-Lactams

-Lactams Rapidly Destroy Bacteria

S. aureus growing normally S. aureus after exposure to penicillin G

Giesbrecht et al., 1998, Microbiology and Molecular Biology Reviews 62: 1371-1414
 And Can Quickly Cure Infection

The fourth patient to receive penicillin G

Before treatment After a few days

 Cell Wall Biosynthesis - the Target of -
Lactams
The bacterial cell wall is built Transpeptidases, also known as
from strands of sugar penicillin-binding proteins (or
molecules that have peptide PBPs) react with one end of the
side chains. peptide side chain (the "acceptor")
to form a covalent intermediate.
 Cell Wall Cross-linking Gives Stability
The covalently attached PBP then Thus, a strong covalent link is built
binds another peptide side chain between neighbouring sugar strands.
(the "donor") and promotes its These peptide cross-bridges are
attack on the previously attached essential for the mechanical strength
acceptor chain. of the cell wall.
-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 covalentintermediate 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.
Inhibition of Cell Division is Especially
Effective
Autolysins S. aureus
Amidases, Lytic transglycosylases

PBP complex

Anchor complex

Cytokinetic Ring
E. coli
 Classes of -Lactam

N
O
R2 R2
R1 R2 R1
X R1 X
R3
N N
O N
O R4
R4 O R3
R4
monobactam penem cephem

All -lactam antibiotics have a strong negative charge at R4.

They do not work against intra-cellular pathogens
 Natural Monobactams
From streptomycetes (Nocardia)
NH2 OH
NH2 OH O
N H H
HOOC N
N
HOOC H O
N
O O OH
N
O OH
N O
Nocardicin A O
Formadicin A HOOC
HOOC

From Gram-negative bacteria (Pseudomonas)

X
R2 R1
Sulfazecins
N O
O SO3H H O
H2N N
N
H
O N
O O O SO3H
MM 42842
 Synthetic Monobactams
O
O
N

OH H
O S N
N
H2N N N
O
H H2N N
N
S O O
COO-
O
N
O SO3H
Oximonam 1985
Aztreonam 1983
O
O
OH OH

O O
N N
O N
O
H NH2 O OH
S N H N
S N H
N N N
O O N H2N
N O O
H2N H2N N S N
O SO3- O O
SO3- O OSO3H

Carumonam 1985 Tigemonam 1987 BAL0030072

 Uses of Aztreonam

Treatment of a variety of infections caused by Gram-negative pathogens.

urinary tract
lower respiratory tract
skin and skin structure
intraabdominal infections
septicemia,
endometritis,
pelvic cellulitis

Highly effective against susceptible bacteria without causing serious

adverse reactions.

Combination therapy required if the presence of Gram-positive organisms

are suspected
 Natural Penems
From fungi (Ascomycetes and Deuteromycetes e.g. Penicillium)
H
R N
Penicillins O

S
Penams Penicillin G O
N
O Penicillin F
O
HO
O
Penicillin V O

From streptomycetes (Streptomyces)

and Gram-negative bacteria R1 Carbapenems
Thienamycins
(Erwinia, Serratia) R2 Carpetimycins
N
O
O
Epithienamycins
OH
HO
Pluracidomycins
R1
X Olivanic acids
Penems R2
N R
N
O
O
O Asparenomycins
HO O
HO

O R Oxapenems
N Clavulanic acid
O
O
HO
 Semi-synthetic Penicillins - 1
The natural penicillins have simple, flexible side chains
They are susceptible to attack by penicillinases
The semi-synthetic penicillins
have bulkier, more rigid side
H chains.
R N S

N They are not susceptible to

O
O
attack by penicillinases and
HO more stable to broad-spectrum
-lactamases.

