Transcript Beta-Lactam Antibiotics - Southern Methodist University

Beta-Lactam Antibiotics
Clinically Important β-Lactam
Antibiotics
Medicinal Chemistry Presentation
David McLeod
Southern Methodist University
Introduction
β-Lactam antibiotics are the most widely produced
and used antibacterial drugs in the world, and have
been ever since their initial clinical trials in 1941.
β-Lactams are divided into several classes based on
their structure and function; and are often named by
their origin, but all classes have a common β-Lactam
ring structure.
History
1928- Alexander Fleming discovers a mold which
inhibits the growth of staphylococcus bacteria
1940- penicillin is isolated and tested on mice by
researchers at Oxford
1941- penicillin mass produced by fermentation for use
by US soldiers in WWII
1950’s- 6-APA is discovered and semi-synthetic
penicillins are developed.
1960’s to today- novel β-lactams/ β-lactamase
inhibitors are discovered and modified from the
natural products of bacteria
Target- Cell Wall Synthesis
The bacterial cell wall is a cross linked polymer called
peptidoglycan which allows a bacteria to maintain its
shape despite the internal turgor pressure caused by
osmotic pressure differences.
If the peptidoglycan fails to crosslink the cell wall will
lose its strength which results in cell lysis.
All β-lactams disrupt the synthesis of the bacterial cell
wall by interfering with the transpeptidase which
catalyzes the cross linking process.
Peptidoglycan
Peptidoglycan is a carbohydrate composed of
alternating units of NAMA and NAGA.
The NAMA units have a peptide side chain which can
be cross linked from the L-Lys residue to the
terminal D-Ala-D-Ala link on a neighboring NAMA
unit.
This is done directly in Gram (-) bacteria and via a
pentaglycine bridge on the L-lysine residue in Gram
(+) bacteria.
Mechanism
Transpeptidase- PBP
The cross linking reaction is catalyzed by a class of
transpeptidases known as penicillin binding proteins
A critical part of the process is the recognition of the
D-Ala-D-Ala sequence of the NAMA peptide side
chain by the PBP. Interfering with this recognition
disrupts the cell wall synthesis.
β-lactams mimic the structure of the D-Ala-D-Ala link
and bind to the active site of PBPs, disrupting the
cross-linking process.
Mechanism of β-Lactam Drugs
The amide of the β-lactam ring is unusually reactive
due to ring strain and a conformational arrangement
which does not allow the lone pair of the nitrogen to
interact with the double bond of the carbonyl.
β-Lactams acylate the hydroxyl group on the serine
residue of PBP active site in an irreversible manner.
This reaction is further aided by the oxyanion hole,
which stabilizes the tetrahedral intermediate and
thereby reduces the transition state energy.
Mechanism of β-Lactam Drugs
The hydroxyl attacks the amide and forms a
tetrahedral intermediate.
Mechanism of β-Lactam Drugs
The tetrahedral intermediate collapses, the amide
bond is broken, and the nitrogen is reduced.
Mechanism of β-Lactam Drugs
The PBP is now covalently bound by the drug and
cannot perform the cross linking action.
Bacterial Resistance
Bacteria have many methods with which to combat the
effects of β-lactam type drugs.
Intrinsic defenses such as efflux pumps can remove the
β-lactams from the cell. β-Lactamases are enzymes
which hydrolyze the amide bond of the β-lactam
ring, rendering the drug useless.
Bacteria may acquire resistance through mutation at
the genes which control production of PBPs, altering
the active site and binding affinity for the β-lactam .
Range of Activity
β-Lactams can easily penetrate Gram (+) bacteria, but
the outer cell membrane of Gram (-) bacteria
prevents diffusion of the drug. β-Lactams can be
modified to make use of import porins in the cell
membrane.
β-Lactams also have difficulty penetrating human cell
membranes, making them ineffective against
atypical bacteria which inhabit human cells.
Any bacteria which lack peptidoglycan in their cell wall
will not be affected by β-lactams.
Toxicity
β-Lactams target PBPs exclusively, and because
human cell membranes do not have this type of
protein β-lactams are relatively non toxic compared
to other drugs which target common structures such
as ribosomes.
About 10% of the population is allergic (sometimes
severely) to some penicillin type β-lactams.
Classes of β-Lactams
The classes of β-lactams are distinguished by the
variation in the ring adjoining the β-lactam ring and
the side chain at the α position.
