当前位置:首页 >> 自然科学 >>

Discovery and Characterization of Potent and Selective Inhibitors against the


UNIVERSITY OF CALIFORNIA, IRVINE

Discovery and Characterization of Potent and Selective Inhibitors against the PEA-hydrolyzing enzyme, N-acylethanolamine-hydrolyzing Acid Amidase

DISSERTATION

submitted in partial satisfaction of the requirements for the degree of

DOCTOR OF PHILOSOPHY in Pharmacology and Toxicology

PR EV

Carlos Efrain Solorzano Say

IE
by 2010

Dissertation Committee: Professor Daniele Piomelli, Chair Professor Paolo Sassone-Corsi Professor Robert Edwards Professor George Chandy, Oversight Member

W

UMI Number: 3404654

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

PR EV

UMI 3404654 Copyright 2010 by ProQuest LLC. All rights reserved. This edition of the work is protected against unauthorized copying under Title 17, United States Code.

ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106-1346

IE

W

PR EV
Chapter 3 ? 2009 Carlos E. Solorzano, PNAS All other materials ? 2010 Carlos E. Solorzano

IE

W

DEDICATIO

To my wife, Elizabeth, To my parents, Carlos and Ruth Solorzano, To my brother Jose Solorzano, To my parents-in-law, Sergio and Elizabeth Montalvo, To my Family and Friends,

And to my Lord and Savior, Jesus Christ.

PR EV
ii

IE

W

TABLE OF CO TE TS Page LIST OF FIGURES LIST OF TABLES ACKNOWLEDGEMENTS CURRICULUM VITAE ABSTRACT OF THE DISSERTATION v vii viii x xiv

CHAPTER 1:

The Fatty Acid Ethanolamides with emphasis on PEA Introduction Discovery Biological Actions Anandamide Oleoylethanolamide Palmitoylethanolamide Cellular Metabolism Biosynthesis Degradation Regulation of PEA during Inflammation Conclusions

1 1 2 3

PR EV

IE
iii

W

7

11 27 27 27 28 29 30 34 35 49

CHAPTER 2:

The -acylethanolamine-hydrolyzing acid amidase (NAAA) Introduction Biochemical Identification Molecular Cloning Regulation and Proteolytic Activation NAAA vs. FAAH Current NAAA inhibitors Conclusions

CHAPTER 3:

Selective NAAA inhibition reveals a key role for endogenous PEA in inflammation Introduction Materials and Methods Results

49 51 68

Discussion Conclusions CHAPTER 4:

74 76

Structure Activity Relationship of β-lactone NAAA inhibitors 109 Introduction Materials and Methods Results and Discussion Conclusions 109 111 119 125 152 152 152 154 157 159 161

CHAPTER 5:

General discussion and future directions Introduction Summary of Results Unresolved Issues Future Directions Conclusion

REFERENCES

PR EV
iv

IE

W

LIST OF FIGURES Page 13

Figure 1.1

Structures of palmitoylethanolamide (PEA), arachidonoylethanolamide (anandamide) and oleoylethanolamide (OEA) Timeline illustrating the research on PEA over the years Metabolism of PEA Regulation of NAPE-PLD/PEA by inflammatory stimuli Enrichment protocol for NAAA from cells or tissue Reactions catalyzed by NAAA and acid ceramidase

Figure 1.2

15

Figure 1.3 Figure 1.4

17 19

Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4

37 39 41 43

Figure 2.5

PR EV

Structure of the NAAA inhibitors, cyclohexyl hexadecanoate and -cyclohexanecarbonyl pentadecylamine Structure of the NAAA inhibitor cyclopentyl hexadecanoate

IE
v

Structure of the FAAH inhibitor URB597

W

45

Figure 3.1 Figure 3.2

Three-dimensional model of NAAA

77 79

Sequence alignment of NAAA and conjugated bile acid hydrolase Site-directed mutagenesis of various amino acid residues in NAAA Characterization of the NAAA inhibitor (S)-OOPP Effect of (S)-OOPP on FAE levels and NAAA activity in RAW264.7 cells and HEK293 cells overexpressing NAAA Effects of (S)-OOPP on endogenous levels of -stearoylceramide in RAW264.7 macrophages