More polar side chains were

introduced to obtain Gram-
negative activity
 Semi-synthetic Penicillins - 2
Increased activity against penicillinase-producing
Gram-positive cocci. Little Gram-negative activity
O O

Methicillin 1961
O

O O
Nafcillin 1963

R2

Oxacillin 1962
R1 O
Cloxacillin (R1 = Cl) 1963
N
O
Floxacillin (R1 = Cl, R2 = F) 1972
 Semi-synthetic Penicillins - 3

Amino-penicillins
Broader spectrum (Gram-positive and Gram-negative)

O Ampicillin 1962

NH2

HO
O
Amoxicillin 1972
NH2
 Semi-synthetic Penicillins - 4
Broad spectrum (“Anti-Pseudomonas”). Stronger Gram-
negative spectrum (some loss of Gram-positive spectrum)

O O
O
Azlocillin 1976
O NH
O NH
O NH Piperacillin 1977
HO O N
N
O
Apalcillin 1986
N
N O N N
H

Carbenicillin 1968
O O
S
O

Ticarcillin 1973
O O
 Uses of Natural Penicillins
Natural penicillins: PenG, (Pen V for oral application)

Streptococcal infections (Scarlet fever, trep throat, erysipelas,

cellulitis, pneumonia, meningitis
(S. pneumoniae, S. pyogenes, Grp B, viridans grp)
Endocarditis
Enterococcal in combination with aminoglycoside
Streptococcal (Grp B Strep, Viridans group – usually PenG)
Menigitis
Neisseria meningitidis high dose Pen G
Syphilis
Treponema pallidum still very sensitive
Listeriosis
Listeria monocytogenes
Anaerobic infections (gas gangrene, tetanus, anthrax)
Clostridum perfringens, C. tetani, Bacillus sp.
 Uses of Penicillinase-stable Penicillins
Methicillin, nafcillin, cloxacillin, floxacillin

Staphylococcal infections:
skin infections (impetigo, boils, carbuncles)
abcesses in organs
pneumonia
prosthetic joint, catheter and artificial valve infections
endocarditis
meningitis (rare)
osteomyelitis (requires v. long therapy)

Streptococcal infections
especially when resistance is suspected or when Staph. is a
possibility
 Uses of Amino-Penicillins
Ampicillin, amoxicillin
Slightly weaker Gram-positive spectrum than Pen V
Gram-negative rods originally better, but now resistance is common

Otitis media – still drug of choice

(S. pneumoniae, Haemophilus influenzae, Moraxella catarrhaliis)
Bronchitis, pneumonia
Enterococcal endocarditis (+ aminoglycoside, drug of choice)
Meningitis – as alternative (+ chloramphenicol for H. influenzae)
UTI: the most common organisms (E. coli, Proteus mirabilis,
Staphylococcus saprophyticus) are covered. Resistance in E. coli.
Prophylaxis for bacterial endocarditis (2gm 1 hr before dental procedure)
Lyme disease (Borrelia burgdorferi) and Erlichiosis (Erlichia chaffeinsis)
STD as an alternative for Neisseria gonorrheae
 Uses of Broad Spectrum Penicillins

Carbenicillin, ticarcillin, azlocillin, piperacillin, apalcillin

Weaker Gram-positive spectrum than Pen V
Gram-negative rods and anaerobes covered. Resistance rising.

Pseudomonas aeruginosa infections (+ aminoglycoside)

Mixed infections

Complicated UTI and prostatitis (carbenicillin)

Surgical propylaxis, especially intra-abdominal & gynecologic

surgery
 Uses of Penicillin Combinations
Amino-penicillins
The amino penicillins are frequently used in combination with -
lactamase inhibitors for treatment of Gram-negative infections in
hospitals, and as broad-spectrum agent in the community

Oral: Amoxicillin and Clavulanate

Otitis media, sinusitis, RTI due to
resistant H. influenzae or M. catarrhalis.
SSTI due to S. aureus, E. coli, Klebsiella
with lactamase

Parenteral: Ampicillin and Sulbactam

SSTI, Intra-abdominal inf., gynecological
inf., [endocarditis (+AG)] S. aureus,
Enterobacteriaceae, Bacteroides fragilis
with lactamase, [enterococci & GrpD strep]
 Uses of Penicillin Combinations
Broad spectrum penicillins
Used parenterally in combination with -lactamase inhibitors for
treatment of penicillin-resistant (due to lactamase) infections in
hospitals

Ticarcillin + Clavulanate
Septicaemia, Lower RTI, Bone & Joint, UTI, gynecologic
infections, mixed infections

