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FUN2: 11:00-12:00
Scribe: Kallie Law
Tuesday, December 9, 2008
Proof: Maggie Law
Dr. Smith
Immunology
Page 1 of 5
Chemotherapy
I. Introduction [S1]:
a. Important part of chemotherapy that will be discussed (including anti parasitic, cancer chemo, antivirals…)
b. Dr. Smith will focus on antibacterials – the most common causes of infections in the eye, gums.
c. Handout is extensive – more in HO than will be covered in class – but it is a summary that Dr. Smith feels will be
the most important features/concepts of chemotherapy pertaining to the major classes of antibacterials in clinical
practice.
d. Major classes – begin with B-lactams, penicillin and cephalosporin.
e. Brief introduction to the history of chemotherapy, discovery of antibacterials.
i. The terms antibacterials and antibiotics are used synonymously, but they are not.
ii. Antibacterial is a more all encompassing term
iii. Antibiotic- a substance produced by one microorganism that inhibits the growth of another microorganism.
True antibiotics are naturally occurring produced by a microorganism.
iv. Concept of antibiotics comes from an earlier idea of antibiosis as a starting point of the lecture, and then
Dr. Smith will focus on the principles of chemotherapy that apply to all chemotherapy – whether it involves
a tumor cell, a virus, a bacteria, a fungus.
v. Will highlight the basic principles of chemotherapy and then B-lactams will be talked about.
vi. Cephalosporin will be covered on Wednesday.
II. Antibiosis [S2]
a. Idea of antibiosis came from studies of microorganisms in soil, closely associated with evolution
b. Idea that there are more microorganisms in soil than realized; the organisms live in soil and compete for a niche
– do this by producing antibiotics that inhibit the growth of other soil organisms
c. [S3] Penicillin- derived from the mold that produces penicillin – naturally occurring mold that Fleming cultured by
accident
i. The penicillin mold accidently contaminated the plate of staphylococcus and Fleming saw it lysed colonies
close to mold.
ii. See big colonies of mold on slide- see that there is faintly appearing lysing colonies of staph close in
proximity to the mold colonies
iii. Fleming interpreted this result in 1928 – dropped the ball – did not have any success.
iv. He did not purify the substance – it was un-isolated, unstable.
v. 10 years later – another group of scientists purified it, injected it into mice, cured mice of bacterial infections
and then went to use in humans who were suffering fatal bacterial infections.
d. Brief summary of the history – most of the antibacterials, antibiotics were discovered in the 40’s and 50’s.
Agents are still in use, but their use is limited because of resistance. (Waites will talk about resistance – there
are diagrams about resistant in HO.)
i. Resistance is the reason why new antibiotics and antibacterials have to be discovered.
ii. New efforts had to be made to find new antibiotics and antibacterials – there will never be an armentarium
to last forever. (takes work, money, political will, advocacy, etc)
III. Principles of chemotherapy [S4]
a. Principles are that you are dealing with 2 living organisms
i. Complex
ii. Used to focusing on patient and drug, now we focus on the patient, the drug and the invading organism.
iii. Invading organism – tumor cell, bacteria, virus, fungus, protozoan
iv. Same principles apply – goal is to eradicate the organism at an acceptable cost in terms of side effects
b. Early efforts to develop antimicrobial agents were plagued by the problem of the agents being more toxic to
patient than microorganism
c. Biggest obstacle to the discovery of an effective, well tolerated antibiotic such as penicillin or a sulfa drug (look
at list of discoveries – 1935 -1st highly effective, well-tolerated microbial agent. It is synthetic – not a true
microbial agent)
d. Problem was that early agents were antiseptics – more toxic to host than invading organisms.
e. Basis for all chemotherapy is selective toxicity.
i. Cornerstone concept – can you exploit a biochemical difference between the invading organism and the
patient?
ii. Biochemical or genetic difference – then tailor an antimicrobial or anti-tumor agent that selectivity kills or
inhibits the growth of the microorganism.
iii. When chemotherapy produces side effects (as anti-tumor agents do) – side effects come from the low
margin of selective toxicity.
iv. Advances in science – understanding of tumor cells and microorganisms have allowed us to target specific
components of tumors and microorganisms.
