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Chemical Evolution of β-
Lactams to Keep Pace with Bacterial
Resistance
Malcolm G. P. PageWisdom Professor of Medicinal Chemistry and Chemical Biology
2Evolution of β-Lactams and Resistance
Penicillin G
Penicillinase
Semi-synthetic penicillins (methicillin)
3rd generation cephalosporinsCephalosporinases
Extended spectrum -lactamasesCarbapenems
Serine carbapenemasesMetallo -lactamases
-lactamase inhibitorsInhibitor-resistant ESBL
Highly resistant transpeptidases
Permeation defects Efflux systems
Gram-positive Bacteria Gram-negative Bacteria
Anti-MRSA cephalosporins
Semi-synthetic penicillins (oxacillin, carbenicillin, piperacillin)
Broad spectrum -lactamases, oxacillinases
1st & 2nd generation
cephalosporins
3-Lactams Rapidly Destroy S. aureus……
Giesbrecht et al., 1998, Microbiology and Molecular Biology Reviews 62: 1371-1414
S. aureus growing normally S. aureus after exposure to penicillin G
4 …… and Can Quickly Cure InfectionThe fourth patient to receive penicillin G
Before treatment After a few days
5-Lactams Inhibit Cell Wall Biosynthesis
-Lactams mimic the peptide side chains. They are bound by PBPs and undergo a reaction similar to that observed with the peptide.
The covalentintermediate formed with a -lactam cannot cross-link the cell wall.Without the cross-bridges, the cell wall remains in a fragile state and it cannot protect the cell against lysis.
0102030405060708090100
1935 1945 1955 1965 1975 1985 1995 2005Year of report
Perc
enta
ge o
f cas
esPenicillin Resistance in Staphylococci
Penicillinase-producing staphylococci
(Pooled US hospital/nursing home reports)
020406080100
0 15000 30000 45000Penicillin production (Tonnes)
Peni
cilli
n re
sist
ance
(%)
Penicillinase 1st report
7Pencillinase-stable β-Lactams
N
S
OHO
O
NH
O
O
O
NO
N
S
OHO
O
NH
O
Methicillin 1962
Oxacillin 1962
N
S
OHO
O
NH
O
Introducing a rigid, bulky substituent close to the acylamino function in penicillin results in poor fit into the penicillinase active site because of clash with the Ω-loop
Moult et al., Biochem. J., 1985
0102030405060708090100
1945 1955 1965 1975 1985 1995 2005Year of report
Perc
enta
ge o
f cas
esEvolution of β-Lactam Resistance in Staphylococci
Penicillinase-producing staphylococci
(Pooled US hospital/nursing home reports)
Methicillin-resistant staphylococci
MRS 1st report
9Methicillin Resistance in Staphylococci is due to an Additional Low Affinity Transpeptidase (PBP2')
PBP 1
PBP 2PBP 3
PBP 2´ PBP 2a mecA gene product
Methicillin-susceptible
Methicillin-resistant
Compound MIC (mg/L)
Penicillin G 1 >64
Cefotaxime 4 >64
Ceftobiprole 0.5 2
Progress of a PBP Inhibitor Project
1990 Decision to start a programme to discover specific inhibitors of lactam-insensitive PBPs
(and modify β-lactams)
MRSA rate in US 12% (1986 data)
Clones S. aureus PBP 2a, S. pneumoniae PBP 2x obtained from expert groups Internal work on enterococcal and Gram-
negative PBPs started after some discussion
10
11Resistance in Pneumococci is due to Altered PBPs
1a1b
2x2a2b
12288 1098 15T 17921 Ap9
Compound MIC (mg/L)
Penicillin G 16 16 16 8 ≤0.06
Cefotaxime 4 2 4 2 ≤0.06
Ceftobiprole 0.5 0.25 0.25 0.5 ≤0.06
Penicillin G binding (at 5 and 1 M for each strain)
12Structure of S. pneumoniae PBP2x
Active site
Flexible domain
Stalk region
Membraneanchor
C-terminal domain
Transpeptidase domain
13Conformation Change in Solution is Linked to AcylationFTIR spectroscopy shows loss of 2°structure concomitant with ester formation
Quench of intrinsic protein fluorescence concomitant with acylation
Chittock et al. (1999) Biochem. J.; Jamin et al. (1993) Biochem. J
14Conformation Change in Crystals Apo enzymeActive site cleft narrow
Methicillin complex (red)Active site opened to fit -lactam
-Lactams Form Stable Acyl-enzymes with PBP 2xNative protein denatures at 49 °C
Carbapenems form the least stable complexes (-1 < Tm < +1 °C)
Penicillins and cephalosporins form complexes with increased thermal stability (+6 < Tm < +10 °C)
Ceftobiprole complex has greatly increased thermal stability (Tm +12 °C)
16Reaction of PBP2x with -Lactams
• Apo-enzyme in closed conformation in crystals
• No structural change prior to acylationsilent event ?open and closed states encompassed by
normal vibrational mode of protein in solution?
