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UNIVERSITY OF CALIFORNIA, SAN DIEGO Anti-inflammatory capabilities of compounds from marine bacteria in a mouse model of allergic inflammation and asthma A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Oceanography by Wendy K. Strangman Committee in charge: Professor William Fenical, Chair Professor Lihini Aluwihare Professor David Broide Professor William Gerwick Professor Victor Vacquier 2007 Copyright Wendy K. Strangman, 2007 All rights reserved. The dissertation of Wendy K. Strangman is approved, and is acceptable in quality and form for publication on microfilm: _____________________________________________ _____________________________________________ _____________________________________________ _____________________________________________ _____________________________________________ _____________________________________________ Chair University of California, San Diego 2007 iii TABLE OF CONTENTS Signature page……………………………………………………..……..…….......... iii Table of Contents………………………………………………………….…………. iv List of Figures……………………………………………………………………….…vi List of Tables………………………………………………………………………...…x List of Abbreviations……………………………………………………….……….…xi Acknowledgements…………………………………………………………………....xii Vita and Publications……………………..…………..………...…...……...….....….xvii Abstract…...………………………………………………………………...........….xviii I. Introduction to the Thesis Research..............................................................…...….....1 I.1. Overview of Asthma……………...……………………………………..……...2 Genetics…………………………………………………………… .………5 Hygiene Hypothesis………………………...……………...……………….5 Cellular Mechanisms and Cytokines…………………………...………..…7 I.2. Historical Summary of Asthma Drugs from Natural Products………..…....…12 I.3. Summary of Current Asthma Drug Therapy………………………….………16 Bronchodilators……………………...…………………………………….17 Anti-inflammatories………………………………………….…………....19 Biologics…………………………………………………………………..22 References…………………………………………………….………..….24 II. Screening a Natural Products Extract Library for In Vitro Inhibition of Pro- Inflammatory Cytokine Production by Ova Stimulated Splenocytes………………27 II.1. Natural Products Drug Discovery: Sources and Strategies….…..………....…28 iv II.2. Development and Application of an In Vitro Assay for Detecting Compounds that Inhibit the Production of Pro-Inflammatory Cytokines….....37 II.3. Hit Identification from the Fenical Laboratory’s Marine Microbial Extract Library ………………………………………………………………….….....45 II.4. Collaborative Efforts to Discover New Inhibitors of Cytokine Production………………………………………………………………….…57 References………………………………………………………………………....61 III. Splenocins: Potent Inhibitors of Allergic Inflammation…….………………........64 III.1. Splenocins A-J: Project Overview.……………………………………….….65 III.2. Splenocins A-J: Isolation and Structure Elucidation………………………...69 III.3. Activity of Splenocins A-J in the Mouse Splenocyte Assay.……...…......….98 III.4. Experimental………………………………….………………………….….105 References…………………….……………………………………………..….…110 IV. Anti-Inflammatory Nor-Diterpenes from Marine Bacteria: Marinenes A and B...112 IV.1. Bacterial Terpene Production……………………….……………...….……113 IV.2. Culturing, Isolation and Structure Elucidation of Marinenes A and B……..118 IV.3. In Vitro Cytokine Inhibition Studies…...…………….……………...…..….133 IV.4. Experimental………………………………………………………………...138 References………………………………………………………….…………..….141 V. Conclusion..…………………………………………………………………….…144 v LIST OF FIGURES Chapter I.1 Figure I.1a. Generalized schematic of pathways involved in the asthmatic cellular cascade……………………………………………………………………..8 Chapter I.2 Figure I.2a. The active components of historically used natural products in the treatment of asthma……………….……….……………..………...……..13 Chapter I.3 Figure I.3a. Structures of some drugs currently prescribed for the treatment of asthma…..……………………………………………….………………..18 Figure I.3b. Corticosteroid carbon skeleton. Arrows indicate structural features with important biological implications.………………………......……....20 Chapter II.2 Figure II.2a. ELISA assay schematic......…..……………………………..…….....…..41 Chapter II.3 Figure II.3a. Known compounds that display activity in the splenocyte assay……..…52 Chapter II.4 Figure II.4a. IC50 values of compounds isolated by collaborators that inhibited IL-5 production of OVA stimulated splenocytes.………..……………....…....58 Figure II.4b. New compounds with selective cytotoxicity against splenocytes…..…...60 Chapter III.1 Figure III.1a. Structures of splenocins A-J (1-10), isolated 8-hydroxy antimycin analogues (11-14), and isolated antimycins (15-18)…………………....67 Chapter III.2 Figure III.2a. Substructures and key HMBC correlations used to define the structure of splenocin A (1)………...........…....…....……………..