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