Vaccines

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Volume 196,920,003
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Antimicrobials
Joseph Vogel MPRB 10220 jvogel_at_borcim.wustl.edu (M
arch 2, 2009)
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• Antimicrobials -- outline
• Development
• Resistance
• New opportunities

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Before Antibiotics

• Sources
• 1900-1970, U.S. Public Health Service, Vital
  Statistics of the United States, annual, Vol. I
  and Vol II
• 1971-2001, U.S. National Center for Health
  Statistics, Vital Statistics of the United
  States, annual National Vital Statistics Report
  (NVSR) (formerly Monthly Vital Statistics
  Report) and unpublished data.
• From Statistical Abstract of the United States
  20042005.

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The advent of antibiotics played a major role in
improving infant mortality and lifespan
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Penicillin The first antibiotic

• Discovered by Alexander Fleming in 1929
• Mass produced in the 1940s in response to the
  war effort
• Howard Florey, Ernst Chain and Norman Heatley
  credited with developing penicillin as a useful
  drug

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Antibiotic Discovery Timeline
Table taken from The Antibiotic Paradox by Dr.
Stuart Levy
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Major Antibiotic Targets
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Christopher Walsh. Nature Reviews Mico. 2003.
165-70.
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Cell wall inhibitors

• b-lactam antibiotics
• Glycopeptides
• Phosphonomycin
• Bacitracin

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?-lactam antibiotics Bind to and inhibit the
activity of transpeptidases that are essential
for crosslinking glycan linked peptide chains in
the bacterial cell wall. Penicillin Ampicillin P
enicillin originally only acted against
Gram-positive bacteria but chemical modifications
have made it functional against Gram-negative
bacteria as well
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Peptidoglycan synthesis
Target of penicillin and glycopeptide antibiotics
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??lactam antibiotics
Q. How might you combat b-lactamases?
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Combating b-lactamases AugmentinTM
Augmentin combination of amoxicillin (b-lactam
antibiotic) clavulanic acid (b-lactamase
inhibitor)
Clavulanic acid
Amoxicillin

• Clavulanic acid binds irreversibly to
  b-lactamase via its b-lactam ring, protecting
  amoxicillin from denaturation
• Augmentin is typically prescribed to treat ear
  infections that do not respond to other
  antibiotics

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Growth factor analogs sulfa drugs-sulfanilamide,
an analog of p-aminobenzoic acid that blocks
folic acid synthesis isoniazid-interferes with
synthesis of mycobacterial specific cell wall
component mycolic acid
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Sulfa drugs
Used to treat a range of infections including
bronchitis, UTIs, and travelers diarrhea.
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Quinolones Synthetic compounds that interfere
with gyrase and prevent packaging of bacterial
DNA Naladixic Acid Ciprofloxin Quinolones
function against a wide range of bacteria, most
famously given prophylactically following
exposure to anthrax
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Aminoglycosides Amino sugars bonded by glycosidic
linkages. Inhibit protein synthesis at 30S
ribosomal subunit Streptomycin Kanamycin Neomycin

Often give as injectables to treat serious/life
threatening infections. They have significant
side effects including hearing loss and seizures,
and are typically used only when no other
antibiotics are effective.
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Macrolide Antibiotics Lactone rings connected to
sugar moieties. Protein synthesis inhibitors
acting at 50S ribosomal subunit Erythromycin
Erythromycin is particularly effective against
Legionella pneumophila, the cause of
Legionnaires disease. Why???
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Tetracycline First broad spectrum antibiotics.
Protein synthesis inhibitors that interfere with
30S ribosomal subunit
Given topically to treat acne and orally to treat
acne and other infections. Typically not given to
young children since they can stain teeth. Also
used to prevent malaria.
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Cephalosporins Act similarly to ?-lactam
antibiotics by binding to transpeptidases and
preventing crosslinking Cephalexin Cephtriax
one Resistant to ?-lactamases
Used to treat a broad range of infections
including pneumonia, and those in bone, ear and
skin
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???
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• Antimicrobials -- outline
• Development
• Resistance
• New opportunities

