UNIVERSITY of CALIFORNIA, SAN DIEGO Discovery And

UNIVERSITY OF CALIFORNIA, SAN DIEGO

Discovery and characterization of calcium-dependent antibiotics via activation of a silent natural product gene cluster

A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy

Chemistry

Kirk Alan Reynolds

Committee in charge:

Professor Bradley Moore, Chair Professor Pieter Dorrestein, Co-Chair Professor Michael Burkart Professor William Fenical Professor Judy Kim

2016

The dissertation of Kirk Alan Reynolds is approved, and it is acceptable in quality and form for publication on microfilm and electronically:

______

______Co-Chair

______Chair

University of California, San Diego

2016

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Dedication

This dissertation is dedicated to my beautiful wife Hannah. Throughout our years together she has endured the late nights, cancelled trips, and the challenges of graduate student life with a grace and poise beyond what I have deserved. While there are teachers, colleagues, friends, and parents who have invested in me throughout my life, it is by Hannah’s generosity of heart that I continue to find my inspiration to endure the most challenging times. This dissertation is dedicated to Hannah whose unconditional love, support, and sacrifice has made it possible for me to achieve the successes that are described herein.

Table of Contents

Signatures Page: ...... iii Dedication ...... iv List of Figures ...... vi List of Tables ...... ix Acknowledgements ...... x Vita ...... xii Abstract of the Dissertation ...... xiii Chapter 1: Introduction to Natural Products ...... 1 Chapter 2: Direct cloning and refactoring of a silent lipopeptides biosynthetic gene cluster yields the antibiotic taromycin A ...... 39 Chapter 3: Isolation and Structure Elucidation of Taromycin B...... 68 Chapter 4: Future Growth of Tar Cloning and the Taromycin Series ...... 100

List of Figures Figure 1.1: Historically relevant terrestrial natural products...... 2 Figure 1.2: Marine natural products and analogues that are currently used in the clinic or are in phase I/II clinical trials ...... 3 Figure 1.3: Examples of aminoglycoside antibiotics isolated from actinobacterial sources ...... 7 Figure 1.4: Peptides offer a diverse collection of antibiotics ...... 10 Figure 1.5: Sequenced genome of Streptomyces coelicolor ...... 16 Figure 1.6: Gene cluster organization for the expression and production of

Epothilone A (R=H) and B (R=CH3) ...... 19 Figure 1.7: (A) Coelichelin (17) one of the earliest secondary metabolites isolated from genome mining of S. coelicolor. (B) Oxidation of epi-isozizaene (18) into albaflavenol by cytochrome P-450...... 21 Figure 1.8: Gene cluster organization for the putative lodopyridone biosynthetic gene cluster ...... 25 Figure 1.9: Examples of proposed β -carbon chemistry ...... 28 Figure 1.10: Proposed biosynthesis of lodopyridone...... 29 Figure 1.10: Proposed biosynthesis of lodopyridone...... 30 Figure 2.1. Design and strategy of TAR-based cloning and expression ...... 41 Figure 2.2. Gene organization of lipopeptide biosynthetic gene clusters ...... 42 Figure 2.3. Physical maps of the TAR-cloned tar gene cluster and the pKY01- based complementation vectors ...... 43 Figure 2.4. HPLC analysis of the taromycins produced heterologously by S. coelicolor mutants and structures of daptomycin and taromycin A...... 43 Figure S2.1. Tar cloning of the taromycin biosynthetic gene cluster (tar) ...... 55 Figure S2.2. Restriction maps of the TAR-cloned tar gene cluster ...... 56 Figure S2.3. HPLC-MS analysis of the taromycins produced by S. coelicolor M1146/pCAP01-tarM2 ...... 57 Figure S2.4 Effect of genetic complementation with tar regulatory genes in S. coelicolor M1146 / pCAP01-tarM1 (∆tar19 sarp, ∆tar20 luxR ...... 58 Figure S2.5. Effect of genetic complementation of the tar regulatory genes in S. coelicolor M1146 / pCAP01-tarM2 (∆tar20 luxR) ...... 58