Piperacillin + tazobactam
Appendicitis, peritonitis, SSTI, endometritis, pelvic
inflammatory disease, community-acquired pneumonia
 Adverse Effects of Penicillins
Allergic
Anaphylaxis up to 0.4%, most common with PenG
Contact dermatitis, 4-10%, most common with amino-penicillins
Rare: serum sickness, hemolytic anemia (PenG high dose)
Idiopathic reactions
Rash, Fever, urticaria, 4-10%, most common with amino-penicillins
GI tract
Diarrhea, enterocolitis, up to 5%, most common with ampicillin
Hematologic
Neutropenia, up to 4%, PenG, oxacillin, piperacillin
Platelet dysfunction, 3%, carbenicillin
Rare: hemolytic anemia PenG
Hepatic
Elevated enzymes, 1-4%, Oxacillin, nafcillin, carbenicillin

Rare: electrolyte distrubances, seizures, bizarre sensation, interstitial nephritis

hemorrhagic cystitis
-Lactamase Inhibitor Combinations
Clinically approved used in combination with a -lactamase
labile
-lactam antibiotic
Penam sulfones Clavulanate
Class A + (C + D) -lactamases Class A + (D) -lactamases

O H
H O
O OH
S
N
Sulbactam N
O
O COOH
COOH

O
H O
N All have intrinsic activity against
Tazobactam S N
Acinetobacter sp.
N
N
O
COOH
 N Erwinia, Serratia
Natural Carbapenems
O
O
HO
Pluracidomycins
Thienamycins Streptomyces pluracidomyceticus
OH
Streptomyces cattleya
H
O O SO3-
N O
S
N
O
S S OH
N N
O NH2 O
O O
HO O
HO O
HO
PS-6, PS-8
O
Streptomyces fulvoviridis O
OH H OH H
N N

S S
N N
O
O Epithienamycins O
O O
HO Streptomyces olivaceus HO
Carpetimycins
S. fulvoviridis Streptomyces griseus
O O
O SO3- H O SO3- H
N N

S S
N Olivanic acids N
O
O O
O Streptomyces olivaceus O
HO HO
 Synthetic Carbapenems
Labile to renal dehyropeptidase
OH
OH Imipenem 1983 Panipenem 198?
Used with cilastatin S Used with betamipron
N
S O
N
O O N NH
HO
O N
HO H
NH

Stable to renal dehyropeptidase

O NH2

OH Meropenem 1992
N
H
S
N
O Ertapenem 2001 O
N O H
HO O N
+ O
OH N
N
OH
Biapenem 2001 N
S
N H
S
O N
O O
HO
O
HO
 Spectrum of Synthetic Carbapenems

Gram-positive bacteria (similar to 1st gen. Cephalosporins)

Streptococcus (not PenR), Staphylococcus (not MRS)
Listeria monocytogenes,
Enterococcus faecalis (bacteriostatic, use +AG)

Gram-negative bacteria
Enterobacteriaceae, Pseudomonas, Acinetobacter
Haemophilus sp., Neisseria, Moraxella

Anaerobes
Gram-positive anaerobes: Actinomyces, Clostridria
Gram-negative anaerobes: Bacteroides
 Uses of Synthetic Carbapenems

Empiric therapy for bacteremia + another antibiotic such

as an aminoglycoside (AG) or vancomycin

Nosocomial lower RTI

Serious intra-abdominal infections (+ macrolide or

tetracycline if Chlamydia is likely)

Pseudomonas aeruginosa infections (+ AG)

Gram-negative osteomyelitis

Meningitis
 Adverse Effects of Carbapenems

Well-tolerated

Some cross-reaction with penicillin-allergy

GI tract problems (1-2%). Pseudomembraneous colitis (0.1%)

Seizures (0.3 to 1 % with imipenem, unlikely with

meropenem)

Enterococcal superinfection (less than with 3rd gen.