Figure 3.3

81

Figure 3.4 Figure 3.5

83 85

Figure 3.6

87

Figure 3.7

Effects of (S)-OOPP on carrageenan-induced neutrophil migration in mice Hematoxylin and eosin staining of infiltrating cells in subcutaneous sponges Effects of (S)-OOPP on carrageenan-induced plasma extravasation in mice and pro-inflammatory gene expression in RAW264.7 macrophages Antiinflammatory effects of PEA and synthetic PPAR-α agonist GW7647 Effects of the FAAH inhibitor URB597 on PEA levels and carrageenan- or LPS-induced inflammation Anti-inflammatory effects of (S)-OOPP in mice following spinal cord injury

89

Figure 3.8

91

Figure 3.9

93

Figure 3.10

95

Figure 3.11

97

Figure 3.12

W

99

Figure 4.1 Figure 4.2

Schemes used for the synthesis of NAAA inhibitors Possible mechanisms for covalent modification of NAAA by lactone derivatives Docking of (S)-OOPP (7a) into the catalytic site of NAAA Putative tetrahedral intermediate resulting from attack of rat cysteine 131 onto 7a

IE
vi

Figure 3.13

Effects of (S)-OOPP on inflammatory markers in spinal cord tissue of spinal cord injury (SCI) mice

101

126 130

Figure 4.3

PR EV

132

Figure 4.4

134

Figure 4.5

Characterization of NAAA inhibition by 7h in vitro Effects of compounds 7a and 7h on PEA levels and carrageenan-induced leukocyte infiltration Superposition of -(2-oxo-3-oxetanyl)amides employed for 3D-QSAR analysis Summary of SARs for β-lactone inhibitors of NAAA

136

Figure 4.6

138

Figure 4.7

140

Figure 4.8

142

LIST OF TABLES Page Table 1.1 Table 1.2 Table 1.3 Table 2.1 Table 3.1 Table 3.2 Table 3.3 Table 4.1 Physiological roles of anandamide Feeding-related pharmacological properties of OEA Pharmacological properties of PEA Substrate selectivity of NAAA Strucuture of compounds tested against NAAA Structure of compounds for SAR study Activity of (S)-OOPP on various lipases Inhibitory potencies (IC50) of various 2-Oxo-3-oxetanyl compounds against NAAA Inhibitory potencies (IC50) of -(2-Oxo-3-oxetanyl) arylamides against NAAA 21 23 25 47 103 105 107 144

Table 4.2

IE
vii

W

147

Table 4.3

PR EV

Effects of compound 7h on Michaelis–Menten constant (Km) and maximal rate of reaction (Vmax) of recombinant NAAA

