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Bacterial DiseasesBubonic Plague Tuberculosis Cholera
Sepsis Lyme Disease
Antibiotics
1929 – Penicillin discovered
1933 – Sulfa drugs synthesized
1969 – US surgeon Gen “end infectious diseases???
Today – bacteria with multi-drug resistance. Concern over resistance to ‘last resort’ antibiotics.
1865 – Pasteur - Decay due to living organisms
1867 – Lister – phenol is disinfectant
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Enterococcus faecalis
A leading cause of hospital infections
vanomycin = antibiotic of last resort
E faecalis resistant strains for years
can transfer resistance genes to Staphylococcus aureus in lab - MRSA
virulent cause of pneumonia, endocarditis, sepsis etc.
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Examples of Antibiotic Targets
Cell Wall Formation - penicillin, cephalosporins, vancomycin
Replication – novobiocin & DNA Gyrase
Transcription – rifampicin & RNA Pol
Translation – puromycin & ribosome ‘A’
Folate biosynthesis – sulfa drugs & DHPS
Fatty Acid synthesis – triclosan & enoyl reductase
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Killing Bacteria without Resistance
Drastically alter Bacterial environment so that multiple systems become inoperative. Therefore, many genes would have to mutate to cause resistance.
Bleach (NaOCl) – Oxidize multiple targets in bacteria
Detergents/soap/alcohol – disrupt membrane
Heat/pH extremes - denature proteins
UV irradiation – grossly damage DNA
Antimicrobial Peptides (AMPs) – lyse membranes
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Bacteria chromosome
plasmids
Plasmids in bacteria often contain genes critical for …..antibiotic resistance, toxins, natural product metabolismF factor plasmid (for sexual transmission of plasmids)
Bacteria can transfer antibiotic resistance plasmids between species
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Practices that Foster Resistance1. taking antibiotics for non-bacterial illness
2. not taking all of antibiotic
3. non-human use of antibiotics antibiotics as growth promoters in animals
Resistant Bacteria ― strategies
1. mutated target enzyme – evasion strategy
2. enzyme to destroy antibiotic – attack strategy
3. efflux channel – bailout strategy
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Fighting Back at Resistant Bacteria
3. Find new targets for Drugs
4. Find new classes of drugs
2. Develop ‘co’-drugs
1. Develop new drugs for same targets
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Sulfthiazole resistance ― case study
1985 – 5 isolates of resistant Streptoccoccus Pyogenes saved from patients in Sweden Hospital
1990’s – Genomes from normal and resistant isolates compared – highly mutated genes cloned & expressed in E. coli.
DHPS gene found to be mutated. (evasion strategy)
Pathway genes: folC - folE - folP - folQ - folK
folE = GTP cyclohydrolasefolQ = dihydroneopterin aldolasefolK = hydroxymethydihydropterin pyrophosphatase converts GTP into dihydropteridin unit
folP = DHPS (dihydropteroate synthetase) adds PABA unit
folC = dihydrofolate synthetase adds glutamate unit
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H2N- -COOH
H2N-
N
NN
HN
O OHN- -C- NH-CH-COO-
CH2
CH2
COO-
Dihydrofolate Biosynthesis
Pathway genes: folC - folE - folP - folQ - folK
folE = GTP cyclohydrolasefolQ = dihydroneopterin aldolasefolK = hydroxymethydihydropterin pyrophosphatase converts GTP into dihydropteridin unit
DHPS DHFS
PABA → → dihydrofolate
folP = DHPS (dihydropteroate synthetase) adds PABA unit ― 16% divergence
folC = dihydrofolate synthetase adds glutamate unit
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NH2
O=S=O | NH2
sulfanilamide
sulfathiazole
NH2
N-H
N S
O=S=O
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E. Coli - DHPS
sulfonamide
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E. Coli - DHPS
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KM(inhib) = KM (1 + [I]/Ki)
KM Ki G1 (suscep) 0.7mM 0.2mM G56 (res) 2.5mM 27.4mMDifference 3.6x 137x
DHPS Kinetics
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They are analogs of the peptide component of the bacterial cell wall
penicillins and cephalosporins are antibiotic classes that possess lactam ring
b-lactamases of varying specificities are often found in ‘R’ plasmids of resistant bacteria.
penicllin and b-lactams inhibit the cell wall synthesis in bacteria
Lactams contain a 4-membered ring with an amide nitrogen and a keto group.
b-lactamases destroy b-lactams by cleaving (O=C ― N) in lactam structure. Attack strategy destroys antibiotic before it can kill bacteria .
