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Latent antibiotics and the potential of the arylomycin class of natural products

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Peter Andrew Smith
Abstract:
Antibiotics are among the greatest contributions of science to the promotion of human health; however, the efficacy of the current antibiotic armamentarium is threatened by the rapid emergence of multi-drug resistant pathogens. As such, novel antibiotics are needed to ensure the continued efficacy of antibiotic therapy. The arylomycin class of natural products binds and inhibits type I signal peptidase, an essential enzyme involved in protein secretion, but has little to no antibiotic activity against many pathogenic bacteria. Herein, I identify naturally occurring mutations in SPase that are responsible for this innate bacterial resistance. These mutations appear to be akin to the specific resistance mechanisms that evolve during clinical antibiotic use, s that arylomycin resistance may have evolved as part of the ongoing microbial arms race. Importantly, based on previous successful efforts to modify antibiotics to overcome clinically evolved resistance, the arylomycins may be prime candidates for chemical optimization into useful therapeutics. I further validate the therapeutic potential of the arylomycins by demonstrating their efficacy against clinical isolates of an important class of bacterial pathogens, coagulase-negative staphylococci. I investigate the cellular stresses induced by arylomycin mediated inhibition of SPase and the factors that ultimately lead to cell death, as well as the activity of the arylomycins in combination with clinical antibiotics. I also characterize the ability of the arylomycins to inhibit the higher order secretion processes required for conjugal DNA transfer. Finally I examine the antibiotic activities of arylomycin derivatives and begin to establish structural activity relationships that lay the ground work for future improvements to this unique scaffold. In total this work establishes the potential of the arylomycin natural products as legitimate candidates for development into clinical antibiotics and in doing so suggests a new approach to antibiotic discovery based on the identification of compounds that have evolved to bind essential bacterial targets but are currently limited due to resistance or imperfect binding.

T ABLE OF C ONTENT S

T ABLE OF F IGURES ................................ ................................ ................................ ............

VIII

T ABLE OF T ABLES

................................ ................................ ................................ ...............

XI

L IST OF A BBREVIATIONS

................................ ................................ ................................ ....

XII

A BSTRA CT

................................ ................................ ................................ ........................

XIII

CHAPTER

ONE

Antibiotic Discovery: Past Successes, Current Challenges, Modern Approaches, and Future Opportunities

................................ ................................ ................................ ................................ .............

1

CHAPTER

TWO

Broad - Spectrum Antibiotic Activity of the Arylomycin Natural Products is Masked by Natural Target Mutations

................................ ................................ ................................ ........................

36

INTRODUCTION

................................ ................................ ................................ ................................ ....

38

MATERIALS AND METHODS

................................ ................................ ................................ .................

40

RESULTS

................................ ................................ ................................ ................................ ...............

49

DISCUSSION

................................ ................................ ................................ ................................ .........

56

CHAPTER

THREE

In Vitro Activity of the Arylomycins against Staphylococcus epidermidis

and other Coagulase - Negative Staphylococci

................................ ................................ ................................ ..........

83

INTROCUTION

................................ ................................ ................................ ................................ ......

85

MATERIALS AND METHODS

................................ ................................ ................................ .................

87

vi

RESULTS

................................ ................................ ................................ ................................ ...............

90

DISCUSSION

................................ ................................ ................................ ................................ .........

94

CHAPTER

FOUR

In Vitro Time - kill and Antibiotic Synergism Studies on the Arylomycins

................................ ...............

115

INTRODUCTION

................................ ................................ ................................ ................................ ..

117

MATERIAL AND METHODS

................................ ................................ ................................ .................

119

RESULTS

................................ ................................ ................................ ................................ .............

124

DISCUSSION

................................ ................................ ................................ ................................ .......

127

CHAPTER

FIVE

Inhibition of Type I Signal Peptidase by an Arylomycin Natural Product

Antibiotic Attenuates Conjugal DNA Transfer

................................ ................................ ................................ ........

149

INTROCUTION

................................ ................................ ................................ ................................ ....

151

MATERIALS AND METHODS

................................ ................................ ................................ ...............

153

RESULTS

................................ ................................ ................................ ................................ .............

