Antimicrobial Drugs: Discovery, design & Resistance

what were traditional remedies for infection

• application of mouldy bread on wounds

• bloodletting (withdrawing blood to treat or prevent illness)

• plants for malarial fevers: Cinchona sp and Artemisia annua

timeline of modern antimicrobial chemotherapy

what is the germ theory (discovery of infective diseases)

Louis Pasteur showed that germs causes the fermentation/decay of organic substances and proved that germs caused diseases

what is the Koch’s postuates 

what was the first antimicrobial agent

• Salvarsan was introduced against T. pallidum (syphillis) by Paul Ehrlich

• Ehrlich introduced the concept of selective therapy using compounds that selectively target the disease, whilst having no effect on healthy tissues

how were sulfonamides developed

• by Gerhardt Dogmagk

• Pontosil is the first drug effective against systemic bacterial infection 

• Pontosil is a prodrug of sulfonamide, which must be activated by gut microflora 

what was the first antibiotic discovered

penicillin

• Alexander Fleming noticed that bacterial growth was inhibited around a mould contamination → identified penicillin G 

• - 6APA became the building block for semisynthetic penicillins 

what is an antibiotic

a chemical produced by microorganisms (natural products) able to affect the growth of other microorganisms

what is an antibacterial

a chemical that kill bacterial cells (bactericidal) or inhibit their growth (bacteriostatic)

what is an antimicrobial

General term for any chemotherapeutic agent active against microbial infection (bacteria, viruses, fungi…..)

what is antifective

General term for any chemotherapeutic agent active against an infective disease

how were antibiotics developed from systematic screening

• Selman Waksman screened soil bacteria and fungi as a source of antibiotics.

• This led to the discovery of streptomycin and the class of aminoglycosides

• Streptomycin was the first agent active against tuberculosis

what is the golden age of antibiotic discovery

• in the mid 40s to mid 60s, antibiotics were discovered by systematic screening → 7000 antibiotics discovered

• the improvement era (till 80s) → New antibiotics mainly introduced via semi-synthesis (improvement of spectrum, ADME, etc)

how is antimicrobial resistance an issue

• Resistance to antimicrobial agents became a wide-scale issue.

  • Use of antimicrobials HAD to be limited

  • Minor improvements of current structures inadequate to circumvent the problem

• It becomes progressively harder to identify new structures against a background of known but not useful species

  • Pharmaceutical companies stopped investing because of low revenues

how do we discover antibiotics nowadays

• Genomics (and other –omics) to identify new targets

• Target-based high-throughput screening (target validation or deconvolution)

• Structure-based discovery

how are natural products and ethnopharmacology are a prolific source of (potential) active principles

bacterial cell

what is the gram staining method 

1. application of purple dye

2. application of mordant to help dye stay

3. wash with alcohol (some bacteria retain purple colour → gram positive)

4. application of counterstain (bacteria that was washed away stains pink → gram negative)

why do bacteria react differently to the gram-staining dyes

due to the chemistry of the bacteria cell wall

• gram positive cell wall has a plasma membrane and thick peptidoglycan layer outside of its membrane

• gram negative cell wall has a plasma membrane, a thin peptidoglycan layer outside of its membrane and an outer hydrophobic membrane

how are antibacterial agents tested 

1. Disk Diffusion Test (Kirby–Bauer Test)

• Each disc is soaked with an antibiotic.

• Drug diffuses into the agar → bacteria fails to grow near discs containing effective antibiotics.

2. Broth Microdilution Test (MIC Determination)

• Bacterial growth is monitored across the gradient of drug concentrations.

• Used to determine the Minimum Inhibitory Concentration (MIC) → the lowest concentration that prevents visible growth.

