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Chemical biology of tetracycline antibiotics
1
Bijan Zakeri and Gerard D. Wright
Abstract: For more than half a century, tetracycline antibiotics have been used to treat infectious disease. However, what
once used to be a commonly prescribed family of antibiotics has now decreased in effectiveness due to wide-spread bacte-
rial resistance. The chemical scaffold of the tetracyclines is a versatile and modifiable structure that is able to interact with
many cellular targets. The recent availability of detailed molecular interactions between tetracycline and its cellular targets,
along with an understanding of the tetracycline biosynthetic pathway, has provided us with a unique opportunity to usher
in a new era of rational drug design. Herein we discuss recent findings that have clarified the mode of action and the bio-
synthetic pathway of tetracyclines and that have shed light on the chemical biology of tetracycline antibiotics.
Key words: antibiotic, tetracycline, biosynthesis, resistance.
Resume : Depuis plus dun demi-sie`cle, les antibiotiques de la famille des tetracyclines ont ete utilises pour traiter les ma-
ladies infectieuses. Cependant, la prescription generalisee de cette famille dantibiotiques en a diminue lefficacite a` cause
dune resistance bacterienne repandue. Lechafaudage chimique des tetracyclines est une structure versatile et modifiable
qui est capable dinteragir avec plusieurs cibles cellulaires. La recente disponibilite de donnees detaillees des interactions
moleculaires de la tetracycline et ses cibles cellulaires conjuguee a` une meilleure comprehension de la voie de biosynthe`se
de la tetracycline nous a fourni une opportunite unique dinaugurer une nouvelle e`re de conception rationnelle de medica-
ments. Nous discutons ci-dessous des recentes decouvertes qui ont permis de clarifier le mode daction et la voie de bio-
synthe`se des tetracyclines, et qui ont fait la lumie`re sur la chimie biologique des antibiotiques de la famille des
tetracyclines.
Mots-cles : antibiotique, tetracycline, biosynthe`se, resistance.
[Traduit par la Redaction]
Introduction
Tetracyclines (Fig. 1) were first discovered in 1948 by
Benjamin Duggar (Duggar 1948) as natural products pro-
duced by species of Streptomyces, and have since proven to
be an economically valuable drug class over the past 6 dec-
ades. The highly modified chemical scaffold of the tetracy-
clines affords them versatility and allows them to interact
with a variety of biological targets. A remarkable feature of
tetracyclines is the presence of keto-enol functional groups
on one face of the scaffold, providing them with the ability
to chelate divalent cations, which plays a prominent role in
their biological functions. Although the chemical biology of
tetracycline antibiotics has been extensively studied, there
are still many aspects of their structureactivity relationships
that are poorly understood.
Streptomyces is a genus of soil-dwelling bacteria that pro-
duce chemically diverse natural products, including tetracy-
clines. As a result, tetracyclines can be easily and cost
effectively isolated by fermentation, which has contributed
to their extensive use in human therapy, veterinary medi-
cine, animal growth promotion, and aquaculture (Chopra
and Roberts 2001). Tetracyclines are broad-spectrum antibi-
otics that act against gram-positive and gram-negative bacte-
ria, the intracellular pathogens chlamydiae, mycoplasmas,
and rickettsiae, as well as eukaryotic protozoan parasites
(Roberts 2003). In addition to their antimicrobial activities,
they have also been prescribed for many noninfectious con-
ditions since the early 1950s. Their clinical applications in-
clude their use as metal chelating ionophores, in the
inhibition of inflammation, in proteolysis, in angiogenesis,
and as anti-apoptotic agents (Ross et al. 1998; Sapadin and
Fleischmajer 2006). Furthermore, this chemical scaffold is
being investigated for its anti-cancer and anti-metastatic po-
tential, in which tetracycline analogues are used as potent
inhibitors of matrix metalloproteinases. The extensive use
of this class of compounds in medicine and agriculture is
paralleled by production levels measured in tons per year.
This has contributed to the emergence of wide-spread bacte-
Received 15 October 2007. Revision received 12 December
2007. Accepted 19 December 2007. Published on the NRC
Research Press Web site at bcb.nrc.ca on 27 March 2008.
B. Zakeri and G.D. Wright.
2
Department of Biochemistry and
Biomedical Sciences, DeGroote School of Medicine, McMaster
University, 1200 Main St. W, Hamilton, ON L8N 3Z5, Canada.
1
This paper is one of a selection of papers published in this
Special Issue, entitled CSBMCB Systems and Chemical
Biology, and has undergone the Journals usual peer review
process.
2
Corresponding author (e-mail: wrightge@mcmaster.ca).
124
Biochem. Cell Biol. 86: 124136 (2008) doi:10.1139/O08-002 # 2008 NRC Canada
rial resistance and a corresponding decline in the use of tet-
racycline antibiotics for human therapy.