150

ACK OWLEDGME TS I am deeply indebted with my graduate school mentor, Professor Daniele Piomelli, for his support, guidance, patience, and trust throughout my graduate training at the University of California, Irvine. Having majored in chemical engineering as an undergraduate, I was less knowledgeable regarding basic biological principles than my peers in the department of pharmacology at the beginning of my journey as a graduate student. As a rotating student in Dr. Piomelli’s lab, I immediately knew that I wanted to become a permanent member of his team because I knew that I would have the greatest potential for growth working with such a great scientist, and friendly, dynamic team. It must have taken a lot of hopeful thinking and bravery on his behalf, however, to agree to take me on as a graduate student. In any case, I am thankful for the opportunity Dr. Piomelli gave me to train with him, and could not have any further reaffirmed the initial sentiments that I felt as a rotating student. I am also thankful for other mentors that I have had as a graduate student, including Professors Paolo Sassone-Corsi, Robert Edwards, Leslie Thompson, and George Chandy, who kindly served on my thesis committee and offered insightful advice and comments. In particular, Professors Sassone-Corsi, Emiliana Borrelli and our Graduate Dean, Dr. Frances Leslie, were invaluable mentors and sources of advice as I thought about the next step in my career, the postdoc. I am grateful to past and present members of the Piomelli lab – Drs. Chenggang Zhu, Giuseppe Astarita, Natalia Battista, Jin Fu, Kwan-Mook Jung, Jason Clapper, Alvin King, Natalia Realini, Ana Guijarro, Guillermo Moreno, Emmanuel Dotsey, Ernest Fung and Regina Mangieri, for help with experiments, for training me in various techniques, and for their friendship. A great portion of the projects that I have worked on have been performed in collaboration or have been complemented by Dr. Chenggang Zhu’s work. I am thankful to Nana Barsegyan, Alyssa DerMugrdechian, Cherine Moazam, and Yasaman Zahedi, undergraduate students who have worked with me in the lab and given me the opportunity to mentor them. Finally, from the Piomelli lab, I would like to thank Fariba Oveisi and Jennifer Lockney for managing and administrating the lab in a mostprofessional way that has made it easy and productive to conduct experiments. I am thankful to our collaborators, Drs. Marco Mor, Alessio Lodola, and Silvia Rivara (all from the Univ. of Parma, Italy); Drs. Giorgio Tarzia, Andrea Duranti, Francesca Antonietti and Andrea Tontini (all from the Univ. of Urbino, Italy); and Dr. Salvatore Cuzzocrea (Univ. of Messina, Italy). All of the chemical synthesis (Univ. of Urbino), molecular modeling (Univ. of Parma) and spinal cord injury work (Univ. of Messina) has been possible because of their work. I would also like to thank the Allegiance for Graduate Education and the Professoriate (AGEP), an organization funded by the National Science Foundation, for financial support to attend scientific conferences and for providing a network and home for minority graduate students at UCI. In particular, I would like to thank Dr. Frances Leslie, Raslyn Rendon, Daniel Fabrega, Carlos Santana, and Mariana Garcia, for their hard work to make this organization existent and productive. I would also like to thank the National Institutes of Health, the Sandler Asthma Foundation, the Graduate Division

PR EV

IE
viii

W

PR EV
ix

IE

W

at UCI, the President’s Dissertation Year Fellowship, the Miguel Velez Graduate Fellowship, and the Henry Wood Elliot Foundation for funding. I am thankful to the administrative staff of the Department of Pharmacology, in particular, Pam Bhalla. Finally, I could not have completed my graduate education without God or my family. I thank God for the life, health and Salvation, which have been given to me as a gift. I thank my parents for the sacrifices they have made to provide me with opportunities that they did not have; and for inculcating in me a good work ethic, and for teaching me to be perseverant and to have a positive outlook, despite the situation at hand. I thank my brother Jose for always been a loyal and sincere friend, with a kind heart; and my cousin Aaron for being a good friend. I thank the Montalvo family, in particular, my parents-in-law, for the love, support and trust they have granted me, which have been a tremendous help in my career as well as personal life. I thank the Say Guerra, Say Solis, Solorzano De Leon, and Solorzano Engle families for love and support throughout my entire life. Finally, I am deeply thankful for and to my wife, Elizabeth, who continuously loves and supports me. She is my best friend and has been an incredible support and companion for me as we both have set out to complete our PhD’s. I am better because of her and could not have completed my PhD without her.

CURRICULUM VITAE Carlos E. Solórzano Education Ph.D. Pharmacology and Toxicology Jun 2010 University of California, Irvine (UCI) Advisor: Daniele Piomelli, Ph.D. Dissertation title: “Discovery and Characterization of Potent and Selective Inhibitors against the PEA-hydrolyzing enzyme NAAA B.S. Chemical Engineering California State Polytechnic University, Pomona Publications Solórzano, C., Zhu, C., Battista, N., Astarita, G., Lodola, A., Rivara, S., Mor, M., Russo, R., Maccarrone, M., Antonietti, F., Duranti, A., Tontini, A., Cuzzocrea, S., Tarzia, G., and Piomelli, D. “Selective NAAA inhibition reveals a key role for endogenous PEA in inflammation,” Proceeding of the ational Academy of Sciences 2009, 106(49):20966-71. Solórzano, C., Antonietti, F., Duranti, A., Tontini, A., Rivara, S., Lodola, A., Vacondio, F., Tarzia, G., Piomelli, D., and Mor, M. Synthesis and StructureActivity Relationships of -(2-Oxo-3-oxetanyl)amides as -Acylethanolaminehydrolyzing Acid Amidase Inhibitors. Submitted. Zhu, C., Solórzano, C., Sahar, S., Sassone-Corsi, P., Piomelli, D. Proinflammatory stimuli control NAPE-PLD expression in macrophages. Submitted. Awards and Honors
May 2005