• Conformation adopted in acylated state dependent on the acylating agent
• Complexes with most -lactams can be obtained by soaking crystals
17Three Major Steps in the Transpeptidase Reaction(s)Binding Acylation Lysis
The acyl-enzyme complex is the critical covalent intermediate in transpeptidation and for inhibition.
Conformational change closes the active site around the peptide or inhibitor, preventing hydrolysis of the acyl-enzyme.
E + L1 E.L1 E-I E-I.L2 E.P E + P L2
Which steps are modified in resistant mutants?
Reaction of PBP2x-505 with β-Lactams
Laboratory mutant isolated by R. HackenbeckResistance attributed to a single point mutation in the
pbp2x geneDescribed as “zero affinity” in PBP2x
18
Affinity not greatly decreased (Kapp 15 μM cf 10 μM)
Acylation rate greatly decreased(kac 0.03 s-1 cf 0.8 s-1)
Progress of a PBP Inhibitor Project
1992 An hypothesis is born
MRSA rate in US 20%
Routine assay of binding to purified soluble constructs of S. aureus PBP 2a, S. pneumoniae PBP 2x (and mutants) and E. faecium PBP5
Studies with PBP2x mutants suggested needed to increase reactivity of β-lactams, optimize productive binding and gain interactions in the amino-peptide (acceptor) binding pocket
19
20Resistance of E. faecium is due to an Endogenous Low Affinity PBP, its Hyperexpression and Mutation
Compound MIC (mg/L)Ampicillin 2 >32 >32Cefotaxime 16 >32 >32Ceftobiprole 1 8 >32
PBP 1
PBP 2PBP 3PBP 4
PBP 5
21Structure of E. faecium PBP5R
Membraneanchor
Stalk region
Flexible domain110 residues
Transpeptidase domain
Active site
N-terminal domain of unknown function
22Amino-acid Substitutions between Ef PBP5S and Ef PBP5R
Multiple substitutions, and one insertion
Reaction slowed
Apparent affinity decreased
23Kinetics of Structural Changes Induced by Reaction with Penicillin Followed by by FTIR
Acyl-enzyme ester C=O
Penicillin C=O
Protein amide C=O
•Structural change concomitant with -lactam opening•Formation of intermediate species
24Differential Scanning Calorimetry of E. f. PBP 5
Native protein denatures at 44 °C
All -lactams form less-stable complexes
Two acyl-enzyme complexes can be distinguished:
Tm -2 °C formed first
Tm -10 °C formed later
25Conformation change accompanying acylation by penicillin GApo enzymeActive site closed PenG complex
(red)
26Opening of the Active Site Cleft Restructures the Catalytic Centre
Lysine comes into alignment
with serine
Complete network of hydrogen bonds established
Serine is activated and positioned for attack on incoming -lactam
27Reaction of PBP5R with -Lactams
• Apo-enzyme in closed and inactive conformation in crystals
• Structural change in solution is concomitant with acylation – no pre-transition
• Intermediate acyl-enzyme conformation clearly detectable
• Final conformation adopted in acylated state dependent on the acylating agent
Progress of a PBP Inhibitor Project
1994 β-Lactams accepted again
MRSA rate in US 32%
Vinyl cephalosporins based on cefdinir
have the desired increase in reactivityExtension of the 3’ side chain increases rate of
reaction (and apparent affinity for PBP2x mutants)
28
29Methicillin Resistance in Staphylococci is due to an Additional Low Affinity Transpeptidase (PBP2')
PBP 1
PBP 2PBP 3
PBP 2´ PBP 2a mecA gene product
Methicillin-susceptible
Methicillin-resistant
Compound MIC (mg/L)
Penicillin G 1 >64
Cefotaxime 4 >64
Ceftobiprole 0.5 2
30Conformation Changes of PBP 2a during Reaction with Penicillin in Solution
17051715172517351745
Time (s)
0 100 200 300
Abs
orba
nce
(mA
uni
ts)
0
2
4
6Penicillin C=O
Acyl-enzyme ester C=O
1760 cm-1
1732 cm-1
1717 cm-1
0
0.1
0.2
0.3
0.4
0.5
1500155016001650170017501800
Two conformations of ester C=O
Minor structural changes
Wavelength (nm)200 225 250
Circ
ular
dic
hroi
sm (m
deg)
-20
0
FTIR
UV CD
31Protein Fluorescence Changes Suggest Different Events During Acylation
Time (s)
0 200 400
Rel
ativ
e Fl
uore
scen
ce (%
)
50
60
70
80
90
100
110
a
bc
d
e
Benzyl penicillin
PiperacillinImipenem
Cephalothin
Vinyl cephalosporin
The acyl-enzyme formed with penicillins exhibits increased tryptophan fluorescence
Many compounds have little effect on tryptophan fluorescence
