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Mechanistic Studies of Beta- Lactamase
Welcome back. In the last session we just started the topic that how the bacteria acquires
resistance against the β-lactam antibiotics. And one of the major cause for that is that the
bacteria produces enzymes which are classified as β-lactamases and these β-lactamases
are so named because they hydrolyze the β-lactam ring. And if you can break the β-
lactam ring, then all the β-lactam antibiotics are going to lose their activity.
So, now the question is what is the difference between transpeptidase and β-lactamase?
That should be made very clear. Transpeptidase is a serine based enzyme, where the
serine attacks the β-lactam ring, breaks the β-lactam ring, makes an acyl-enzyme
complex and that stays there for a long time. On the other hand, there are different
classes of β-lactamases; one of them utilizes a serine. So, if it utilizes a serine, again the
mechanism is virtually same, the serine attacks the β-lactam ring, forms the acyl-enzyme
(Refer Slide Time: 01:49)
However now, the attack by water is very fast. So, this is the penicillin and β-
lactamase hydrolyzes the β-lactam ring. So, the OH attacks the carbonyl, this breaks,
there is substituent here. So, O then the enzyme and then you have NH S and this. In case
of β-lactamase, now water immediately comes, very fast and frees the β-lactamase
enzyme and leave the hydrolyzed penicillin which is ineffective. For transpeptidase, this
next reaction; that means, attack by water is very slow. So, this is the difference and this
difference is causing havoc; that a β-lactamase enzyme has a very high turnover number.
So, basically high turnover number means 1 molecule of β-lactamase can hydrolyze
several molecules of penicillin. So, that makes it ineffective. And this is the major cause
of concern in today’s bacterial infection scenario.
(Refer Slide Time: 03:33)
Because the β-lactamase enzymes are grouped into four classes; class A, class B, class C
and class D. At least for today at this point of time, these are the four different β-
lactamases. What is class A? Class A is basically the gram negative organisms, which
were earlier treatable with ampicillin or amoxicillin type of drugs; that means, this class
A β-lactamase hydrolyzes ampicillin or amoxicillin by a serine, the active site has a
Class B hydrolyzes penicillins, it hydrolyzes cephalosporins; but the most important is
that it hydrolyzes the carbapenems. We have not talked about carbapenems, I have
showed you only the structure the skeleton; but carbapenems are powerful drugs which
are in the market now. It is one of the last defenses that we have; that if the infection is
very resistant, then people start giving the carbapenems. But there are now enzymes,
which are called class B enzymes, which utilizes a metal ion to hydrolyze the β-lactam
So, it is not the serine OH, it is now a metallo β-lactamase. Then class C basically
hydrolyzes the cephalosporin molecules and class D hydrolyzes the oxacillin; that is
another type of penicillin which was in the market to have, which has got very good
resistance earlier against this β-lactamases. But now there are enzymes which are called
class D β-lactamases that hydrolyzes very good effective antibiotics like oxacillin.
Which is a penicillin by the way, but it has got a different section.
So, these are the four classes; out of these, most difficult one is this class B, because it is
a metallo β-lactamase. So, its mechanism is different from the other three and it
extremely difficult to treat this type of infections which are caused by microorganisms
producing class B. And, I think you should know this that one bacterial strain which
produces the class B metallo β-lactamase enzyme is what is called the NDM bacteria,
NDM harboring bacteria. What is NDM? New Delhi β-lactamase.
So, this strain was originated maybe 10 years back, New Delhi metallo β-lactamase. So,
any NDM strain it will be Klebsiella pneumoniae is the one which is very dangerous; it
causes pneumonia and they are now becoming very difficult to treat because of this
NDM harboring bacteria. What is NDM, New Delhi metallo β-lactamase?
So, you see that gone are those era that when we are really safe, because we had many
antibiotics in our hand; like erythromycin is one group of macrolide antibiotics,
ciprofloxacin that is another group. I initially, I showed you one slide where the different
targets have been shown; that some drugs target the nucleic acid, some target the
transcription, some target the translation protein synthesis, some targets this synthesis of
the cell wall.