? Henry Wood Elliot Award May 2010 Department of Pharmacology, Univ. of California, Irvine ? President’s Dissertation Year Fellowship Oct 2009 – Jun 2010 ? Best Oral Presentation Award in Pharmacology/Immunology Oct 2009 Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) ? Second Place Poster Award Aug 2009 Cannabinoid Signaling in the CNS – Gordon Research Conference ? Carl Storm Travel Award (Gordon Research Conference) Jul 2009 ? Miguel Velez Graduate Fellowship Dec 2007 ? National Association of Corrosion Engineers Scholarship Recipient Jun 2005 ? President’s Council Scholarship Finalist May 2002 x

PR EV

IE

W

? Chevron Scholarship Recipient May 2001, 2003 ? Society of Hispanic Professional Engineers Scholarship Recipient Feb 2001 ? National Hispanic Recognition Merit Award May 1999 Research Presentations Solórzano, C., Zhu, C., Battista, N., Astarita, G., Lodola, A., Rivara, S., Mor, M., Russo, R., Maccarrone, M., Antonietti, F., Duranti, A., Tontini, A., Cuzzocrea, S., Tarzia, G., and Piomelli, D. Inhibiton of the lipid amidase AAA is antiinflammatory and attenuates spinal cord injury. (2009) Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) Abstract, Dallas, TX, Oct 15 – 18, 2009 (Oral) – Received Best Oral Presentation Award Solórzano, C., Zhu, C., Battista, N., Astarita, G., Lodola, A., Rivara, S., Mor, M., Russo, R., Maccarrone, M., Antonietti, F., Duranti, A., Tontini, A., Cuzzocrea, S., Tarzia, G., and Piomelli, D. Selective AAA inhibition reveals a key role for endogenous PEA in inflammation. (2009) Cannabinoid Signaling in the CNS Gordon Reserch Conference, Biddeford, ME, Aug 2 – 7, 2009 (Poster) – 2nd Place in poster competition. Zhu, C., Solorzano, C., Battista, N., Astarita, G., Lodola, A., Rivara, S., Mor, M., Russo, R., Maccarrone, M., Antonietti, F., Duranti, A., Tontini, A., Cuzzocrea, S., Tarzia, G., and Piomelli, D. Inhibitors of the lysosomal lipid amidase AAA. (2008) Society for Neuroscience Abstracts, Washington, D.C., Nov 15 – 19, 2008 (Poster). Research Interests

? Discovering novel lipid mediators involved in the inflammatory response and neuropathic pain ? Identifying the molecular mechanisms that lead to chronic inflammatory disorders with the aim of developing novel therapeutics Skills ? In vivo pharmacology experience - Animal behavior (inflammation models, feeding) - Drug administration (IP and PO routes) - Surgical techniques in mice - Pharmacokinetics ? HPLC-MS: Developed an LC/MS method to quantify enzymatic activity of novel lipases, as well as to measure the levels of endogenous lipids in tissues ? Enzymology: Michaelis-Menten analysis and dialysis experiments to measure mechanism of inhibition and reversibility of inhibitors ? Cell culture xi

PR EV

IE

W

- Mammalian cells - Human embryonic stem cells ? Molecular cloning and expression techniques ? Western blotting ? RNA isolation and quantitative PCR Work Experience Italian Institute of Technology Oct 2009 – Oct 2010 Drug Discovery and Development Consultant ? Assist in technology transfer and assay development for implementation of drug discovery program Nutrilite Health Products Concentrate Development Engineering Intern Apr – Jul 2005 Jun – Sep 2004

