Abiola Muhammad, ADEOSUN

Department of Biochemistry,
Lead City University,
Ibadan
What are Antibiotics?
 Antibiotics: chemical substances produced by
microorganisms that inhibits the growth or kills other
microorganisms
 Antimicrobial agents: chemical substances from a
biological source or produced by chemical synthesis
that kills or inhibits the growth of microorganisms.
Sources of Antibiotics
 Natural : mainly fungal sources (e.g Benzylpenicillin
and Gentamycin)
 Semi synthetic: chemically altered natural compound
(e.g Ampicillin & Amikacin).
 Synthetic: chemically designed in the lab (e.g
Moxifloxacin and Norfloxacin).
What are the Roles of Antibiotics?
 Bacteriostatic effect: To inhibit multiplication
@ [drug] = MIC minimal inhibitory concentration.
 Bactericidal effect: To kill (destroy) the bacteria
population
@ [drug] = MBC minimal bactericidal concentration.
There is a much closer relationship between the MIC
and MBC values for bactericidal drugs than for
bacteriostatic drugs.
Principle of Selective toxicity: What
is an ideal Antibacterial?
 Selective target – target unique
 Bactericidal – kills
 Narrow spectrum – does not kill normal flora
 High therapeutic index – ratio of toxic level to
 therapeutic level
 Few adverse reactions – toxicity, allergy
 Various routes of administration – IV, IM, oral
 Good absorption
 Good distribution to site of infection
 Emergence of resistance is slow
Antibiotics classification
Antibiotics are usually classified based on their structure , Function and/or spectrum of
activity
1. Structure - molecular structure.
ß-Lactams - Beta-lactam ring
Aminoglycosides - vary only by side chains attached to basic structure

2. Function - how the drug works, its mode of action.

5 functional groups
These are all components or functions necessary for bacterial growth
Targets for antibiotics:
 Inhibitors of cell wall synthesis
 Inhibitors of protein synthesis
 Inhibitors of membrane function
 Anti-metabolites
 Inhibitors of nucleic acid synthesis

3. Spectrum of Activity:
Narrow spectrum
Broad Spectrum
In these discussions, we will primarily use the functional classification, but will point out where
1. Inhibitors of Cell Wall Synthesis:

 Beta-lactams:
 Penicillins
 Cephalosporins
 Monobactams
 Carbapenems
 Glycopeptides
 Fosfomycins
Inhibitors of Cell Wall Synthesis:
The Penicillins
1928 - Alexander Fleming
 Bread mold (Penicillin notatum) growing on petri dish
1939 - Florey, Chain, and Associates
 Began work on isolating and synthesizing large amounts
of Penicillin.
1944 - Used in WWII to treat infections
Late 1940’s - available for general use in US
Penicillins as well as cephalosporins are called beta-lactam
antibiotics and are characterized by three fundamental
structural requirements:
 the fused beta-lactam structure (shown in the blue
and red rings,
 a free carboxyl acid group (shown in red bottom
right),
 one or more substituted amino acid side chains
(shown in black).
 The lactam structure can also be viewed as the
covalent bonding of pieces of two amino acids -
cysteine (blue) and valine (red).
 The beta-lactam nucleus itself is the chief structural
requirement for biological activity;
 metabolic transformation or chemical alteration of this
portion of the molecule causes loss of all significant
antibacterial activity Figure 1: beta lactam structure
Figure 2: The beta lactam ring
Mechanism of Actions of Beta
lactams
 All penicillin derivatives produce their bacteriocidal
effects by inhibition of bacterial cell wall synthesis.
 Specifically, the cross linking of peptides on the
mucosaccharide chains is prevented. If cell walls are
improperly made cell walls allow water to flow into the
cell causing it to burst.
Bacteria Cell Wall Synthesis
 The cell walls of bacteria are essential for their normal
growth and development.
 The peptidoglycan (which provide rigid mechanical
stability) is composed of glycan chains, which are
linear strands of two alternating amino sugars (N-
acetylglucosamine and N-acetylmuramic acid) that are
cross-linked by peptide chains. (NAG-NAM).
 In gram-positive microorganisms, the cell wall is 50 to
100 molecules thick, but it is only 1 or 2 molecules
thick in gram-negative bacteria
Bacteria Cell Wall Synthesis (cont)
The biosynthesis of the peptidoglycan involves about 30 bacterial enzymes and
may be considered in three stages.
 The first stage is precursor formation in the cytoplasm. The product, uridine diphosphate
(UDP)-acetylmuramyl-pentapeptide, called a "Park nucleotide“.