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Antibiotic Resistance Early signs that perhaps
the magic bullet was losing its magic

• Fleming warned in 1945 that the misuse of
  penicillin could lead to selection of resistant
  forms of bacteria (he had derived resistant
  strains in his lab by varying the dosage and
  growth conditions).
• Until the 1950s, penicillin was available over
  the counter in the US increasing the chance of
  misuse
• In 1946, one hospital reported that 14 of staph
  isolated from sick patients were penicillin
  resistant.
• By the end of the decade, the same hospital
  reported that resistance had been conferred to
  59 of the strains of staph studied.
• The first means of combating antibiotic
  resistance was the development of modified
  forms of penicillin--like ampicillin--that were
  resistant to degradation by beta-lactamases.

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• In 2002, the CDC estimated that at least 90,000
  deaths a year in the USA could be attributed to
  bacterial infections, more than half caused by
  bugs resistant to at least one commonly used
  antibiotic
• Serious infections caused by MRSA (methicillin
  resistant Staphylococcus aureus) was close to
  100,000/yr with almost 19,000 related fatalities
• Methicillin introduced in 1959 (semi-synthetic
  penicillins designed in response to penicillin
  resistance)
• However, within 2 years started to see
  resistance due to mecA, which encodes an altered
  PBP (mecA probably came from common skin bacteria
  Staph sciuri).

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Mechanisms of resistance

• Limiting access of the antibiotic
• Enzymatic inactivation of the antibiotic
• Active efflux of the antibiotic
• Modification or protection of the antibiotic
  target

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Mechanisms of resistance

• Limiting access of the antibiotic
• b-lactam antibiotics must transit outer membrane
  to reach PBPs in the inner membrane
• Porins in the outer membrane limit diffusion and
  can mutate to increase resistance
• e.g. vancomycin very effective against Gm() but
  not Gm(-) because it is too bulky to cross the
  outer membrane
• Can also get reduced uptake across the inner
  membrane (important for antibiotics with
  cytoplasmic targets, e.g. ribosomes)
• Not common because some antibiotics are membrane
  permeant (e.g. hydrophobic tetracycline)
• However, aminoglycosides use transporters to get
  into cells bacteria are more resistant when
  grown anaerobically

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Mechanisms of resistance

• Enzymatic inactivation of the antibiotic
• Best examples are b-lactamases
• b-lactamases are secreted into the periplasm by
  Gm(-) into the extracellular fluid by Gm() and
  cleaves the b-lactam ring

Satellites around AmpR colonies
Inhibitor of b-lactamase
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Mechanisms of resistance

• Enzymatic inactivation of the antibiotic
• Aminoglycoside-modifying enzymes inactivate the
  antibiotic by adding a group (phophoryl, adenyl,
  or acetyl group) to the antibiotic
• Chloramphenicol or Kanamycin acetyltransferase --
  adds an acetyl group to the antibiotic
• Streptogramin acetyltransferase -- also add an
  acetyl group
• Oxidation of tetracycline

N-acetyltranferase encoded by a resistance
plasmid can inactivate by attacking free
aminogroup
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Mechanisms of resistance

• Active efflux of the antibiotic
• Efflux pump -- pumps antibiotic out of the
  cytoplasm and prevents it from reaching high
  enough concentration
• First efflux mechanism discovered mediated
  resistance to tetracycline -- cytoplasmic
  membrane protein that catalyzed
  energy-dependent transport of tet out of the cell
• Macrolides can also be pumped out (e.g. Staph)
• Quinolones -- low level resistance
• Streptogramins -- efflux pumps identified

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Mechanisms of resistance

• Modification or protection of the antibiotic
  target
• Resistance to b-lactams
• Alter the penicillin-binding protein (PBP) so it
  no longer binds the b-lactams
• Most common in Gm() -- e.g. mecA confers
  resistance to methicillin in Staph
• Resistance to glycopeptide antibiotics
• Vancomycin prevents peptidoglycan cross-linking
  by binding D-Ala-D-Ala of the muramyl-peptide
  (NAM)
• Can become resistant by replacing D-Ala-D-Ala
  with D-Ala-D-lactate, which does not bind
  vancomycin
• 3 essential enzymes include vanA or vanB (ligase
  that makes D-Ala-D-lactate