? Performed chemical and physical tests on soils to determine their corrosivity ? Performed in-the-field electromagnetic conductivity and four-pin resistivity tests ? Executed and analyzed electrochemical-noise corrosion study in a city water treatment plant Chevron Products Company, El Segundo Maintenance Intern ? ? ? ? Jun – Sep 2001

Activities and Affiliations ? Alliance for Graduate Education and the Professoriate Sep 2008 – present (AGEP) Planning Committee Member, UCI - Workshop Chair - Performed outreach, recruitment, and retention activities working with minority students xii

PR EV

MJ Schiff & Associates, Inc. Corrosion Consulting Engineers Laboratory, Field Technician

IE

? Participated in vertical integration of citrus concentrate products to enhance profit margins and quality control ? Modeled concentrate granulation scale-up to help engineers take processes from the pilot to the manufacturing plant Dec 2002 – Jul 2005

Repaired leaky valves and flanges to AQMD compliance Performed quarterly preventative maintenance on pumps and turbines Performed hydro tests on piping and machinery prior to field installation Disassembled pumps and compressors to replace seals and bearings

W

? ? ? ? ?

- Planned workshops, seminars, and networking opportunities for minority students Graduate Student Representative, Sep 2008 – Jun 2009 Department of Pharmacology Society for Neuroscience Aug 2008 – present Society for the Advancement of Chicanos and Aug 2008 – present Native Americans in Science (SACNAS) Omega Chi Epsilon May 2004 – present Chemical Engineering Honor Society American Institute of Chemical Engineers Sep 2003 – Jun 2004 - Chapter Secretary

Professional Certifications Engineering in training California Board for Professional Engineers and Land Surveyors Jun 2004

PR EV
xiii

IE

W

ABSTRACT OF DISSERTATIO Discovery and Characterization of Potent and Selective Inhibitors against the PEAhydrolyzing enzyme, N-acylethanolamine-hydrolyzing Acid Amidase by Carlos E. Solorzano Doctor of Philosophy in Pharmacology and Toxicology University of California, Irvine, 2010 Professor Daniele Piomelli, Chair

The fatty acid ethanolamides (FAEs) are a family of signaling lipids involved in

best characterized member of this family is anandamide, which exerts its multiple effects by activating cannabinoid receptors, type-1 and type-2. Despite the structural similarity of two other members of this class, palmitoylethanolamide (PEA) and

oleoylethanolamide (OEA), their effects are not mediated by activation of cannabinoid receptors, but rather activation of the nuclear receptor peroxisome proliferator-activated receptor type-alpha (PPAR-α). PEA is a potent anti-inflammatory compound that was first isolated from plant and animal tissues over 50 years ago. Though the

pharmacological actions of PEA are well understood, its endogenous roles remain unclear. In the present dissertation, I used a biochemical and pharmacological approach to elucidate the intrinsic roles of PEA in the regulation of the inflammatory response. In chapter 3, I found that PEA levels in immune cells are markedly reduced by inflammatory stimuli, and in collaboration with medicinal and computational chemists, I xiv

PR EV

IE

regulating various physiological processes such as feeding, pain and inflammation. The

W

discovered the first potent and selective inhibitors of the PEA-hydrolyzing enzyme,

-

acylethanolamine-hydrolyzing acid amidase (NAAA). This class of NAAA inhibitors, derived from a beta-lactone active warhead, normalizes PEA levels in activated immune cells and exerts marked anti-inflammatory effects. In chapter 4, we optimized the

potency of this class of compounds by structure activity relationships, and identified (S)-(2-oxo-3-oxetanyl)-4-biphenylamide, which inhibits NAAA with an IC50 value of 115nM, and blunts inflammatory reactions induced by carrageenan in vivo. Together, insights into the regulation of PEA levels by activated immune cells, and the marked anti-inflammatory effects of agents which prevent PEA hydrolysis, suggest that PEA is an intrinsic anti-inflammatory signal, which negatively regulates the activation of immune cells by inflammatory triggers. This role distinguishes PEA from other known lipid mediators, which either incite inflammatory reactions (i.e. prostaglandins) or terminate it by promoting resolution and tissue healing (i.e. lipoxins and resolvins). My work has contributed to our understanding of the endogenous roles of PEA, and provides the basis for future studies investigating its role in chronic inflammatory conditions, which could result in novel therapeutic strategies against such diseases.