Penicillin
Modification of β-Lactams
β-Lactam type antibiotics can be modified at various
positions to improve their ability to:
-be administered orally (survive acidic conditions)
-be tolerated by the patient (allergies)
-penetrate the outer membrane of Gram (-) bacteria
-prevent hydrolysis by β-lactamases
-acylate the PBPs of resistant species (there are many
different PBPs)
Penicillins- Natural
Natural penicillins are those which can be obtained
directly from the penicillium mold and do not
require further modification. Many species of
bacteria are now resistant to these penicillins.
Penicillin G
not orally active
Penicillin G in Acidic Conditions
Penicillin G could not be administered orally due to
the acidic conditions of the stomach.
Penicillin V
Penicillin V is produced when phenoxyacetic acid
rather than phenylacetic acid is introduced to the
penicillium culture. Adding the oxygen decreases
the nucleophilicity of the carbonyl group, making
penicillin V acid stable and orally viable.
Production
All commercially available β-lactams are initially
produced through the fermentation of bacteria.
Bacteria assemble the penicillin molecule from L-AAA,
L-valine, and L-cysteine in three steps using ACV
synthase, IPN synthase, and acyltransferase.
Modern recombinant genetic techniques have allowed
the over expression of the genes which code for these
three enzymes, allowing much greater yields of
penicillin than in the past.
Penicillin Biosynthetic Pathway
o
Semi-Synthetic Penicillins
The acyl side chain of the penicillin molecule can be
cleaved using enzyme or chemical methods to
produce 6-APA, which can further be used to
produce semi-synthetic penicillins or cephalosporins
75% of the penicillin produced is modified in this
manner.
Penicillins- Antistaphylococcal
Penicillins which have bulky side groups can block the
β-Lactamases which hydrolyze the lactam ring.
Penicillins- Antistaphylococcal
These lactamases are prevalent in S. aureus and S.
epidermidis, and render them resistant to Penicillin
G and V. This necessitated the development of semisynthetic penicillins through rational drug design.
Methicillin was the first penicillin developed with this
type of modification, and since then all bacteria
which are resistant to any type of penicillin are
designated as methicillin resistant. (MRSAmethicillin-resistant S. aureus)
Penicillins- Antistaphylococcal
Methicillin is acid sensitive and has been improved
upon by adding electron withdrawing groups, as was
done in penicillin V, resulting in drugs such as
oxacillin and nafcillin.
Due to the bulky side group, all of the
antistaphylococcal drugs have difficulty penetrating
the cell membrane and are less effective than other
penicillins.
Penicillins- Aminopenicillins
In order to increase the range of activity, the penicillin
has been modified to have more hydrophilic groups,
allowing the drug to penetrate into Gram (-) bacteria
via the porins.
Ampicillin R=Ph
Amoxicillin R= Ph-OH
Penicillins- Aminopenicillins
These penicillins have a wider range of activity than
natural or antistaphylococcal drugs, but without the
bulky side groups are once again susceptible to
attack by β-lactamases
The additional hydrophilic groups make penetration of
the gut wall difficult, and can lead to infections of the
intestinal tract by H. pylori
Penicillins- Aminopenicillins
Due to the effectiveness of the aminopenicillins, a
second modification is made to the drug at the
carboxyl group.
Changing the carboxyl group to an ester allows the
drug to penetrate the gut wall where it is later
hydrolyzed into the more polar active form by
esterase enzymes.
This has greatly expanded the oral availability of the
aminopenicillin class.
Penicillins- Extended Spectrum
Extended spectrum penicillins are similar to the
aminopenicillins in structure but have either a
carboxyl group or urea group instead of the amine
Penicillins- Extended Spectrum
Like the aminopenicillins the extended spectrum drugs
have an increased activity against Gram (-) bacteria
by way of the import porins.
These drugs also have difficulty penetrating the gut
wall and must be administered intravenously if not
available as a prodrug.
These are more effective than the aminopenicillins and
not as susceptible to β-lactamases
Cephalosporins
Cephalosporins were discovered shortly after penicillin
entered into widespread product, but not developed
till the 1960’s.
Cephalosporins are similar to penicillins but have a 6
member dihydrothiazine ring instead of a 5 member
thiazolidine ring.
7-aminocephalosporanic acid (7-ACA) can be obtained
from bacteria, but it is easier to expand the ring
system of 7-APA because it is so widely produced.
Cephalosporins
Unlike penicillin, cephalosporins have two side chains
which can be easily modified. Cephalosporins are
also more difficult for β-lactamases to hydrolyze.
