Mechanism of action of

Antibiotics, Antivirals
Antifungals and
Antimicrobial Resistance

Budiman Bela
T. Mirawati Sudiro

Dept. Mikrobiologi FKUI

References
PR Murray et.al. Medical Microbiology, fourth edition,
Mosby Inc.
KP Talaro. Foundation in Microbiology, 6th ed., 2008
GE Brooks,JS Butel,SA Morse. Jawetz, Melnick and
Adelberggs
Medical Microbiology, ed 24, , Appleton and Lange,
California. 2004
Definition
Antimicrobial resistance:
the ability of microbes (bacteria, viruses,
parasites, or fungi) to grow in the presence of
a chemical (drug) that would normally kill it or
limit its growth.
 exposure selection expansion

sensitive population resistant clones outbreak, epidemic, pandemic

mrsa: a pandemic
http://www3.niaid.nih.gov/topics/antimicrobialResistance/Understanding/drugResistanceDefinition.htm
Accessed March 23, 2009
Antibacterial Agents
Red azo dye protosil:
1935
protection of mice against systemic streptococcal infection
curative in patients suffering from the same infection
Cleavage result in release of p-aminobenzene sulfonamide
(sulfonilamide) that possess antibacterial activity 1st sulfa
drug
Compounds produced by microorganisms that
inhibit the growth of other microorganism
(Antibiotic):
Alexander fleming: the mold Penicillium prevented the
multiplication of staphylococci
1940s: Streptomycin
1950s: Tetracyclines
Antibacterial Agent
New antibacterial agents have been
introduced and have to be continually
developed due to the remarkable ability of
bacteria to develop resistance to newly
introduced agents
Mode of action and target
molecules of antibacterial agents
Inhibition of cell wall synthesis
Inhibition of nucleic acid synthesis
Inhibition of protein synthesis
Antimetabolites
Cell Wall Synthesis
-Beta-lactams
! DNA Replication
-Quinolones
!
-Vancomycin -Metronidazole
-Isoniazid
-Ethambutol
-Cycloserine
-Ethionamide
RNA Synthesis
-Rifampin
!
-Bacitracin -Rifabutin
-Polymyxin
DNA

Ribosomes
50 50 50
30 30 30
Protein synthesis
(50S ribosome)
!
Antimetabolites
-Sulfonamides
! -Chloramphenicol
-Dapsone -Macrolides
-Trimethoprim Protein synthesis -Clindamycin
-Para-aminosalicylic acid (30S ribosome)
! -Streptogramins
-Aminoglycosides
-Tetracyclines
-Oxazolidinone
 Basic Mechanisms of Antibiotic Action
Antibiotic Action

Disruption of Cell Wall ! !

Penicillin Binds PBPs and enzymes responsible for peptidoglycan synthesis
Cephalosporin Binds PBPs and enzymes responsible for peptidoglycan synthesis
Cephamycin Binds PBPs and enzymes responsible for peptidoglycan synthesis
Carbapenem Binds PBPs and enzymes responsible for peptidoglycan synthesis
Monobactam Binds PBPs and enzymes responsible for peptidoglycan synthesis
-lactamase inhibitor/ -lactam Binds -lactamases and prevents enzymatic inactivation of -lactam
Vancomycin Inhibits cross-linkage of peptidoglycan layers
Isoniazid Inhibits mycolic acid synthesis
Ethionamide Inhibits mycolic acid synthesis
Ethambutol Inhibits arabinogalactan synthesis
Cycloserine Inhibits cross-linkage of peptidoglycan layers
Polymyxin Inhibits bacterial membranes
Bacitracin Inhibits bacterial cytoplasmic membrane and movement of peptidoglycan precursors

Inhibition of Protein Synthesis

Aminoglycoside
! Produces premature release of aberrant peptide chains from 30S ribosome
!
Tetracycline Prevents polypeptide elongation at 30S ribosome
Oxazolidinone Prevents initiation of protein synthesis at 30S ribosome
Macrolide Prevents polypeptide elongation at 50S ribosome
Clindamycin Prevents polypeptide elongation at 50S ribosome
Streptogramins Prevents polypeptide elongation at 50S ribosome
 Basic Mechanisms of Antibiotic Action