 The last reaction in the synthesis of this compound is the addition of a dipeptide, D-
alanyl-D-alanine.

 The second stage, UDP-acetylmuramyl-pentapeptide and UDP-acetylglucosamine are

linked to form a long polymer.

 The third and final stage involves the completion of the cross-link. This is accomplished by
a transpeptidation reaction that occurs outside the cell membrane. The transpeptidase
itself is membrane bound. The terminal glycine residue of the pentaglycine bridge is
linked to the fourth residue of the pentapeptide (D-alanine), releasing the fifth residue
(also D-alanine).

 Penicillin binds at the active site of the transpeptidase enzyme that cross-links the
peptidoglycan strands. It does this by mimicking the D-alanyl-D-alanine residues that
would normally bind to this site. Penicillin irreversibly inhibits the enzyme transpeptidase
by reacting with a serine residue in the transpeptidase. This reaction is irreversible and so
the growth of the bacterial cell wall is inhibited.
Bacteria Cell Wall Synthesis (cont):
The PBPs and Binding of Penicillins
 Related targets of penicillins and cephalosporins collectively termed penicillin-
binding proteins (PBPs)

 PBPs functions are diverse: catalyze the transpeptidase reaction, maintam

shape, forms septums during division, Inhibit autolytic enzymes.

 Binding to PBPs results in:

 Inhibition of transpeptidase: transpeptidase catalyzes the cross-linking
of the pentaglycine bridge with the fourth residue (D-Ala) of the
pentapeptide. The fifth reside (also D-Ala) is released during this reaction.
Spheroblasts are formed.
 Structural irregularities: binding to PBPs may result in abnormal
elongation, abnormal shape, cell wall defects.
 Figure 3. The transpeptidase reaction in Staphylococcus
aureus that is inhibited by penicillins and cephalosporins.
 Figure 4. Comparison of the structure and composition of
gram-positive and gram-negative cell walls.
Other Inhibitors of Cell Wall
Synthesis:
Glycopeptide
 Include two compounds with similar structures;
Vancomycin and Teicoplanin
 Teicoplanin not FDA approved in the U.S.
 Both are of high molecular weight (1500-2000 daltons)
 Glycopeptides have a complex chemical structure
 Inhibit cell wall synthesis at a site different than the
beta-lactams
 All are bactericidal
 All used for Gram-positive infections. (No Gram-
negative activity)
 Pharmaceutical research and development has been
very active in this area recently resulting in new
antimicrobials and classification
Vancomyci
In Gram-Positives: The drugs enter without any problem n
because peptidoglycan does not act as a barrier for the
diffusion of these molecules.
In Gram-Negatives: Glycopeptides are of high molecular
weight (1500-2000 daltons), stopping them from passing
through the porins of gram-negative bacteria (i.e.,
glycopeptides have no activity against Gram-negatives).
Other Inhibitors of Cell Wall
Synthesis:
Glycopeptide
MOA
Glycopeptides inhibit the final cell wall stage of the
peptidoglycan synthesis process
The ‘pocket-shaped’ glycopeptide binds the D-ala-D-ala
terminal of the basic sub-unit theoretically waiting to be
incorporated into the growing peptidoglycan
Because it is so bulky, the glycopeptide inhibits the action of
the glycosyltransferases and transpeptidases (which act as a
kind of “cement”) - blocks pentaglycine from joining
molecules, thereby blocking peptidoglycan growth.
Glycopeptides are bactericidal, but slow-acting
Other Inhibitors of Cell Wall
Synthesis:
Fosfomycins
Spectrum of Action
 Fosfomycin: Acts to inhibit cell wall synthesis at a stage
earlier than the penicillins or cephalosporins. FDA
approved 1996.
 It is a broad spectrum agent
Mode of Action:
Inhibits the first step of the peptidoglycan synthesis
process (Actual step of inhibition is not completely
understood)
2. Inhibitors of Protein Synthesis
Aminoglycosides -(Bactericidal) : Gentamicin, Tobramycin, Amikacin