Mechanism of Cephalosporins
The acetoxy group (or other R group) will leave when
the drug acylates the PBP.
Cephalosporins- Classification
Cephalosporins are classified into four generations
based on their activity.
Later generations generally become more effective
against Gram (-) bacteria due to an increasing
number of polar groups (also become zwitterions.)
Ceftazidime (3rd gen) in particular can cross blood
brain barrier and is used to treat meningitis.
Later generations are often the broadest spectrum and
are reserved against penicillin resistant infections to
prevent the spread of cephalosporin resistant
bacteria.
Carbapenems
Carbapenems are a potent class of β-lactams which
attack a wide range of PBPs, have low toxicity, and
are much more resistant to β-lactamases than the
penicillins or cephalosporins.
Carbapenems
Thienamycin, discovered by Merck in the late 1970’s, is
one of the most broad spectrum antibiotics ever
discovered.
It uses import porins unavailable to other β-lactams to
enter Gram (-) bacteria.
Due to its highly unstable nature this drug and its
derivatives are created through synthesis, not
bacterial fermentation.
Carbapenems
Thienamycin was slightly modified and marked as
Imipenem. Due to its rapid degradation by renal
peptidase it is administered with an inhibitor called
cilastatin under the name Primaxin. Imipenem may
cause seizures or sever allergic reactions.
Other modifications of Thienamycin have produced
superior carbapenems called Meropenem and
Ertapenem, which are not as easily degraded by
renal peptidase and do not have the side effects of
Imipenem.
Monobactams
The only clinically useful monobactam is aztreonam.
While it resembles the other β-lactam antibiotics and
targets the PBP of bacteria, its mechanism of action
is significantly different.
It is highly effective in treating Gram (-) bacteria and is
resistant to many β-lactamases
β-Lactamases
β-Lactamases were first discovered in 1940 and
originally named penicillinases.
These enzymes hydrolyze the β-lactam ring,
deactivating the drug, but are not covalently bound
to the drug as PBPs are.
Especially prevalent in Gram (-) bacteria.
Three classes (A,C,D) catalyze the reaction using a
serine residue, the B class of metallo- β-lactamases
catalyze the reaction using zinc.
β-Lactamase Inhibitors
There are currently three clinically available βlactamase inhibitors which are combined with βlactams; all are produced through fermentation.
These molecules bind irreversibly to β-lactamases but
do not have good activity against PBPs. The rings are
modified to break open after acylating the enzyme.
β-Lactam/Inhibitor combinations
Aminopenicillins:
ampicillin-sulbactam = Unasyn
amoxicillin-clavulante = Augmentin
Extended-Spectrum Penicillins
piperacillin-tazobactam = Zosyn
ticarcillin-clavulanate = Timentin
Summary
β-Lactam antibiotics have dominated the clinical
market since their introduction in the 1940’s and
today consist of nearly ¾ of the market.
Development of natural products such as penicillin G
into more potent forms through rational
modification has increased the range of activity of
these drugs, although this has led to some toxicity
problems.
Widespread use of β-lactams has led to the
development of resistant strains, new modifications
are necessary in order for β-lactams to remain viable.
Assigned reading:
Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition.
New York: Oxford University Press, 2009. 388-414. Print.
Optional References/ Reading
Brunton, Laurence L. et al. Goodman and Gillman’s Pharmaceutical Basis
of Therapeutics 11th Edition. McGraw-Hill, 2006 1134- 52. Print.
Bush, Karen. β-Lactamase Inhibitors from Laboratory to Clinic. Clinical
Microbiology Reviews, Jan. 1988, p. 109-123. Web.
Elander, R.P. Industrial production of β-Lactam antibiotics. Journal of
Applied Microbiology and Biotechnology (2003) 61:385–392. Web.
Hauser, Alan R. Antibiotic Basics for Clinicians: Choosing the Right
Antibacterial Agent. Philadelphia: Lippincott, 2007. 18-46. Print.
Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition.
New York: Oxford University Press, 2009. 388-420. Print.
Rolinson, George N. Forty years of β-lactam research. Journal of
Antimicrobial Chemotherapy (1998) 41, 589–603. Web.
Questions
1. What are two ways by which a bacteria could
become resistant to carbapenems?
2. How were the natural penicillins modified to be
orally available?
3. How are extended spectrum penicillins modified to
be orally available?
4. What are two ways that the β-lactam can be
protected from β-lactamases?