So, for different targets, different antibiotics are there; ciprofloxacin, then you have the
erythromycin that is macrolide antibiotics, you have cyclic peptide antibiotics, you have
carbohydrates based antibiotics, amikacin; those type of molecules. Then you have this
β-lactam class of antibiotics and you have vancomycin polymyxin. So, lot of antibiotics
are there, but the problem is that many of these antibiotics have serious side effects;
because they are targeting something which is also there in the host.
So, that is where the problem lies, except for penicillin and possibly vancomycin;
because vancomycin also targets the cell wall biosynthesis. Now let us see the major
problem of this β-lactamase producing microorganisms, they are mainly with a gram
negative bacteria. Gram positive bacteria are easier to treat, because the cell wall is
exposed to the outside world. But in case of the gram negative, first you have this outer
membrane which is hydrophobic; then you have a space which is where these bacteria
produces the beta-lactamase enzyme.
So, they are basically traversing here; they are looking for molecules which come here
and then if it is penicillin, they are going to destroy it, then the cell wall which is much
thinner and actually here is your cell wall; that means, your transpeptidase is here. So,
the problem is, that if penicillin molecule enters it cannot go to the transpeptidase;
because it has to cross the layer, a periplasmic space, where the β-lactamase is present.
So, it is a very difficult or challenging task to treat the gram negative bacteria. So, if
somebody says why gram negative bacteria are difficult to treat; this is because first of
all the molecule has to cross this hydrophobic barrier. And that is difficult, because you
need carrier molecules to take it through the hydrophobic barrier. However, there are
certain channels, because bacteria also need some molecules, some nutrients which has
to be given from outside. So, that goes through pores which are called porins or these are
the channels through which small molecules enter and go there.
So, if penicillin by chance enters here, now the β-lactamase is here; that is going to
destroy the penicillin. So, that was the major problem till late 70s; then cephalosporins
came into the market and lot of new cephalosporins were having resistance to this β-
lactamase producing microorganism. So, cephalosporins became the savior, then first
generation cephalosporin, second generation cephalosporin; but people were running out
of options, because the bacteria is also changing its character.
Changing its characters, so new antibiotics were required, because it felt that yes it is
time that we should discover new antibiotics, or at least we should discover molecules
which takes care of this β-lactamase, ok.
(Refer Slide Time: 12:29)
This is what the reaction is; β-lactamase hydrolyzes the penicillin; I already told you that
there are various mechanisms it can do it by serine or it can do it by a metal ion. Like if it
so; obviously, the metal ion chelates here, like metallo-proteases you know. So,
very similar mechanism, it activates the carbonyl and then this is broken very easily only
So, the enzyme does not have to participate directly, and usually these metallo β-
lactamase have two metal ions; one for the coordination to the carbonyl and another for
this negative charge to be stabilized by the second metal ion. So, what I was telling that
for the late 70s people, these various pharmaceutical companies frantically searched for
molecules which have stability towards β-lactamase.
And what they found a molecule which is known as clavulanic acid. The structure is
again a β-lactam compound with an oxygen and then an exocyclic double bond and this
is what is clavulanic acid. So, what is the difference with penicillin? First of all it is an
oxapenam and then it does not have the side chain here. And it has oxygen and this
oxygen is very funny or very interesting, in the sense that there is a double bond here, so
this has got a very good leaving group character; because it is with this O minus, then it
can resonate with the double bond.
In case of penicillin, where you have sulfur, there is not much stability of the sulfur;
although sulfur is quietly good leaving group, but it is not assisted by any double bond
like this. So, clavulanic acid is a molecule which was discovered again by Beecham
pharmaceuticals and they found that this molecule is a very potent inhibitor of β-
lactamase. But at that time, metallo β-lactamase was not there, protein inhibitor of β-
lactamase means they are serine class of beta lactamase; and that was prevalent at that
However, this it has very weak antibacterial activity. So, it is an inhibitor of β-lactamase,
but it is not a good antibiotic. So, if you take clavulanic acid, it will not treat you;
because it will just inhibit the β-lactamase that is all. But the cell wall biosynthesis will
take place because it is not a good antibiotic, if it is not a good antibiotic; that means, it
targets the β-lactamase enzyme, but it does not target the transpeptidase.
