bLactams

1
b-Lactams
2
b-Lactams Rapidly Destroy Bacteria
S. aureus after exposure to penicillin G
S. aureus growing normally
Giesbrecht et al., 1998, Microbiology and
Molecular Biology Reviews 62 1371-1414
3
And Can Quickly Cure Infection
The fourth patient to receive penicillin G
After a few days
Before treatment
4
Cell Wall Biosynthesis - the Target of b-Lactams
Transpeptidases, also known as penicillin-binding
proteins (or PBPs) react with one end of the
peptide side chain (the "acceptor") to form a
covalent intermediate.
The bacterial cell wall is built from strands of
sugar molecules that have peptide side chains.
5
Cell Wall Cross-linking Gives Stability
The covalently attached PBP then binds another
peptide side chain (the "donor") and promotes its
attack on the previously attached acceptor chain.
Thus, a strong covalent link is built between
neighbouring sugar strands. These peptide
cross-bridges are essential for the mechanical
strength of the cell wall.
6
b-Lactams Inhibit Cell Wall Biosynthesis
b-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 b-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.
7
Inhibition of Cell Division is Especially
Effective
Autolysins Amidases, Lytic transglycosylases
S. aureus
PBP complex
Anchor complex
Cytokinetic Ring
E. coli
8
Classes of b-Lactam
monobactam
penem
cephem
All b-lactam antibiotics have a strong negative
charge at R4. They do not work against
intra-cellular pathogens
9
Natural Monobactams
From streptomycetes (Nocardia)
Nocardicin A
Formadicin A
From Gram-negative bacteria (Pseudomonas)
Sulfazecins
MM 42842
10
Synthetic Monobactams
Oximonam 1985
Aztreonam 1983
BAL0030072
Carumonam 1985
Tigemonam 1987
11
Uses of Aztreonam
12
Natural Penems
From fungi (Ascomycetes and Deuteromycetes e.g.
Penicillium)
Penicillins Penicillin G Penicillin
F Penicillin V
Penams
From streptomycetes (Streptomyces) and
Gram-negative bacteria (Erwinia, Serratia)
Carbapenems Thienamycins Carpetimycins Epithien
amycins Pluracidomycins Olivanic
acids Asparenomycins Oxapenems Clavulanic
acid
Penems
13
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 chains. They are not susceptible to
attack by penicillinases and more stable to
broad-spectrum b-lactamases. More polar side
chains were introduced to obtain Gram-negative
activity
14
Semi-synthetic Penicillins - 2
Increased activity against penicillinase-producing
Gram-positive cocci. Little Gram-negative
activity
Methicillin 1961 Nafcillin 1963 Oxacil
lin 1962 Cloxacillin (R1 Cl)
1963 Floxacillin (R1 Cl, R2 F) 1972
15
Semi-synthetic Penicillins - 3
Amino-penicillins Broader spectrum (Gram-positive
and Gram-negative)
Ampicillin 1962 Amoxicillin 1972
16
Semi-synthetic Penicillins - 4
Broad spectrum (Anti-Pseudomonas). Stronger
Gram-negative spectrum (some loss of
Gram-positive spectrum)
Azlocillin 1976 Piperacillin 1977 Apalcillin
1986 Carbenicillin 1968 Ticarcillin 1973
17
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 Ent
erococcal in combination with aminoglycoside Str
eptococcal (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.
18
Uses of Penicillinase-stable Penicillins
Methicillin, nafcillin, cloxacillin,
floxacillin Staphylococcal infections skin
infections (impetigo, boils, carbuncles) abcesse
s in organs pneumonia prosthetic joint,
catheter and artificial valve infections endocar
ditis meningitis (rare) osteomyelitis
(requires v. long therapy) Streptococcal
infections especially when resistance is
suspected or when Staph. is a possibility
19
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
20
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
21
Uses of Penicillin Combinations Amino-penicillins
22
Uses of Penicillin Combinations Broad spectrum
penicillins
23
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
24
b-Lactamase Inhibitor Combinations
Clinically approved used in combination with a
b-lactamase labile b-lactam antibiotic
Clavulanate Class A (D) b-lactamases
Penam sulfones Class A (C D) b-lactamases
Sulbactam
All have intrinsic activity against Acinetobacter
sp.
Tazobactam
25
Natural Carbapenems
Erwinia, Serratia
Pluracidomycins Streptomyces pluracidomyceticus
Thienamycins Streptomyces cattleya
PS-6, PS-8 Streptomyces fulvoviridis
Epithienamycins Streptomyces olivaceus S.
fulvoviridis
Carpetimycins Streptomyces griseus
Olivanic acids Streptomyces olivaceus
26
Synthetic Carbapenems
Labile to renal dehyropeptidase
Panipenem 198?
Imipenem 1983
Used with betamipron
Used with cilastatin
Stable to renal dehyropeptidase
Meropenem 1992
Ertapenem 2001
Biapenem 2001
27
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
28
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
29
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)
30
Reactions of Carbapenems
Carbapenems have two features that contribute to
stability towards b-lactamase
The hydroxyethyl group
Chemical rearrangement
slow
stable
31
Natural Cephems
Cephalosporin C Acremonium sp. (Fungi)
Cephamycin C (R NH2) Streptomyces sp.
(Bacteria) Cephabacines (R
oligopeptide) Lysobacter lactamgenans Xanthomonas
lactamgenans
Chitinovorin A Flavobacterium sp. (Bacteria)
32
Classification of Semi-synthetic Cephalosporins