Antibiotic Action

!
Inhibition of Nucleic Acid Synthesis !
Quinolone Binds subunit of DNA gyrase
Rifampin Prevents transcription by binding DNA-dependent RNA polymerase
Rifabutin Prevents transcription by binding DNA-dependent RNA polymerase
Metronidazole Disrupts bacteria DNA (is cytotoxic compound)

Antimetabolite
Sulfonamides
Dapsone
! Inhibit dihydropteroate synthase and disrupt folic acid synthesis
Inhibits dihydropteroate synthase
Trimethoprim Inhibits dihydrofolate reductase and disrupts folic acid synthesis !
 Mechanisms of resistance
Intrinsic resistance (chromosomal)
Acquired resistance
Gain of function properties
new enzymatic activity
phosphorylation, acetylation, pump, etc.
Mutation to existing proteins
alteration of ribosome subunits, cell wall
permeability, etc.
Inhibition of Cell Wall Synthesis
Interference with bacterial cell wall
synthesis
The most common mechanism of antibiotic
-lactam antibiotics:
Constitute the majority of cell wall-active
antibiotics
Penicilins, Chepalosporins, Cephamycins,
Carbapenems, Monobactams, -lactamase
inhibitors
 Inhibition of Cell Wall Synthesis

Other antibiotics that interfere with

bacterial cell wall synthesis
Vancomycin:
Bacitracin
Antimycobacterial agents:
Isoniazid
Ethambutol
Cycloserine
Ethionamide
Inhibition of Cell Wall Synthesis by
Beta Lactam Antibiotics
Peptidoglycan layer:
Major structural component of bacterial cell
walls
! Basic structure:
A chain of 10 to 65 disaccharide residues
consisting of alternating molecules of:
N-acetylglucosamine and
N-acetylmuramic acid

! Chains of disaccharide residues are cross-

linked with peptide bridges rigid mesh
coating for bacteria
http://en.wikipedia.org/wiki/File:Gram-positive_cellwall-schematic.png
 Inhibition of Cell Wall Synthesis by Beta Lactam Antibiotics

Penicillin-binding proteins (PBPs):

Specific enzymes that build the peptidoglycan layer of
bacterial cell wall and can be bound by -lactam
antibiotics:
Transpeptidases
Carboxypeptidases
Endopeptidases
-lactam antibiotics generally act as bactericidal
agents:
Exposure of growing bacteria to -lactam antibiotics
binding with PBPs in the growing bacterial cell wall
inhibition of synthesis but not turnover (degradation) of
peptidoglycan bacterial cell death
Mechanisms of Bacterial Resistance
towards Beta Lactam Antibiotics
1. Prevention of interaction between
antibiotic and target PBP:
2. Decreased binding of antibiotic to PBP
3. Hydrolysis of antibiotic by -lactamases
 Mechanisms of Bacterial Resistance towards Beta Lactam Antibiotics

1. Prevention of interaction between antibiotic and

target PBP:
Only seen in gram negative bacteria (particularly
Pseudomonas species):
Posession of outer membrane that overlies the
peptidoglycan layer
Penetration of -lactam antibiotics into gram-
negative bacilli requires transit through the outer
membrane pores:
Changes in the outer membrane pore proteins
(porins) alteration of the size or charge of the
porin channel exclusion of the antibiotic
 Mechanisms of Bacterial Resistance towards Beta Lactam Antibiotics

2. Decreased binding of antibiotic to PBP:

! Formation of modified PBP that fails to bind
to -lactam antibiotics but contributes to the
synthesis of the peptidoglycan layer
Origin of modified PBP:
Mutation in the PBP gene:
Streptococcus pneumoniae resistant to penicillins
Acquisition of a new PBP:
introduction of Escherichia coli PBP into
Staphylococcus aureus confers resistance to oxacillin
 Mechanisms of Bacterial Resistance towards Beta Lactam Antibiotics