Cephalosporins)
 Reactions of Carbapenems
Carbapenems have two features that contribute to stability towards -
lactamase O

N O

The hydroxyethyl group

O
O N
S H
H
O
N
S
O
NO
O O Ser64 O
Asn152 O Ser64
O O
S Asn152 O
O
O O OH
N
H
H N H
H

R' R'
R R
R
R' OH OH
N N N
O O H O H
O O OH O
OH
O ENZ ENZ
OH

Chemical rearrangement
H
R'
slow H
R'
R R
OH OH
N N
O O
O OH O
O
ENZ
ENZ
stable OH
O
O
Natural Cephems
H
N S
H2N
O
N O
Cephalosporin C
O Acremonium sp. (Fungi)
O
O O
O

O
H Cephamycin C (R = NH2)
N O
H2N S Streptomyces sp. (Bacteria)
O
N O R
O Cephabacines (R = oligopeptide)
O
O O Lysobacter lactamgenans
Xanthomonas lactamgenans
O
O
O
H
H2N N NH
S Chitinovorin A
O Flavobacterium sp. (Bacteria)
N OH
O

O O
Classification of Semi-synthetic
Cephalosporins
Gram-positive Bacteria Gram-negative Bacteria

Methicillin Methicillin Ampicillin Ampicillin

sensitive resistant sensitive resistant

1st Generation +++ - + -

2nd Generation ++ - ++ -

3rd Generation + - +++ +

4th Generation + - +++ ++

Anti-MRSA
+++ ++ +++ +(+)
cephalosporins
 1st Generation Cephalosporins
H
N S
S
O + Cephaloridine 1964
N N
O parenteral
O O
H
N S
S
O Cephalothin 1965
N O
O parenteral
NH2 O
O O
H
N S
O
N Cephalexin 1967
O oral
N O O

S H
N S Cephapirin 1970
O parenteral
N O
O
O
O O
 2nd Generation Cephalosporins
N
N H
N S
N N
O
Cefazolin 1971
O
N S N
N parenteral
S
O O
NH2
H
N S
HO
O
Cefamandole 1973
N S N
O N parenteral
N N
O O

NH2
H
N S Cefaclor 1976
O
N oral
O Cl

O O
NH2
H
HO
N S Cefprozil 1990
O
N
oral
O

O O
 Semi-synthetic Cephamycins
H
N O
S
S
Cefoxitin 1973
O
N O NH2 parenteral
O
O
O O
O O
H2N
S
O
S H Cefotetan 1983
N O
S
parenteral
O
N S N
O
N
N
O O N

N S H
N O
S
O
N S N Cefmetazole 1987
O
N
N
N
parenteral
O O
 Semi-synthetic Cephems
Carbacephem
NH2
H
N Loracarbef 1989
O oral
N
O Cl

O O

Oxacephamycin
O
O Moxalactam 1980
H O parenterral
N O
HO
O
N S N
O
N
N
O O N
 3rd Generation Cephalosporins
N O

N
H
N Cefotaxime 1979
S
S
O
parenteral
H2N N O
O
O O
O O

N O Ceftazidime 1980
N
H
N
parenteral
S
S
O +
H2N N N Very strong coverage of Pseudomonas
O

O O

N O
H
N N S Ceftriaxone 1981
S parenteral
O
H2N N S N O
O
N Very long half-life
O O N O
H
 Oral 3rd Generation Cephalosporins
O
Cefixime 1987
O

N O
H
N N S Specific transport by peptide carrier
S
O
H2N N
O

O O

N O

N
H
N Cefpodoxime Proxetil 1988
S
S
O
H2N N O
O
Prodrug, taken up by passive diffusion.
O O
Unstable ester readily hydrolyzed in serum
O O O
 4th Generation Cephalosporins

Cefepime 1987
N O parenteral
H
N N S
S
O +
H2N N N
O

O O

N O
Cefpirome 1989
H
N N S parenteral
S
O +
H2N N N
O

O O
 Uses of Cephalosporins

1st Generation: infections with lactamase-producing strains

RTI with Staph & Strep., UTI, SSTI

2nd Generation
CAP, SSTI, UTI, RTI, mixed infection, surgical prophylaxis

3rd Generation
Gram-negative septicaemia, Pseudomoans infections
Gram-negative meningitis, gonorrhea (single shot)
UTI, Gram-negative osteomyelitis
Lyme disease (ceftriaxone)

4th Generation
Similar to 3rd generation, especially if ESBL are suspected
Adverse Reaction of Cephalosporins
Well-tolerated