156

DISCUSSION

................................ ................................ ................................ ................................ .......

161

CHAPTER

SIX

Antibiotic Activities of Arylomycin Derivatives Designed for Improved Potency and Spectrum

................................ ................................ ................................ ................................ ................

180

INTRODUCTION

................................ ................................ ................................ ................................ ..

182

MATERIALS AND METHODS

................................ ................................ ................................ ...............

186

RESULTS

................................ ................................ ................................ ................................ .............

189

DISCUSSION

................................ ................................ ................................ ................................ .......

192

APPENDICES

Combating Bacteria and Drug - Resistance by Inhibiting Mechanisms of Persistence and

vii

Adaptation

................................ ................................ ................................ ................................ .............

213

Curiculum Vitae

................................ ................................ ................................ ................................ ......

251

viii

T ABLE OF F IGURES

CHAPTER

ONE

Figure. 1.1. Novel s ystemic antibacterial entities approved by the US Food and Drug Administra t i on

................................ ................................ ................................ ................................ .........

20

Figure. 1.2. Iterative evolution of resistance could drive the evolution of a small molecule inhibitor

................................ ................................ ................................ ................................ ...................

21

Figure. 1.3. Chemical structures of the arylomycin class of natural products

................................ .........

22

CHAPTER

TWO

Figure. 2.1.

Chemical composition of the arylomycin class of natural product antibiotics

.....................

63

Figure. 2.2. Fitness cost of arylomycin resistance conferring mutations in Staphylococci strains

................................ ................................ ................................ ................................ .......................

64

Figure. 2.3. Growth rates and arylomycin C 16

sensitivitie s of E . coli

strains harboring various amino acid s

at SPase residue 84

................................ ................................ ................................ .

65

Figure. 2.4. Physical and biochemical evidence for the p roposed mechanism of arylomycin resistance

................................ ................................ ................................ ................................ .................

66

Figure. 2.5. Affinity of arylomycin C 16

for soluble N - terminally truncated E .

coli

SPase

.........................

67

Figure. 2.6. Distribution of SPase with Pro 29

(or Pro 31 ) residues based on bacterial 16S rRNA phylogenies

................................ ................................ ................................ ................................ .....

68

Figure. 2.7. Phylogenies of SPase genes from five bacterial phyla

................................ ..........................

69

Figure. 2.8. Phylogenetic reconstruction the evolution of Pro 29

within Staphylococcaceae SPases

................................ ................................ ................................ ................................ ......................

70

CHAPTER

THREE

Figure. 3.1. Structures of arylomycins

................................ ................................ ................................ .....

98

Figure. 3.2. CoNS Phylogenies confirmed by dnaJ sequencing.

................................ ...............................

99

ix

Figure. 3.3. Diagram of the S. epidermidis genomic region that encodes SpsIB

................................ ....

100

Figure. 3.4. Phylogenetic relationship, SPase sequence, and arylomycin susceptibility of CoNS species

................................ ................................ ................................ ................................ ..........

101

Figure. 3.5. Distribution of arylomycin sensitivities for the examined CNS species ...............................

102

Figure. 3.6. Alignment of SPase proteins from CNS isolates displaying unusual sensitivities

................

103

Figure 3.7. Distribution of arylomycin susceptibilities of unspeciated CoNS isolates obtained from the University of California, San Diego Medical Center.

................................ ................

104

CHAPTER

FOUR

Figure. 4.1.

Chemical composition of the arylomycin class of natural product antibiotics

...................

135

Figure. 4.2. Sequence map of pTetBHR2 - LepB

................................ ................................ ......................

136

Figure. 4.3. Time - kill curves of actively growing E . coli

and S . aureus ................................ ....................

137

Figure. 4.4. Time - kill curves of quiescent E . coli

and S . aureus .

................................ .............................

138

F igure. 4.5. Regulated ectopic u nder - expression of E . coli

SPase

................................ .........................

139

Figure. 4.6. Summary of E . coli

checkerboard MIC experiments

................................ ...........................

140

Figure. 4. 7 . Summary of S. aureus checkerboard MIC experiments

................................ .....................

