5/24/2017
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“Tuberculosis: An Introduction for Medicinal Chemists” Carl Nathan of Weill Cornell Medicine and Christopher Boyce of Merck 13
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Upcoming ACS Webinars www.acs.org/acswebinars Thursday, June 1, 2017
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Antibacterial Agents and Their Mechanisms Penicillin, Vancomycin, Daptomycin
Cell Envelope Homeostasis Streptomycin, Erythromycin, Rifampicin Chloramphenicol, Tetracycline
Transcription
DNA
Replication Ciprofloxacin
RNA
Translation
Protein
Proteolysis No Drugs!
There are no clinically used antibacterial drugs that target protein turnover!
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Chaperone Proteases: Validated Antibacterial Drug Targets
ClpP Peptidase
20S Proteasome 19
Chaperone Proteases: Validated Antibacterial Drug Targets
ClpP Peptidase
20S Proteasome 20
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ClpXP Protease: A Proteolytic Machine
• •
• •
ClpXP plays a critical role in protein quality control in bacteria. ClpP is a tetadecameric peptidase with a barrel-shaped structure with physically sequestered active sites. ClpX (or ClpC) is an accessory ATPase that unfolds substrates for ClpP-catalyzed proteolysis. ClpP and ClpX are either required for virulence (S. aureus, S. pneumoniae) or for viability (M. tuberculosis) Baker, A. Sauer, R. Annu. Rev. Biochem. 2011. 80: 587-612
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Chemistry of the ClpXP Protease
Catalytic Hydrolysis of Peptides
ClpP Peptidolytic Chamber
ClpP is a serine protease.
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Antibacterial Drug Leads that Target the ClpXP Protease Cyclomarin A1 (2011) Lassomycin (2014) b-lactones (2006)
Phenyl Esters (2015)
Ecumicin (2015)
ADEPs (2005) Sclerotomide (2016)
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Mechanistic studies and development at Bayer Healthcare AG 24
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Cyclic Acyldepsipeptides (ADEPs)
A54556 A (R= CH3) A54556 B (R= H)
Enopeptin A (R= CH3) Enopeptin B (R= H)
Acyldepsipeptidolactones produced by Streptomyces hawaiiensis and Streptomyces sp. RK-1051. Potent bactericidal activity against Gram-positive bacteria. 25
Selected ADEP-Susceptible Bacteria Troublesome Gram-Positive Bacteria
Methicillin-Resistant Staphylococcus aureus
VancomycinResistant Enterococcus faecium
Multi-Drug Resistant Mycobacterium tuberculosis
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ADEPs Bind and Activate the ClpP Peptidase
ADEPs
Brötz-Oesterhelt, Nature Medicine, 2005
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ADEPs Activate the ClpP Peptidase
Folded Protein
ClpP
Proteolysis
Folded Protein
ClpP + ADEP
Peptide Fragments
ADEPs can stimulate protein degradation in the absence of chaperones.
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Treatment with of S. aureus with ADEP decreased the abundance of ~400 proteins by >2-fold!
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Mechanism of ClpP-Activation by the ADEPs Top View
Side View
Li, Chem. Biol. 2010
ADEPs bind at the ClpP monomer interfaces and block interactions with the accessory ATPases.
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ADEPs Alter ClpP’s Quaternary Structure
apo-ClpP tetradecamer (top view)
ClpP tetradecamer in complex with ADEPs (top view)
ADEPs bind at ClpP subunit interfaces and induce expansion of the axial pores. Lee, B. et al. Nat Struct Mol Biol 2010, 17, 471-478.
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ADEPs Mimic a Loop of ClpX Accessory ATPase
IGF or LGF Loop Pore-2 Loop N-terminal Loop
Hydrophobic Cleft Baker and Sauer, Biochimica et Biophysica Acta, 2012
Li, D. S. et al. Chem. Biol. 2010, 17, 959 32
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ADEPs Prevent Association of ClpP with ClpX
ClpX
ClpX
ClpP
ClpP ADEPs 33
Pharmacological Properties of ADEPs O N O O
O NH O O N
N
O N H
O HN O
HO N H
O
Enopeptin B
MIC against methicillin-resistant S. aureus is 8 g/mL Poor efficacy in mouse models of infection
Hinzen, ChemMedChem, 2006
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Medicinal Chemistry Optimization of ADEPs O N O O
O
O NH O O
N H
N
N
O HN
HO N H
O
O
Enopeptin B
MIC against methicillin-resistant S. aureus is 8 g/mL Poor efficacy in mouse models of infection
ADEP 4
MIC against methicillin-resistant S. aureus is 0.6 g/mL Excellent efficacy in mouse models of infection 35
Hinzen, ChemMedChem, 2006
Medicinal Chemistry Optimization of ADEPs O N O O
O NH O O N
N
O N H
O HN O
HO N H
O
Enopeptin B
MIC against methicillin-resistant S. aureus is 8 g/mL Poor efficacy in mouse models of infection
ADEP 4
MIC against methicillin-resistant S. aureus is 0.6 g/mL Excellent efficacy in mouse models of infection Hinzen, ChemMedChem, 2006
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In Vivo Efficacies of ADEP4 and Zyvox® Control Zyvox treated ADEP 4 treated
In mouse models of S. aureus sepsis, ADEP 4 outperforms Zyvox®, a clinically used anti-bacterial drug. Hinzen, ChemMedChem, 2006
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ADEPs act synergistically in combination with antibacterial drugs. 38
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Medicinal Chemistry Optimization of ADEPs O N O O
O NH O O N
N
O N H
O HN
HO N H
O
O
Enopeptin B
MIC against methicillin-resistant S. aureus is 8 g/mL Poor efficacy in mouse models of infection
Pipecolic Acid Rigidifies Macrocycle
ADEP 4
MIC against methicillin-resistant S. aureus is 0.6 g/mL Excellent efficacy in mouse models of infections Hinzen, ChemMedChem, 2006
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Hypothesis: Macrocycle rigidification reinforces a bioactive conformation and lowers the entropic cost of ClpP binding.