• darker colour → higher number of bacteria

what are the three main approaches (targets) for antibacterial agents 

• interfere with cell wall synthesis or maintenance 

• interferes with nucleic acid synthesis 

• interferes with protein synthesis 

what classes of antibiotics inhibit the synthesis of the cell wall 

• β-lactams

  • penicilllins

  • cephalosporins

  • carbapenems

  • monobactams

• vancomycin

• daptomycin

• polypeptides

  • bacitracin

  • cholestin

how do antibiotics inhibit nucleic acid synthesis 

• inhibit DNA gyrase +/- e.g. topoisomerase IV and quinolones

• Inhibit folate synthesis e.g. sulfonamides and trimethoprim

• create free radicals e.g. nitroimidazoles and nitrofurans

what antibiotics inhibit protein synthesis

on 50 sub unit

• macrolides

• clindamycin 

• linezolid

• streptogramins

• chloramphenicol 

on 30s sub unit 

• aminoglycosides

• tetracylines

• tigecylin 

what are the main agents that impair cell wall synthesis

• β-lactams

• Glycopeptides

• Peptides

• Cycloserin

what are β-lactams (impair cell wall synthesis)

• first antibiotic discovered

• Wide class (antibiotic and semisynthetic products)

• Generally bactericidal (kills)

• includes penicillins, carbapenems, cephalosporins, monobactams 

• structure  → four member ring 

how is the peptidoglycan layer formed in bacterium (mechanism of action)

• bacterium synthesises a dimer

• red and purple squares represent sugars (N-acetylglucosamine and N-acetylmuramic acid), which form the base of the peptidoglycan layer

• NAM has a short peptide chain, branches on one side with 5/6 glycines

• enzymes polymerize repeating NAM–NAG–NAM–NAG units into long carbohydrate strands → proteoglycan layer is formed by layers of these species

• the bacterium has an enzyme called transpeptidase to form cross links between two different chains → one amino acid from a layer is removed → formation of a bond between one layer and the next

what is the mechanism of action of β-lactams, carbepenems, cephalosporin, penicillin etc against the proteoglycan/peptidoglycan wall

• inhibition of the last step of the biosynthesis of the proteoglycan (peptidoglycan) layer of the cell wall by inhibiting transpeptidase 

• this prevents formation of the cross-linking → cell wall is unstable and not functional → bacterium dies

how does transpeptidase cross linking occur (mechanism of action)

• transpeptidase has a serine amino acid and lysine with a positive charge in its active site

• when peptide chain arrives, the OH on serine reacts with D-alanine residue on peptide → D-Alanine is released

• peptide chain is attached temporarily to the active site of the enzyme, to serine

• when another peptide chain arrives to make the cross link, its glycine amino group reacts with the carbonyl group on the peptide that is attached to serine

• this promotes detachment of the original peptide chain species from serine

• the enzyme’s serine is now restored to its free Ser–OH state → new peptide links form the final peptidoglycan cross-link between the two glycan strands.

how does beta lactam inhibition occur 

• beta lactam has a free carboxylic acid, its pH is negatively charged due loss of proton

• negative charge on beta lactam and positive charge of lysine on the enzyme 

• four ring structure of beta lactam opens up and is attacked by the OH of serine 

• incoming peptide chain can’t react anymore as enzyme is blocked 

Driving force of the reaction is the ring strain of the lactam (4-member rings have strained bond angles = unstable)

what kind of inhibiton is beta lactam

• suicide inhibition

• beta lactams act as suicidal substrates to block the active site of the enzyme 

what are penicillins

• the first beta-lactams to be discovered 

• hard to isolate it as it is acid labile → easily destroyed in acidic environment

what are the different penicillins

• penicillin G

• 6-amino penicillin 

• penicillin V

what is penicillin G

• obtained from Penicillum sp.

• ACID-LABILE → not stable in gastric environment → has to be administered parenterally, not orally 

what is 6-amino penicillin 

• obtained by hydrolysis from penicillin G

• starting material for semi-synthesis of penicillin as you are able to obtain a large amount of 6-amino penicillin and synthesise it 

• similar structure to penicillin G but chain ends at the NH group 

what is penicillin V

• obtained from Penicillum sp. with phenoxyacetic acid in culture medium

• similar structure to penicillin G, but has an extra O 

• this increased stability means  it is stable at gastric pH → can be taken orally

what happens when you have two fused rings for penicillin

• for penicillin to work, you need two fused rings: beta lactam and thioazolidine (five member ring)

• by changing the substituent on position 6, you can alter activity

  • if we want penicillin to be active against gram-negative bacteria, R group needs to be very hydrophilic

  • for gram positive, you need very hydrophobic substituents 

  • electron-withdrawing groups increase acidic stability of the species → drug can be administered orally 