Penicillin inhibits last connection in making bacterial cell wall … Glycopeptide Transpeptidase
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Glycopeptide transpeptidase
b-lactam antibiotic
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Glycopeptide transpeptidase
b-lactam antibiotic
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Polysaccharide
X-X-X-A-A
X-X-X-A-A
G-G-G-G-G
G-G-G-G-G
X-X-X-A-A
X-X-X-A-A
G-G-G-G-G
G-G-G-G-G
Peptidoglycan
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Bacterial Cell Wall Completion
X-X-X-A
X-X-X-A
G-G-G-G-G
G-G-G-G-G
G-G-G-G-G
G-G-G-G-G
X-X-X-A-A
X-X-X-A-A
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R C = O H - N S CH3
C - C C - CH3
C - N C O COO-
CH3 CH3
- N - C - C - N - C O COO-
penicillin
-D-Ala-D-Ala
mimics AAseq of peptidelinker
b-lactamase
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R C = O H - N S CH3
C - C C - CH3
C - N C O COO-
penicillin
O
CH3
C - C C - CH3
C - N C O COO-
O
clavulanate
given along with penicillin it will inhibit penicillinase
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OH
OO
Cl
Cl
NNCO
OHOH
OH
N
O
HOOC
O
N
O
OH
N
ON-CH3
O
NH2
HO
Vancomycin binds to D-Ala – D-Ala peptide unit
Resistance due to target mutation in peptidoglycan – D Ala to D – lactate giving 3x less drug affinity due to missing H-bond. replacing C=O with CH2 produces 100x activity to mutant retains only 3% activity to sensitive bacteria. C&E News Feb 13, 2006
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Vancomycin (blue)
D-Ala – D-Ala
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Efflux Pumps ― bailout strategy
Many efflux pumps expel a broad range of compounds – may have normal anti-toxin function.
efflux pump inhibitors, like b-lactamase inhibitors, could well be analogs of the original antibiotic and have mild antibiotic activity as well.
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E. Coli ACRB
Multi-drug efflux transporter
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Efflux Pumps
antibiotic
bacteria cell membrane
antibiotic target
effluxpump
EP inhibitor
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Pdb – 2f2m EmrEtetraphenylphosphonium
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Cl
Cl
ClO
O
Triclosan
inhibits enoyl reductase
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Fatty Acid Synthesis
acetylCoA + HCO3- + ATP malonyl CoA +ADP
acetylCoA + ACP acetyl-ACP + CoA
malonylCoA + ACP malonyl-ACP + CoA
acetyl-ACP +malonyl-ACP acetoacetyl-ACP + CO2 + ACP
acetoacetyl-ACP + NADPH hydroxybutyryl-ACP + NADP+
hydroxybutyryl-ACP Crotonyl-ACP + H2O
Crotonyl-ACP + NADPH butyryl-ACP + NADP+
Enoyl-ACP reductase
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Enoyl reductase (step in fatty acid synthesis)
triclosan
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Enoyl reductase (step in fatty acid synthesis)
triclosan
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Parikh et. al. (2000) Biochemistry 39, 7645-7650
Triclosan inhibits enoyl-ACP reductase from Mycobacterium Tuberculosis Ki ~ 0.22 mM for crotonyl-ACP & NADH
Y158 F Ki ~ 47 & 36 mM
M161 V triclosan resistant Ki ~ 4.3 mM also less sensitive to isoniazid
triclosan could stimulate TB resistant strains of mycobacterium
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New Antibiotics
oxazolidimones (linezolid) – binds 30S subunit of ribosome and prevents mRNA & fMet-tRNA binding.
gemifloxacin – DNA gyrase inhibitor used on respiratory tract infections
daptomycin – blocks peptidoglycan and lipoteichoic acid synthesis (cell wall formation) works on vanomycin resistant enterococci
BPI Protein - (bacterial permeability increasing) naturally found in bactria killing wbc’s – good in combo
Antimicrobial Peptides – defensins & protegrins may function as voltage-gated pores specific for
acidic phospholipids found only in bacteria
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New Targets for Antibiotics
sortase – cleaves loosely bound surface proteins in gram (+) bacteria to activate infectivity proteins. (doesn’t kill bacteria)
deformylase – removes formyl group from amino end of bacterial polypeptides – includes actinonen (natural cpd)
Efflux Pump Inhibitors