Modul Biomol UIN, Resistance to Antibiotics 2010

8/8/2019 Modul Biomol UIN, Resistance to Antibiotics 2010

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Mechanism of action ofAntibiotics, Antivirals

Antifungals and

Antimicrobial Resistance

Budiman Bela

T. Mirawati Sudiro

Fera Ibrahim

Dept. of Microbiology,

FKUI


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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

Adelbergs Medical Microbiology, ed 24, , Appleton and

Lange, California. 2004


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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.


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exposure selection expansion

sensitive population resistant clones outbreak, epidemic, pandemic


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mrsa: a pandemic


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http://www3.niaid.nih.gov/topics/antimicrobialResistance/Understanding/drugResistanceDefinition.htmAccessed March 23, 2009


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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 sulfadrug

Compounds produced by microorganisms thatinhibit the growth of other microorganism

(Antibiotic): Alexander fleming: the mold Penicillium prevented themultiplication of staphylococci

1940s: Streptomycin

1950s: Tetracyclines


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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


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Mode of action and target

molecules of antibacterial agents

Inhibition of cell wall synthesis

Inhibition of nucleic acid synthesis Inhibition of protein synthesis

Antimetabolites


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50

30

50

30

50

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Antimetabolites

-Sulfonamides-Dapsone

-Trimethoprim

-Para-aminosalicylic acidProtein synthesis

(30S ribosome)

-Aminoglycosides

-Tetracyclines

-Oxazolidinone

Protein synthesis

(50S ribosome)

-Chloramphenicol-Macrolides

-Clindamycin

-Streptogramins

RNA Synthesis

-Rifampin

-Rifabutin

DNA Replication

-Quinolones

-Metronidazole

Cell Wall Synthesis

-Beta-lactams

-Vancomycin

-Isoniazid

-Ethambutol

-Cycloserine

-Ethionamide

-Bacitracin

-PolymyxinDNA

Ribosomes

!

!

!

!

!

!


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Basic Mechanisms of Antibiotic Action

Antibiotic Action

Disruption of Cell Wall

Penicillin

Cephalosporin

Cephamycin

Carbapenem

Monobactam

F-lactamase inhibitor/ F-lactam

Vancomycin Isoniazid

Ethionamide

Ethambutol

Cycloserine

Polymyxin

Bacitracin

Binds PBPs and enzymes responsible for peptidoglycan synthesis





Binds F-lactamases and prevents enzymatic inactivation ofF-lactam

Inhibits cross-linkage of peptidoglycan layersInhibits mycolic acid synthesis

Inhibits mycolic acid synthesis

Inhibits arabinogalactan synthesis

Inhibits cross-linkage of peptidoglycan layers

Inhibits bacterial membranes

Inhibits bacterial cytoplasmic membrane and movement of peptidoglycan precursors

Inhibition of Protein Synthesis

Aminoglycoside

Tetracycline

Oxazolidinone

Macrolide

Clindamycin

Streptogramins

Produces premature release of aberrant peptide chains from 30S ribosome

Prevents polypeptide elongation at 30S ribosome

Prevents initiation of protein synthesis at 30S ribosome




!

!

!

!


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Basic Mechanisms of Antibiotic Action

Antibiotic Action

Inhibition of Nucleic Acid Synthesis

Quinolone

Rifampin Rifabutin

Metronidazole

Binds E subunit ofDNA gyrase

Prevents transcription by binding DNA-dependent RNA polymerasePrevents transcription by binding DNA-dependent RNA polymerase

Disrupts bacteria DNA (is cytotoxic compound)

Antimetabolite

Sulfonamides

Dapsone

Trimethoprim

Inhibit dihydropteroate synthase and disrupt folic acid synthesis

Inhibits dihydropteroate synthase

Inhibits dihydrofolate reductase and disrupts folic acid synthesis

!!

!

!


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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 wallpermeability, etc.


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Inhibition of Cell Wall Synthesis

Interference with bacterial cell wall

synthesis

The most common mechanism of antibiotic F-lactam antibiotics:

Constitute the majority of cell wall-active

antibiotics

Penicilins, Chepalosporins, Cephamycins,

Carbapenems, Monobactams, F-lactamase

inhibitors


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Inhibition of Cell Wall Synthesis

Other antibiotics that interfere with

bacterial cell wall synthesis

Vancomycin: Bacitracin

Antimycobacterial agents:

Isoniazid

Ethambutol

Cycloserine

Ethionamide


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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 meshcoating for bacteria

!

!


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http://en.wikipedia.org/wiki/File:Gram-positive_cellwall-schematic.png


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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 F-lactamantibiotics:

Transpeptidases

Carboxypeptidases

Endopeptidases

F-lactam antibiotics generally act as bactericidal

agents: Exposure of growing bacteria to F-lactam antibiotics bindingwith PBPs in the growing bacterial cell wall inhibition ofsynthesis but not turnover (degradation) of peptidoglycan bacterial cell death


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Mechanisms of Bacterial Resistance

towards Beta Lactam Antibiotics

1. Prevention of interaction between

antibiotic and target PBP:

2.D

ecreased binding of antibiotic to PBP3. Hydrolysis of antibiotic by F-lactamases


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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 F-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


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2. Decreased binding of antibiotic to PBP:

Formation of modified PBP that fails to bind

to F-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 ofEscherichia coliPBP into

Staphylococcus aureus confers resistance to oxacillin

!


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3. Hydrolysis of antibiotic by F-lactamases:

Inactivation ofF-lactam antibiotics

There are 200 different F-lactamases: Specific for penicilins: penicilinases

Specific for cephalosporins: cephalosporinases

Broad range of activity: capable of inactivating most F-lactam antibiotics

Extended-spectrumF-lactamases (ESBLs): Commonly encoded on plasmids can be

transferred from organism to organism : Causingmuch difficulties and severely limit the empirical useofF-lactam antibiotics in some hospitals


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Mechanisms of Methicillin

Resistance The staphylococcal beta-lactamase protein, which

cleaves the beta-lactam ring structure, confersresistance to penicillin but not to semi-syntheticpenicillins such as:

methicillin, oxacillin, or cloxacillin. Acquisition of the mecA gene, which codes for thepenicillin binding protein PBP2a, complete resistance toall beta-lactam antibiotics including the semisyntheticpenicillins.

PBP2a has a very low affinity for beta-lactam antibiotics,and is thought to aid cell wall assembly when the normalPBPs are inactivated.

The mecA gene is found on a large mobile geneticelement called the staphylococcal chromosomalcassette mec (SCCmec).


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Glycopeptides

A complex glycopeptide that disrupts cell wallpeptidoglycan synthesis in growing gram-positivebacteria

Interacts with D-alanine-D-alanine termini of the

pentapeptide side chainsinterferes sterically with theformation of the bridges between the peptidoglycan

chains

Used for the management of infections caused byoxacillin-resistant staphylococci and other gram-positive bacteria resistant to F-lactam antibiotics

Inactive against gram-negative bacteria: Molecule is too large to pass through the outer membrane and

reach the peptidoglycan target site


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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


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Glycopeptides

Acquired resistance to Vancomycin:

Plasmid mediated

Enterococcus faecium and Enterococcus faecalis

A potential threat to the usefulness of vancomycinfor the treatment of enterococcal infections

There is a concern that if these genes are

transferred to staphylocci (proven by lab

experiments) highly resistant and virulentorganism will emerge: Vancomycin resistant

Methicillin resistant Staphylococcus aureus


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Polypeptides

Bacitracin:

Mixture of polypeptides

Inhibits cell wall synthesis by interfering with

dephosphorylation and the recycling of the lipidcarrier responsible for moving the peptidoglycan

precursors through the cytoplasmic membrane to

the cell wall

Also damage the bacterial cytoplasmic membraneand inhibit RNA transcription

Resistance:

Failure of the antibiotic to penetrate into the bacterial cell


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Polypeptides

Polymixins:

Cyclic polypeptides

Inserted into bacterial membranes by interactingwith lipopolysaccharides and the phospholipids inthe outer membrane increased cell permeability cell death

Most active against gram-negative bacilli sincegram-positive bacteria do not have

an outer membrane

Example:

Polymyxin B and E (colistin)


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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


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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 ofD-alanine-Dalanine synthetase and

alanine racemase that catalyze cell wall synthesis


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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


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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 thepremature release of the ribosome from mRNA


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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


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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 outermembrane porin protein OmpfF low level resistance tothe 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 activeefflux 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 thatprotect the 30S ribosome antibiotic can still bind to theribosome but protein synthesis is not disrupted

Enzymatic modification of the antibiotic


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Oxazolidones

Representative: Linezolid

Narrow-spectrum class of an antibiotics that block

initiation of protein synthesis by interfering with theformation 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


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Chloramphenicol Also disrupts protein synthesis in the human bone

marrow cells and can produce blood dyscrasias

Binding reversibly to the peptidyl transferasecomponent of the 50S ribosomal subunit blockingpeptide elongation

Resistance: Plasmid-encoded chloramphenicol acetyltransferase

catalyze the acetylation of the 3-hydroxy group ofchloramphenicol incapable of binding to the 50S subunit

Chromosomal mutations (less common) alter the outermembrane porin proteins gram-negative bacilli becomeless permeable


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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 bythe antibiotic

Destruction of the lactone ring by erythromycin esterase

Active efflux of the antibiotic from the bacterial cell


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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


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Inhibition of Nucleic Acid Synthesis

Quinolones

Synthetic chemotherapeutic agents

Inhibition of enzymes required forDNA

replication, recombination and repair: DNA gyrases or topoisomerases:

Resistance (chromosomally mediated), 2mechanisms:

Alteration of alfa subunit ofDNA gyrase Decreased drug uptake:

Changes in porin proteins on the bacterial surface


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Some bacteria Interact of with each other to form a stickyweb of bacteria and polysaccharides called a biofilm, whichadheres to a surface within a host (example: dental plaque,on catether, pacemakers, intravenous devices, etc)

Bacteria in infectious biofilm tend to be 100x more drugresistant 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 certainresistance mechanisme, e.g. drug pump.

Biofilm


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Budiman Bela, T. Mirawati Sudiro, Fera Ibrahim

INHIBITION OF VIRAL REPLICATION

BY ANTIVIRAL AGENTS


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The progress in development of antiviral chemotherapyThe progress in development of antiviral chemotherapy

is much slower than that of antibacterial drugsis much slower than that of antibacterial drugs

Viruses are obligate intracelluler parasitesViruses are obligate intracelluler parasites VirusesViruses

use the host cells biosynthetic machinery and enzymesuse the host cells biosynthetic machinery and enzymes

-------- it is more difficult to inhibit viral replicationit is more difficult to inhibit viral replication

without any toxicity to the host cellswithout any toxicity to the host cells

INTRODUCTIONINTRODUCTION


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Early antiviral drugs: targeted cells with extensiveEarly antiviral drugs: targeted cells with extensiveDNA and RNA synthesisDNA and RNA synthesis

Newer antiviral drugs are targeted toward :Newer antiviral drugs are targeted toward :

-- viralviral--encoded enzymesencoded enzymes-- Structures of the virus that are important forStructures of the virus that are important for

replicationreplication

The activity of antiviral drugs is generally limited toThe activity of antiviral drugs is generally limited to

specific families of viruses:specific families of viruses:

Example:Example:

anti reverse transcriptase for therapy of HIVanti reverse transcriptase for therapy of HIV

infectioninfection


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Resistance to antiviral drugs:Resistance to antiviral drugs:

is becoming more problematic due to theis becoming more problematic due to the

higher rate of longhigher rate of long--term treatment of someterm treatment of some

patientspatientsExample:Example:

resistance toward antiretroviral drugs inresistance toward antiretroviral drugs in

people with AIDSpeople with AIDS

injudicious use of oseltamivir (Tamiflu)injudicious use of oseltamivir (Tamiflu)may induce resistance of H5N1 influenzamay induce resistance of H5N1 influenza

virus towards the drugvirus towards the drug


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Viral protein synthesis as antiviral target ???Viral protein synthesis as antiviral target ???


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SintesisSintesis protein virusprotein virus sebagaisebagai target antiviral ?target antiviral ?

SasaranSasaran antivirus yangantivirus yang kurangkurang baikbaikSEBABSEBABtidaktidak memungkinkanmemungkinkan inhibisiinhibisi selektifselektif terhadapterhadap

sintesissintesis protein virus (protein virus (replikasireplikasi virusvirus memanfaatkanmemanfaatkan

ribosomribosom dandan mekanismemekanisme sintesissintesis selsel pejamupejamu))

!


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e.g.

- Inactivation of protein that activates antiviral

drugs in the cells. E.g. thymidine kinase

- Change in drug target

e.g. : reverse transcriptase, protease, GP41

Mechanism of antiviral resistance


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Nucleoside analog can do selective inhibition, because:Nucleoside analog can do selective inhibition, because:1. It binds vial DNA polymerase better than cellular1. It binds vial DNA polymerase better than cellular

DNA polymeraseDNA polymerase

2. DNA synthesis in infected cells is faster than non2. DNA synthesis in infected cells is faster than non--

infected cellsinfected cells

NucleosideAnalogNucleosideAnalog


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Analog nukleosidaAnalog nukleosida

Acyclovir, Valacyclovir, Penciclovir dan Famciclovir:Acyclovir, Valacyclovir, Penciclovir dan Famciclovir:-- Memiliki rantai samping asiklik (bukan gula ribosa atau deoksiribosa)Memiliki rantai samping asiklik (bukan gula ribosa atau deoksiribosa)

-- Bersifat selektif terhadapBersifat selektif terhadap HSVHSV (virus herpes simpleks) atau(virus herpes simpleks) atau VZVVZV (virus(virusvaricella zoster),varicella zoster), karenakarena kedua virus herpes tersebut menyandikedua virus herpes tersebut menyandi kinasekinase

timidintimidin-- Kinase timidin virus mengaktivasi obat melalui fosforilasi (inisiasiKinase timidin virus mengaktivasi obat melalui fosforilasi (inisiasi

fosforilasi)fosforilasi)

enzimenzim--enzimenzim sel pejamu melanjutkan proses pembentukan menjadisel pejamu melanjutkan proses pembentukan menjadibentuk difosfat kemudian ke bentuk trifosfatbentuk difosfat kemudian ke bentuk trifosfat

-- Pada sel tidak terinfeksi obat ini terdapat dalam bentuk tidak aktif karenaPada sel tidak terinfeksi obat ini terdapat dalam bentuk tidak aktif karena

tidak terjadi inisiasi fosforilasitidak terjadi inisiasi fosforilasi

-- Bentuk trifosfat berkompetisi dengan guanosin trifosfat:Bentuk trifosfat berkompetisi dengan guanosin trifosfat:

-- menghambat polimerasamenghambat polimerasa-- terminasi perpanjangan rantai DNA virusterminasi perpanjangan rantai DNA virus

-- Digunakan 100x lebih banyak oleh DNA polimerasa virus dibanding olehDigunakan 100x lebih banyak oleh DNA polimerasa virus dibanding oleh

DNA polimerasa selDNA polimerasa sel


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Analog nukleosidaAnalog nukleosida

Ganciclovir:Ganciclovir:

-- Aktif terhadapAktif terhadap CMVCMV

-- CMV tidak menyandi kinase timidin, tetapi dapat melakukanCMV tidak menyandi kinase timidin, tetapi dapat melakukan

fosforilasifosforilasi GCV olehGCV oleh suatu kinase protein yang disandisuatu kinase protein yang disandi

oleh virus inioleh virus ini-- Digunakan 30x lebih banyak oleh DNA polimerasa virusDigunakan 30x lebih banyak oleh DNA polimerasa virus

dibanding oleh DNA polimerasa seldibanding oleh DNA polimerasa sel

-- Digunakan dalamDigunakan dalam terapi antitumor dengan terapi genterapi antitumor dengan terapi gen


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NONNUCLEOSIDE

REVERSETRANCRIPTASEINHIBITORS


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Targets of anti-HIVdrugs


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ANTI VIRAL THERAPYANTI VIRAL THERAPY

NUCLEOSIDEANALOGNUCLEOSIDEANALOGAcyclovir (Acycloguanosine)Acyclovir (Acycloguanosine)

Lamivudine (3TC)Lamivudine (3TC)

RibavirinRibavirin

Vidarabine (Adenine arabinoside)Vidarabine (Adenine arabinoside)

Zidovudine (Azidothymidine =AZT)Zidovudine (Azidothymidine =AZT)

NUCLEOTIDEANALOGNUCLEOTIDEANALOG

Cidofovir (HPMPC)Cidofovir (HPMPC)

NONNUCLEOSIDEREVERSETRANSCRIPTASEINHIBITORSNONNUCLEOSIDEREVERSETRANSCRIPTASEINHIBITORS

NevirapineNevirapine

PROTEASEINHIBITORSPROTEASEINHIBITORS

SaquinavirSaquinavir

IndinavirIndinavir

RitonavirRitonavir


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AMANTADINE & RIMANTADINEAMANTADINE & RIMANTADINE

inhibit viral uncoatinginhibit viral uncoating InfluenzaA virus, profilaksisInfluenzaA virus, profilaksis

FOSCARNET (Phosphonoformic acid = PFA)FOSCARNET (Phosphonoformic acid = PFA)

inhibits viral DNA polymerase and reverse transcriptaseinhibits viral DNA polymerase and reverse transcriptase

METHISA

ZONEMETHISA

ZONE

inhibits final step in viral replicationinhibits final step in viral replication immature viralimmature viral

particleparticle, non infectious (poxvirus), non infectious (poxvirus)

OSELTAMIFIR (Tamiflu)OSELTAMIFIR (Tamiflu)

inhibits neuraminidase of influenza virusinhibits neuraminidase of influenza virus inhibitsinhibits

buddingbuddingFUZEONFUZEON

inhibits attachment ofGP41 HIV to cellular receptorinhibits attachment ofGP41 HIV to cellular receptor

OTHER TYPES OF ANTIVIRAL DRUGSOTHER TYPES OF ANTIVIRAL DRUGS


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INTERFERONINTERFERON


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Modul Biomol UIN, Resistance to Antibiotics 2010

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