3. Hydrolysis of antibiotic by -lactamases:

Inactivation of -lactam antibiotics
There are 200 different -lactamases:
Specific for penicilins: penicilinases
Specific for cephalosporins: cephalosporinases
Broad range of activity:
capable of inactivating most -lactam antibiotics
Extended-spectrum -lactamases (ESBLs):
Commonly encoded on plasmids can be
transferred from organism to organism : Causing
much difficulties and severely limit the empirical use
of -lactam antibiotics in some hospitals
 Mechanisms of Methicillin
Resistance
The staphylococcal beta-lactamase protein, which
cleaves the beta-lactam ring structure, confers
resistance to penicillin but not to semi-synthetic
penicillins such as:
methicillin, oxacillin, or cloxacillin.
Acquisition of the mecA gene, which codes for the
penicillin binding protein PBP2a, complete resistance to
all beta-lactam antibiotics including the semisynthetic
penicillins.
PBP2a has a very low affinity for beta-lactam antibiotics,
and is thought to aid cell wall assembly when the normal
PBPs are inactivated.
The mecA gene is found on a large mobile genetic
element called the staphylococcal chromosomal
cassette mec (SCCmec).
Glycopeptides
A complex glycopeptide that disrupts cell wall
peptidoglycan synthesis in growing gram-positive
bacteria
Interacts with D-alanine-D-alanine termini of the
pentapeptide side chains interferes sterically with
the formation of the bridges between the peptidoglycan
chains
Used for the management of infections caused by
oxacillin-resistant staphylococci and other gram-
positive bacteria resistant to -lactam antibiotics
Inactive against gram-negative bacteria:
Molecule is too large to pass through the outer membrane and
reach the peptidoglycan target site
 Glycopeptides

Intrinsic resistance to Vancomycin:

Due to pentapeptide termination in D-
alanine-D-lactate does not bind
vancomycin:
Leuconostoc, Lactobacillus, Pediococcus and
Erysipelothrix
D-alanine-D-serine termination of
peptapeptide:
Enterococcus gallinarum, Enterococcus
casseliflavus
 Glycopeptides

Acquired resistance to Vancomycin:

Plasmid mediated
Enterococcus faecium and Enterococcus faecalis
A potential threat to the usefulness of vancomycin
for the treatment of enterococcal infections
There is a concern that if these genes are
transferred to staphylocci (proven by lab
experiments) highly resistant and virulent
organism will emerge: Vancomycin resistant
Methicillin resistant Staphylococcus aureus
Polypeptides

Bacitracin:
Mixture of polypeptides
Inhibits cell wall synthesis by interfering with
dephosphorylation and the recycling of the lipid
carrier responsible for moving the peptidoglycan
precursors through the cytoplasmic membrane to
the cell wall
Also damage the bacterial cytoplasmic membrane
and inhibit RNA transcription
Resistance:
Failure of the antibiotic to penetrate into the bacterial cell
 Polypeptides
Polymixins:
Cyclic polypeptides
Inserted into bacterial membranes by interacting
with lipopolysaccharides and the phospholipids in
the outer membrane increased cell permeability
cell death
Most active against gram-negative bacilli since
gram-positive bacteria do not have
an outer membrane
Example:
Polymyxin B and E (colistin)
Isoniazid, Ethionamide, Ethambutol
and Cycloserine
Cell wall-active antibiotics for treatment
of mycobacterial infections
Isoniazid (INH):
Affect the synthesis of mycolic acid
Disruption of:
The desaturation of the long-chain fatty acids
The elongation of fatty acids and hydroxy lipids
 Isoniazid, Ethionamide, Ethambutol and Cycloserine

Ethionamide:
Derivative of Isoniazid
Blocks mycolic acid synthesis
Ethambutol:
Interferes with the synthesis of arabinogalactan in
the cell wall
Cycloserine:
Inhibition of D-alanine-Dalanine synthetase and
alanine racemase that catalyze cell wall synthesis
 Inhibition of Protein Synthesis
Aminoglycosides:
Example:
Streptomycin, neomycin, kanamycin, tobramycin
Amikacin : synthetic derivatives of kanamycin
Netilmicin: synthetic derivatives of sisomycin
Able to pass through:
Bacterial outer membrane (in gram-negative
bacteria)
Cell wall
Cytoplasmic membrane
 Inhibition of Protein Synthesis

Aminoglycosides:
Active site:
Cytoplasm
Binds to the 30S ribosomal proteins
Attachment to the ribosomes has two effects:
Production of aberrant proteins as the result of
misreading of the messenger RNA (mRNA)
Interruption of protein synthesis by causing the
premature release of the ribosome from mRNA
 Inhibition of Protein Synthesis

Aminoglycosides:
Bactericidal:
Able to bind irreversibly to ribosomes
Penetration through the cytoplasmic membrane:
Aerobic, energy dependent process
Anaerobic bacteria are resistant to aminoglycosides
Streptococci and Enterococci:
Cell wall of these bacteria can not be penetrated by
aminoglycosides
Treatment with aminoglycoside therefore requires an
inhibitor of cell wall synthesis (e.g. penicillin,
ampicillin, vancomycin)
 Inhibition of Protein Synthesis