Figure S2.6 Schematic diagram for construction of the codon redressed tar20 luxR regulatory gene ...... 59 Figure S2.7 Effect of genetic complementation of the codon redressed tar20 luxR regulatory gene...... 59 Figure S2.8 HSQC spectra of daptomycin aromatic region (d6-DMSO, 600 MHz) ...... 61 Figure S2.9 HSQC spectra of taromycin A aromatic region (d6-DMSO, 600 MHz) ...... 62 Figure S2.10. MS and MSn analysis of taromycin A. MS 2 ...... 63 Figure S2.11 MS and MSn analysis of taromycin ...... 64 Figure S2.12 HPLC analysis of the marinopyrrole A produced by S. coelicolor mutants ...... 65 Figure 3.1: lipopeptides showcasing fatty acid side chain diversity as well as diverse amino acid sequences...... 68 Figure 3.2: Chromatograph of the taromycin series produced by M1146-M1 showing 10 distinct taromycin compounds ...... 71 Figure 3.3: MS and MSn analysis of taromycin B ...... 73 Figure 3.4: Selected 2D NMR correlations for taromycin B side chain...... 75 1 Figure S3.1: H NMRspectrum of taromycin B (d6-DMSO, 600 MHz)...... 82

Figure S3.2: COSY NMR spectrum of taromycin B (d6-DMSO, 600 MHz)...... 83

Figure S3.3: TOCSY NMR spectrum of taromycin B (d6-DMSO, 600 MHz). .... 84

Figure S3.4: HSQC NMR spectrum of taromycin B (d6-DMSO, 600 MHz)...... 85

Figure S3.5: HMBC NMR spectrum of taromycin B (d6-DMSO, 600 MHz) ...... 86

Figure S3.6: HSQC spectra of daptomycin aromatic region (d6-DMSO, 600 MHz) ...... 87

Figure S3.7: HSQC spectra of taromycin B aromatic region (d6-DMSO, 600 MHz) ...... 88 Figure S3.8: HPLC chromatogram showing rotomeric nature of the taromycin A molecule ...... 89 Figure S3.9: HPLC chromatogram showing rotomeric nature of the taromycin B molecule ...... 91

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Figure 4.1: Deacylation of N-Orn-Boc-taromycin series to N-Orn-Boc-taromycin nucleus ...... 101

Figure 4.2: Direct production of taromycin nucleus ...... 101

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List of Tables Table 1.1: Evolution of resistance to clinical antibiotics...... 14 Table 1.2: AntiSMASH gene cluster assignment for Saccharomonospora sp. CNQ-490...... 24 Table 1.3: Detailed analysis of putative lodopyridone genes: Size, proposed role, and known similar protein ...... 26 Table S2.1. Strains and Plasmids Used in this study ...... 52 Table S2.2. Deduced function of ORFs in the tar locus in Saccharomonospora sp. CNQ-490...... 53 Table S2.3. NMR correlations of the daptomycin Kyn and Trp residues (d6- DMSO, 600 MHz) ...... 60 Table S2.4. NMR correlations of the taromycin A 4-Cl-Kyn and 6-Cl-Trp residues (d6-DMSO, 600 MHz) ...... 60 Table S2.5. Retention times (in min) of taromycin A and daptomycin constituent amino acids ...... 62 Table S2.6. Taromycin A and daptomycin minimum inhibitory concentration ..... 64

Table 3.1: NMR table for taromycin B side chain (d6-DMSO, 600 MHz) ...... 75 Table S3.1: Selected 2D NMR correlations for taromycin B (aromatic region) .. 80 Table S3.2: MS and MSn analysis of taromycin B ...... 81 Table S3.3: Taromycin A, taromycin B, and daptomycin minimum inhibitory concentrations ...... 81

Acknowledgements

I would like to thank my advisor Professor Bradley S. Moore for taking me on as a Ph.D. student in spite of my unique situation as a graduate student in the

Department of Chemistry and Biochemistry. Prof. Moore has provided an incredible environment to thrive in as both a scientist and as a person. His collaborative spirit promotes a work environment where graduate students and postdoctoral scholars exchange ideas freely, leading to high quality research and training. I would also like to thank Dr. Kazuya Yamanaka for his collaboration on the taromycin project as well as training in genetics and molecular biology.

Additionally, I would like to thank Dr. Leonard Kaysser, Dr. Roland Kersten, Dr.

