Antimicrobial Drugs: Discovery, design & Resistance

timeline of modern antimicrobial chemotherapy

what is the germ theory (discovery of infective diseases)

Louis Pasteur showed that germs cause 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

• Prontosil 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 

• 6-APA 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 anti-infective

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 a prolific source of (potential) active principles

bacterial cell

what is the gram staining method 

1. application of purple dye

2. application of iodine mordant to help dye stay

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

4. application of safranin counterstain (bacteria that waswashed 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 (purple)

• gram negative cell wall → has a plasma membrane, a thin peptidoglycan layer outside of its membrane and an outer hydrophobic membrane (red/pink)

how are antibacterial agents tested 

1. Disk Diffusion Test (Kirby–Bauer Test)

• Each disc is soaked with an antibiotic.

• Effective drug diffuses out → bacteria fails to grow near discs

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

• glycopeptides: vancomycin

• lipopeptides: daptomycin

• polypeptides

  • bacitracin

  • cholestin

how do antibiotics inhibit nucleic acid synthesis 

• inhibit DNA gyrase and/or topoisomerase IV e.g. 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

• tigecyline

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

• 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 → 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 called dihydrothiazine

• 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 position 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 sulphonic 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 Staphylococcus aureus infections (gram positive)

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

what is bacitracin

• Polypeptide complex produced by Bacillus 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 Streptomyces 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, cycloserine and bacitracin

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

• bacitracin blocks 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

• Tetracyclines

• Oxazolidinones

• Macrolides

• Chloramphenicol

ATOM-C

how is protein synthesis inhibited in bacteria

act on the ribosome

• Oxazolidinones → bind to 50s subunit

• tetracylcine → blocks tRNA binding

• chloramphenicol → blocks peptide chain transfer

• macrolides and aminoglycosides → block translocation

what are aminoglycosides (bactericidal)

• Isolated from Streptomyces species 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 to allow 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 (bacteriostatic)

• Erythromycin isolated from Streptomyces erythreus

• Inhibit translocation by binding to 50s subunit of ribosome

• Class has >40 compounds

• broad spectrum of action resembles that of penicillin (G+ cocci and bacilli, G- cocci) but also mycoplasma. Not active on G- bacilli

• many 6-member rings, macro rings, ketone and lactone function

what is Chloramphenicol (bacteriostatic)

• Originally isolated from S. venezuela now synthetic.

• Binds to 50s subunit preventing the elongation of the peptide chain.

• Potent and broad spectrum. Used in typhoid and for eye infections

• Very toxic to the bone marrow

• Toxic metabolism in babies (grey baby syndrome)

what are Oxazolidinones (bacteriostatic)

New class of synthetic antibacterial agents

• Bind to the 50s subunit and prevent the ribosome from assembling (50s to 30s)

• Active orally against G+ (including MRSA)

what agents damage or impair the synthesis of DNA

• Sulfonamides

• Sulfones

• Trimethoprim

• Rifamycins

• Quinolones

• Nitroimidazole

SSTRQN

how do agents inhibit nucleic acid synthesis

Either inhibit enzymes involved in DNA/RNA synthesis or cause direct damage to DNA

• polymerase/gyrase/topoisomerase

• direct DNA damage

what are Quinolones (bactericidal)

• Very wide class of synthetic antibacterial agents

• MOA: bind to topoisomerase IV and gyrase, preventing supercoiling of bacterial DNA

• Active against G+ and G-

• Used in urinary tract infections

• two fused ring, C=O group, COOH group, double bond in ring, N atom

• examples: ciprofloxacin and nalidixic acid

what are Nitroimidazoles (bactericidal)

• Synthetic antibacterial agents (originally antiprotozoal for parasites)

• causes direct damage to DNA

• Nitro group essential for activity, can’t modify

• nitro group undergoes reduction steps to form amino group → Form oxygen radical species toxic to DNA

what are rifamycins (bactericidal)