PR EV

IE
xv

W

Chapter 1 The Fatty Acid Ethanolamides with emphasis on Palmitoylethanolamide
Introduction The fatty acid ethanolamides (FAEs) are a class of lipid messengers involved in the regulation of various physiological functions. Unlike water-soluble cellular

messengers, the fatty acid ethanolamides are not stored in synaptic vesicles, but are synthesized and released on-demand from membrane precursors stored in the cell

FAE, is one of the two best-characterized endogenous agonists of cannabinoid receptors. In contrast, and despite their structural similarity, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) exert their effects through activation of the nuclear receptor peroxisome-proliferator activated receptor-α (PPAR-α). The chemical structures of anandamide, PEA and OEA are shown in Figure 1.1. In this chapter, I first review the discovery of the FAEs and then discuss their roles in mammalian organisms as well as their biosynthetic and catabolic pathways. Finally, in the metabolism section, I discuss the regulation of PEA levels by activated macrophages. This is the project of a former graduate student in the lab, Dr. Chenggang Zhu, though I also contributed to the results in a significant way. I chose to describe these results briefly because they complement my work, and altogether, provide a more holistic picture of the roles of PEA in inflammation. The whole of my thesis has consisted in investigating the endogenous roles of PEA, and therefore, particular attention will be devoted to this molecule.

PR EV

IE
1

W

membrane. Arachidonoylethanolamide (also known as anandamide), the best known

Discovery The FAEs can be found in plants and animal tissues, where they participate in the regulation of various physiological functions including appetite, pain, mood, inflammation and systemic immunity (Chapman, Tripathy et al. 1998; Piomelli 2003). The identification of the FAEs dates back more than 50 years when a member of this class, PEA, was found to be the active substance responsible for the anti-inflammatory effects of peanut oil, egg yolk, and soybean lecithin (Kuehl 1957). This finding fueled the search for PEA in mammalian cells, which occurred soon thereafter (Bachur 1965). Despite the potent anti-inflammatory effects of PEA, which were documented in various animal and human studies (Masek, Perlik et al. 1974; Kahlich, Klima et al. 1979; Berdyshev 1998; Costa, Conti et al. 2002; Lo Verme, Fu et al. 2005; Genovese, Esposito et al. 2008; De Filippis, D'Amico et al. 2009), interest in the physiology of PEA and the FAEs remained minimal until the identification of anandamide as an endogenous ligand for cannabinoid receptors (Devane, Hanus et al. 1992). Soon after the molecular cloning of cannabinoid receptors (Matsuda, Lolait et al. 1990; Munro, Thomas et al. 1993), which bind the active constituent in marijuana, ?9tetrahydrocannabinol (?9-THC) (Adams 1942; Gaoni and Mechoulam 1964), a quest for their endogenous ligands began. Devane and colleagues succeeded in isolating an active fraction from porcine brain that competitively decreased binding of a specific cannabinoid probe, and elucidated the structure of this compound (Devane, Hanus et al. 1992). Following identification of anandamide as an integral component of the

endocannabinoid system (Devane, Hanus et al. 1992), renewed interest in the

PR EV

IE
2

W

biochemistry and function of other FAEs arose. A timeline describing the discovery and progress by studies on the pharmacology of PEA is shown in Figure 1.2.

Biological Actions Despite the previously held belief that lipid-derived molecules exert their physiological effects through non-specific interactions with phospholipids and components of the cellular membrane thereby affecting its fluidity (Hillard, Harris et al. 1985), it is now known that various classes of lipids, including the prostaglandins and leukotrienes serve as signaling molecules through their high-affinity and specific interactions with proteins (Flower 2006). The FAEs are not an exception, regulating various physiological functions by interacting with cellular proteins in a selective manner (Lo Verme, Fu et al. 2005; Mackie and Stella 2006).