Roland Winter, and Dr. Vinayak Agarwal for their mentorship at the lab bench throughout my time in the Moore group. While the members of the Moore group have changed over the years, it remains a place of scientific curiosity, excellent training, and a fantastic group of people. To all those who have aided me during my graduate school career, thank you.

Chapter 2, in full, is a reprint of materials as it appears in “Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A” in Proc. Natl. Acad. Sci. USA, 2014, Yamanaka, K.,

Reynolds, K. A., Kersten, R. D., Ryan, K.S., Gonzalez, D. J., Nizet, V.,

Dorrestein, P. C., Moore, B. S. The Dissertation author was one of two equally contributing primary investigators and authors of this paper.

Kazuya Yamanaka (K.Y.) was responsible for the genetic and molecular biology contributions to this work (gene cluster identification and capture). Kirk

A. Reynolds (K.A.R) was responsible for the organic and analytical chemistry contributions to this work (isolation and structure elucidation of the taromycin molecule). Roland D. Kersten (R.D.K.) provided assistance with mass spectrometry instrumentation. Kaity S. Ryan (K.S.R.) contributed to the identification of the silent gene cluster prior to this study. David J. Gonzaelz

(D.J.G.) and Prof. Victor Nizet (V.N.) contributed the bioactivity assays for the taromycin molecule. Prof. Pieter C. Dorrestein (P.C.D) contributed new mass spectrometry tools. Prof. Bradley S. Moore (B.S.M.) K.Y., K.A.R., R.D.K.,

D.J.G., V.N. analyzed data and K.Y., K.A.R., and B.S.M. wrote the paper.

Chapter 3, in part is currently being prepared for submission for publication of the material. Reynolds, K.A.; Moore, B.S. The dissertation author was the primary investigator and author of this material.

Vita

2016 Ph.D., Chemistry

Supervisor: Prof. Bradley S. Moore University of

California at San Diego, San Diego, CA

2012 M.S., Chemistry University of California at

San Diego, San Diego, CA

2010 B.S., Chemistry Westmont College,

Santa Barbara, CA

PUBLICATIONS

Yamanaka, K.; Reynolds, K.A.; Kersten R.D.; Ryan K.S.; Gonzalez D.J.; Nizet, V.; Dorrestein P.C.; Moore, B.S. Direct Cloning and Refactoring of a Silent Lipopeptide Biosynthetic Gene Cluster Yields the Antibiotic Taromycin A. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 1957.

Molinski, T.F.; Reynolds, K.A.; Morinaka, B.I. Symplocin A, a Linear Peptide from the Bahamian Cyanobacterium Symploca sp. Configurational Analysis of N,N-Dimethylamino Acids by Chiral-Phase HPLC of Naphthacyl Esters. J. Nat. Prod. 2012, 75, 425.

Molinski, T.F.; Ko, J.; Reynolds, K.A.; Lievens, S.C.; Skarda, K.R. N,N’- Methyleno-didemnin A from the Ascidian Trididemnum solidum. Complete NMR Assignments and Confirmation of the Imidazolidinone Ring by Strategic Analysis of 1JCH. J. Nat. Prod. 2011, 74, 882.

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Abstract of the Dissertation

Discovery and characterization of calcium-dependent antibiotics via

activation of a silent natural product gene cluster

Kirk Alan Reynolds

Doctor of Philosophy in Chemistry

University of California, San Diego, 2016

Professor Bradley S. Moore, Chair

Professor Pieter C. Dorrestein, Co-Chair

The field of natural products has enjoyed great success in identifying novel molecules from both the terrestrial and marine environments. These novel chemical discoveries have gone on to inspire pharmaceutical and biosynthetic chemists to understand the complex relationships between novel chemistry and the producing organism, the respective microbiome, and human disease. Many modern natural product success stories have started in the field or in cultivation

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rooms leading to novel chemistry with incredible diversity in chemical structure and bioactivity. While these traditional approaches to natural products have been invaluable to the field, the addition of genome sequencing has furthered our understanding of the chemical potential of marine microbes.

Reported herein is a brief synopsis of the history of marine natural products and its further complementation by the genetic sequencing revolution.