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Mechanisms of resistance

• Modification or protection of the antibiotic
  target
• Resistance to glycopeptide antibiotics
• Vancomycin prevents peptidoglycan cross-linking
  by binding D-Ala-D-Ala of the muramyl-peptide
  (NAM)
• Can become resistant by replacing D-Ala-D-Ala
  with D-Ala-D-lactate, which does not bind
  vancomycin
• 3 essential enzymes include
• vanA or vanB ligase that makes D-Ala-D-lactate
• vanH lactate dehydrogenase that makes lactate
  from pyruvate
• vanX cleaves D-Ala-D-Ala but not
  D-Ala-D-lactate (have to get rid of D-Ala-D-Ala
  otherwise vancomycin can still bind)

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How to select for resistant bacteria

• Taking antibiotics unnecessarily
• Antibiotics are useful in x lt 20 of patients
  seen with an infectious disease. However, they
  are prescribed 80 of the time!
• Not finishing the prescribed course of
  antibiotics
• Below optimal dosage is insufficient to eliminate
  pathogen and selects for resistant strains
• However, not everyone agrees (Science. 2008.
  321 356)
• A few existing studies suggests that the
  necessary length of therapy is surprisingly short
  (e.g. UTIs can be treated with 1-3 day course of
  antibiotics and vast majority of patients with
  pneumonia get better after 2-3 days)
• MORE RESEARCH IS NEEDED

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Chart from the CDC indicating when to prescribe
antibiotics
As you can see the answer is RARELY!!!
http//www.cdc.gov/drugresistance/community/tools.
htm
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Antibiotic resistance can then be transferred via
standard routs of gene swapping
Conjugation-Transformation-Transduction
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Conjugation
Salyers and Whitt, Microbiology (2001)
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Generalized Transduction
Salyers and Whitt, Microbiology (2001)
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Transformation
Natural Competence occurs in Streptococcus
pneumoniae, Haemophilus influenzae, Bacillus
subtilis
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Horizontal transfer is not species specific!!
Salyers and Whitt, Microbiology (2001)
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Relationship between antibiotic use and frequency
of resistant bacteria
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Drug resistant gonorrheae
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• Antimicrobials -- outline
• Development
• Resistance
• New opportunities

45

• Drugs for bad bugs confronting the challenges
  of antibacterial discovery
• Most large pharm companies and many biotechs have
  left the area of antimicrobials (many reasons
  but major one was not enough )

Payne et al. Nature Reviews Drug Discovery 6,
2940 (January 2007) doi10.1038 / nrd2201
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• Drugs for bad bugs confronting the challenges
  of antibacterial discovery
• Most large pharm companies and many biotechs have
  left the area of antimicrobials (many reasons
  but major one was not enough )
• 1995 determination of the Haemophilus influenzae
  genome changed everything
• GlaxoSmithKline (GSK) spent 7 years (1995-2001)
  evaluating more than 300 genes for potential as
  targets and showed that 160 were essential genes
• 67 high-through-put screens (HTS) of individual
  targets were run against the SmithKline Beecham
  compound collection (260,000-530,000 compounds)
• Only 16 HTS gave rise to hits and only 5 resulted
  in leads

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• Drugs for bad bugs confronting the challenges
  of antibacterial discovery
• Also ran 2 whole-cell antibacterial screens
• Wild type antibiotic sensitive strain of Staph
  aureus
• Wild type (efflux competent) strain of E. coli
• Up to 500,000 synthetic compounds were screened
  at a concentration of 10 mM
• Results
• No exploitable hits for the E. coli screen (many
  nuisance compounds)
• 300 antibacterials against Staph (however most
  were ruled out as non-specific membrane-active
  agents (detergents and uncouplers)