MLSK (Macrolides, Lincosamides, Streptogramins, Ketolides) (Bacteriostatic) –

Erythromycin, Clindamycin, Quinupristin-Dalfopristin (Synercid), Clarithromycin,
Azithromycin, Telithromycin

Tetracyclines (Bacteriostatic) – Tetracycline, Doxycycline, Minocycline

Glycylcyclines - Tigecycline

Phenocols (Bacteriostatic), Chloramphenicol

Oxazolidinones – Linezolid (Bactericidal for Streptococci; Bacteriostatic for Enterococcus

and Staphylococci)

Ansamycins - Rifampin
(Bacteriostatic or Bactericidal depending on organism and concentration)
Inhibitors of Protein Synthesis
These classes interferes with ribosomes

Most are bacteriostatic

Resistance to tetracycline and Macrolide is common.

Overview of Protein Synthesis

Figure 5: Overview of protein synthesis

Tetracyclines
Analogs; Doxycycline Minocycline, and Tigecycline

 Enter microorganisms in part by passive diffussion, and in

part by active transport
 Binds to 30S subunits & blocks the binding of amino acyl
tRNA to the acceptor site on the mRNA-ribosome complex.
 Active against; many gram+ve & gram –ve, rickettsiae,
chlamydiae, mycoplasmas.
Macrolides
Characterized by Macrocyclic lactone rings + deoxy sugars

 Prototype: Erythromycin (from streptomyces erythreus)

 Semisynthetic derivatives: clarithramycin , ketolides and azithromycin

 Inhibit 50S ribosomal RNA near peptidyl transferase centre, thereby preventing peptide
chain elongation by blocking of polypeptide exit tunnel. As a result,m pepidyl tRNA is
dissociated from the ribosome
 Active against: pneumococci, streptococci, staphylococci, H. Pyroli, Ricketssia spp,
chlamydia spp
 Hemophilus influenza and Campylobacter are less susceptible

 Resistances: Usually plasmic encoded, reduced permeability of membrane, active efflux,

or by production of esterases (by enterobaceriaceae) that hydrolyzes macrolides
 Action of clindamycin and streptogramins is related to that of erythromycin
Chloramphenicol
 Chloramphenicol binds reversibly to the 50S subunit
of the bacterial ribosome and inhibit peptide bond
formation
 Bacteriostatic broad-spectrum antibiotic that is active
against both aerobic and anaerobic gram +ve & gram -
ve organisms.
 It is active also against Rickettsiae but not Chlamydiae.
 Clinically significant resistance is due to production of
chloramphenicol acetyltransferase, a plasmid-encoded
enzyme that inactivates the drug.
MOA of MLSK

 Translation:
1.Initiation 2. Elongation 3Termination
Figure 6: MOA OF MLSK (Macrolides, Lincosamides,
Streptogramins, Ketolides)
3. Inhibitors of Membrane
Function
 Lipopeptides
 Polymyxins (A,B,C,D, and E)
 • Polymyxin B and E can be used therapeutically
 • Polymyxin B – derived from Bacillus polymyxa var.
aerosporus
 • Polymyxin E – derived from Bacillus polymyxa var.
colistinus = Colistin. Colistin exsists as two forms:
Colistin sulfate – intestinal infections, topical, powders, media
Colistimethate sodium – most active, effective form
 Cyclic Lipopeptides

 • All Bactericidal
Polymyxin Mode of Action
 Target =Membrane phospholipids (lipopolysaccharides (LPS) and
 lipoproteins)
 1. Outer and Cytoplasmic Membrane Effect:
 Polymyxins are positively charged molecules (cationic) which are attracted to the
negatively charged bacteria.
 The negative charge of bacteria is due to LPS in the outer membrane and the
peptidoglycan (notably the teichoic acid).
 The antibiotic binds to the cell membrane, alters its structure and makes it more
permeable. This disrupts osmotic balance causing leakage of cellular molecules,
inhibition of respiration and increased water uptake leading to cell death.

 The antibiotic acts much like a cationic detergent and effects all membranes similarly.
Toxic side effects are common.