Chapter 1 details the power of sequencing information in finding new and previously inaccessible natural products. Following this introduction, Chapter 2 describes the discovery of a “silent” biosynthetic gene cluster with similarities to the gene cluster associated with the potent antibiotic daptomycin. The isolation of this gene cluster by the novel methodology transformation-associated- recombination (TAR) cloning is then described. The activation of this gene cluster by regulator knockout is then detailed as well as the isolation and structure elucidation of the novel antibiotic taromycin A. Chapter 3 then delves further into the taromycin series with the isolation and structure elucidation of taromycin B. The unexpected rotomerization of taromycin B and the isolation and structure elucidation challenges produced as a result are discussed.

Comparative bioactivity results reveal the more potent of the two novel antibiotics and these results are discussed in the historical context of daptomycin bioactivity optimization. Chapter 4 concludes this dissertation with a discussion of possible future studies with the taromycin series, TAR cloning, and genome mining as a source for novel natural products.

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Chapter 1: Introduction to Natural Products

Nature’s use of small molecules to communicate, attack, and defend is among the most fascinating achievements of evolution. The questions of novel chemical structure, biosynthesis, and human application continue to make the study of natural products a rich and exciting field. The path of human application of potent natural products can be found throughout ancient times and across cultures.1 Mesopotamian, Chinese, Greek, and later European “pharmacists” were well versed in the use of a diverse collection of herbs, foliage, and soil to treat the sick. Some of the early ailments treated included sore throat, colds, and inflammation using oils from early terrestrial, medicinal plants including

Cupressus sempervirens (Cypress) as well as Commiphora species (myrrh).2

1.1 Historically significant terrestrial natural products

Some of the earliest characterized terrestrial natural products have become staples of modern medicine (Figure 1).3 Salicin, isolated from the bark of the tree Salix alba, can be processed into salicylic acid. Synthetic acetylation of salicylic acid yields acetylsalicylic acid (1), commonly known as Aspirin.

Another example comes from the poppy Papaver somniferum, from which the potent pain killer morphine (2) is extracted. Finally, the antibiotic penicillin (3) isolated from the fungus Penicillium notatum represented the promise of natural products as a legitimate source of pharmaceuticals and kicked off the golden age of antibiotics in the 1940’s.4 These representative natural products illustrate the great diversity found in structure and bioactivity in terrestrial secondary

2 metabolites. With such a wealth of chemistry found on land, the natural evolution of the field shifted towards the marine environment as a source for new natural products.

Figure 1 .1: Historically relevant terrestrial natural products displaying a wide range of structural diversity (1-3). Compound 4 shows the high degree of halogenation observed in the marine environment.

1.2.1 Early examples of marine natural products

With the advent of self-contained underwater breathing apparatus

(SCUBA), the marine environment became available as a source of novel marine natural products beginning in the 1950s. Some of the earliest marine natural products observed were highly halogenated aromatic molecules. The high propensity for brominated and chlorinated molecules became a hallmark of the earliest isolated marine natural products. Hexabromo-2,2’-bipyrrole (4) isolated in 1974 from a marine Chromobacterium displayed the striking contrast between marine and terrestrial natural products.5 Compound 4 and other secondary metabolites also observed alongside showed a high degree of halogenation relative to natural products found in terrestrial environments. The incorporation of chlorine and bromine into natural product scaffolds by the producing organisms has led to a chemical diversity rarely seen on land.6 The original investigators of compound 4 and related compounds also noted modest antibiotic

3 activity, lending early credibility to the marine environment as a source for novel, bioactive natural products.

1.2.2 Marine natural products as anticancer drugs

The promise of bioactivity and new pharmaceuticals drove isolation chemists to continue scouring the world’s oceans. New molecules with unprecedented molecular scaffolding and bioactivity were discovered establishing the ocean as an untapped source for novel drug discoveries

(Figure 2).

