 1,3-cyclopentanedione; DMD – dimedone; MA - Medrum’s acid; 2-ACP – 2-acetylcyclopentanone.

Also shown is 2,5-

hexanedione (HD) a -diketone used as a negative control in our studies. Figure 3. 1,3-Dicarbonyl compounds protect cultured MN9D cells against H2O2- (A) and acrolein- (B) induced lethality. Cell were incubated with putative cytoprotectant (100-750 µM) for 1 hr followed by exposure to graded concentrations of H2O2 (A; 50-750µM x 24 hrs) or acrolein (B; 0.5-150µM x 24 hrs). In each study, cell survival was measured by direct counting and concentration-response data were fitted by nonlinear regression analysis. The respective concentrations producing 50% cell lethality (LC50s) were calculated by the Cheng-Prusoff equation. In either study, an LC50 for 2-ACP could not be calculated because lethality did not decrease below 50% over the respective concentration ranges. (C) Plot of log[SH/SHo] versus time (s) illustrating the effects of each 1,3-dicarbonyl or phytopolyphenol on the rate (nmol/s) of acrolein-induced sulfhydryl loss (n=3 experiments).Abbreviations: AcAc – acetylacetone; TFPD - 1,1,1,-trifluoro-2,4-pentanedione; DEM – diethylmalonate; CPD – 1,3-cyclopentanedione; DMD – dimedone; MA - Medrum’s acid; 2-ACP – 2-acetylcyclopentanone. Also shown is 2,5hexanedione (HD) a non-enolate forming -diketone used as a negative control in our studies.

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Figure 4. (A.) Dose-dependent hepatoprotective effects of i.p. 2-ACP administered 10 min prior to unclamping the portal circulation (reperfusion phase). IRI-induced plasma appearance was measured for alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in rat (n=10-15 /group). Data are expressed as mean activity ± SEM and joining lines indicate statistically significant differences in treatment groups at ** p

Enolate-Forming Compounds as a Novel Approach to Cytoprotection

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