FUN2: 11:00-12:00
Scribe: Kallie Law
Tuesday, December 9, 2008
Proof: Maggie Law
Dr. Smith
Immunology
Page 2 of 5
1. We can then achieve a high margin of selective toxicity
2. It is an ongoing process – could be better
IV. Drug-Pathogen-Patient [S5]
a. Triad of interactions is what we have to consider.
b. Selective toxicity is the corner stone
c. Emphasis in the diagram – comes from the immune response and host defense, which is critically important.
d. Without the mounting of an effective host immune defense response, most infective agents will not be
eradicated simply by using a chemotherapeutic agent, even if it is bactericidal
i. That is one that kills bacteria; still not able to eradicate the infection when the patient’s immune system is
compromised.
ii. However effective a chemotherapeutic agent is, still dependant on immune system of patient
iii. Studies of anti-tumor agents, antimicrobial agents – one of the confounding parts of the research is that
some proportion of individuals that are suffering from cancer or infection can recover spontaneously without
treatment. This is dependent on the host immune system.
V. Classification of Antibacterial drugs [S6]
a. Classification of bacterial agents is based on their target in the bacteria.
b. Most sensible way of classifying antibacterial agents – put into classes and understand how they work, if they
will be toxic
i. Bactericidal – kill bacteria
ii. Bacteria static – inhibit growth. What is necessary to produce eradication in most cases, providing the
immune system is functional
c. Based on targets:
i. Cell wall synthesis inhibitors
1. B-lactams
2. Cepholosporin
3. Penicillin
4. Vancomycin – important cell wall synthesis inhibitor
ii. Protein synthesis in the bacteria
1. Occurs via the ribosomes that translate the messenger RNA into protein.
2. Ribosomes are targets of many antibacterials including – erythromycin, cholorihphencial,
clindomycin, tetracycline and aminoglycosides such as streptomycin, tobramycin, gentamicin, etc
3. Is that a reasonable target? – will the bacteria ribosome give selective toxicity?
4. Don’t humans have ribosomes? Yes, but there is much evolution between human ribosome and
bacterial ribosomes so we are able to exploit biochemical, molecular difference between the
ribosomes
5. Therefore, there are many effective agents that target the bacterial ribosome and don’t inhibit protein
synthesis in the ribosomes of our cells; that is the basis of selective toxicity inhibitors. Target the
components unique to bacteria.
iii. DNA gyrase – only exists in bacteria – not in humans
1. Unwinds the bacterial DNA
2. Bacterial chromosome is a closed circle; human chromosomes are linear, double stranded DNA
wrapped around nucleosomes.
3. Bacterial chromosome is round, super twisted and to be replicated, it has to be unwound.
4. DNA gyrase is target by quinolones, nalidixic acid
iv. Cell membrane – another target. Comes into play with antifungals. Targets the fungal membrane
1. Have some antifungals that target to the sterol component of the fungal membrane
2. Sterol in our cells is mostly cholesterol, but fungus does not have cholesterol, has a different sterol.
3. That is how the membrane antifungals are able to be selectively toxic
4. In general, antibacterial agents that target the bacterial membrane have low levels of selective
toxicity; can also be harmful to our cells because they disrupt the membrane in our cells to some
extent
d. Can expect and predict a cell wall synthesis inhibitor – highly selectively toxic for bacteria.
e. A cell membrane targeted antibacterial – not so selectively toxic.
i. Can predict on the basis of knowing the target how likely the selective toxicity is.
f. Next slides are being skipped – in HO – elaborate on what was already discussed. Skip [S7,8]
VI. Gram Stain [S9]
a. Gram stain is retained in the gram + because of the cell wall being 50-100 strands of PG thick
b. Gram – is only a few strands of PG thick
c. Gram stain is useful to distinguish the two major classes of bacteria that may be involved in an infection. [S10]
FUN2: 11:00-12:00
Scribe: Kallie Law
Tuesday, December 9, 2008
Proof: Maggie Law
Dr. Smith
Immunology
Page 3 of 5
i. It is a microscopic staining mechanism for distinguishing gram – and +; works because the cell wall is so
different in the 2 bacteria classes
1. Positive wall is 50-100 PG molecules thick
VII. Bacterial cell wall synthesis [S11]
a. Target of B-lactams.
b. Will talk about penicillin, much of what is talked about will apply to the cephalosporins. The common
denominator is the B-lactam ring. It is 4 membered ring.
c. It is a square. The B-lactam ring is the cornerstone of the penicillin and cephalosporin.
i. Has a nitrogen atom and an oxygen
d. A picture of a beta lactam ring says 1,000,000 words.
e.