ET-743 (5) Aplidine (6)

Halichondrin B (7) Eribulin (8)

Figure 1.2: Marine natural products and analogues that are currently used in the clinic or are in phase I/II clinical trials

In 1969, one of the early pharmaceutical success stories began with the observation of potent anticancer activity from extracts of the sea squirt

Ecteinascidia turbinate.7 Analytical chemistry techniques at the time, however,

4 did not allow for researchers to identify the most active component. It was not until 1983 when Dr. Kenneth Rinehart identified and characterized the structure of ecteinascidin-743/ET-743 (5) as the most bioactive component.8 The structure of 5 features a complex fused ring system that is connected by a thioether bridge to create a 10-member lactone ring. Even with the structure solved, however, the limited amount of ET-743 produced by the sea squirt did not allow for clinical trials. This was overcome in 1996 when Dr. E.J. Corey established the first total synthesis of ET-743 and opened the door for clinical trials.9 In 2007, ET-743 was approved by the European Medicines Agency for the treatment of soft-tissues sarcomas, becoming the first marine natural product to be approved for the treatment of cancer.10

The cyclic depsipeptide aplidine (6) is another excellent example of a marine natural product as a lead drug candidate. Isolated in 1988 from the tunicate Aplidium albicans, aplidine shares much of its structure with that of the potent depsipeptide didemnin B. Didemnin B itself was the first marine natural product to enter phase I and II clinical trials.8 The mechanism of action of didemnin B has been difficult to determine.10 Investigations have shown didemnin B inhibits palmitoyl protein thioesterase noncompetitively, though this activity does not account for the nanomolar activity observed in didemnin B.10–12

Though its mechanism of action and activity showed initial promise, didemnin B was eventually phased out of clinical trials due to cardiotoxicity. Aplidine, however, continues to show excellent bioactivity against non-Hodgkins

5 lymphoma. Due to the structure similarities, it is believed that the aplidine shares its mechanism of action with didemnin B.

Marine sponges have also become a great source of chemical diversity and bioactivity. While halichondrin B (7) itself did not progress to approval for use in the clinic, its potent bioactivity inspired the synthetic community to investigate its structure-function characteristics. Isolated in 1986 from the sponge Halichondria okadai, 7 showed sub-ng/mL activity against B-16

Melanoma cells.13 The small concentrations of 7 available from sponge, combined with the promising bioactivity, led to an effort from the synthetic community to provide a viable source of natural product for clinical trials. The first total synthesis of 7 was completed by Dr. Yoshito Kishi in 1992, requiring approximately 90 steps from commercially available material.14 During the course of the total synthesis, a second study focused on the synthetic analogues of 7. These experiments determined that the eastern hemisphere of the molecule was responsible for the observed bioactivity15. This observation greatly decreased the synthetic requirements for the drug candidate leading to a cheaper synthetic candidate. The synthetic macrocyclic analogue eribulin (8) was approved for clinical use by the Food and Drug Administration in 2010, becoming the first approved drug derived from a sponge secondary metabolite.

1.3.1 Streptomyces as a source of natural products

As explored earlier, the sources of marine natural products vary extensively in the marine ecosystem (sponge, tunicate, sea squirt, etc.). As cultivation and sequencing technology improves, however, the true sources of

6 these bioactive natural products become more clear. Recent work by Sherman et al. has shed light on the origin of ET-743, indicating a symbiotic relationship between bacterium and tunicate host.16 Evidence also shows a similar story for the didemnin series, indicating that many natural products share a common source in marine bacteria.17 Among the most prolific producers of bioactive bacterial natural products are from the genus Streptomyces. These aerobic,

Gram-positive actinobacteria are well known for their robust secondary metabolism.18 The Streptomyces genus contains over 500 known species based on 16S rRNA gene sequence analysis19. These bacteria are responsible for approximately two-thirds of the clinically viable antibiotics from microbial origin.20,21 The antibiotics produced by the Streptomyces genus cover a range of classes and mechanisms of action described below. Each class contains many examples of antibiotics that were discovered using activity guided isolation techniques. These antibiotic natural products offered exciting possibilities towards a continuous supply of bioactive compounds. Their applicability, however, became short lived due to the constant threat of resistance development.

1.3.2 Highlighted Class of antibiotics: Aminoglycosides

The aminoglycosides gained attention in 1944 with the potent antibiotics streptomycin (9), kanamycin (10), neomycin (11), and gentamicin (12) (Figure 3).