Allergic: rash (1-3%), anaphylaxis (rare), low cross-reactivity

with penicillin-allegy

Phelbitis, pain on IM injection

Hypoprothombinemia – associated with methylthiotetrazole ring

20-60% of patients

GI complaints (5-10%)

Elevated liver enzymes (5-10%)

Displacement of bilirubin from albumin (ceftriaxone)

Cholecystitis (precipitation in bile: ceftriaxone)

 Evolution of -Lactam Resistance
Gram-positive Bacteria Gram-negative Bacteria
staphylococci, enterococci, pneumococci Klebsiella, Acinetobacter, Pseudomonas
Stenotrophomonas

Anti-MRSA cephalosporins Inhibitor-resistant

ESBLA -lactamase inhibitors
Permeation defects Efflux systems
Metallo -lactamases Carbapenemases
Carbapenems
Extended spectrum -lactamases
Cephalosporinases
3rd Generation cephalosporins
Highly resistant 1st & 2nd generation
transpeptidases cephalosporins Broad spectrum -lactamases,
Semi-synthetic oxacillinases
penicillins
(methicillin) Semi-synthetic penicillins (oxacillin,
carbenicillin, piperacillin)
Penicillinase

Penicillin G
 Resistance in S. aureus

100 S. aureus (hospital)

Penicillinase
90
Resistance (% of isolates)

Introduction S. aureus (community)

80 Penicillinase
70
First resistance S. aureus (hospital)
60
Methicillin-resistant
50

40

30
S. aureus (community)
20 Methicillin-resistant
10

0
1938 1954 1970 1986 2002 S. aureus
Year Vancomycin-resistant
Methicillin Resistance in S. aureus is due
to an Additional Transpeptidase (PBP)
Methicillin-susceptlible Methicillin-resistant

PBP 1
Member of a small sub-
PBP 2
Changes in PBP 2´ family of type B PBPs
PBP 3
expression of found in staphylococci,
PBP 2 and enterococci and bacilli.
PBP4 All have low reactivity
can modulate towards many -
sensitivity lactams.
towards some
-lactams Next closest sequence
PBP 4
homology is to E. coli
PBP2
MIC <0.1 mg/L MIC >500 mg/L
 Structure of PBP 2´
Active site Flexible domain
110 residues

Stalk region

Membrane
Transpeptidase domain
anchor
The Highly Resistant Transpeptidases
of Gram-positive Bacteria
S. pneumoniae PBP 2x
Similar in structure to essential
-lactam-sensitive
transpeptidases
(30% sequence similarity)
S. aureus PBP 2’
Acylation very slow
( t 1/2 30 min cf 30s)

Very low affinity

(KS 10-1000 M cf 1-10 M)

Deacylation faster
(t 1/2 2 hr cf 2 days)

Much lower occupancy of active site

Active Site Accessibility in PBP 2´ is
Critical

Resting
The active site cleft is
too narrow for -lactams
to enter and is closed by
PBP side chains

Active
PBP domains
move, separating
the side chains
and opening the
cleft wide enough
for a -lactam to
enter
 Cephalosporins Active Against PBP2´

+
N
Ceftaroline (Forest)
O
N
N TAK-599 [T-91825] Peninsula (Takeda)
S H H
O N S Yoshizawa et al. Journal of Antibiotics (2002), 55(11),
-O N N 975-992.
P O
N
HO H N
O S S
COO-

RWJ-442831 [RWJ-54428] JnJ

Cl
N
OH (MC-02,479 Microcide)
S H H Hecker et al. Journal of Antibiotics (2000), 53(11), 1272-1281
N S
N
H2N O
N
O O O S
Cl COO- NH3+
N S
O H

BMS-247243 Bristol Myers Squibb

S H
N
H Singh et al., Organic Process Research & Development
Cl S
(2000), 4(6), 488-497.
O
N S
O O
+ +
COO- N N
 Cephalosporins Active Against PBP2´
OH
N
S H
TOC-39 Taiho Pharmaceuticals
H
N S + O Hanaki, H., et al. Antimicrobial Ag.
N
O
N Chemotherapy (1995), 39(5), 1120-6.
H2N
N NH2
O S
COO-