• Isolated from S. mediterranei

• Active on Gram positives

• Binds to DNA-dependent RNA polymerases

• Used against bacterial diarrhoea and E. coli infection

what are sulfonamides (bacteriostatic)

• Class of antibacterials derived from Prontosil (red structure)

• inhibit dna synthesis without interfering with the enzymes directly

• First effective chemotherapeutics for systemic infections

• Big reduction in mortality caused by bacterial infections

• Superseded by penicillins as they are more active

• prontosil when adminstered systemically releases sulfonamide

• acidic group on right loses proton due to presence of electron attractive group

mechanism of action of sulfonamides

bacteria synthesise their own dihydropteorate, a prescursor of tetrahydrofolate (co-factor of the enzyme that allows antibiotic to pass into bacterial cells to produce DNA bases from RNA)

• first start with species with two phosphate groups

• dehydropteorate synthetase removes the phosphate groups and attaches para-aminobenzoic acid to make dihydropteorate

• glutamic acid residue attached

• reduction → double bond on second ring is lost to tetrahydrofolate, co enzyme F

sulfonamide interferes with removing phosphate groups and attaches para-aminobenzoic acid to make dihydropteorate → compete against para-aminobenzoic acid as they are structurally similar → need specific groups, but can modify

what are Trimethoprim and Sulfones (bacteriostatic)

Main antimicrobial agents that interfere with CoF synthesis → antimetabolites

• trimethoprim → Interferes with dihydrofolate reductase. Used in conjunction with sulfonamides

• sulfones → Used in the treatment of leprosy.Mechanism of action still debated but, evidences of similarity with sulfonamides

what is a virus

genetic material packed in proteins (reproduction occurs only via colonisation of a host cell)

• hides from the immune system → harder to single out with therapeutic agent

• there is a long lag time between time of infection and symptoms (e.g., HIV, rabies)

what is the structure of viruses

• spikes are proteins

• function of genetic material → propagates the virus and gives the target cell instructions to make more viruses dentinal to the entered virus

what happens when humans meet viruses

• Some can pass from animals to humans (zoonoses)

• Virus can be air-, food-, water-borne, by direct contact or through vectors (e.g., ticks)

• Humankind has been challenged by several viral epidemics/pandemic

• Best approach = vaccination (less effective for rapidly mutating viruses)

what are the characteristics of antiviral therapy

• disrupt stage of virus life cycle

• bear little resemblance to human proteins (+ selectivity)

• common to a variety of viruses (+ broad spectrum)

• important for early stages of viral life (- symptoms/spreading)

what is the cycle of viral infection

• virus docks through cell wall structure with help of spike protein

• fuse with membrane with help of spike protein

• virus uncoats and releases genetic material into nucelus, if DNA reverse transcription occurs to become DNA

• DNA replication occurs → transcription to make viral proteins

• protein synthesis → virus ressembled with the copied DNA, spikes etc

• budding and released to infect new cells

antiviral agents can block any of these steps in the cycle

what are the classes of antiviral agents

• Inhibitors of DNA polymerase

• Inhibitor of reverse transcriptase (RNA into DNA)

  • Nucleoside reverse transcriptase inhibitors

  • Non-nucleoside reverse transcriptase inhibitors

• Protease inhibitors

  • Aspartyl protease inhibitors (HIV)

  • Neuraminidase inhibitors (influenza)

  • NS3-4A protease inhibitors (hepatitis C)

• Interferon

• Uncoating/fusion inhibitors

what are inhibitors of DNA polymerase

• Nucleoside-like structures which are phosphorylated, incorporated in growing DNA chain to lead to chain interruption

• MOA; they resemble the nuceloside structure → phosphorylated with a kinase enzyme(three phospahte groups) → becomes part of DNA chain → their structure prevents the chain from growing