Recently many advances have been made in elucidating
the structureactivity relationships, resistance mechanisms,
and biosynthetic pathways of tetracyclines. The availability
of high resolution crystallographic structures of tetracycline
bound to the 30S ribosomal subunit and a detailed under-
standing of the tailoring steps in the biosynthetic pathways
will result in exciting new strategies for the rational design
of novel tetracycline derivatives. Herein we review new
findings that shed light on how tetracyclines interact with
their cellular targets and the secondary metabolic pathways
that lead to their production from simple precursor mole-
cules.
Tetracycline antibiotics
The first members of the tetracycline antibiotics to be de-
scribed, chlortetracycline (CTC) and oxytetracycline (OTC)
(Fig. 1), were discovered in the late 1940s during the early
period of the Golden Age of antibiotic discovery as a result
of natural product screening programs. These compounds
are broad-spectrum antibiotics that act against a plethora of
microbes from diverse physiological, ecological, and genetic
backgrounds, making them attractive to the pharmaceutical
industry. Not only are they relatively easy and inexpensive
to produce, but they do not cause severe side effects and
have favourable oral bioavailability and pharmacokinetic pa-
rameters (Agwuh and MacGowan 2006). As a result of their
impressive bioactivity, tetracyclines were swiftly promoted
through clinical trials (Duggar 1948). All of these attributes
made tetracyclines a favourable choice of drug as a first-line
defence against bacterial infections. Soon thereafter, many
more members of this group of antibiotics were isolated as
fermentation products produced by soil-dwelling organisms,
making tetracyclines a large family of antimicrobials. At
about the same time, Stokstad et al. observed that the inclu-
sion of fermentation products from the CTC producer Strep-
tomyces aureofaciens accelerated the growth of poultry,
which demonstrated the potential of these compounds as
growth promoting agents in the agricultural industry (Stok-
stad et al. 1949). Naturally, the extensive use of tetracy-
Fig. 1. Selected members of the tetracycline family of antibiotics.
Zakeri and Wright 125
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2008 NRC Canada
clines, both in the clinic and in agriculture, imposed a mas-
sive selection pressure for resistant isolates. Not surpris-
ingly, by 1953 the first tetracycline-resistant strains of
Shigella dysenteriae were isolated, and soon afterwards in
1955, multidrug resistant Shigella species that were resistant
to tetracycline, chloramphenicol, and streptomycin were dis-
covered (Watanabe 1963; Roberts 1996).
To keep pace with the rapid emergence of resistant patho-
gens, new tetracyclines were sought out; however, new tet-
racyclines from traditional microbial sources proved
difficult to find. As a result, we entered a golden age of
antibiotic medicinal chemistry, during which, scientists
tried to improve potency and evade resistance mechanisms
by chemically modifying antibiotic scaffolds (Wright 2007).
This resulted in the development of semisynthetic second-
generation tetracyclines, which include doxycycline and
minocycline (Fig. 1). These compounds are more lipophilic
than their parent compounds, and as a result, they have bet-
ter absorption and pharmacokinetic parameters (Agwuh and
MacGowan 2006). Recently, third-generation tetracyclines,
which include the semisynthetic glycylcyclines and amino-
methylcyclines, have been developed (Sum and Petersen
1999; Agwuh and MacGowan 2006), some of which are
currently in clinical trials (e.g., Paratek Pharmaceuticals,
www.paratekpharm.com/pt_tet_inhib.html). Tigecycline is a
member of the glycylcyclines, and was approved for clinical
use by the US Food and Drug Administration in 2005. It
contains an N-alkyl-glycylamido group at position 9 on the
scaffold of minocycline (Fig. 1). This modification results
in a more efficient interaction with the ribosome and the
evasion of classic tetracycline-resistance mechanisms such
as efflux pumps and ribosomal protection proteins (vide in-
fra) (Slover et al. 2007).
Tetracyclines and indeed, many other natural products,
often possess very complex chemical structures that are pro-
duced by a large number of enzymes in a "conveyor-belt"
fashion. Once the scaffolds of these compounds are pro-
duced, many regio-specific tailoring reactions position func-
tional groups at strategic location, allowing the compounds
to interact with biological targets via hydrogen bonding. As
a result, tetracyclines contain several chiral centres and
many chemically reactive substituents, which makes their
total chemical synthesis challenging. Furthermore, their
chemical sensitivity to acidic and basic media, which cause
the molecules to undergo chemical transformations, further
hinders synthetic approaches (Muxfeldt et al. 1979). For
these reasons, it would seem that the ideal method of devel-
oping novel tetracycline derivatives would be through semi-
synthetic approaches. Nevertheless, successful attempts at
the total synthesis of tetracyclines have been made. In 1979,
Muxfeldt et al. (Muxfeldt et al. 1979) reported the total syn-
thesis of OTC from a tetralone aldehyde producing 47 other
precursor molecules before isolating OTC in an unspecified
yield. Further improvements to this approach have been
made recently by Myers group. In 2005 they reported the
synthesis of ()-tetracycline from benzoic acid in 17 steps
with an overall yield of 1.1% (Charest et al. 2005a). In the
same year, they also reported the synthesis of ()-doxycy-
cline, also from benzoic acid, with an 8.3% yield over 18
steps (Charest et al. 2005b). However, total syntheses of
such complex molecules are time consuming, labour inten-
sive, and expensive, which highlights the advantages of
semisynthetic modifications for the improvement of antibiot-
ics.