140

CHAPTER

FIVE

Figure. 5.1.

Chemical composition of the arylomycin class of natural product antibiotics

...................

165

Figure. 5.2. Inhibition of SXT conjugal transfer from wild - type E . coli

................................ ..................

166

Figure. 5.3. I nhibit SXT conjugation

by other SPase inhibitors

................................ ..............................

167

Figure .

5.4.

Inhibition of SXT conjugal transfer from arylomycin sensitive E . coli

................................ .

168

Figure. 5.5. SPase overexpression attenuates inhibition of SXT transfer

................................ ..............

169

Figure .

5.6 .

Arylomycin C 16

inhibits the conjugal t ransfer of multiple elements

................................ ..

170

Figure. 5.7.

Effect of other antibiotics on SXT conjugation

................................ ................................ ...

171

Figure 5.8 .

Inhibition of translation ameliorates arylomycin C 16

inhibition of SXT conjugation

................................ ................................ ................................ ................................ ............

172

x

CHAPTER

SIX

Figure. 6.1.

Chemical structures of the arylomycin class of natural products

................................ ......

199

Figure. 6.2. Comparison of arylomycin A 2

and substrate binding

................................ .........................

200

Figure. 6.3.

Hydrogen bonds between SPase and arylomycin A 2

................................ ..........................

201

Figure. 6.4. Antibiotic activity of arylomycin C 16

and arylomycin B - C 16

against B . brevis

......................

202

APPENDIX

Figure. 7.1. Emergence of mutation mediated resistance

................................ ................................ ....

234

Figure. 7.2. Horizontal tra nsfer of resistance and virulence factors

................................ .....................

235

Figure. 7.3. Tolerance to antimicrobial therapy

................................ ................................ ....................

236

xi

T ABLE OF T ABLES

CHAPTER

TWO

TABLE 2.1.

MICs of arylomycin C 16

for bacterial strains

with and without Pro 29 ................................ .....

71

TABLE 2.2.

Conser v ation of core SPase regions

................................ ................................ ......................

72

TABLE 2.3.

SPase genotype/ arylomycin sensitivity association

in different species

...............................

73

TABLE 2.4.

Primers used within this study

................................ ................................ ..............................

74

CHAPTER

THREE

TABLE 3.1.

Primers used in this study

................................ ................................ ................................ ...

1 05

TABLE 3.2.

Accession numbers of DNA sequences

................................ ................................ ................

106

TABLE 3.3. MICs of coagulase - negative strains to arylomycin C 16

and vancomycin

.............................

107

TABLE 3.4.

SPase proteins

within Staphylococci species.

................................ ................................ ......

108

CHAPTER

FOUR

TABLE 4.1. Effects of SPase expression levels on E . coli

arylomycin C 16 sensitivity

...............................

142

TABLE 4.2. MICs of antibiotics alone and FIC indexes of antibiotic

combination s

................................

143

CHAPTER

FIVE

Table 5.1.

Strains and plasmids used in this study

................................ ................................ ................

173

CHAPTER

SIX

TABLE 6.1. Antibiotic activities

of arylomycin N - terminal alkyl chain derivatives

................................ .

204

TABLE 6.2.

Antibiotic activities of arylomycin N - terminal peptide

chain derivatives ............................

205

TABLE 6.3.

Antibiotic activities of arylomycin P3 derivatives .

................................ ...............................

206

TABLE 6.4.

Effects of naturally occuring macrocycle modification on antibotic activity .

......................

207

xii

L IST OF A BBREVIATIONS

SPase

b acterial type I signal peptidase also known as leader peptidase

lepB

gene encoding Escherichia

coli

SPase protein

MIC

minimal inhibitor concentration

CFU

colony forming units

Km

kanamycin

Cm

chloramphenicol

Ap

ampicillin

St

streptomycin

Sp

spectinomycin

Gm

gentamicin

Cp

ciprofloxacin

Rif

rifampicin

T c

tetracycline

xx r

resistance to the antibiotic xx

aTc

anhydrotetracycline

DMSO

d imethyl sulfo xide

TSB

tryptic soy broth

TSA

trypitic soy agar

LB

L uria broth

LA

L uria agar

MHBII

cation - adjusted Mueller Hinton broth

MHAII

cation - adjus ted Mueller Hinton agar

k d

dissociation constant

xiii

A BSTRACT

Antibiotic s

are among

the greatest contribution s

of science to the promotion of human health ; h owever, the efficacy of the current antibiotic armamentarium is threatened by the rapid emergence of multi - drug resistant pathogens . As such ,