The acyl-enzymes formed with vinyl cephalosporins have strongly quenched tryptophan fluorescence
32
Native protein denatures at Tm 45.8 °C
Most acyl-enzyme complexes are less stable:
Imipenem: Tm 36.5 °C (Tm –9.3 °C)
Benzyl penicillin Tm 41.3 °C (Tm –4.5 °C)
The vinyl cephalosporin acyl-enzyme is more stable:
Tm 47.5 °C (Tm +1.7 °C)
The Acyl-enzymes Formed with -Lactams have Different Thermal Stabilities
Success of a PBP Inhibitor Project
1996 RO-63-9141 synthesized
MRSA rate in US 41% (1994 data)
33
Broad-spectrum activity against many pathogens that cause pneumonia, including MRSA, VRSA, PRSP, strains of Enterobacteriaceae and Pseudomonas species
34Structure of S. aureus PBP 2a
Membraneanchor
Stalk region
Flexible domain
Transpeptidase domain
Active site
N-terminal domain of unknown function
Lim & Strynadka, 2002
35Conformation Change Induced in Crystals by Reaction with Penicillin
Apo enzyme Penicillin G complex Active site closed Active site opened
36Opening of the Active Site Cleft Restructures the Catalytic Centre
Similar to Ef PBP5R but not as extreme
• Lysine comes into alignment with serine
• Complete network of hydrogen bonds established
• Serine is activated and positioned for attack on incoming -lactam
37Conformation Changes of PBP 2a during Reaction with β-Lactams in Solution
17051715172517351745
Time (s)
0 100 200 300
Abs
orba
nce
(mA
uni
ts)
0
2
4
6Penicillin C=O
Acyl-enzyme ester C=O
1760 cm-1
1732 cm-1
1717 cm-1
0
0.1
0.2
0.3
0.4
0.5
1500155016001650170017501800
Two conformations of ester C=O
Minor structural changes
Wavelength (nm)200 225 250
Circ
ular
dic
hroi
sm (m
deg)
-20
0
FTIR
UV CD
Conformation Changes in Solution during Reaction of PBP2a with Ceftobiprole Observed by FTIR•Only one conformation of ester C=O at 1712 cm-1
• Narrower band and lower wavenumber suggest more hydrogen bonding in oxyanion hole than with penicillin G
•Perturbation at 1697 cm-1 concomitant with ester formation
• Change of peptide amide C=O absorption suggests conformation change during acyl-enzyme formation
•Perturbation at 1632 cm-1 follows acylation, as 1697 cm-1 absorption relaxes to ground state
• Changes in peptide amide C=O absorption bands suggest a conformation rearrangement leading to a second acyl-enzyme species
38
Major Absorbance Changes During the Reaction of PBP2a with Ceftobiprole are Concomitant with Acylation
39
Time (s)0 50 100 150 200 250
Abs
orba
nce
chan
ge
0
0.02
0.04
318 nm
370 nmTime (s)
0 500 1000 1500 2000
Inco
pora
tion
or h
ydro
lysis
(m
ol/m
ol p
rote
in)
0
0.2
0.4
0.6
0.8
1
1.2
Ceftobiprole Binds with an Unexpected Second Non-covalent Intermediate
E.L Rapid non-covalent binding of ceftobiprole. Hydrophobic contacts
Covalent reactionOpening of -lactam ring and formation of E-IFurther structural changes (FTIR)
Three phases of change in protein fluorescence correspond to absorption changes
E.L* Second non-covalent intermediate with decreased absorbance and first FTIR signalHydrogen-bonding increased ► occluded state?
A318
F273/340
40
Summary 41
E + L E.L E.L* E-I t1/2 < 5 ms t1/2 = 0.8 s t1/2 = 36 s
• Ceftobiprole binds rapidly to PBP2a, initially mostly with charge-charge and hydrophobic interactions Also observed with penicillin G
• Structural changes (conformation selection?) tighten binding through increased H-bonding and form a productive complex Not observed with penicillin G
• Rapid acylation and formation of a stable acyl-enzyme complex Acylation by penicillin G is slow and results in an unstable acyl-enzyme
complex
• Rapid and stable acylation of PBP2a by ceftobiprole are also demonstrable in growing cells, with similar kinetics, and account for the strong bactericidal activity of ceftobiprole against MRSA
Acknowledgements 42
Roche & Basilea Birmingham UniversityIngrid Heinze-Krauss Christopher WhartonPaul Hebeisen Kanjanu ThumanuChristian Hubschwerlen Alan WilkinsonPeter AngehrnFranck DanelClothilde DantierMalgosia KaniaJohn GoodallFritz WinklerGlenn Dale
University of British ColumbiaNatalie Strynadka
Mike GretesAndrew Lovering
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