b5 lecture slides (cmf) folate antagonists (2)

B5 Therapeutic Classes of Drugs: FolateAntagonists (Mode of Action and Infections)

B31BFI Bacterial and Fungal Infections

Dr Felicity Rose (UK)Dr Chee-Mun Fang

Objectives

26/03/2015 B31BFI Bacterial and Fungal Infections 2

At the end of this part of the lecture you should .

Understand and be able to describe how bacteria utilise folateand how this differs for mammalian cells.

Understand how antibiotics can selectively interfere with thisprocess in bacteria.

Understand the microbiology of infections that this class ofantibiotics are used to treat using urinary tract infection as anexample.

Describe the mechanisms of resistance to folate antagonists.

Classes of Antibiotics


KEYNo brackets anti-bacterial agents() anti-mycobacterial agents[] anti-fungal agents{} anti-protozoal agents

Bacterial Cell

From Hugo and Russell Pharmaceutical Microbiology 8th Edition Chapter 12, Figure 12.1: p 201

Folate


Folate (aka folic acid & vitamin B9) isan important co-factor in all livingcells.

Folate is not biologically active itself,but it provides important moleculesupon its metabolism.

In mammals , it is important duringphases of rapid cell division, such asin infancy and pregnancy.

The reduced form, tetrahydrofolate(THF) functions as a carrier of singlecarbon fragments used synthesiseadenine, guanine, thymine &methionine for DNA synthesis.

Folate synthesis in bacteria:1. Synthesis of dihydropteroicacid from one molecule ofpteridine & one molecule of p-aminobenzoic acid (PABA).2. Glutamic acid then added toform dihydrofolic acid.3. Dihydrofolic acid reduced totetrahydrofolate bydihydrofolate reductase.

Folate Utilization


From Hugo and Russell Pharmaceutical Microbiology 8th Edition Chapter 12, Figure 12.7: p 213

Folate utilization in bacteriaand mammalian cells isfundamentally different.

Bacteria (& protozoa) areunable to take up exogenousfolate and so they mustsynthesise it themselves.

Mammalian cells do notsynthesise dihydrofolate, andso it is taken up through thediet and converted totetrahydrofolate in the liver.

Folate Metabolism


DHF - dihydrofolateDHFR - dihydrofolate reductaseTHF - tetrahydrofolate reductaseSHT serine hydroxymethyl transferasedUMP - deoxyuridine monophosphatedTMP - deoxythymidine monophosphate

P

dUMP

DNA synthesis

Thymidylate-Synthetase

H

H

Addition 2 protons

CH2

HAddition C from

conversion of serine toglycine

Loss ofmethylgroup

todUMP

P

H3C

Folic acidFolic acid DHFDHF THFTHFDHFRDHFR

5,10-methyleneTHF

5,10-methyleneTHF

NADPH+H+ NADP+Ser Gly

SHT

NADPH+H+ NADP+

Conversion of tetrahydrofolate back to dihydrofolicacid the reaction repeats

dTMP

Antibiotics:sulfonamides


Sulphonamides (aka sulfo drugs) are structuralanalogues of pABA.

Inhibit incorporation of pABA intodihydropteroic acid.

Their importance in clinical use has decreaseddue to bacterial resistance.

New antibiotics are also generally more activeand less toxic further supporting theirreplacement.

Example drugs in this class aresulfamethoxazole and sulfadiazine.

The first step in the synthesis of folate is theformation of dihydropteroic acid from onemolecule of pteridine & one molecule of p-aminobenzoic acid (PABA).

Sulphonamides inhibit this reaction by actingas pABA analogues.

Sulfamethoxazole can be administrated with trimethoprim as they exhibit synergisticactivity (co-trimoxazole).

Co-trimoxazole only prescribed for treating respiratory and urinary tract infectionswhere there is good evidence for its use.

Antibiotics:trimethoprim


Trimethoprim is a selective inhibitor of bacterialDHFR.

Acts as a structural analogue of dihydropteroicacid portion of the dihydrofolate substrate.

Trimethoprim fits into active site of DHFRnormally occupied by DHF forming stronghydrogen bonds with amino acid residues &water molecules lining the site.

Trimethoprim can be used alone for urinary andrespiratory tract infections amongst others.

Trimethoprim is a selective inhibitor ofbacterial DHFR by acting as a structuralanalogue of dihydropteroic acid portion (bluebox below) of the dihydrofolate substrate.