novel antibiotics are needed to ensure the continued efficacy of an tibiotic

therapy. The arylomycin

class of natural products

bind s

and inhibits type I signal peptidase, an essential enzyme

involved in protein secretion, but ha s

little

to no

antibiotic activity against many pathogenic bacteria. Herein, I identify naturally occurring mutations in SPase that are responsible for this innate bacterial resistance . These mutations appear to be akin to the specific resistance mechanisms that evolve during clinical antibiotic use , s

that arylomycin resistance may

have evolved as part of the ongoing microbial arms race. Importantly,

based on previous success ful efforts to modify

antibiotics to overcome clinical ly evolved

resistance , the arylomycins may be prime candidates for chemical optimization

into useful ther apeutics . I further validate the therapeutic potential of the arylomycins by demonstrating their efficacy against clinical isolates of an important class of bacterial pathogens, coagulase - negative staphylococci. I

investigate the cellular stresses induce d by arylomycin mediated inhibition of SPase

and the factors that ultimately lead to cell death, as well as the activity of the arylomycin s

in combination with clinical antibiotics . I

also

characterize the ability of the arylomycin s

to inhibit the higher order secretion processes

required for conjugal DNA transfer . Finally I

examine the antibiotic activities of arylomycin derivatives

and begin to establish structural activity relationships that lay

the ground work for future improv ements to this unique sc affold.

In total this work establishes the potential of the arylomycin natural products as legitimate candidates for development into clinical antibiotics and in doing so suggests a new

xiv

approach to antibiotic discovery based on the identification of compo unds that have evolved to bind essential bacterial targets but are currently l imited due to resistance or imperfect binding .

1

CHAPTER ONE

Antibiotic Discovery: Past Success es , Modern Approaches, Current Challenges, and Future Opportunities

2

A

B RIEF H ISTORY OF A NTIBIOTICS AND A NTIBIOTIC R ESISTANCE

In an event of historic serendipity, Alexander Fleming discovered the antibiotic properties of secretions from a mold st rain that he termed penicillium

( 1 ) . Less than twe nty years later, the isolation, characterization, and mass production of penicillin, the small molecule responsible for this remarkable antibacterial activity, aided the Allied forces in their victory in World War II and ushered in the era of antibacterial

chemotherapy. Although the penicillins were neither the first potent small molecule antibiotic to be discovered or used in humans

( these distinctions belong to the synthetic sulfonamide class of antibiotics ),

the superior potency and spectrum

and the red uced toxicity of the penicillins revolutionized antibiotic therapy. Perhaps even more important than the therapeutic impact of the penicillins, was the paradigm shift precipitated by the realization that microbial natural products might provide a rich sou rce of antibacterial agents. In 1943, the discovery of the aminoglycoside antibiotic streptomycin by Albert Schatz and Selman Waksman marked the beginning of the „golden age‟ of antibiotic discovery

( 2 ) , during which a variety of novel classes of antibiotics were discovered from the bacterial secretions , particularly those from bacteria of the genus Streptomyces ( 3 ) . During the following two decades, microbial fermentation extracts were screened in simple whole - cell bacterial growth assays, which resulted in the discovery of almost every class of broad - spectrum antibiotic known to day including, tetracyclines, cephalosporins, macrolides, glycopeptides, rifamycins, and lincosamides ( 4 ) .