After *40 years of use in the clinic, tetracyclines began
to decline as first-line antibiotics. This decline correlated
with certain key events. First, in the early 1980s, the mecha-
nism by which the Escherichia coli transposon Tn10 confers
tetracycline resistance was identified. This led to the identi-
fication of several tetracycline-resistance determinants that
functioned by reducing intracellular tetracycline concentra-
tions through active efflux (Ball et al. 1980; McMurry et al.
1980). Second, several members of the fluoroquinolone class
of antimicrobials emerged and received FDA approval, lead-
ing to their subsequent clinical use. Although they function
by inhibiting DNA gyrase activity, the fluoroquinolones,
like the tetracyclines, are broad-spectrum antimicrobials and
were therefore an attractive alternative to tetracyclines.
These events contributed to a general decline over the past
few decades in the clinical use of tetracyclines for the treat-
ment of common infectious diseases.
Nevertheless, tetracyclines are still potent inhibitors of
bacterial protein synthesis and are amenable to chemical
modification. An understanding of the mode of action of tet-
racyclines at the molecular level will no doubt provide use-
ful information that will enable the production of novel
compounds that can bypass resistance mechanisms.
Mode of action
Tetracyclines impart their broad spectrum antibiotic activ-
ities through both bacteriostatic and bactericidal modes of
action. The clinically used compounds are bacteriostatic and
their mode of action has been well characterized, whereas
the action of bactericidal tetracyclines, such as chelocardin
(Fig. 1), is poorly understood (Schnappinger and Hillen
1996). Recently, Kohanski et al. have shown that the cellu-
lar killing capabilities of three major classes of bactericidal
antibiotics (quinolones, b-lactams, and aminoglycosides) can
be partly credited to the production of toxic hydroxyl radi-
cals (Kohanski et al. 2007). Although there is no evidence
of hydroxyl radical formation by bactericidal tetracyclines,
it does warrant further investigation. The antimicrobial func-
tion of tetracycline has been attributed to the binding of the
bacterial 30S ribosomal subunit near the A site and subse-
quent inhibition of protein synthesis by the prevention of
aminoacylated-tRNA docking (Maxwell 1967; Brodersen et
al. 2000). However, there is considerable biochemical evi-
dence for the presence of one strong and several minor bind-
ing sites for tetracycline in the ribosome, which has led to
discrepancies in the mode of action and the functions of the
minor binding sites reported in the literature (Tritton 1977;
Epe and Woolley 1984). The recent revelation of the molec-
ular interactions between tetracycline and the ribosome
through X-ray crystallography has clarified their mode of
action.
Uptake
To gain access to the ribosome, tetracyclines must first
traverse biological membranes and do so in both gram-posi-
tive and gram-negative organisms. It has long been known
that tetracyclines chelate divalent cations, commonly mag-
126 Biochem. Cell Biol. Vol. 86, 2008
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2008 NRC Canada
nesium, and this ionic interaction is pivotal for their cellular
functions (White and Cantor 1971). Likewise, the ionic state
and charge of tetracyclines plays a key role in drug uptake.
The mechanism of tetracycline uptake has been reviewed
elsewhere (Nikaido and Thanassi 1993; Schnappinger and
Hillen 1996) and will therefore not be discussed in detail
here. Briefly, to enter the periplasm of gram-negative bacte-
ria, tetracyclines cross the outer membrane by passing
through porin channels while in complex with Mg
2+
. Subse-
quently, the tetracyclineMg
2+
complex dissociates, thereby
allowing tetracycline to simply diffuse through the cytoplas-
mic membrane to enter the cytosol, where it once again che-
lates an Mg
2+
ion to bind the ribosome (Schnappinger and
Hillen 1996).
Mode of action
Tetracyclineribosomal interactions have been extensively
studied since the 1960s through in vitro biochemical analy-
sis, mechanisms of action of tetracycline derivatives, and
UV-induced cross-linking studies. Yet many unanswered
questions remained as to how tetracycline prevents the bind-
ing of aminoacylated-tRNA to the A site or whether it inter-
acts with the RNA or protein components of the ribosome.
Many of these questions were answered when two groups
independently published detailed crystal structures of tetra-
cycline in complex with the 30S ribosomal subunit of Ther-
mus thermophilus. The first model was published by
Brodersen et al. in 2000 (Brodersen et al. 2000), in which
they found two binding sites for tetracycline on the small ri-
bosomal subunit. Soon thereafter, Pioletti et al. (2001) pub-
lished another structure that depicted six tetracycline binding
sites on the 30S ribosome (named Tet-1 to Tet-6, based on
decreasing relative occupancy) where sites Tet-1 and Tet-5
corresponded to the primary and secondary binding sites of
the Brodersen et al. model, respectively. In both models, the
highest occupied binding site was near the A site, consistent
with the reported mode of action.
The Tet-1 tetracycline binding pocket measures 20 A
wide and 7 A