Trimethoprim

Gram positive Gram negativeMRSA Staphylococci Streptococci Ef Anaerobes Coliforms Resp Pyo ESBL Atypicals

Trimethoprim

Urinary Tract Infection


Urinary tract infection (UTI) is an infection thataffects the urinary tract.

An infection of the lower urinary tract (bladder) isknown as a cystitis. Symptoms include:

painful urination and either frequent urinationor the urge to urinate (or both).

An infection of the upper urinary tract (kidneys) isknown as pyelonephritis. Symptoms include:

fever and lower back pain in addition to thesymptoms of a lower UTI.

The main causative micro-organism of both upperand lower UTI is Escherichia coli (can be caused byother bacteria, viruses or fungi but this is rare).

UTI occur more commonly in women than men andrecurrences are common.

The urinary organ system (the kidneys, theureters, the bladder and the urethra)produces, stores, and eliminates urine fromthe body. The urinary tract in males andfemales are similar except that the urethrain women is shorter making women moresusceptible to infection.

Image taken from http://universalium.academic.ru

Escherichia coli


Picture taken from Bauman Microbiology withDiseases by Taxonomy; 3rd Edition, Chapter 20:

p 580.

Escherichia coli is a gram-negative, coliform, rod-shaped enteric bacteria.

They form part of the natural flora of mammaliangastrointestinal tract.

E. coli often causes UTI (from entry from theintestinal tract to the urinary tract) andgastroenteritis (from eating infected foodcontaminated with pathogenic strains).

Pathogenicity mediated by three antigens: Polysaccharide shell. O-polysaccharide. Lipid A (causes toxic shock).

Escherichia coli cont


Virulence factors include:

Outer capsule - protection from phagocytosis.

Fimbriae and adhesins attachment toepithelial cells.

Exotoxins

Iron-binding proteins (siderophores) acquireiron needed for growth.

Haemolysins release iron from blood cells.

Type III secretion system builds andinsertion needle to inject proteins into hostcells.

T3SS needle complex

Antibiotic therapy for UTI


Gram positive Gram negativePyelonephritis /

Upper UTIMRSA Staphylococci Streptococci Ef Anaerobes Coliforms Resp Pyo ESBL Atypicals

Trimethoprim

Nitrofurantoin

Gentamicin

Co-amoxiclav

Cefuroxime

Ertapenem

Trimethoprim usual choice for simple UTI (nitrofurantoin 2nd line) Urosepsis/pyelonephritis IV co-amoxiclav /cefuroxime or gentamicin

Resistance to trimethoprim


Resistance to trimethoprim occursas a result of:

Over-production of DHFR bybacteria.

Acquisition of the dfr geneencoding a resistant form ofthe enzyme most commonlyDHFR type I, II or V (amongstenterobacteria).

This image shows antibiotic sensitivity testing of E. coli isolated from a urine sample from a patient with a UTI. Itcan be seen that the bacterium is resistant to trimethoprim (W; blue arrow), the first line antibiotic for UTI, butsensitive to nitrofurantoin (F; red arrow), the second line antibiotic for UTI).

Watch the video in Moodle that shows how the causative microorganism of a UTI is identified in a clinicalmicrobiology laboratory and antibiotic sensitivity determined.

Image used with permission from the Clinical Microbiology Laboratories, University Hospitals Trust, Nottingham.

Key Points


Folate provides small carbon fragments (via an intermediate tetrahydrofolate)essential for DNA synthesis.

Bacteria synthesise folate; mammals take up folate in the diet.

Sulphonamides are structural analogues of pABA; inhibit incorporation of pABAinto dihydropteroic acid.

Reduced clinical use of sulphonamides due to bacterial resistance.

Trimethoprim fits into active site of DHFR normally occupied by DHF.

Trimethoprim predominantly used to treat UTIs (mainly caused by E. coli)

Resistance to trimethoprim arises due to increased production of DHFR or aresistant form of the enzyme.

References and Further Reading


Bauman Third Edition

Chapter 20

Hugo and Russell Eighth Edition

Chapters 7, 11 -13

BNF latest edition

5. Infections

Thanks to Tim Hills Lead Pharmacist Antimicrobials and Infection Control, Nottingham UniversityHospitals for the antibiotic spectrum charts.

b5 lecture slides (cmf) folate antagonists (2)

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