The rapid development and remarkable success of these natural product antib iotics transformed the opinions of the scientific and medical community, and in 1967, the US Surgeon General W. H. Stewart is infamously reported to have commented that, “The time has come to close the book on infectious diseases. We have basically wiped o ut

3

infection in the United States”. T oday , however, infectious

disease remains the second largest cause of death world - wide, and in the United States, bacteria are the number one cause of death by infectious agent ( 5 ) . Indeed, in the United States, deaths caused by the methicillin resistant Staphylococcus

aureus

( MRSA) outnumber those caused by HIV/AIDS ( 6 ) . The difficulties encountered in treating infectious disease are primarily due to the wide - spread evolution of antibiotic resistance. Although originally underappreciated,

the threat posed by the evolution and antibiotic resistance

w as already apparent to Alexander Fleming ,

who noted in his Nobel Prize acceptance speech that, “… there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non - lethal quantities of the drug make them resistant.”

( 7 ) . Today, as a result of the extreme selective pressures exerted by antibiotic use and misuse in medicine and agriculture, the evolution of antibiotic resistance now threatens to bring an end to the antibiotic era. A number of pathogens are again difficult to treat, including the Gram - positive organism MRSA, as well as

fluoroquinolone resistant Pseudomonas

aeruginosa

and cephalosporin resistant Escherichia

coli ( 8 ) , and the emergence of pan - antibiotic resistant bacter ia appears imminent

( 9 , 10 ) .

M ECHANISMS OF A NTIBIOTIC R ESISTANCE

Bacteria possess a plethora of mechanisms to evade the activity of antibiotics, first among which are the intrinsic permeability barriers provided by bacterial membranes ( 11 , 12 ) . The cytoplasmic membrane constitutes the primary permeability barrier of Gram - positive bacteria and prevents the diffusive entry of charged or highly polar compounds, but provides lit tle protection against many nonpolar antibiotics and no

4

protection against cell wall inhibitors such as penicillin and vancomycin that inhibit targets on the outer surface of the cell . Gram - negative bacteria possess an additional lipid bilayer or „outer - m embrane‟ that is composed of densely packed lipopolysaccharides and provides a more effective permeability barrier against both polar molecules that exceed ~600 Da and non - polar compounds

( 13 , 14 ) . Promiscuous low - affinity efflux pumps act synergistically with the Gram - negative outer - membrane to further reduce the intracellular con centrations of many toxic molecules including antibiotics ( 15 ) . This dual arch itecture affords Gram - negative bacteria an intrinsic resistan ce

to many antibiotics that are effective against Gram - positive organisms . In addition to the intrinsic and relatively non - specific resistance provided by the cell membrane

( s), highly specific „endogenous resistance‟ can evolve via mutations within the target of an antibiotic that reduce inhibitor binding without significantly affecting cellular function ( 16 , 17 ) .

Additionally, „exogenous‟ or „positive function‟ resis tance can a rise through the acquisition of enzymes that modify

( kinases, adenylases, methylases), degrade

( β - lactamases), or efflux antibiotic s

with high activity and specificity as well as those that modify the antibiotic target

( 18 ) .

T HE S PREAD OF A NTIBIOTIC R ESISTANCE

The dramatic increase in antibiotic resistant infections is due in part to the clonal spread of resistant bacteria, much like the pandemic spread of viruses. Such clonal expansion, as observed in the global spread of multidrug resistant S . aureus,

results

in the vertical dissemination of target mutations and enzyme mediated resistance mechanisms ( 19 , 20 ) . However, the interspecies transfer of DNA encoding the proteins responsible

5

for exogenous resistance, or horizontal gene transfer

( HGT), provides a more pervasive and versatile mechanism for the spread of resistance. HGT can occur through a number of distinct processes, including the uptake of DNA present in the environment, virus - mediated DNA transfer, and DNA transfer mediated by d irect cell - to - cell contact ( 21 ) . This latter mechanism is further potentiated by plasmids and other mobile genetic elements that simultaneously act as reservoirs for the accumu lation of multiple resistance genes and actively spread these genes to new hosts ( 22 , 23 ) . Furthermore, because multiple resistance elements are often genetically linked on a single mobile element, multidrug resistant phenotypes can rapidly spread to new species. Consequently, HGT is recognized as one of the most important factors leading to the rapid spread of antibiotic resistance throughout bacterial

populations and the subsequent failure of antibiotic therapy ( 24 ) .