So, quite interesting molecule, but then people are very clever at Beecham. So, what they
thought that better take a penicillin which is hydrolyzed by β-lactamase and mix it with
clavulanic acid. If you take this combination; then what will happen? Since clavulanic
acid has got very high affinity for β-lactamase, so it is going to stop the β-lactamase from
attacking the penicillin.
And, so the β-lactamase is busy with the clavulanic acid and by that way penicillin
sneaks through and then attacks the transpeptidase and stops the transpeptidase enzyme.
In fact, this combination is one of the lifesaving drugs today; it is called
augmentin, which is a mixture of amoxicillin and clavulanic acid. So, it is a combination
of drugs, and clavulanic acid targets β-lactamase, takes care of the β-lactamase; and
amoxycillin now attacks the transpeptidase.
More interesting is that, in presence of clavulanic acid, amoxicillin becomes very
powerful antibiotic; means earlier whatever the antibacterial activity amoxicillin had
alone, when there was no β-lactamase, if you have to take 500 milligram to have the
same effect now in presence of clavulanic acid, you need much less amount. That means,
amoxicillin not only now targets the transpeptidase in presence of clavulanic acid, its
power also is increased in presence of clavulanic acid this is called synergistic effect.
Synergistic effect is, that suppose you have a drug A and that has got a potency say P1
and now you mix that with B; now the potency is say 10 P1; that means, 10 times more
potent in presence of B. How that does happen? Synergism can be very easily described
by comparing with a real life scenario. This is not chemistry; but it is it can explain this
very well, the synergism. You know Lionel Messy, everybody knows him, he is a very
good footballer, and he plays for Barcelona and scores many goals.
But when he plays for Argentina, he has not produced that form which he showed in
Barcelona. Now instead of the molecule, you have the same man Messy, he plays, so
well in Barcelona, but not that well for Argentina. So, there must be some synergistic
effect going on when he plays for Barcelona and that is called synergistic effect; if you
are a football fan you know that there is a there are players whose names are Iniesta and
Xavi; they are the clavulanic acid of Barcelona.
So, they supply balls which Messy can convert into goals. So, that is this synergistic
effect, that in presence of good footballers the other player shows much better efficiency
than when those players are not there. So, clavulanic acid and penicillin combination is
basically a miracle.
(Refer Slide Time: 20:05)
But always remember, bacteria is also very clever; clavulanic acid was there. So,
everybody thought that now the answer is there, but actually this battle between bacteria
and human being will continue forever.
Because bacteria wants to survive, we also want to have a healthy life. So, bacteria wants
to mutate, have different strategy and then like this advent of the metallo β-lactamase
where clavulanic acid does not work. So, for treating the infection by metallo beta-
lactamase, carbapenem was introduced. I think, I have the structure of carbapenem
(Refer Slide Time: 20:47)
Let us see, this I told you about the about cephalosporin how it works.
(Refer Slide Time: 20:59)
This is the Carbapenem that is in the market today. So, carbapenem antibiotics have
different heterocyclic ring here and in the side chain it is always hydroxymethyl group.
So, there are drugs which are called meropenem, ertapenem. Variations depending on
. So, lot of research is being spent on developing new types of carbapenem, but
these are the ones which are in the market today.
Remember this alone can withstand the β-lactamase. This alone can target the
transpeptidase, the same molecule takes care of, the β-lactamase, does not get hydrolyzed
and then by the same molecule, the transpeptidase is inhibited. But today at 2019, the
scenario is that, because of this advent of NDM strains, carbapenems are also not
effective against this. Carbapenems also failed, it does not work. So, then what is the
answer? This situation is very bleak; if people have this NDM infection then unless we
get new antibiotics, I think the last answer is polymyxin.