Anandamide

Anandamide binds cannabinoid receptors of the type-1 and 2 (CB1 and CB2 receptors, respectively). CB1 receptors are primarily and abundantly found in the brain, whereas CB2 receptors are mainly expressed in cells of the immune system. CB1 and CB2 receptors are seven transmembrane spanning G-protein coupled receptors (GPCR) that couple to the Gi/o subtype of G-proteins that inhibit adenylyl cyclase, leading to decreased cellular concentrations of cAMP (Devane, Dysarz et al. 1988; Matsuda, Lolait et al. 1990; Munro, Thomas et al. 1993). Activation of cannabinoid receptors decreases calcium channel activity and increases potassium channel activity resulting in reduced cellular excitability (Mackie and Hille 1992; Mu, Zhuang et al. 1999). Consistent with

PR EV

IE
3

W

the effects exerted by ?9-THC or synthetic cannabinoid agonists, administration of anandamide decreases spontaneous motor activity, lowers rectal body temperature, and produces analgesia (Crawley, Corwin et al. 1993; Fride and Mechoulam 1993). The roles played by endogenous anandamide, however, can not be discerned by its pharmacological administration. Anandamide is rapidly hydrolyzed by the intracellular membrane-bound serine hydrolase, fatty acid amide hydrolase (FAAH) (McKinney and Cravatt 2005). FAAH activity was first discovered in brain microsomes (Deutsch and Chin 1993; Desarnaud, Cadas et al. 1995) and was later cloned from rat liver plasma membranes (Cravatt, Giang et al. 1996). The molecular identification of FAAH opened up many experimental

possibilities to probe into the endogenous roles of anandamide. Indeed, discovery of the potent and selective FAAH inhibitor, URB597, has allowed for the elucidation of various roles played by anandamide (Kathuria, Gaetani et al. 2003). Administration of the FAAH inhibitor URB597 to animals has revealed that anandamide signaling regulates mood, pain, anxiety, and responses to stress (Kathuria, Gaetani et al. 2003; Bortolato, Mangieri et al. 2007; Russo, Loverme et al. 2007). For instance, administration of URB597 to rodents elevates brain anandamide levels by about 3-fold and exerts anxyolitic-like effects in the elevated plus maze, which are blocked by the CB1 antagonist, rimonabant (Kathuria, Gaetani et al. 2003). In the chronic mild stress model of depression,

administration of URB597 attenuates the loss in body weight and sucrose intake induced by this paradigm with an efficacy comparable to that of the tricyclic anti-depressant imipramine (Bortolato, Mangieri et al. 2007). Other FAAH inhibitors besides URB597 have been developed, and they have been used to unmask the actions of endogenous

PR EV

IE
4

W

anandamide (Trang 2007). At present, FAAH inhibitors are the focus of many studies to evaluate their efficacy as analgesics, and thus far, the results are very promising (Russo, Loverme et al. 2007; Trang 2007). A summary of the physiological roles of anandamide is found in Table 1.1.

Oleoylethanolamide A second FAE that has attracted much attention is oleoylethanolamide (OEA). When administered as a drug, OEA reduces feeding and body mass by activating the ligand-activated transcription factor PPAR-α (Rodriguez de Fonseca, Navarro et al. 2001; Fu, Gaetani et al. 2003). These effects are mediated through the peripheral nervous system and result in activation of brain regions involved in the regulation of feeding and satiety such as the paraventricular nucleus and the nucleus of the solitary tract (Rodriguez de Fonseca, Navarro et al. 2001). More interesting is the fact that OEA metabolism is tightly regulated in the proximal small intestine by feeding: OEA levels increase postprandrially, and they are lowered during starvation or between meals (Fu, Gaetani et al. 2003; Fu, Astarita et al. 2007). These findings suggest that OEA signaling at PPAR-α serves as a sensor for food intake. Indeed, a recent study showed that OEA synthesis in the proximal small intestine is induced by dietary fatty acids, but not protein or sugars, and that OEA signaling via PPAR-α serves as a satiety signal (Schwartz, Fu et al. 2008). Table 1.2 lists the pharmacological properties of OEA.