Because antibiotic use provides the initial selection pressure for the fixation of resistance - conferring mutations within a population or for dissemination of res istance genes via HGT, the cessation of antibiotic use could be expected to induce a reversion to the original antibiotic sensitive phenotype , particularly given the fitness cost that is often associated with either endogenous or exogenous resistance ( 25 , 26 ) . Indeed ,

cycling between different classes of antibiotics on the decade s

time scale has been proposed as a strategy to reduce resistance ( 27 ) . Unfortunately, the fitness costs associated with resistance conferring mutations are often alleviated by additional “ compensatory ”

mutation

( s) that not only restore fitness while maintaining the resistant phenotype, but also reduce the fitness of t he sensitive phenotype ( 28 - 31 ) . Resistance encoded by mobile genetic elements is often similarly stable in the absence of antibiotic selection, due to

6

element encoded toxin - antitoxin systems, which kill cells that fail to inherit the element upon cell division ( 32 ) . Additionally, because multiple resistance elements are frequently located on a single element, the use of one antibiotic can ensure the maintenance of resistance to unrelated agents ( 33 ) . This relatively unidirectional progression from a n antibiotic

sensitive phenotype to resistant one further compounds the spread of antibiotic resistance.

R ESISTANCE O RIGI NATING FROM A NTIBIOTIC P RODUCING O RGANISMS

The acquisition of exogenous resistance through HGT and the evolution

of endogenous resistance via t arget mutations have been responsible for the rapid increase in antibiotic resistance within pathogenic bacterial species, but the evolution of the enzymes that efflux, degrade, or modify antibiotics with high specificity and activity would be expected to take a significantly longer period of time. Regardless of their subsequent spread, it is interesting to consider the origins of the se

resistance conferring enzymes. An early explanation was provided by Julian Davies, whose landmark 1973 paper noted the s imilarities between the protein responsible for aminoglycoside resistance in clinical isolates and the enzyme that affords self - protection to the bacteria that naturally produce these molecules

( 34 ) . Numerous resistance elements have since been discovered in other antibiotic producing organisms, and the se elem ents

are often closely genetically linked to the antibiotic biosynthesis operons. These observations

seem to confirm

an ironic scenario in which the microorganisms that produce so many of the clinically useful antibiotics are also the origin of many antibiotic resistance

mechanisms

( 35 - 39 ) . Indeed, the frequency of antibiotic resistance within Streptomyces , which are by far the most

7

prolific genus of antibiotic producing bacteria ( 3 ) , is remarkabl y

high; in one study, Streptomyces soil isolates were found to be on average resistant to 7 of 21 antibiotics examined with some isolates harboring resistance to as many as 15 antibiotics ( 40 ) .

W IDESPREAD A NTIBIOTIC R ESISTANCE AND S YNTHESIS :

T HE B ACTERIAL A RMS R ACE

Despite a resurgence of interest in the field, the selective pressures responsible for the widespread presence of antibiotic resistance outside of the clinical setting remain uncertain ( 41 ) . The widespread contaminati on of ecosystems with manmade antibiotics ( 42 )

and the use of antibiotic resistant bacteria as bio - insecti cides ( 43 )

have certainly contributed to resistance within environmental bacteria. But significant evidence is emergin g to suggest that antibiotic resistance is far more ancient and widespread than once thought. To begin with, antibiotic resistance has been observed at high frequencies in bacterial isolates from diverse species that do not naturally produce antibiotics a nd from bacteria isolated from environments

that are untainted by human kind

( 44 - 46 ) . The absence of

contamination by manmade antibiotics suggests the presence of natural selective pressures for the evolution and spread of resistance. Not only are these resistance genes widespread, they appear to be ancient, long preceding the evolution of hu man s

much l ess the industrialized production of antibiotics. Vancomycin resistance enzymes are widespread among soil bacteria, and although their protein sequences are highly conserved, the GC contents of their encoding genes are extremely different ( 47 ) , and molecular clock analysis suggests that they have been evolving for over 200 million years ( 48 ) . Similarly the β - lactamase enzymes that degrade penicillins appear to have evolved from penicillin binding proteins

( the targets of the penicillins),

and the extreme

8

sequence difference between the se

two protein families suggests that this divergence may

have occurred over 1 billion years ago ( 49 - 51 ) .