But polymyxin is very toxic also. So, the patient may not survive, that is the problem; but
polymyxin can kill the NDM strain, but the problem is that because of the toxic side
effect, the patient may not survive. So, things are very grim, unless we have some
mechanism to take care of these NDM problems. So, that is carbapenem again.
(Refer Slide Time: 23:09)
Now, the I told you that there are some drugs like aztreonam; this is also the one which is
prescribed in case of NDM this metallo β-lactamase, MBL-infections; aztreonam is one.
Now, you can actually ask that why, because earlier you have been told that it has to be a
bicyclic network, to increase the electrophilicity of this carbonyl carbon.
But if you do not have bicyclic framework, how come that this is also opening very
rapidly? You see the group here, you can bypass the bicyclic framework by putting an
electron withdrawing group here; if you put an electron withdrawing group, then also,
that is going to activate the opening of the β-lactam ring and exactly nature has done that
by putting the sulphonic acid group here.
So, that is an electron withdrawing group and that helps the carbonyl to be attacked by
the enzyme, that serine in the transpeptidase. There is another group of antibiotics which
are called nocardicins. This is also a mono-cyclic compound and they are also having
antibacterial activity so; that means, the myth that it has to be bicyclic is not true. They
are called mono bactams because they have only the β-lactam group.
So, the mono-bactams are there which can show activity, but in order to do that, it is
better that they have an electron pull from the nitrogen.
(Refer Slide Time: 25:13)
Every time you cannot use clavulanic acid, there you have to always have different
varieties of inhibitors of β-lactamase. Clavulanic acid is very good; that is true; again it is
a microorganism made compound.
Now there are synthetic compounds, sulbactam; what they did? It is basically a
penicillin without any side chain, I think the numbering goes like this, but we will have
to check that whether different text follows different numbering. But whatever you
follow, this becomes 6 in penicillin and this becomes 7 in cephalosporin.
Now, this is oxidized to a sulfone, sulbactam is a penicillin sulfone. So, what happens if
you make a sulfone; that means you are making this a good leaving group, much better
than your only sulphur? Because that is similar to a withdrawing group, it is now
pushing, so if the nucleophile attacks here, that goes there, that opens up. Now question
is after opening up what happens, I will go to tell you next. In clavulanic acid, the same
thing happens; this is a very good leaving group, so that leaves; but what happens after
that, why the enzyme is inhibited; that is the million dollar question.
You have another compound avibactam, which is entirely different; but again it is a
bicyclic compound and again the nitrogen is attached to a sulphone. So, this is opened up
by the nucleophilic serine –OH in transpeptidase or in β-lactamase. So, they are actually
good β-lactamase inhibitor; do not get confused. Some molecules which are β-lactamase
inhibitors they are very weak antibiotics like clavulanic acid, like sulbactam, like
avibactam, like tazobactam; these are only beta lactamase inhibitors except the
Carbapenems are the ones which are not attacked by β-lactamase; or even if they are
attacked by β-lactamase, the β-lactam enzyme is inhibited; at the same time they are very
good antibiotics. So, that has to be very clear. So, now, the question is what is the
mechanism of this inhibition of β-lactamase by clavulanic acid?
(Refer Slide Time: 28:05)
We take the clavulanic acid as the example. So, in clavulanic acid molecule, I right here
N O, forget about the stereochemistry for the time being; double bond CH2OH, this is
clavulanic acid. So, the first β-lactamase attacks the carbonyl, this goes here comes back
and that opens up.
So, from here we can start. So, the serine has attacked and opened the clavulanic acid β-
lactam ring; then what happens, then as I told you that this is a very good leaving group
that oxygen, so the lone pair goes here that comes here and this takes up a proton.
So; that means, you get this iminium ion. Now there are several mechanisms, because it
is all done by mass spec and some IR spectroscopy, several mechanisms were
investigated. So, what they did? They added that β-lactamase and tried to identify
different metabolites which are obtained from clavulanic acid by mass spec; and one of
the mechanisms says that you get once this happens. So, you get O enzyme; and then you
have this iminium ion NH plus then you have CO2H here you have a carbonyl here and
you have this OH; that is the same compound which is written here. Now there are many
things that can happen, this is a β-keto acid.