  • bulky group increase activity of β-lactam against bacteria (β-lactamase resistance)

what is β-lactamase

an enzyme that bacteria make to break down and inactivate β-lactam

what are cephalosporins

• Cephalosporium acremonium identified as antibiotic-producing microorganism
• Cephalosporin C isolated and identified first
• Contain a β-lactam ring → same family as penicillin

what is the structure of cephalosporins

• similar to penicillin with two fused rings, containing a beta lactam

• however, other ring is a six member ring with a double bond

• substituents can be changed (as seen in diagram)

• altering group on position 7 creates a drug that is less susceptible to beta lactamase

what are the different generations of cephalosporins

people have worked a lot on this drug 

• 1st generation → 1000 less active than penicillin but broader spectrum of action: active against both both G⁺ and G^-

• 2nd generation → methoxy group (OCH₃) in 7 causes resistance to β-lactamase. Broad spectrum 

• 3rd generation → methoxy group (OCH₃) in position 7 causes resistance to β-lactamase and change to left chain on diagram, more active against G^-

• 4th generation → hydrophilic/heterocyclic substituent in 7 to increase penetration in G^- cells

what are carbapenems

• resemble cephalosporins and penicillin with the beta lactam ring, but five-member ring has a C and double bond

• double bond makes beta lactam ring more reactive → more prone to reacting with active site of transpeptidase enzyme

• no amide

• very broad spectrum of action

• active against P. aeruginosa

• e.g.: thienamycin

what are monobactams

• single beta lactam ring

• negative charge given by the sulfonic group

• the only class of beta lactams that doesn’t have a fused ring

• narrow spectrum but active against P. aeruginosa

what are polypeptides and glycopeptides

• examples are vancomycin, cycloserin, bacitracin

• impair cell wall synthesis, but in a different way compared to beta lactams

what is vancomycin

• Glycopeptide isolated from S. orientalis

• Very effective against S. aureus infections (gram positive)

• Replaced by methicillin, back in use against MRSA → only used as LAST resort

what is bacitracin

• Polypeptide complex produced by B. subtilis

• Dispensed as a powder (highly unstable in solution)

• Not absorbed in GI tract and hard to penetrate through tissues → • Active principle in TOPICAL OTC preparations

• Wide spectrum

what is cylcoserin

• Isolated from S. garyphalus

• active site inhibitor → inhibits two key enzymes by mimicking their natural substrate (D-alanine)

  • Alanine racemase

  • D-Ala–D-Ala ligase

• Second line treatment of tuberculosis

mechanism of action of vancomycin, clyclosrin and bacitracin

• vancomycin prevents the monomer attaching to the cell wall → prevents the process transglycosidation. works in the periplasmic space (space between cell membrace and cell wall), similar to beta lactams

• bacitracin blocks the recycling of carrier lipid, which helps assemble the membrane. has to get inside the cell wall

• cycloserin prevents synthesis of double alanine

what agents impair protein synthesis

• Aminoglycosides

• Macrolides

• Chloramphenicol

• Oxazolidinones

• Tetracyclines

how is protein synthesis inhibited in bacteria

act on the ribosome

• Oxazolidinones → bind to 50s subunit

• tetracylcine → blocks tRNA binding

• chloramphenicol → blocls peptide chain transfer

• macrolides and aminoglycosides → block translocation

what are aminoglycosides (bactericidal)

• Isolated from Streptomyces sp. during a systematic screening

• Good activity against aerobic Gram- (systemic infections)

• Poorly absorbed (< 1%), used to treat GI tract infections

• Ototoxic (ear) and nephrotoxic (kidney) → limited their use

• protonated(positively charged) at physiological pH → outer cell wall of gram-negative is hydrophobic, but porins are hydrophilic allows them through

• has many basic groups

examples: streptomycin and gentamycin C1a

what are tetracyclines (bacteriostatic)

• first one was Aureomycin isolated from Streptomyces aureofaciens during a systematic screening

• Inhibit the attachment of aminoacyl-tRNA to 30s subunit of ribosome

• Most prescribed class after penicillins

• Very broad spectrum of action (G+, G-, mycoplasma…)

• 4 fused rings, can't remove/modify certain groups, some need a basic group due to PKA

what are macrolides

Erythromycin isolated (1952) from Streptomyces erythreus

• Inhibit translocation by binding to 50s subunit of ribosome

• Class counts >40 compounds

• Spectrum of action resembles that of penicillin (G+ cocci and bacilli, G- cocci) but also

mycoplasma. Not active on G- bacilli