Further supporting nature ‟ s ro le in the evolution of resistance, the fascinating non - ribosomal peptide synthesis and polyketide synthesis pathways that produce almost all of the natural product antibiotics are also ancient and widespread . Molecular clock analysis indicates that the pa thways for streptomycin and erythromycin biosynthesis are more than 610 and 880 and million years old ( 48 ) , and approximately 10% of Streptomyces isolates

produce the aminoglycoside antibiotic streptothricin, while 1% produ ce streptomycin, and 0.1% produce tetracycline ( 52 ) . Although humans have prod uced antibiotics at seemingly large quantities, it is worth considering that the total number of prokaryotes on the earth is estimated to exceed 4

x

10 30

cells consisting of more than 3

x

10 17

grams of biomass ( 5 3 ) . Given the number of bacteria, the frequency of antibiotic production, and the evolutionary timescales involved, perhaps the assumption that humans are the primary cause of antibiotic resistance is as presumptuous as the prediction that mankind could

permanently eradicate infectious disease.

Thus an emerging explanation for naturally occurring antibiotic resistance is perhaps the most intuitive one: antibiotic resistance may have evolved as part of a microbial arms race, in response to

the selective pressure imposed by naturally produced antib iotics. It should be noted that

dissenting explanations for widespread resistance also exist. The most prevalent is based on the observation that antibiotics elicit changes in bacterial physiology at concentrati ons below those required to inhibit growth

or induce death , and proposes that these molecules evolved to communicate rather than kill. Within this context „antibiotic resistance‟ could be a form of signal modulation ( 54 - 56 ) .

9

However, due to the pathways and the targets that they inhibit, the natural product antibiotics used in the clinic are poorly suited to act as signaling , and while this evidence may be anecdotal, after viewing a crystal structure of an essential transpepti dase irreversibly acylated by a cephalosporin or measuring the rapid cell membrane depolarization induced by daptomycin, this evidence is convincing nonetheless.

N ATURAL P RODUCTS TO S YNTHETIC L IBRARIES AND B ACK A GAIN

Toward the end of the twentieth c entury, as novel broad - spectrum natural product antibiotics bec ame increasingly rare

and resistant pathogens increasingly widespread, technological advances in drug discovery promised to meet the need for novel therapeutics through a two pronged approach.

New targets that appeared to be essential and conserved were identified by mining the ever increasing

number of fully sequenced bacterial genomes ( 57 , 58 )

and were validated using novel genetic methodologies such as antisense gene knockdown and regulated target expression ( 59 - 61 ) . T hose targets that were amenable to enzymatic assay were then purified and screened against synthetic small molecule libraries using automat ed high - throughput screening technologies. As an added advantage, it was expected that these fully synthetic molecules, never having been present in nature, would be less susceptible to natural resistance mechanisms. Unfortunately, despite significant ef forts by multiple major pharmaceutical companies, this approach largely failed, and as a result the number of antibiotic s

introduced into the clinic has been falling at the same time that antibiotic resistance has become more widespread

( Fig .

1. 1 ). As a r epresentative example, GlaxoSmithKline recently reported the comprehensive results from a seven year effort to target approximately 300 novel

10

essential bacterial genes ( 62 ) . Seventy high - throughput screening campaigns were initiated on the most promising targets, from which only five leads were delivered, a success rate that is four to five times lower than that of other therapeutic areas and financially unsustainable. Mor eover, of these five early leads none are currently under development as antibiotics. Analysis of the available data from other pharmaceutical companies suggests that this level of difficulty in discovering novel antibiotics is common ( 62 ) .