Gram-positive Bacteria
Gram-negative Bacteria
Methicillinresistant
Methicillinsensitive
Ampicillinsensitive
Ampicillinresistant
1st Generation

-

-
-
2nd Generation


-
3rd Generation

-


4th Generation

-


Anti-MRSA cephalosporins



()
33
1st Generation Cephalosporins
Cephaloridine 1964 parenteral
Cephalothin 1965 parenteral
Cephalexin 1967 oral
Cephapirin 1970 parenteral
34
2nd Generation Cephalosporins
Cefazolin 1971 parenteral
Cefamandole 1973 parenteral
Cefaclor 1976 oral
Cefprozil 1990 oral
35
Semi-synthetic Cephamycins
Cefoxitin 1973 parenteral
Cefotetan 1983 parenteral
Cefmetazole 1987 parenteral
36
Semi-synthetic Cephems
Carbacephem
Loracarbef 1989 oral
Oxacephamycin
Moxalactam 1980 parenterral
37
3rd Generation Cephalosporins
Cefotaxime 1979 parenteral
Ceftazidime 1980 parenteral Very strong
coverage of Pseudomonas
Ceftriaxone 1981 parenteral Very long
half-life
38
Oral 3rd Generation Cephalosporins
Cefixime 1987 Specific transport by peptide
carrier
Cefpodoxime Proxetil 1988 Prodrug, taken up by
passive diffusion. Unstable ester readily
hydrolyzed in serum
39
4th Generation Cephalosporins
Cefepime 1987 parenteral
Cefpirome 1989 parenteral
40
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
41
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)
42
Evolution of b-Lactam Resistance
Gram-positive Bacteria staphylococci,
enterococci, pneumococci
Gram-negative Bacteria Klebsiella, Acinetobacter,
Pseudomonas Stenotrophomonas
Inhibitor-resistant ESBLA
Anti-MRSA cephalosporins
b-lactamase inhibitors
Permeation defects Efflux systems
Carbapenemases
Metallo b-lactamases
Carbapenems
Extended spectrum b-lactamases
Cephalosporinases
3rd Generation cephalosporins
1st 2nd generation cephalosporins
Highly resistant transpeptidases
Broad spectrum b-lactamases, oxacillinases
Semi-synthetic penicillins (methicillin)
Semi-synthetic penicillins (oxacillin,
carbenicillin, piperacillin)
Penicillinase
Penicillin G
43
Resistance in S. aureus
S. aureus (hospital) Penicillinase
Resistance ( of isolates)
S. aureus (community) Penicillinase
Introduction
First resistance
S. aureus (hospital) Methicillin-resistant
S. aureus (community) Methicillin-resistant
S. aureus Vancomycin-resistant
Year
44
Methicillin Resistance in S. aureus is due to an
Additional Transpeptidase (PBP)
Methicillin-resistant
Methicillin-susceptlible
PBP 1 PBP 2 PBP 3 PBP 4
Member of a small sub-family of type B PBPs found
in staphylococci, enterococci and bacilli. All
have low reactivity towards many b-lactams. Next
closest sequence homology is to E. coli PBP2
PBP 2
Changes in expression of PBP 2 and PBP4 can
modulate sensitivity towards some b-lactams
MIC lt0.1 mg/L
MIC gt500 mg/L
45
Structure of PBP 2
Flexible domain 110 residues
Active site
Stalk region
Membrane anchor
Transpeptidase domain
46
The Highly Resistant Transpeptidases of
Gram-positive Bacteria
S. pneumoniae PBP 2x
Similar in structure to essential
b-lactam-sensitive transpeptidases (30 sequence
similarity) Acylation very slow ( t 1/2 30 min
cf 30s) Very low affinity (KS 10-1000 mM cf
1-10 mM) Deacylation faster (t 1/2 2 hr cf 2
days)
S. aureus PBP 2
Much lower occupancy of active site
47
Active Site Accessibility in PBP 2 is Critical
Resting
The active site cleft is too narrow for b-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 b-lactam to
enter
48
Cephalosporins Active Against PBP2
Ceftaroline (Forest) TAK-599 T-91825 Peninsula
(Takeda) Yoshizawa et al. Journal of Antibiotics
(2002), 55(11), 975-992.
RWJ-442831 RWJ-54428 JnJ (MC-02,479
Microcide) Hecker et al. Journal of Antibiotics
(2000), 53(11), 1272-1281
BMS-247243 Bristol Myers Squibb Singh et al.,
Organic Process Research Development (2000),
4(6), 488-497.
49
Cephalosporins Active Against PBP2
TOC-39 Taiho Pharmaceuticals Hanaki, H., et al.
Antimicrobial Ag. Chemotherapy (1995), 39(5),
1120-6.
LB11058 LG Life Sciences
Ceftobiprole BAL5788 BAL9141 Basilea
Pharmaceutica (Ro-63-9141 Roche) Hebeisen et
al. J. Med. Chem.
50
Anti-MRSA Carbapenems
Banyu
Merck
Meiji Seika Kaisha ME1036
Roche/Sumitomo
Sankyo CS-023 (Roche)
51
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
52
Resistance in Gram-negative Pathogens
Antibiotic susceptibility in isolates from a
hospital burns unit in Turkey
-
-
53
b-Lactam Resistance in Gram-Negative Bacteria
Lack of activity is due to the interplay of
several distinct mechanisms, each capable of
conferring high-level resistance
Mutated porins
Efflux systems
Multiple b-lactamases
Mutated PBPs
54
Mutated or Alternative Porins
Prevalent in Enterobacteriaceae (Enterobacter,
Klebsiella). The water channel through which
b-lactams pass is relatively narrow. Changes in
expression of porins in favor of those with
narrower pores alters influx of bulky
b-lactams. Point mutations add bulk and charge
interactions that further restrict the channel
R82
G119E
55
Bypassing Porin
Siderophore Uptake Systems Transport Larger
Molecules
PenG
OmpF
OmpF
56
Siderophore b-Lactams