Aminoglycosides:
Resistance (3 ways):
Mutation of the ribosomal binding site:
Relatively uncommon
Occurs in the genus Enterococcus
Decreased uptake of the antibiotic into the bacterial cell:
Observed in Pseudomonas
More commonly seen with anaerobic bacteria
Enzymatic modification of the antibiotic:
Modification occurs through: phosphorylation, adenylation and
acetylation of the amino and hydroxyl groups of the antibiotic
 Inhibition of Protein Synthesis

Tetracyclines
Broad spectrum, bacteriostatic antibiotics
Binding reversible to the 30S ribosomal subunits
Blocking the binding of aminoacyl-transfer RNA (tRNA) to the 30S
ribosome-mRNA complex
Resistance:
Decreased penetration of the antibiotic into the bacterial cell:
Mutations in the chromosomal gene encoding the outer membrane porin protein
OmpfF low level resistance to the tetracyclines as well as to other antibiotics
(e.g. beta-lactams, quinolones, chloramphenicol)
Active efflux of the antibiotic out of the cell:
A variety of genes in different bacteria control the active efflux of the tetracyclines
from the cell
The most common cause of resistance
Alteration of the ribosomal target site:
Production of proteins similar to elongation factors that protect the 30S ribosome
antibiotic can still bind to the ribosome but protein synthesis is not disrupted
Enzymatic modification of the antibiotic
 Inhibition of Protein Synthesis

Oxazolidones
Representative: Linezolid
Narrow-spectrum class of an antibiotics that block
initiation of protein synthesis by interfering with the
formation of the initiation complex at the 30S
ribosomal subunit cross-resistance with other
protein inhibitors does not occur can be used for
treatment of bacterial strains (Staphylococci,
streptococci and enterococci) that are resistant to
penicillins, vancomycin and the aminoglycosides
 Inhibition of Protein Synthesis

Chloramphenicol
Also disrupts protein synthesis in the human bone
marrow cells and can produce blood dyscrasias
Binding reversibly to the peptidyl transferase
component of the 50S ribosomal subunit blocking
peptide elongation
Resistance:
Plasmid-encoded chloramphenicol acetyltransferase
catalyze the acetylation of the 3-hydroxy group of
chloramphenicol incapable of binding to the 50S subunit
Chromosomal mutations (less common) alter the outer
membrane porin proteins gram-negative bacilli become
less permeable
 Inhibition of Protein Synthesis

Macrolides
Bacteriostatic
Basic structure:
Macrocyclic lactone ring bound to two sugars
Reversible binding to the 50S ribosome blockage
of polypeptide elongation
Resistance:
Methylation of the 23S ribosomal RNA prevents binding by
the antibiotic
Destruction of the lactone ring by erythromycin esterase
Active efflux of the antibiotic from the bacterial cell
 Inhibition of Protein Synthesis

Clindamycin:
Blocks protein elongation by binding to the
50S ribosome
Inhibits peptidyl transferase by interfering with
the binding of the amino acid-acyl-tRNA
complex
Resistance:
Methylation of the 23S ribosomal RNA
Inhibition of Nucleic Acid Synthesis
Quinolones
Synthetic chemotherapeutic agents
Inhibition of enzymes required for DNA
replication, recombination and repair:
DNA gyrases or topoisomerases:
Resistance (chromosomally mediated), 2
mechanisms:
Alteration of alfa subunit of DNA gyrase
Decreased drug uptake:
Changes in porin proteins on the bacterial surface
Biofilm
Some bacteria Interact of with each other to form a sticky
web of bacteria and polysaccharides called a biofilm, which
adheres to a surface within a host (example: dental plaque,
on catether, pacemakers, intravenous devices, etc)
Bacteria in infectious biofilm tend to be 100x more drug
resistant to free bacteria caused by:
-Microbes are protected by the thick impenetrable nature of
extracellular matrix
- bacteria slow their growth and less active
- microbes communicate in mass regulation of certain
resistance mechanisme, e.g. drug pump.
INHIBITION OF VIRAL REPLICATION BY
ANTIVIRAL AGENTS