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• Drugs for bad bugs confronting the challenges
  of antibacterial discovery
• 70 HTS campaigns run between 1995-2001 (67 target
  based and 3 whole cell) --gt 5 leads were
  delivered
• Success rate is 4-5 fold lower than for targets
  from other therapeutic areas at the time
• Each HTS campaign costs over US 1 million
• Other companies did just as bad
• X gt 125 antibacterial screens on 60 different
  antibacterial targets were run by 34 different
  companies
• Few/no credible developmental candidates

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• Drugs for bad bugs confronting the challenges
  of antibacterial discovery
• Why not work???
• Consequence of the lack of chemical diversity
  screened at the time?
• Asking for too much (e.g. broad spectrum
  antibiotics)
• New approach
• 2002 GSK overhauled its antibacterial research
  strategy
• Shifted to a select number of programs with
  late-stage leads
• Focused on novel, chemical structures -- not
  targets -- whose members had excellent in vitro
  and in vivo antibacterial activities

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• Lipinski's Rule of Five
• Rule of thumb to evaluate druglikeness, or
  determine if a chemical compound with a certain
  pharmacological or biological activity has
  properties that would make it a likely orally
  active drug in humans
• Rule was formulated by Christopher Lipinski in
  1997 (based on observation that most medication
  drugs are relatively small and lipophilic
  molecules)
• Rule describes molecular properties important
  for a drug's pharmacokinetics in the human body,
  including their absorption, distribution,
  metabolism and excretion (ADME)
• Modification of the molecular structure often
  leads to better drugs

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• Lipinski's Rule of Five
• Lipinski's rule says an orally active drug has
  no more than one violation of the following
  criteria
• Not more than 5 hydrogen bond donors (nitrogen
  or oxygen atoms) with one or more hydrogen
  atoms)
• Not more than 10 hydrogen bond acceptors
  (nitrogen or oxygen atoms)
• A molecular weight under 500 daltons
• An octanol-water partition coefficient log P of
  less than 5
• Note that all numbers are multiples of five,
• which is the origin of the rule's name (damn
  clever!)

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Natural antibiotics tend to be large
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Chemical diversity of antibacterial is different
to other drugs (more hydrophilic and slightly
larger)
Payne et al. Nature Reviews Drug Discovery 6,
2940 (January 2007) doi10.1038 / nrd2201
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Other approaches in academics e.g. Pilicides
Rationally designed small compounds inhibit
pilus biogenesis in uropathogenic bacteria.
Pinkner et al. PNAS. 2006 10317897-902.
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Cholera toxin secretion causes intestine to lose
water and induces strong and often lethal diarrhea
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Small-Molecule Inhibitor of Vibrio cholerae
Virulence and Intestinal Colonization (Hung et
al. Science. 2005. 310670)

• Screened 50,000 compound library for those that
  inhibit cholera toxin production
• One compound shown to inhibit ToxT (an activator
  needed for Ctx production)
• Compound (termed Virstatin) had no effect on
  Vibrio cholerae or other bacterial growth in
  vitro
• But when tested in a mouse model, Virstatin
  reduced bacterial levels 1000 fold and reduced
  pathology
• Proof of principle for a pathogen specific
  compound
• Unfortunately many strains of cholera naturally
  express a Virstatin-resistant ToxT or use
  alternative mechanisms to activate Ctx

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An Inhibitor of FtsZ with Potent and Selective
Anti-Staphylococcal Activity (Haydon et al.
Science. 2008. 3211673)

• Created a class of small synthetic
  antibacterials (e.g. PC190723), which inhibits
  FtsZ and prevents cell division
• PC190723 has potent and selective in vitro
  bactericidal activity against staphylococci,
  including methicillin- and multi- drug-resistant
  Staphylococcus aureus
• Putative inhibitor-binding site of PC190723 was
  mapped to region of FtsZ analogous to the
  Taxol-binding site of tubulin
• PC190723 was efficacious in an in vivo model of
  infection, curing mice infected with a lethal
  dose of S. aureus.

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An Inhibitor of Gram-Negative Bacterial
Virulence Protein Secretion (Felise et al. Cell
Host Microbe. 2008. 4325)

• High-throughput screen for inhibitors of type
  III secretion systems (T3SS)
• This is your assigned reading!