.
f. B-lactam ring is the heart of penicillins and cephalosporins---how are these inactivated and destroyed by
bacteria that are resistant to them?---by hydrolysis of the beta lactam ring by bacterial enzymes called betalactamases; these are bacterial enzymes that add water across the ring and break the bond and destroy the
antibacterial activity of the penicillins and cephalosporins.
g. Target is cell wall synthesis—cell wall is composed of peptidoglycan—this means peptide (which is amino acids
just like how proteins contain amino acids---peptides are small numbers of amino acids.
h. Glycan is the sugar component of the cell wall—it’s a linear polymer of peptidoglycan units that makes the
spaghetti that encases the bacterium and confers upon the bacterium its characteristic shape like spherical for
streptococcus or cylinder-shape for bacillus—this is determined by the cell wall.
i. The bacteria synthesize the cell wall through an elaborate process; it’s like being in outer space and you are in a
space capsule--if you have a hole in your capsule, you lose all the oxygen so you have to make a new capsule
to make a new home for your offspring—you must build them a space capsule without allowing your space
capsule to be compromised in any way.
j. Bacteria can do this in a remarkably short time—every 20 minutes under optimal conditions they can divide and
produce a new space capsule without at all risking their safe environment; this is miraculous and is
accomplished by enzymes.
k. The enzymes are the targets of the penicillins and cephalosporins---the enzymes go by a generic name of PBPs
(penicillin binding proteins)---targets of the penicillins and cephalosporins.
l. One class of PBPs is called transpeptidases---these cross link the cell wall which is what is really critically
important for maintaining the integrity of the cell wall and the bacteria; bacteria accumulate all types of salts,
building blocks, amino acids, proteins, nucleic acids etc.—these are all packed into a bacteria in a little space—
this means compared to the environment in which they live, there is tremendous osmotic pressure caused by
the water trying to diffuse in to equilibrate the concentration of water on the inside and outside.
m. A little nick in the cell wall will cause lysis of the bacteria due to the tremendous pressure that is build up on the
cytoplasmic membrane of the bacteria.
VIII. [S12] Cross-linking peptidoglycan
a. Transpeptidases is one of the major targets of the penicillins and cephalosporins.
b. Other targets are cell wall synthesizing proteins, enzymes---it’s a dynamic process of cell wall synthesis and
degradation---nicks are life-threatening.
c. Transpeptidases is a major target, but not the only target. Transpeptidase is involved in cross linking (a peptide
bond formed between the peptide (amino acid component) of adjacent building blocks---glycines will be linked to
the 2nd to the last D-ala in the adjacent peptidoglycan unit the cell wall.
d. This is what the penicillin and cephalosporins do---prevent cross linking.
IX. [S13] Penicillin mimics D-alanyl
a. Prevent cross linking by using molecular mimicry---mimicking an endogenous molecule.
b. D-ala D-ala on the right have the same bond angles as the critical penicillin on the left. The beta lactam ring
component of penicillins and cephalosporins mimics the D-ala D-ala component of the cell wall.
X. [S14] Penicillin Acylates the active
a. Transpeptidase mistakes penicillins for its substrate of d-ala d-ala involved in the cross link.
FUN2: 11:00-12:00
Scribe: Kallie Law
Tuesday, December 9, 2008
Proof: Maggie Law
Dr. Smith
Immunology
Page 4 of 5
b. Penicillins and cephalosporins can be known as suicide substrates—biochemical nomenclature-- (this means
that penicillin/cephalosporin binds to the active site or center of the transpeptidase and chemically reacts and
permanently destroys the active center of the transpeptidase or the other PBPs.
c. It’s the covalent bond between the penicillin and the transpeptidases or another PBP that permanently
inactivates the PBP including the transpeptidase. This is a powerful mechanism of action.
d. The penicillins and cephalosporins are not simple molecules just sitting around doing nothing---they are ready to
spring into action and it’s like they have accumulated potential energy (energy to react) and are like a coiled
spring and go right to the heart of the molecule (the active center).
XI. [S15] Antibiotic Mechanism
a. It’s irreversible.
XII. [S16] Mechanism of Penicillin verus cycloserine
a. Just about the contrast between penicillins and cephalosporins and another cell wall inhibitor called cycloserine.
b. Cycloserine is an antibacterial agent that used as a 2nd or 3rd line drug to treat TB.
c. It blocks cell wall synthesis and is NOT chemically reactive, it just binds; it blocks the synthesis of the D-alanine
that the bacteria need to crosslink the cell wall.
d. Penicillins and cephalosporins are much more effective because cycloserines just reversibly inhibit this process
of cross linking and just blocks the production of the d-ala chain on the peptide part of the wall.
e. That’s what the diagram is about.