The failure

of modern antibiotic d iscovery efforts appear s

to be attributable to both the libraries and the screening methodologies employed. First, synthetic libraries have been designed largely to conform to Lipinski‟s rule of five ( 63 ) , and the molecules contained therein proved to be remarkably poor at inhibiting microbi al targets. Indeed antibiotics tend to be larger, more complex, and more polar than the therapeutics that modulate human targets

( 64 ) . Second, inhibition of a purified enzyme using a biochemical assay routinely failed to translate into antibacterial activity against whole cells . This failure appears to stem in large part from the inability of the identified inhibitors

penetrate bacterial membranes ( 62 ) . The emerging conclusion from these and other efforts is that eons of evolution have tailored natural product antibiotics with the molecular features

( size, charge, total hydrophobicity, and distribution of hydrophobicity) required to penetr ate bacterial membranes and inhibit bacterial targets.

The currently exploited antibacterial targets may also have properties that make them uniquely suited for broad - spectrum antibacterial therapy ( 65 ) . First , enzymes that are conserved in all bacteria may nonetheless prove to be non - essential or poorly conserved in some species ( 60 ) .

Nonetheless, all clinical antibiotics inhibi t universally

11

essential targets.

Additionally,

the targets of broad - spectrum antibiotics must present a highly conserved binding site of sufficient size and composition that a suitable small molecule can bind with high affinity. The presence of a conserv ed binding site does not always correlate directly with overall sequence conservation and may not be apparent from sequence analysis alone, which makes target prediction difficult.

Again, the targets of known broad - spectrum agents present suitably attract ive binding sites.

In this light, it is not surprising that the most successful strategy to date in the fight against antibiotic resistance has been to derivatize known scaffolds to overcome the resistance that has emerged following their clinical use. D ue to the immutable nature of these pockets, resistance often evolves through mutations located at periphery of the binding site, or through modifying enzymes, and as such ,

resistance can be overcome without changing the core pharmacophore responsible for the broad - spectrum activity of the scaffold ( 66 ) .

Given the failure of the high - throughput, biochemical assay - based scree ning of synthetic libraries, efforts to identify novel antibiotic scaffolds have refocused on screening natural products in whole cell assays ( 67 ) . However, the majority of broad - spectrum natural product antibiotics appear to have been discovered; the low hanging fruit was picked during the golden age of antibacterial discovery. Based on historical trends, Baltz et al at Cubist Phar maceuticals have estimated that identifying a single new broad - spectrum antibiotic will require screening extracts from tens of millions of previous ly

uncultured microbes ( 52 ) . To pursue this brute force approach, micro - fermentation techniques have been developed to provide access to unprecedented numbers of natural products, a nd novel screening strains that are resistant to all known antibiotic classes have been engineered to enable high - throughput screening with low re -

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Full document contains 267 pages
Abstract: Antibiotics are among the greatest contributions of science to the promotion of human health; however, the efficacy of the current antibiotic armamentarium is threatened by the rapid emergence of multi-drug resistant pathogens. As such, novel antibiotics are needed to ensure the continued efficacy of antibiotic therapy. The arylomycin class of natural products binds and inhibits type I signal peptidase, an essential enzyme involved in protein secretion, but has little to no antibiotic activity against many pathogenic bacteria. Herein, I identify naturally occurring mutations in SPase that are responsible for this innate bacterial resistance. These mutations appear to be akin to the specific resistance mechanisms that evolve during clinical antibiotic use, s that arylomycin resistance may have evolved as part of the ongoing microbial arms race. Importantly, based on previous successful efforts to modify antibiotics to overcome clinically evolved resistance, the arylomycins may be prime candidates for chemical optimization into useful therapeutics. I further validate the therapeutic potential of the arylomycins by demonstrating their efficacy against clinical isolates of an important class of bacterial pathogens, coagulase-negative staphylococci. I investigate the cellular stresses induced by arylomycin mediated inhibition of SPase and the factors that ultimately lead to cell death, as well as the activity of the arylomycins in combination with clinical antibiotics. I also characterize the ability of the arylomycins to inhibit the higher order secretion processes required for conjugal DNA transfer. Finally I examine the antibiotic activities of arylomycin derivatives and begin to establish structural activity relationships that lay the ground work for future improvements to this unique scaffold. In total this work establishes the potential of the arylomycin natural products as legitimate candidates for development into clinical antibiotics and in doing so suggests a new approach to antibiotic discovery based on the identification of compounds that have evolved to bind essential bacterial targets but are currently limited due to resistance or imperfect binding.