……73 vi Figure III.2b. Expansion of part of the gCOSY NMR spectrum for splenocin A(1) in CDCl3. Key COSY correlations for the assemblage of substructure (i) are noted.…………………………………………....…74 Figure III.2c. Expansion of part of the gHMBC NMR spectrum for splenocin A (1) in CDCl3. Key HMBC correlations for the assembly of substructure (ii) are noted..……………………………………………..76 Figure III.2e. Relative configuration of splenocin J (10). Arrows indicate key NOESY correlations…………...…………………………………….....85 Figure III.2f. NOESY spectrum for splenocin J (10). Key correlations are noted…..………………………………………………………………..86 Figure III.2g. Proposed mechanism for deformylation and subsequent acylation of splenocin J(10)……….…...…………………………………………….88 Figure III.2h. The structures of splenocin J derivatives from Mosher acylation and the differentiation of 1H NMR chemical shift values between S and R Mosher esters (∆S-R). (i) Splenocin J (10) 2', 3'-di-Mosher derivatives and ∆S-R values. (ii) Splenocin J (10) 2', 3', 7-tri- Mosher derivatives and ∆S-R values. (iii) Tri-Mosher ∆S-R - di-Mosher ∆S-R......91 Figure III.2i. Orientations of L (i) and D (ii) threonine as would be seen with cis-relative configurations within the dilactone ring…..…...……….….92 Figure III.2j. Representative CD spectra of splenocin structural groups indicating configuational similarity……………...……...………………………....94 Figure III.2k. Conformational structure (i) and vector diagram (ii) for splenocins D-I (4-9) and 10g illustrating the observed CD Cotton effect.……........95 Figure III.2l. Reaction scheme for the benzoylation of splenocins I (9) and J (10) into identical tri-benzoylated products (9b, 10g) and corresponding CD spectra…………….……………………………………..………....96 Chapter III.3 Figure III.3a. Interleukin 5 (IL-5) levels of cultured splenocytes. (A) unstimulated control. (B) OVA-stimulated control. (C) OVA- stimulated + Splenocin B (2) (5nM). (D) OVA-stimulated + dexamethasone (5nM)………...………………………...……………100 vii Figure III.3b Splenocyte cytokine inhibition: Levels of Th2 (IL-5, IL-13) and APC related (IL-1, TNF-α) cytokines of OVA-stimulated splenocytes incubated with either splenocins B or G (2 or 7) at 10nM, or dexamethasone at 10nM..…………………………...….......102 Chapter IV.1 Figure IV.1a. Some known pure terpenes produced by bacteria……………..….......116 Figure IV.1b. Structures of marinenes A and B…………...……………..........…......117 Chapter IV.2 Figure IV.2a. Proton NMR spectrum of marinene A in acetone-d6…….…….……...120 Figure IV.2b. Expansion of TOCSY NMR spectrum. Key 1H-1H correlations are noted. Spin systems are indicated by bold lines in structure inset..….123 Figure IV.2c. Key HMBC correlations for establishing the planar structure of marinene A…………………………………………………………….124 Figure IV.2d. gHMBC NMR spectrum of marinene A. Key correlations are noted…………………………………………………………………...125 Figure IV.2e. Structures of Trifoliones………………….……………………………126 Figure IV.2f. Relative configuration of marinene A. Arrows indicate key NOESY correlations………………………………………..………….126 Figure IV.2g. NOESY spectrum for marinene A. Key 1H-1H NOESY correlations are noted…………………………………………..……...127 Figure IV.2h. Structure and relative configuration of marinene B. Arrows indicate key NOESY correlations……………………...……………...130 Figure IV.2i. IR spectra of marinenes A and B………………………………………131 Chapter IV.3 Figure IV.3a. Non-cytotoxic inhibition of IL-5 production in OVA stimulated splenocytes by Marinenes A and B…………..………………………..135 viii Figure IV.3b. Inhibition of OVA-stimulated splenocyte APC (IL-1) and Th2 (IL-5 and IL-13) cytokine production by marinenes A and B at 8 µg/ml………………………………………………………………...136 Figure IV.3c. Structure of acanthoic acid……………………………………..……...137 ix LIST OF TABLES Chapter II.1 Table II.1a. New Chemical Entities and Medical Indications by Source of Compound (Edited from complete reference courtesy of Dr. David Newman, personal communication).6 (B) = biologics including large peptides and proteins, (N) = Natural Products, (ND), = Natural Product Derivatives – molecules that have semi-synthetic modifications to a natural product, (S) = Synthetic molecules, (S*) = Synthetic molecules with a natural product derived pharmacaphore, and (V) = Vaccines……………………..……………………...…….........30 Chapter II.3 Table II.3a. HCT-116 cell survival data for marine microbial extracts exhibiting greater than 75% IL-5 inhibition and less than 50% cytotoxicity in the splenocyte assay……………...………………….……………………......48 Table II.3b. Microbial strains and compounds observed and/or isolated from them.…55 Chapter III.2 Table III.2a. NMR data from splenocins A-C (1-3) in CDCl3..…...……….…..….…..71 Table III.2b. NMR data from splenocins D-H (4-8) in CDCl3………………...….…...79 Table III.2c. NMR data from splenocins I and J (9 and 10) in CDCl3.……....…….….82 Table III.2d.