Budiman Bela, T. Mirawati Sudiro

INTRODUCTION

The progress in development of antiviral chemotherapy

is much slower than that of antibacterial drugs
Viruses are obligate intracelluler parasites Viruses
use the host cells biosynthetic machinery and enzymes
---- it is more difficult to inhibit viral replication
without any toxicity to the host cells
 INTRODUCTION

Early antiviral drugs: targeted cells with extensive

DNA and RNA synthesis
Newer antiviral drugs are targeted toward :
- viral-encoded enzymes
- Structures of the virus that are important for
replication
The activity of antiviral drugs is generally limited to
specific families of viruses:

Example:
anti reverse transcriptase for therapy of HIV
infection
 INTRODUCTION

Resistance to antiviral drugs:

is becoming more problematic due to the

higher rate of long-term treatment of some
patients
Example:
resistance toward antiretroviral drugs in
people with AIDS

injudicious use of oseltamivir (Tamiflu)

may induce resistance of H5N1 influenza
virus towards the drug
Can we consider protein synthesis as antiviral target
for inhibition of viral replication ?
!
!
!
 !
Protein synthesis is not an ideal target for inhibition of viral
viral replication SINCE it is not possible to achieve selective
inhibition targeted to synthesis of viral protein
(viral replication involves the host cell ribosome and mechanism
for protein synthesis)
Mekanisme terjadinya resistensi
a.l.:

- Inaktivasi protein yang mengaktifkan antivirus dalam sel,

misal : kinase timidin
- Perubahan target obat
Misal : reverse transcriptase, protease, GP41
Analog nukleosida
Dapat melakukan inhibisi selektif karena:
1. Berikatan lebih baik dengan polimerasa DNA virus dibanding
polimerasa DNA sel
2. Obat digunakan lebih ekstensif pada sel terinfeksi, karena sintesis
DNAnya lebih cepat dibanding yang tidak terinfeksi
 Analog nukleosida

Acyclovir, Valacyclovir, Penciclovir dan Famciclovir:

- Memiliki rantai samping asiklik (bukan gula ribosa atau deoksiribosa)
- Bersifat selektif terhadap HSV (virus herpes simpleks) atau VZV (virus
varicella zoster), karena kedua virus herpes tersebut menyandi kinase
timidin
- Kinase timidin virus mengaktivasi obat melalui fosforilasi (inisiasi
fosforilasi)
enzim-enzim sel pejamu melanjutkan proses pembentukan menjadi
bentuk difosfat kemudian ke bentuk trifosfat
- Pada sel tidak terinfeksi obat ini terdapat dalam bentuk tidak aktif karena
tidak terjadi inisiasi fosforilasi
- Bentuk trifosfat berkompetisi dengan guanosin trifosfat:
- menghambat polimerasa
- terminasi perpanjangan rantai DNA virus
- Digunakan 100x lebih banyak oleh DNA polimerasa virus dibanding oleh
DNA polimerasa sel
 Analog nukleosida

Ganciclovir:
- Aktif terhadap CMV
- CMV tidak menyandi kinase timidin, tetapi dapat melakukan
fosforilasi GCV oleh suatu kinase protein yang disandi
oleh virus ini
- Digunakan 30x lebih banyak oleh DNA polimerasa virus
dibanding oleh DNA polimerasa sel
- Digunakan dalam terapi antitumor dengan terapi gen
 NON NUCLEOSIDE
REVERSE TRANCRIPTASE INHIBITORS
Target obat anti HIV
 KEMOTERAPI ANTI VIRUS

NUCLEOSIDE ANALOG
Acyclovir (Acycloguanosine)
Lamivudine (3TC)
Ribavirin
Vidarabine (Adenine arabinoside)
Zidovudine (Azidothymidine = AZT)

NUCLEOTIDE ANALOG
Cidofovir (HPMPC)

NON NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS

Nevirapine

PROTEASE INHIBITORS
Saquinavir
Indinavir
Ritonavir
 ANTI VIRUS TIPE LAIN
! AMANTADINE & RIMANTADINE
menghambat uncoating virus
virus Influenza A, profilaksis
FOSCARNET (Phosphonoformic acid = PFA)
menghambat DNA polimerase virus dan
reverse transcriptase
METHISAZONE
menghambat tahap akhir replikasi virus partikel
virus immature, non infeksius poxvirus
OSELTAMIFIR (Tamiflu)
menghambat neuraminidasa virus influenza hambat
perakitan/budding
FUZEON
menghambat perlekatan GP41 HIV pada reseptor seluler
INTERFERON