So, decarboxylation can take place and if you look at this slide, decarboxylation has
taken place here, so it can decarboxylate. So, what they got? They got this type of
species, which means, it is just only three carbon; but without going into such details
what is generally believed is that this iminium ion now tautomerize into the
enamine. You know the enamine- imine tautomerism; that means, double bond is here
now and this becomes NH and CO2H and then the rest carbonyl and this OH.
Now, this has become a good Michael acceptor,. So, what will happen? The enzyme
which is acyl enzyme, there are many things that can happen. First of all, if I ask you
which ester is hydrolyzed faster? A propionic ester or an acrylic ester? The answer is that
the acrylic ester will be slower because of this conjugation.
So, that reduces the electrophilicity of this carbon. So, now, when the double bond is
here, the enzyme cannot free it because, hence the hydrolysis will be slow. Further, the
enzyme is actually a very big molecule; see what happens here, here it is only written as
E and Z; but actually the enzyme is like this, whatever is happening is inside the cavity
of the enzyme. So, then the double bond, then you have this NH and then other part of
the clavulanic acid. So, what can happen, this is a very good Michael acceptor.
So, if there is a nucleophilic amino acid in the enzyme that can come and attack this
carbonyl and that undergoes a 1,4-addition. And finally, what you get is a covalent
attachment with the enzyme. So, even if the enzyme, now frees here, water comes and
hydrolyzes this; this is already attached, the other portion of the enzyme is attached to
the hydrolyzed clavulanic acid. So, that means, it is covalently linked to the that
modified clavulanic acid, so; that means, it is irreversibly inhibited.
(Refer Slide Time: 32:53)
I can again write it in a better way, let me see whether there is any blank, yes. So, again I
write this is your clavulanic acid, by the way this is one of the mechanism because the
mass spec has given many peaks. Of course, that does not tell you, because in mass spec,
there are may be fragmentation also that is not the sacrosanct that that has to happen. So,
now, you have a β-lactamase like this; I write the big enzyme now and an XH here.
So, this is going to hydrolyze, that goes here, in one step you can show it and that comes
here. So, if that be the case; that means, now XH and you have O, then the CO, then you
have double bond N; what you have is a double bond here and then a carbonyl here
CO2H here and then this CH2CH2OH. Now this tautomerize into O CO double bond here,
then actually you should not write any stereochemistry here; it could be cis or trans NH,
and then you have this CO2H CO and CH2CH2OH.
So, what I am saying now XH is present. So, X now attacks here and forms the covalent
connection. So, even if it is hydrolyzed, the enzyme stays with this hydrolyzed
So, this is an irreversible inhibition, because covalent bonding is formed. Now the
question is whether it a suicide inhibitor or is it an active site directed irreversible
inhibition? This is a suicide inhibition, because the molecule has been accepted first as
the normal substrate what the β-lactamase does; then it is transformed into a reactive
species and that reacts with the active site, active site amino acid residues.
So, that is the best example of a suicide inhibition. So, this is the mechanism and all
penicillin that sulfone whatever I said, tazobactam or your sulbactam they all work on
this principle; that the β- lactamase comes attacks this, this opens up, and that goes out.
As soon as this forms imine, that tautomerize to the enamine and then the enzyme active
site adds on to the double bond. So, the trick to develop a new β-lactamase inhibitor, is to
have a good leaving group at this site.
However molecule has to be accepted by the enzyme. Clavulanic is such a molecule,
penicillin derivative is such a molecule. So, we have now done the penicillin chemistry
in quite detail that how penicillin works and what are the different modifications, what
are the different natural products which contain β-lactam, where you can use them, then
what is the genesis of resistance in against the β-lactam antibiotics, and then how to
overcome them, and how to utilize β-lactamase inhibitor. So, next we will go to other
topics in the next session let. So, this session ends here, ok.
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