Cl OH
N
OH
S H H
N S

H2N
N
O
N LB11058 LG Life Sciences
N
O S N NH2
COO-

OH
N O
N O
S H O
H O
N S N O
N
H2N O N
N Ceftobiprole
O
COO-
O BAL5788 [BAL9141] Basilea
Pharmaceutica (Ro-63-9141
Roche)
Hebeisen et al. J. Med. Chem.
 “Anti-MRSA” Carbapenems
O
S +
HO H H N N
+
N NH2
S +
N
+ N NH2
N O
H H O
Banyu O
HO
O

N
N
S
Merck O
O O NH2
O
O +
O O N

HO H H
S

N N
N
Meiji Seika Kaisha
O ME1036
NH O
O

N NH
H H S O H
HO O N
S NH2
N
N Roche/Sumitomo HO H H
N N
O H
S
O
O
O
N
Sankyo CS-023
O (Roche)
O
 Ceftobiprole Fits Tightly into the Active Site
Cleft

Strong
interactions
with the lining
of the cleft Deep
penetration of
acyl-amino
side chain into
the widened
cleft and close
interaction with
the protein
Covalent
attachment to
the protein at
the active
centre serine

Lovering et al., ECCMID 2006

 Resistance in Gram-negative Pathogens
Antibiotic susceptibility in isolates from a hospital burns unit in Turkey
Species IMP CA CR AZT CT TIC/Cla Amikasi Gentamici Cipro
Z O X v n n

Pseudomonas 44 30 1 12 3 22 16 7 29
aeruginosa

Proteus 77 - 22 61 11 94 77 33 -
mirabilis

Providencia  
68   - 6 6 6 75 43 - 75
stuartii

Klebsiella 75 16 41 41 25 16 75 33 100
pneumoniae

Escherichia 60 -  20 20 60 40 60 20 60
coli
Acinetobacter 54 9 18 18 18 27 18 18 45
sp
 -Lactam Resistance in Gram-Negative
Bacteria
Lack of activity is due to the interplay of several distinct COO-

O
mechanisms, each capable of conferring high-level S
N
H
H2N
resistance N
N

O
N
O O
S
O O

Mutate
d porins
Efflux systems

Multiple -
lactamase
Mutated s
PBPs O
COO-

N
S O
H2N H
N O
S
N N
O H O
O O
 Mutated or Alternative Porins
Prevalent in Enterobacteriaceae (Enterobacter, Klebsiella).

The water channel through which -lactams pass is relatively

narrow. Changes in expression of porins in favor of those with
narrower pores alters influx of bulky -lactams.

Point mutations add bulk and charge interactions that further

restrict the channel
R82

G119E
 Bypassing Porin

Siderophore Uptake Systems Transport Larger Molecules

PenG
OmpF

OmpF
 Siderophore -Lactams

O
Catechol cephalosporins
O
O - elevated MICs in tonB and cir/fiu
N
H2N S
H
OH mutants
N
N S OH - inactivation in blood by COMT
O H
N N
O S
O O
O O
O
O
HN
O
O
N OH
H2N S O
H HN
N O OH
O
Pirazmonam N
O
N
H
- rapid selection of tonB O
N N
S
N N
O OH
mutants O
O O
N
H
N N
H2N
S O N
O OSO3H
BAL0030072
- less propensity to select tonB mutants
BAL0030072 has high affinity for multiple targets
Binds to penicillin-binding proteins PBP 1a, 1b and 2 as well as PBP 3
(target of aztreonam)

Inhibition of P. aeruginosa PBPs

BAL30072 (mg/L)
0.25 0.5 1 2 4 8 16 0

1a
1b
2

IC50
 Rapid onset of lysis, instead of filament
formation
E. coli growing normally

After treatment with BAL0030072

After treatment with Aztreonam

 Loss of Selective Porin D2

Resistance to carbapenems, especially imipenem, in

Pseudomonas aeruginosa is associated with the loss of
OprD (porin D2).

Meropenem may select for loss of OprD, but many

isolates remain susceptible.

OprD has been implicated in transport of basic amino

acids and small peptides containing basic amino acids.

May have a protease domain.