Pipecolic Acid Rigidifies Macrocycle
ADEP 4
Free Energy of Binding DG= DH- TDS 40
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Crystal Structure of a Synthetic ADEP
ADEP exhibits trans-annular hydrogen bonding. Hinzen, ChemMedChem, 2006
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Structure of ADEP-ClpP Peptidase Complex
In complex with ClpP, ADEPs exhibit transannular hydrogen bonding. Predisposition for ClpP binding. Lee, B. et al. Nat Struct Mol Biol 2010, 17, 471-478
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Assessing Hydrogen-Bonding via Deuterium Exchange Experiments
Hydrogen bonding should attenuate the rate of exchange of the amide hydrogen atoms with deuterium in a deuterated solvent.
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Observing Deuterium Exchange via NMR t0
T15 min
T35 min
T55 min
T95 min
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Seven ADEP analogs were synthesized and evaluated. 47
Macrocycle Rigidification Strategy Serine vs. allo-(2S, 3S)-threonine
Pipecolate (Pip) vs. 4-methyl-Pip 48
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Conformationally Constrained ADEP Analogs Pip + allo-Thr
Pip + Ser
Natural ADEP
4- methyl Pip + Ser
4-methyl Pip + allo-Thr
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ADEP Deuterium Exchange (2 mM ADEP in CD3OD) 1 0.9
Fraction Hydrogenated
0.8 0.7 0.6 0.5 0.4
T1/2 = 25 min 0.3 0.2 0.1 0 0
50
100
150
200
250
Time (minutes)
300
350
400
450
500 50
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ADEP Deuterium Exchange (2 mM ADEP in CD3OD) 1 0.9
Fraction Hydrogenated
0.8 0.7 0.6 0.5
T1/2 = 63 min
0.4 0.3 0.2 0.1 0 0
50
100
150
200
250
300
350
400
450
500 51
Time (minutes)
ADEP Deuterium Exchange (2 mM ADEP in CD3OD) 1 0.9
Fraction Hydrogenated
0.8 0.7 0.6 0.5
T1/2 = 115 min
0.4 0.3 0.2 0.1 0 0
50
100
150
200
250
Time (minutes)
300
350
400
450
500 52
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ADEP Deuterium Exchange (2 mM ADEP in CD3OD) 1 0.9
Fraction Hydrogenated
0.8 0.7 0.6 0.5
T1/2 = 1737 min
0.4 0.3 0.2 0.1 0 0
50
100
150
200
250
300
350
400
450
500 53
Time (minutes)
ADEP Deuterium Exchange (2 mM ADEP in CD3OD) 1 0.9
Fraction Hydrogenated
0.8 0.7 0.6 0.5
T1/2 = 1178 min 0.4 0.3 0.2 0.1 0 0
50
100
150
200
250
Time (minutes)
300
350
400
450
500 54
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Summary of Deuterium Exchange Rates Pip + Ser
Pip + allo-Thr
Natural ADEP
T1/2 = 63 min
T1/2 = 25 min
4- methyl Pip + Ser
T1/2 = 115 min
T1/2 = 1737 min
4-methyl Pip + allo-Thr
T1/2 = 1178 min 55
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ClpP Activation Assay
Fluorogenic Decapeptide
• Titration of purified E. coli ClpP with ADEP induces degradation of a fluorogenic peptide in vitro • ADEP-ClpP affinities are inferred from differences in rates of peptidolysis at various [ADEP] 57
Titration Curves for ClpP Activation by ADEPs
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Pip + Ser
Pip + allo-Thr
Natural Macrocycle T1/2 = 63 min Kapp= 2.93 M 4- methyl Pip + Ser
T1/2 = 1737 min Kapp = 1.26 M 4-methyl Pip + allo-Thr
T1/2 = 25 min Kapp = 7.48 M
T1/2 = 115 min Kapp = 3.01 M
T1/2 = 1178 min Kapp= 1.11 M 59
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Natural Macrocycle
Pip + Ser
Pip + allo-Thr
T1/2 = 63 min Kapp= 2.93 M MIC = 0.0008 g/mL
T1/2 = 1737 min Kapp = 1.26 M MIC =