• Catechol cephalosporins
• elevated MICs in tonB and cir/fiu
• mutants
• - inactivation in blood by COMT

• Pirazmonam
• rapid selection of tonB
• mutants

• BAL0030072
• less propensity to select tonB mutants

57
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
3
4
IC50
58
Rapid onset of lysis, instead of filament
formation
E. coli growing normally
After treatment with BAL0030072
After treatment with Aztreonam
59
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.
60
Efflux Systems
Enterobacteriaceae TolC, AcrA,B
Pseudomonas aeruginosa
MexA,B-OprM exports certain b-lactam
antibiotics e.g. carbenicillin cefoperazone cef
otaxime meropenem
61
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
62
b-Lactamases
Class C Cephalosporinases Inhibitor-resistant Can
confer resistance to carbapenems
Class A Many known inc. Cephalosporinases Carbapen
emases Inhibitor-resistant
Class D Increasing Inhibitor-resistant ESBL
and Carbapenemase variants
Class B Broad spectrum inc. carbapenems
and inhibitors of serine Enzymes.
63
Reactions of Penam sulfones
Irreversible inhibition
Stable inhibition
64
Hot Spots in Class A "ESBLs"
W-loop
b3-b4
b5
a1-b1
104
69
182
TEM
SHV
65
Substitutions in a1-b1 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
66
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 98283-288, Chen et
al (2005) J Mol Biol 348349-63.
67
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
68
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 TEM-54 L TEM-79 G
244
69
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)
70
Substitutions in the W-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
71
Substitutions in the b3 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 SHV-2,2A,3,4,5,7,9,10,12,15,20,21
,22, 30,34,39,45,46,55,64,66,86 S
SHV-13,18,29 A 240 TEM-5,10,24,27,28,42,46,4
7,48,49,61, 68,71,72,85,86,91,93,101,114,121,1
36 SHV-4,5,7,9,10,12,15,18,22,31,45,46,
55,64,66 K SHV-86 R
237 238
240
72
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
73
Experimental b-Lactamase Inhibitors
Bridged monobactams Class C
Penam sulfones Class A C (D)
Bunyak Class A C B
BAL0010078 Roche/Basilea
Roche
Methylgene Class A C D
Pfizer
Aventis AVE 1330A Class A C
BRL 42715 SKB Class A C (D)
Wyeth Class A C
74
Reactions of Clavulanic Acid
Stable inhibition
Irreversible inhibition
75
Reactions of Penam sulfones
Irreversible inhibition
Stable inhibition
76
Reactions of Exomethylene Penams
Analogues of the asparenomycins