 Efflux Systems
Enterobacteriaceae Pseudomonas aeruginosa

TolC, AcrA,B MexA,B-OprM exports

certain -lactam
antibiotics

e.g. carbenicillin
cefoperazone
cefotaxime
meropenem
 Mutated Penicillin-binding Proteins

PBP 2 mutants commonly associated with resistance in Neisseria sp.

May involve point mutations, insertions or chimaeric proteinsformed by
inter-specific exchange of DNA.

PBP3 mutants. Rare in Haemophilus influenzae in Europe and US,

more common in Japan

Occasional in P. aeruginosa (PBP 3), Acinetobacter (PBP 3), most

often associated with carbapenem usage
 -Lactamases

Class A Class C
Many known inc. Cephalosporinases
Cephalosporinases Inhibitor-resistant
Carbapenemases
Inhibitor-resistant Can confer resistance
to carbapenems

Class D
Class B Increasing
Broad spectrum inc. Inhibitor-resistant
carbapenems and
inhibitors of serine ESBL and
Enzymes. Carbapenemase
variants
 Reactions of Penam sulfones
X
Irreversible
X X
inhibition
O 70
O O
S O
S O
O O
N
N
O H OH
70 70
OH O
HO O
O

X
-O2S O
O N
OH
S 70
O O
O
O
70 N
OH H OH
HO
O
-O2S O
N
OH X
HO
O
Stable inhibition
-O2S O
N
H OH
-O2S 70
O O
O
-O2S N
O H OH
HO O
H2N HO
OH O
O
 Hot Spots in Class A "ESBLs"
35 1-1 -loop 3-4 5
69 104
30
182
25
Incidence (%)

20

15
10 3-4
5
1-1
0
1 51 101 151 201 251
Residue number
69
TEM 104
SHV 5

-loop
 Substitutions in 1-1 Region
None of the substitutions significantly affect kinetic parameters

Position Found in substitution

21 TEM-4 derivatives F
TEM-67 I

35 SHV-2A derivatives Q
TEM-130 P

39 TEM-2 derivatives K

43 SHV-7, 14, 18, 29, 30, 34 S

May effect stability, folding or secretion of (pre-)protein

 Substitutions at Position 182

• Substitution of methionine in TEM with threonine (18x)

or isoleucine (2x)

• Effect on kinetic parameters insignificant

• Has been suggested that

these substitutions
affect dynamics of folding
or thermal stability and thus may
compensate deleterious
effects of other substitutions
Sideraki et al. (2001) PNAS 98:283-288,
Chen et al (2005) J Mol Biol 348:349-63.
 Substitutions at Position 69

Responsible for an inhibitor-resistant phenotype, as single

mutations or in association with others leading to extended
spectrum

Substitution of methionine by a more

hydrophobic residue

Isoleucine SHV-49, TEM-40

Leucine TEM-33, 35, 39, 45, 50, 77,

80, 81, 109, 125

Valine TEM-34, 36, 78, 82, 97

 Substitutions at Arg244

Responsible for an inhibitor-resistant phenotype, as

single mutations or in association with others leading
to extended spectrum

Substitution of arginine by a
residue with a shorter side chain
disrupts hydrogen-bond pattern

TEM-30, 44,58,74,77,99,121 [S]

TEM-31,65,67,73 [C]
TEM-51,145,146 [H] 244
TEM-54 [L]
TEM-79 [G]
 Substitution at Position 104
Substitution of glutamate by lysine

One of the commonest substitutions: found in 41 TEM

derivatives
TEM-3, 4, 6, 8, 9, 13, 15, 16, 17, 18, 21, 22, 24, 26, 43, 46, 50, 56, 60, 63, 66, 87,
88, 89, 92, 94, 106, 107, 109, 111, 113, 121, 123, 124, 129, 130, 131, 133, 134,
138

Rare as a single mutation but frequently

combined with other substitutions:
164 (19/42)
182 (12/20)
237 (5/9)
238 (19/32)
240 (3/22)
265 (5/20)
 Substitutionsin the-loop
Substitution of arginine 164 by serine (23x), histidine (16x) or
cysteine (3x)
The commonest substitution: found in 42 TEM derivatives
TEM-5, 6, 7, 8, 9, 10, 11, 12, 16, 24, 26, 27, 28, 29, 43, 46, 53, 60, 61, 63, 75, 85,
86, 87, 91,102, 107, 109, 114, 115, 118, 121, 125, 129, 130, 131, 132, 133, 134,
136, 143, 147