XIII. [S17]: Antibiotic Activity
a. Look up and pay attention too—no class time will be spent here to define these terms.
XIV.
[S18] [S19] skipped
a. Broad and narrow antibiotics are pretty obvious; some antibacterials act on a wide range of bacteria, while some
are focused on a specific subclass of bacteria.
b. Bacterial cidal vs. Bacterial static
c. More important terms---minimal inhibitory concentration, minimal bacterial cidal concentration---important terms
used to quantity antibacterial activity of antimicrobial agents.
d. Randomly starts talking about aminoglycosides: there is a tiny bit about these on the skipped slides: There is
also time dependent vs. concentration dependent---interesting difference between the beta lactams and the
aminoglycosides.
e. Aminoglycosides block protein synthesis in the bacteria; the only protein synthesis inhibitors that are able to kill
bacteria; most protein synthesis inhibitors like tetracyclines, erythromycins, clindamycin, and so on inhibit the
growth, but do not kill.
f. The killing depends on the concentration of the aminoglycoside and how high it is above the minimum bacterial
cidal concentration.
g. Constrast with a time dependent killing that goes along with the beta lactams; it’s not concentration dependent
it’s a matter of how long the bacteria have been exposed to the beta lactam.
h. Knowing the properties of an antimicrobial agent, it’s possible to tailor the dosing interval so you can optimize
the killing of the microorganism and minimize the side effect.
i. Take advantage of the this post-antibiotic effect with the aminoglycosides that persists for some time after the
drug is removed or falls below its effect concentration which in this case would be the MBC.
j. Aminoglycosides are quite toxic and have to be administered in the hospital to minimize damage (side effects)
to things like the inner ear or kidney failure.
XV. [20] Resistance
a. Three major mechanisms of resistance
b. Alteration of the drug target, decreased accumulation of the drug by the bacteria or thirdly, inactivation.
c. Inactivation of the penicillin or cephalosporins by the beta lactamases is the major mode of resistance.
XVI.
[S21] Beta Lactam Antibacterials
a. Reads off the names from slide.
XVII. [S22] Beta Lactams
a. Structures are illustrated here…all have a reactive beta lactam ring responsible for the antibiotic activity.
XVIII. {S23] Penicillin V Potassium
a. Major classes of penicillins
b. How simple the structure of the original penicillin is---There is Pen. V and Pen. G that were the two original and
naturally occurring penicillins; V as in victory and G as in……Go Dawgs!
c. Have the beta lactam ring here that is fused to a sulfur containing ring that is not essential…go back to previous
slide and see that the sulfur ring is not necessary.
d. All the beta lactams are effective antibacterials.
e. Pen V is usually administered as a potassium salt—has an ionized group called a carboxyl group that is carbon
with 2 oxygen atoms; one of the oxygen atoms carries a negative charge.
FUN2: 11:00-12:00
Scribe: Kallie Law
Tuesday, December 9, 2008
Proof: Maggie Law
Dr. Smith
Immunology
Page 5 of 5
f. Naturally occurring penicillins in use are salts and the potassium form is the preferred form of penicillin.
XIX.
[S24] Five Major Classes of Penicillins
a. Very important diagram that organizes the penicillins into the 5 major classes---know what distinguishes one
class from another
b. The 1st class is the originally occurring one---The Pen G and Pen V---principally active against G+ cocci---like
staph and strep and are destroyed by the penicillinases as are all the other classes of penicillins except for the
Class 2 (only one that as a class is resistant to the enzymes that destroy the penicillins—the penicillinases).
c. Beta lactamases is the generic term that includes penicillianases but also includes cephalosporinases, the
bacterial enzymes that can destroy cephalosporins.
d. Class 2 is active chiefly against only gram positives and these are also active against those gram positive
bacteria that produce pencillinase; Class one is destroyed by penicllinases.
e. Class 3, 4, and 5 are easy to distinguish---as you go down the classes, you get increased activity with gram
negatives…class 4 has more activity than class 3 and class 5 has more activity than class 4…so class 5 has the
most activity against gram negatives.
f. As you go down the classes, you are also losing activity against the gram positives.
g. Class 3, 4, 5 are destroyed by pencilliniases---all have tiny modifications like adding a nitrogen atom or an
amino group and get activity against a gram negatives! Fantastic!