Palmitoylethanolamide

PR EV

IE
5

W

PEA’s discovery was the effort of a search for the anti-inflammatory factor in dried egg yolk, peanut oil and soybean lecithin (Long 1956; Kuehl 1957). Soon after its discovery, the pharmacological properties of PEA began to be investigated in various animal models of inflammation, and it was briefly used to treat influenza symptoms in humans (Masek, Perlik et al. 1974). The pharmacological effects of PEA include antiinflammation (Kuehl 1957; Mazzari, Canella et al. 1996; LoVerme, La Rana et al. 2005), analgesia (Calignano, La Rana et al. 1998; Jaggar, Hasnie et al. 1998), anti-epilepsy and neuroprotection (Lambert, Vandevoorde et al. 2001; Franklin, Parmentier-Batteur et al. 2003). The anti-inflammatory and analgesic effects of PEA have been found in our lab to be mediated through activation of PPAR-α (Lo Verme, Fu et al. 2005; LoVerme, Russo et al. 2006). Though best known for its roles in the transcriptional regulation of lipid metabolism (Schoonjans, Staels et al. 1996), PPAR-α has been also implicated in the inflammatory process, which it may influence by modulating the activities of nuclear factor (NF)-κB, activating protein (AP)-1, and inhibitor of κB kinase complex (Glass and Ogawa 2006). In transactivation assays, PEA activated PPAR-α in vitro with an EC50 value of ~3?M. Furthermore, the anti-inflammatory and analgesic effects of PEA were absent in mice lacking PPAR-α (Lo Verme, Fu et al. 2005). These findings are consistent with the fact that 1) peroxisome proliferators such as the fibrates, which also activate PPAR-α, have anti-inflammatory effects in addition to their well-known lipid-lowering effects (Lo Verme, Fu et al. 2005), and 2) mice lacking PPAR-α have an exacerbated response to inflammatory stimuli compared to wild-type mice (Devchand, Keller et al. 1996; Cuzzocrea, Mazzon et al. 2006).

PR EV

IE
6

W

The functions of PEA in the central nervous system (CNS), where PEA is present at high levels (Cadas, di Tomaso et al. 1997), have also been investigated with growing interest. Evidence suggests that PEA is an antiepileptic and neuroprotective compound (Lambert, Vandevoorde et al. 2001; Sheerin, Zhang et al. 2004). In one study, PEA protected against maximal electroshock and pentylenetetrazol-induced seizures (Lambert, Vandevoorde et al. 2001). Recently, administration of PEA was shown to reduce

neuronal damage induced by spinal cord injury (Genovese, Esposito et al. 2008). Table 1.3 summarizes the pharmacological effects of PEA administration in animals.

Metabolism Biosynthesis

FAEs are produced from membrane phospholipids by two main enzymatic reactions. The first reaction is the transfer of a fatty acid from the sn-1 position of phosphatidylcholine to the free amine group of phosphatidylethanolamine resulting in the formation of an -acyl phosphatidylethanolamine (NAPE). This reaction is catalyzed by a Ca2+-dependent -acyltransferase in the brain and possibly by a Ca2+-independent -

acyltransferase in peripheral tissues (Piomelli 2003; Jin, Okamoto et al. 2007). The second step in FAE biosynthesis has been proposed to occur through one of three mechanisms: 1) the phospholipase D-mediated cleavage of NAPE by a NAPE-specific phospholipase D (NAPE-PLD) (Cadas, Gaillet et al. 1996; Okamoto, Morishita et al. 2005) resulting in the formation of FAE and phosphatidic acid; 2) the dual deacylation of NAPE by α/β hydrolase-4 (ABHD4) followed by the glycerophosphodiesterase mediated release of FAE and phosphoglycerol (Sun, Tsuboi et al. 2004; Simon and Cravatt 2006);

PR EV

IE
7

W


赞助商链接
相关文章:
EGFR晶体
selective EGFR kinase inhibitors against EGFR T790M...Structure-Based Approach for the Discovery of ...s as potent ErbB-2/EGFR dual kinase inhibitors....
更多相关标签: