*ANTIBACTERIAL CHEMOTHERAPY

SARATH T M AMRITA UNIVERSITY

*Since peptidoglycan essential for bacterial survival

*Its biosynthesis is the major target of most antibiotics

*The largest and widely used bacterial cell wall synthesis inhibitors is the β lactam antibiotics

*It inhibits transpeptidases enzyme, which inhibits peptide cross linking.

* PEPTIDOGLYCAN BIOSYNTHESIS

*Peptidoglycan biosynthesis occurs in three main steps

1. Monomer synthesis2. Glycan polymerization3. Polymer cross linking

*The first stage is intracellular and involves murein monomer synthesis from amino acids and sugars

*The second and third stage involves the export of murein monomers to the surface of inner membrane, followed by their polymerization into linear peptidoglycan polymers and their cross linking into two dimensional lattices and three dimensional mats

Glucose

amidation+phosryln Glucosamine-1-

P

acetylCoA UDP

GlmU

UDP-NAG

PEP

MurA MurB

Phosphomycin

UDP-NAM

A DE K/DAP

MurEMurC MurD

UDP-NAM

DE

A

K

UDP-NAMA

DEK

DA

DA

A A

Alan

ine

race

mas

eD-

A-D-

A sy

nthe

tase

Mur

F

Cycloserine

STAGE-1: MUREIN MONOMER SYNTHESIS

cytoplasm

MurEMurD

4

5

*The “murein monomer "is a disaccharide comprising Nacetylglucosamine connected via a beta linkage to the C4 hydroxyl of N-acetyl muramic acid

*The first phase of peptidoglycan synthesis takes place in the cytoplasm and involves the conversion of UDP-N-acetyl glucosamine (UDP-NAG), to UDP-N-acetyl muramic acid.

*MurA , also known as enolpyruvate transferase, transfers enolpyruvate from phospho enolpyruvate (PEP) to UDPNAG to form UDP-NAG pyruvate enol ether.

*The flavoenzyme MurB (also known as UDP-NAG enolpyruvate reductase) reduces the double bond to produce UDP-NAM

6

*MurC , MurD , and MurE sequentially add the amino acids L-alanine, D-glutamate, and a diamino acid—either L-lysine or diaminopimelic acid (DAP) —to UDP-NAM.

* DAP differs from lysine in having a carboxyl group as well as an amine on the side chain.

* Most Gram- positive bacteria use L-lysine, whereas a minority of Gram-positive and all known Gram-negative bacteria use DAP.

*DAP is not found in humans, therefore it offers a unique target for antibiotics.

7

*Peptide formation continues with the addition of D- alanyl- D- alanine dipeptide (D- ala-D- ala) to the growing chain.

*The dipeptide is synthesized from two molecules of L- alanine in two reactions.

*The first reaction requires the transformation of L-alanine to D- alanine.

*This reaction is catalyzed by the enzyme alanine racemase.

*In the second reaction, an ATP- dependent enzyme called D-ala-D-ala synthetase joins the two alanines together.

*The resulting D- ala-D- ala dipeptide is added to the UDP- NAM tripeptide by MurF

Cell memb

Periplasmic space

cytoplasm

BPUDP-NAM

ADEK

DA

DA

UMPBP-NAM

ADEK

DA

DA

MraY

UDP-NAGUDP

BP-NAM-NAGA

DEK

DA

DA

BP-NAM-NAG BPA

DEK

DA

DA

G 5

Gly

tRNA

tRNA

MurG

NAM-NAGA

DEK

DA

DA

G 5

NAM-NAG – NAM-NAGA

DEK

DA

DA

G 5

ADEK

DA

DA

G 5

vancomycin

Transglycosylase

bacitracin

STAGE-2: MUREIN MONOMER TRANSLOCATION & POLYMERIZATION8

9

*The second phase of peptidoglycan synthesis takes place on the inner surface of cytoplasmic membrane and begins with the transfer of UDP- NAM peptide to a phospholipid carrier embedded in the membrane.*This carrier is called Bactoprenyl phosphate, because murein

monomers are assembled on it, delivered by it to the surface of plasma membrane, and then released in a process that regenerates the carrier for further cycles of reaction*The reaction by which UDP- NAM peptide is anchored to this carrier

lipid is mediated by an integral protein called MraY.* Once the NAM peptide is anchored to the carrier lipid, a membrane

associated enzyme called MurG catalyzes the transfer of NAG to the C4 hydroxyl of the NAM sugar to produce a lipid anchored NAM- NAG disaccharide commonly known as LIPID II *Polymerization is catalyzed by enzymes called Peptidoglycan

glcosyltransferases (PGTs)

NAM-NAGA

DEK

DA

DA

G G G G G

NAM-NAGA

DEK

DA

DA

G G G G G

NAM-NAGA

DEK

DA

DA

G G G G G

NAM-NAGA

DEK

DA

G G G G G

transpeptidase

ß-lactams

STAGE-3: POLYMER CROSS LINKING 10

11

*In the final stage of cell wall synthesis, murein chains are cross linked to one another by enzymes called transpeptidases (TPs).

*TPs were first identified as the molecular targets of Penicillin, so they are also called Penicillin- binding proteins.

*The PGT domain couples murein monomers to produce glycan strands.

*These oligosaccharide chains must then be cross linked to produce the murein found in bacterial cell wall.

12

* INHIBITORS OF MUREIN MONOMER SYNTHESIS

PHOSPHOMYCIN AND PHOSMIDOMYCIN

*Inhibit murein monomer synthesis by inhibiting the synthesis of UDP- NAM from UDP- NAG.*Phosphomycin is a PEP analogue that inhibits bacterial enolpyruvate

transferase by covalent modification of enzyme’s active site.*PEP is a key intermediate in mammalian glycolysis, but these agents

does not interfere with carbohydrate metabolism in human cells.*This selectivity of antibacterial action is likely caused by structural

differences between the mammalian and bacterial enzymes that act on PEP.*PHOSMIDOMYCIN another PEP analogue acts by the same

mechanism as Phosphomycin.

13

CYCLOSERINE

*Cycloserine, a structural analogue of D-ala, is a second line agent used to treat multi drug resistant M. tuberculosis infection.

*Cycloserine inhibits both alanine racemase that converts L- Ala to D- Ala and the D-Ala-D-Ala ligase that joins together two D-Ala molecules.

*Cycloserine is an irreversible inhibitor of these enzymes and binds to them more tightly than the natural substrate D-Ala.

*Resistance of Cycloserine includes over expression of alanine racemase and mutations in alanine uptake system.

14

BACITRACIN

*It is a peptide antibiotic that interferes with the dephosphorylation of Bactoprenyl diphosphate.

*It is notable among the anti cell wall agents for having a lipid as target.

*Bacitracin inhibits dephosphorylation by forming a complex with bactoprenyl diphosphate that involves bacitracin’s imidazole and thiazoline rings.

*Due to its significant kidney, neurological and bone marrow toxicity, bacitracin is not used systemically.

*It is most commonly used topically for superficial dermal or ophthalmologic infections.

15

* INHIBITORS OF MUREIN POLYMERIZATION

VANCOMYCIN

*Vancomycins are glcopeptides with bactericidal activity against Gram- positive rods and cocci.*These agents interrupt cell wall synthesis by binding tightly to the D-Ala-

D-Ala terminus of the murein monomer unit, inhibiting peptidoglycan polymerization and thereby blocking addition of murein units to the growing polymer chains.*Toxicity of vancomycin causes this agents to be used only when an

infection is found to be resistant to other agents.*Its toxicity includes skin flushing or rash *Vancomycin has also been associated with nephrotoxicity and ototoxicity,

particularly when other ototoxic or nephrotoxic medications such as gentamycin are co administered.

16

* INHIBITORS OF POLYMER CROSS LINKING

β- lactam Antibiotics (Penicillin, Cephalosporin, Monobactams, Crabapenems)

*Largest and most widely prescribed class of antibiotics that inhibits bacterial cell wall synthesis.*The different agents in this class vary in chemical structure and

consequently in spectrum of action.*But all β- lactams share the same antibiotic mechanism of action:

inhibition of murein polymer cross linking*Chemically, the key to the mechanism of action is the presence of a four

membered β lactam ring.*This ring makes every β lactam a structural analogue of terminal D-Ala-

D-Ala dipeptide.*B-lactams have affinity for several or specific transpeptidases-

17

* ANTI MYCOBACTERIAL AGENTS

Ethambutol, Pyrazinamide and Isoniazid

*Used widely in treating tuberculosis*Ethambutol, a bacteriostatic agent, decreases arabinogalactan synthesis

by inhibiting the arabinosyl transferase that adds arabinose units to the growing arabinogalactan chain.*Pyrazinamide and Isoniazid inhibit mycolic acid synthesis. *Pyrazinamide is a pro drug; it must be converted to its active form,

pyrazinoic acid, by the enzyme pyrazinamidase.*Pyrazinoic acid inhibits FAS1, the enzyme that synthesizes the fatty

acid precursors of mycolic acid.*Isoniazid targets FAS2 complex.

18

RESISTANCE----

ETHAMBUTOL

*Mutation/over expression of target enzymes

ISONIAZID

*Mutation in catalase- peroxidase which is needed for the activation of the drug

19

* AGENTS ACTING ON BACTERIAL CELL MEMBRANE

*primarily used for resistant gram negative infections.

*Polymyxin B binds to the cell membrane and alter its structure making it more permeable.

*They are cationic polypeptides which make electrostatic & hydrophobic interactions with anionic components like phospholipids & lipopolysaccharide

*Little to no effect on gram-positive since cell wall is too thick to permit access to membrane.

20

* INHIBITORS OF PROTEIN SYNTHESIS

21

* TRANSCRIPTION INHIBITORS

RIFAMPIN

* Semisynthetic derivative of naturally occurring antibiotic Rifamycin B

* Exerts bactericidal activity against mycobacteria by forming a highly stable complex with the DNA- dependent RNA polymerase, thereby inhibiting RNA synthesis.

* The drug targets β subunit of bacterial RNA polymerase

* Rifampin permits the initiation of transcription, but then blocks elongation once the length of nascent RNA reaches 2 to 3 nucleotides.

*Used in combination with Isoniazid

*Another example is Streptolydigins

22

* INHIBITORS OF TRANSLATION

AMINOGLYCOSIDES (Streptomycin)

*Used mainly in treating infections caused by Gram negative bacteria*Aminoglycosides bind to 16s rRNA of 30s subunit and elicit

concentration- dependent effects on protein synthesis.*At low concentrations, aminoglycosides induce ribosomes to misread

mRNA during elongation, leading to synthesis of proteins containing incorrect amino acids.*Aminoglycosides interfere with mRNA decoding function of 30s

subunit.

23

*RESISTANCE----

*Plasmid encoded transferase enzyme which inactivates the drug

*Alteration or elimination of porins or other proteins needed for drug transport

*Mutation of target site

24

* TETRACYCLINE

*Tetracyclines binds reversibly to the 16s rRNA of the 30s subunit and inhibits protein synthesis by blocking the binding of aminoacyl tRNA to the A site on the mRNA- ribosome complex.*This action prevents the addition of further amino acids to nascent

peptide.

RESISTANCE---

*Plasmid encoded drug efflux pumps

*Production of proteins that interfere with the binding of tetracycline to ribosome

*Enzymatic inactivation of drug

25

* CHLORAMPHENICOL

*Binds to 23SrRNA & inhibits peptide bond formation (interferes with the peptidyl transferase activity of 23srRNA)

*Because of serious toxicities used only if safer alternatives are not available

*Used occasionally for typhoid, bacterial meningitis , rickettsial disease etc.

RESISTANCE----

*Plasmid encoded acetyl transferases inactivate the drug

26

* MACROLIDES (erythromycin)

*Binds to 23S rRNA of 50s subunit

*Block the exit tunnel from which the nascent peptide emerge ( block translocation step)

*Important in treating pulmonary diseases (e.g.: Legionnaire’s disease)

RESISTANCE---

*Mutation in target site

*Increased drug efflux activity

*Methylase production by gram positives (modifies ribosomal target)

INHIBITORS OF NUCLEOTIDE SYNTHESIS

27

Steps in nucleotide synthesis

Synthesis of ribonucleotidesReduction of ribonucleotides to deoxyribonucleotidesConversion of dUMP to dTMP

28

PURINES

PYRAMIDINES

base

ribonucleoside

ribonucleotide

deoxyribonucleoside

deoxyribonucleotide

adenine

adenosine

AMP deoxyadenosine

dAMP

guanine

guanosine

GMP deoxyguanisine

dGMP

cytosine cytidine CMP deoxycytidine dCMP

uracil uridine UMP deoxyuridine

dUMP

thymine

deoxythymidine dTMP - -

reductionNUCLEOTIDES

29

Target the DNA synthesis ( S-phase of cell cycle)Applied in both infectious & neoplastic diseases