Aminoglycosides are structurally distinguished by the presence of one or more aminated sugars joined in glycosidic linkages to a dibasic cyclitol.22 This modified sugar moiety gives the aminoglycosides their potency against

7 prokaryotic Gram-negative bacteria. The polar nature of the aminoglycosides allow for self-promoted uptake into the Gram-negative bacterium outer membrane. The aminoglycoside movement across the inner cytoplasmic membrane is dependent upon electron transport and is the rate limiting step for antibiotic activity.22 Once inside the cytosol, aminoglycosides bind to the 30S subunit of the ribosome. This binding does not prevent protein expression but impairs the proofreading mechanisms associated with protein synthesis.22 These misfolded proteins then weaken the cells, causing a cascade effect eventually resulting in cell death.

Streptomycin (9) Kanamycin (10)

Neomycin C (11) Gentamicin (12) Figure 1.3: Examples of aminoglycoside antibiotics isolated from actinobacterial sources

By the mid-1950’s, resistance to 9 and other aminoglycosides was widespread.23 These resistance mechanisms can be divided into three major categories. The first method of resistance involves mutation in the ribosomal protein S12 that prevents binding of 9.24 The modified ribosomal protein is unaffected by streptomycin leading to properly folded proteins. While this mutation is significant, it mainly confers resistance only to 9 and is not a main contributor to general aminoglycoside resistance.22 The second resistance mechanism works by reducing the drug uptake by decreasing the membrane permeability. While the underlying molecular mechanism for membrane impermeability remains unknown, evidence suggests a combination of membrane protein changes and changes in gene regulation have decreased the effectiveness of aminoglycosides.22 Bacterial multidrug resistant proteins have also been shown to transport a broad range of drugs out of the cell, further conferring resistance.25 This resistance is more significant than the ribosomal protein mutation as it affects all aminoglycosides. The third resistance mechanism observed for aminoglycosides comes from aminoglycoside modifying enzymes. In this mechanism, the amine and/or hydroxyl functionalities of the aminoglycoside are covalently modified, rendering the antibiotic unable to bind effectively to the ribosome.22 The three types of enzymes involved in the modification are N-acetyltransferases, O-phosphotransferases, and O- adenyltransferases. N-acetyltransferases acetylates the amine, while O- phosphotransferases phosphorylate hydroxyl groups. O-adenyltransferases confer resistance by adenylating hydroxyl groups and is the least common form

9 of modification of aminoglycosides.26 These enzymes are believed to occur naturally in many bacterial strains and there is evidence that they are overexpressed when the host bacteria is under selection pressure from aminoglycosides.27 In concert, these three mechanisms of resistance greatly reduced the usefulness of the aminoglycoside as potent, long term antibiotics.

1.3.3 Highlighted Class of antibiotics: Peptides as antibiotics

Peptidic antibiotics can be further refined into three subclasses: glycopeptides, polypeptides, and lipopeptides (Figure 4). Each of these classes of antibiotics can be further subdivided based on structure and activity.

Glycopeptides are characterized structurally by a cyclic or polycyclic non- ribosomal peptide chain that undergo glycosylation.28 The glycopeptidic drug vancomycin (13) established glycopeptides as a potent class of Gram-positive antibiotics.

Isolated in 1952 by scientists at Eli Lilly from a sample of Streptomyces orientalis, 13 quickly gained attention for its antibiotic activity.29 Though extremely potent, vancomycin treatment also led to severe side effects and was eventually replaced by moderate and less toxic antibiotics. Vancomycin saw a resurgence in clinical use in the 1980’s due to a combination of new clinical relevance and the rise of resistant pathogens. The early history of vancomycin made it an ideal candidate for the treatment of pseudomembranous enterocolitis.

This condition is associated with pathogenic bacteria growing throughout the lower digestive system. The two bacteria Clostridium difficile and

Staphylococcus aureus were identified as the main sources of this disorder.30

Vancomycin became a potent candidate due to a combination of bioactivity and lack of absorbance in the intestines. The second cause for the increase in vancomycin application was the advent of methicillin-resistant S. aureus (MRSA), and later, penicillin-resistant Streptococcus pneumoniae.29 The first human cases of MRSA were observed in Detroit hospital patents in early 1982.31 This new, resistant pathogen led to further use of vancomycin, which saw a 100-fold increase in use by the end of the 1980’s.29

Vancomycin (13) Actinomycin D (14)

Daptomycin (15)

Figure 1.4: Peptides offer a diverse collection of antibiotics: vancomycin (glycopeptide), actinomycin D (Polypeptide), and daptomycin (lipopeptide).