Occurs as a single mutation or

frequently with substitutions
at other loci:
104 (19/41 237 (8/9) 238 (3/33)
240 (14/23) 265 (7/20)
In crystal structures, alters the
hydrogen-bonding pattern in
side-chain binding pocket
 Substitutionsin the3 Strand
Substitutions at 238 and 240 common in TEM and SHV

Position Found in [substitution]:

237 TEM-5,24,86,114,121,130,131,136 [T] TEM-22 [G]

238 TEM-3,4,8,15,19,20,21,22,25,42,47,48,49,50,52,66,68,71,72
88,89,92,93,94,101,107,112,113,120,
123,134,136 [S]
TEM-111 [D] 237
SHV-2,2A,3,4,5,7,9,10,12,15,20,21,22, 238
30,34,39,45,46,55,64,66,86 [S]
SHV-13,18,29 [A]

240 TEM-5,10,24,27,28,42,46,47,48,49,61,
68,71,72,85,86,91,93,101,114,121,136
SHV-4,5,7,9,10,12,15,18,22,31,45,46,
55,64,66 [K] 240
SHV-86 [R]
 Substitutions at Gly 238
TEM-52 vs TEM-1

Loop 238−243 is shifted by as much as

2.8 Å
widening the opening to the active site
• might accomodate cephalosporin
side chains better

Movement of loop 238−243 moves Glu

240 out of the active site
• reduces the possibility of
interference with substrate or
inhibitor binding

Orencia et al., (2001) Nature Structural Biology  8, 238 - 242

 Experimental -Lactamase Inhibitors
Penam sulfones Bunyak Bridged
Class A + C + (D) Class A + C + B monobactams
O
H
O O N O O Class C
BAL0010078 S HS S N
S N

Roche/Basilea N N
N
N
H H

O N N
O
COOH
O Roche O SO3-
O

O O Methylgene
Pfizer HO S N
Class A + C + D
N
Cl
O O O
N N S O
O
O N P
N H
O OH
F
O H
S S
O O
S N

N
N N O Aventis AVE 1330A
O
O H
S
BRL 42715O Class A + C
O O
O SKB O N
N NH2
Wyeth O Class A + C + (D)
O N
Class A + C O
-O3SO
 Reactions of Clavulanic Acid
130 130 130
130 OH OH OH
OH
O OH k2 2
O O O
N O OH O O
O O N
O HN 70 OH O HN
70 OH -O O 70 70
OH
O -O O
-O
S. aureus -O O

k1 and
TEM-2 130
OH
130
OH
O
130 O H
OH O O N

N OH
O OH
70 70 O
-O
O

70 OH

130 OH 130
OH 3

O O O
H
N
O

OH
Stable inhibition
O HN
70 OH 70 O

130 130 OH 130 130 130

OH

O O O
OH O O O O OH
O

70 O HO O 70 O O HO
70 O 70 70

Irreversible
TEM-2
130 130
inhibition
O O

70 O 70 OH

OH
 Reactions of Penam sulfones
X
Irreversible
X X
inhibition
O 70
O O
S O
S O
O O
N
N
O H OH
70 70
OH O
HO O
O

X
-O2S O
O N
OH
S 70
O O
O
O
70 N
OH H OH
HO
O
-O2S O
N
OH X
HO
O
Stable inhibition
-O2S O
N
H OH
-O2S 70
O O
O
-O2S N
O H OH
HO O
H2N HO
OH O
O
 Reactions of Exomethylene Penams
Analogues of the asparenomycins
O OH O OH O O
OH
O O O O
-O2S O SO2-
S S
N O -N O N O -N
O O O O
O ENZ O O
O ENZ HO ENZ HO
HO HO

O OH O

O O SO2- O SO2-
SO2-

O N O N O N
O H O H O H
O O O
ENZ HO ENZ HO ENZ HO

N N N
N N N
N N N N
N N
S -S
S
O S
N O N O O N O
O O O O
HO HO N
O ENZ
HO ENZ ENZ H
H+