 Little or no effect on Gram-positives since the cell wall is too thick to permit access to the
membrane.
 Gram-positives are naturally resistant.
 4. Antimetabolites
 Folate Pathway Inhibitors:
Sulfonamides, Trimethoprim/Sulfamethoxazole
 The drug resembles a microbial substrate and competes with
that
 substrate for the limited microbial enzyme
 The drug ties up the enzyme and blocks a step in metabolism

Figure 7: Competitive Antagonism

Synthesis of Tetrahydrofolic Acid

Humans do not
synthesize folic acid.
Good selective target

Sulfonamides
(sulfadiazine,
sulfamethoxazole,
sulfadoxine)
Bacteriostatic
Introduced in 1930’s –
first effective systemic
antimicrobial agent
Used for treatment of
acute, uncomplicated
UTI’s
Trimethoprim/Sulfamethoxaz
ole
TMP/SXT is bactericidal
Broad spectrum
Synergistic action
Figure 8: synthesis of THF
Anti-metabolite (conts)
 The combination SXT (thrimethoprim-sulfamethoxazole)
is synergistic and the association provides a bactericidal
effect

 Natural Resistance
Enterococcus – low level and poorly expressed
S. pneumoniae
Ps. aeruginosa (impermeability)
5. Inhibitors of Nucleic Acid
Synthesis (Qunolones & Furanes)
Quinolones:
Humans do synthesize DNA - shared process with
bacteria
Do tend to see some side effects with Quinolones
Some drugs withdrawn from market quickly
All are bactericidal
Quinolones
Mode of Action
 Small and hydrophilic, quinolones have no problem
crossing the outer membrane.
 They easily diffuse through the peptidoglycan and the
cytoplasmic membrane and rapidly reach their target.
 Target = Topoisomerases (DNA-gyrase)
 Rapid bactericidal activity
Quinolones inhibit DNA synthesis
Mode of Action
 A typical E. coli’s chromosome is 1400 microns long (1 micron in diameter
when supercoiled), enough to fit in the E. coli’s bacterial cells which is 2-3
microns long
 The bacterial chromosome consists of a single circle of DNA
 DNA is double-stranded forming a left-handed double helix
 All topoisomerases ( which are involved in DNA replication,
transcription and recombination) can relax DNA but only gyrase which
carry out DNA supercoiling.
 The main quinolone target is the DNA gyrase which is responsible for
cutting one of the chromosomal DNA strands at the beginning of the
supercoiling process. The nick is only introduced temporarily and later
the two ends are joined back together (i.e., repaired).
 • The quinolone molecule forms a stable complex with DNA gyrase
thereby inhibiting its activity and preventing the repair of DNA cuts
Resistance to Quinolones
 Natural Resistance
 Gram Positives – 1st generation quinolones
 S.pneumoniae – decreased activity to Ofloxacin and
 Ciprofloxacin
 Ps. aeruginosa – decreased activity to Norfloxacin and
 Ofloxacin
Furanes
 Nitrofurantoin

Mode of Action:
 The drug works by damaging bacterial DNA.

 In the bacterial cell, nitrofurantoin is reduced by flavoproteins (nitrofuran

reductase). These reduced products are are highly active and attack ribosomal
proteins, DNA, respiration, pyruvate metabolism and other macromolecule
within the cell.
 It is not known which of the actions of nitrofurantoin is primarily responsible
for its bactericidal acitivity.
 Natural Resistance:
Pseudomonas and most Proteus spp. are naturally resistant.
 Questions?
Bibliography
 Katzung, B.G. Basic and Clinical Pharmacology, 12th
Edition, Chapters 44 and 45, p774-783. D
 Avis, B. D., Chen, L. L. & Tai, P. C. Misread protein creates
membrane channels: an essential step in the bactericidal
action of aminoglycosides. Proc. Natl Acad. Sci. USA 83,
6164–6168 (1986).
 Wise, E. M. Jr & Park, J. T. Penicillin: its basic site of action
as an inhibitor of a peptide cross-linking reaction in cell
wall mucopeptide synthesis. Proc. Natl Acad. Sci. USA 54,
75–81 (1965).
 Davis, B. D. Mechanism of bactericidal action of
aminoglycosides. Microbiol. Rev. 51, 341–350 (1987).