With the increased use of vancomycin, resistance to its unique mechanism of action became widespread by 1987.32 Vancomycin mechanism of action involves disruption of cell wall synthesis by binding to the terminal D-alanyl-D- alanine moiety of the cell wall. This binding prevents the cell wall peptidoglycan polymers from crosslinking with each other, causing the cell to lyse. Vancomycin resistance is categorized into six patterns, denoted VanA through VanG resistance. These denotations are assigned based on whether resistance is induced or constitutive, as well as the additional susceptibility to the antibiotic teicoplanin.29 While six resistance categories exist, vancomycin resistance is conferred by one of two possible pathways. In both pathways the peptidoglycan polymer intermediates are modified from the susceptible D-alanyl-D-alanine. In the cases of VanA, VanB, and VanD resistant bacteria, the D-alanyl-D-alanine residue is replaced by D-alanyl-D-lactate.33 In VanC, VanE, and VanG resistant bacteria, D-analyl-D-alanine is replaced by D-analyl-D-serine in the peptidoglycan intermediate. Vancomycin binding affinity for serine and lactate is considerably less than for alanine, leading to normal cell wall synthesis.33

Polypeptides are a broad class of antibiotics and are characterized by non-ribosomally synthesized polypeptide chains. A natural product in this class includes the antibiotic and cytotoxic compound actinomycin D (14). Originally isolated in 1940 from Actinomyces antibioticus, the actinomycin series were identified as potent antibiotic as well as cytotoxic compounds.34 The cause of this duel functionality comes from the molecular target of the compound and the mechanism of action. 14 congregates within the DNA transcriptional complex

12 and binds to the DNA preparing to be transcribed. This binding to DNA interferes with the elongation of RNA by immobilizing the transcription complex and terminates DNA transcription.35 The mechanism of action of 14 is somewhat unique among other polypeptides, including bacitracin, colistin, and polymyxin B.

While many of the polypeptide mechanisms of action are unknown, there is some evidence that their activity is derived from interactions with the cell membrane.36

This mechanism of action is highly similar the lipopeptide class of antibiotics.

Lipopeptides are characterized by a non-ribosomally synthesized peptide appended to a lipid side chain. Lipopeptide antibiotics contain both polar and non-polar regions that yield potent bioactivity. Examples of lipopeptides include the surfactins, daptomycin (15), and the echinocandins. Daptomycin has enjoyed great commercial success since its isolation by researchers at Eli Lilly and Company in the early 1980’s.37 Isolated from Streptomyces roseosporus, a series of lipopeptide antibiotics caught the attention of researchers. Following bioactivity optimization, the peptide nucleus containing the C10 saturated side chain was found to yield the most potent antibiotic.38 15 is used to treat Gram- positive infections and its distinct mechanism of action makes it a useful antibiotic against other drug-resistant bacteria. The bioactivity of 15 is calcium dependent and derived from the C10 lipid tail. Once inside the cell membrane, 15 undergoes calcium facilitated conformation change and begins to accumulate.

This accumulation results in deformation and leakage leading to loss of ions and depolarization across the membrane.39

Resistance to daptomycin has been observed in isolated situations, though it is believed resistance will continue to develop as daptomycin is used clinically.39 15 resistance is commonly observed in bacteria that are also resistant to vancomycin. It is hypothesized that vancomycin resistant bacteria cell walls are increasingly less permeable to small molecules.39 With the cell wall being the primary target of both antibiotics, this lack of permeability towards vancomycin is readily applied to 15. Daptomycin resistance has not yet reached the same tipping point observed in vancomycin, and its effectiveness against

Gram-positive bacterial infections continues to be exploited.

1.3.4 Summary and conclusions regarding natural products as antibiotics

Microbial natural products have provided a wealth of antibiotics covering a diverse class of molecules as well as a range of targets. While the importance of these antibiotics cannot be overstated, there are also concerning observations.

First, it is well established that the golden age of antibiotics occurred during the

1940’s and 1950’s.40 From that defined golden age to present day, the discovery of new classes of antibiotics has been sparse. The last several decad