30

PURINE SYNTHESIS PYRAMIDINE SYNTHESIS

Aminoacids PRPP

IMP

Folate

Folate precursors

AMP GMP

amination

oxidation

dAMP dGMP

(reduction)

UMP CMPamination

dUMP

dTMP dCMP

reductionreduction

methylation

NUCLEOTIDE SYNTHESIS

Aspartate

Orotate

Carbomyl phosphate

PRPP

31

In the de novo purine synthesis pathway, two separate steps utilize 10-formyl-THF.

32

Folate-requiring reactions, collectively referred to as one-carbon metabolism, include those involved in phases of amino acid metabolism, purine and pyrimidine synthesis, and the formation of the primary methylating agent, S-adenosylmethionine (SAM )

methyltransferase reactions

10-formyl-THF

33

INHIBITORS OF FOLATE METABOLISM

BACTERIA

BACTERIA & HUMANS

Pteridine+ PABA

Dihydropteroic acid

Pteridine+ PABA

Dihydropteroic acid

dihydrofolate

Dihydropteroate synthase

sulfonamides

glutamate

tetrahydrofolate

DHFR

dTMP

5 FUFlucytosine

TrimethoprimMethotrexate

Pyrimethamine

TS

34

SULFA DRUGS

• Sulfonamides & sulfones

• PABA analogues

• Inhibits dihydropteroate synthase &prevents folic acid synthesis in bacteria

• Bacteriostatic

• Highly selective drugs---mammalian cells do not express dihydropteroate synthase

35

RESISTANCE---

• Overproduction of endogenous PABA (70 fold more than normal)

• Mutation in target enzyme

• Decreased permeability of bacterial membranes to sulfonamides

• Sulfones –used in leprosy treatment (Dapsone)

36

METHOTREXATE ( MTX)

• DHFR inhibitor

• Folate analogue– close structural resemblance with natural substrate

• Used in cancer chemotherapy

• Rapidly growing cancer cells have increased need of purines & thymidylate

• Malignant cells more susceptible to apoptosis inducing effects of MTX

37

methotrexate pyrimethamine

trimethoprim

38

TRIMETHOPRIM

Folate analogueInhibits bacterial DHFR

PYRIMETHAMINE

Folate analogueInhibits parasitic DHFROnly effective drug against toxoplasmosisMost common drug for malaria also

39

5-FLUROURACIL (5-FU) possibility1

• 5-FU is converted into FdUMP (5-fluro-2’-deoxyuridylate)

• which in turn inhibit TS

• Cells undergo thymine less deathPossibility2

• 5-FU can be metabolized to FUTP( flox uridine triphosphate)

• FUTP can be incorporated into mRNA in place of UTP

• Interferes with mRNA processing

40

• Toxic effect of 5-FU can be either single/combinational

• TS inhibition is considered as the dominant mechanism

• Mostly used as anti neoplastic drug

FLUCYTOSINE

• Highly selective anti fungal drug

41

pm

Flucytosine

Cytosine deaminase

5-FU

5-FdUMP

dUMP dTMPThymidylate synthase

MECHANISM OF ACTION OF FLUCYTOSINE42

IMP Adenylo succinate

AMP

ATP

dAMP

dATP

DNA

Ribonucleotide reductase

Hydroxyurea

6-Mercaptopurine

XMPIMPDH

GMP

dGMP

dGTP

DNA

Ribonucleotide reductase

INHIBITORS OF PURINE NUCLEOTIDE SYNTHESIS

ASS

43

6- MERCAPTOPURINE (6-MP)

• Inosine analogue (contains S at C-6)

• Inhibits inosine monophosphate dehydrogenase & adenylo succinate synthase

• Major application in acute lymphoblastic leukemia

HYDROXYUREA

• Inhibits ribonucleotide reductase by scavenging a tyrosyl radical from the active site of the enzyme

44

UMP UTP CTP

dCTP

Ribonucleotide reductase

DNA

HydroxyureadUMP

Ribonucleotide reductase

dTMP

dTTP

DNA INHIBITORS OF PYRAMIDINE SYNTHESIS45