• example: aciclovir, red bit is not included in the drug, but is in deoxyguanosine

how is aciclovir modified

has poor oral bioavailabilty → given topically

• valociclivir has improved oral bioavailabiltiy

• more hydrophobic → faster onset and longer duration

examples of other DNA polymerase inhibitors

• contain the OH group, unlike aciclovir

• they have the OH group that is needed for growing the chain

• however, the structures alter pairing of complementary bases → more prone to damage and errors

• they are substrates for the virus and host thymidylate/thymidine kinase → high possibility of toxicity to host enzyme → the DNA that is synthesised by the host gets affected

• some viruses don’t produce thymidine kinase → use an analogue of the next step, thymidylate kinase analogue

foscarnet → phosphate analogue that prevents the attachment of phosphates on thre molecule. however it can’t distinguish between the viral enzyme and host enzyme

what are inhibitors of reverse transcriptase

inhibit reverse transcriptase from translating RNA into DNA, includes

• Nucleoside reverse transcriptase inhibitors

• Non-nucleoside reverse transcriptase inhibitors

what are Nucleoside reverse transcriptase inhibitors (NRTI)

Nucleoside structures: phosphorylated, incorporated in growing DNA chain (chain interruption), same as DNA polymerase inhibitors

• PROBLEM: in some cases (HIV) the virus does not produce TK → Host enzymes is used to activate NRTI instead→ less selectivity as all cells are affected

• caution: reverse transcriptase is still a DNA polymerase, which humans have → Selective affinity for RT vs. DP of the host is vital

what are Non-nucleoside reverse transcriptase inhibitors (NNRTI)

Hydrophobic molecules that bind to allosteric binding in reverse transcriptase, a site other than the active site

• Binding blocks active site into inactive conformation

• Punctual mutation (K103N) cause onset of rapid resistance to NNRTI

• X-ray crystallography helped site-targeted rational design

• NNRTI are part of highly active antiretroviral therapy against HIV

what are protease inhibitors

Series of derivatives active against viral proteases, including

• Aspartyl protease inhibitors (HIV)

• Neuraminidase inhibitors (influenza)

• NS3-4A protease inhibitors (hepatitis C)

virus produces protease to help enter the cell

what are inhibitors of HIV protease

• No need to be metabolised (unlike NRTI), hence easier to evaluate activity in vitro

• Target enzyme is aspartyl protease, which cleaves viral pro-enzymes to release mature, active viral enzymes

• Designed based on the structure of the substrate (peptides) to block protease so proteins don’t get cut

• however, Instability and poor bioavailability (typical of peptide drugs)

• Rapid onset of resistance to these drugs as virus easily mutates

example: saquinavir (Roche)

how were HIV protease inhibitors designed to look similar to the natural substrate for the enzyme aspartyl protease, but different enough to then prevent enzyme from working

Major medicinal chemistry effort aimed at

1) Improving affinity for enzyme (resemble the substrate transition state)

2) Improving stability to proteases (eliminate peptide bonds)

3) Improving oral bioavailability

4) Reduce molecular weight

5) Avoid resistance

the first protease inhibitor was Saquinavir

examples of attempted improvements for HIV protease inhibitors, compared to Saquinavir

• restraining its conformation (to the "active" shape to bind more strongly) → Lopinavir and Ritonavir

• non-peptide species (to prevent hydrolysis) → Tipranavir

• smaller → Nelfinavir

• Better fit to enzyme aspartyl protease → Indinavir

• 1 dose/day → Atazanavir

what is HAART

highly active anti-retroviral therapy → combination therapy to treat HIV

• HIV mutates and becomes resistance to drugs quickly

• HIV has no cure → anti-HIV drugs are for lifetime and have toxic side effects

• examples of HAART are:

  • 2 nucleoside reverse transcriptase inhibitors (NRTI) + 1 protease inhibitor (PI)

  • 1 nucleoside reverse transcriptase inhibitor (NRTI) + 2 protease inhibitor (PI)

  • 2 nucleoside reverse transcriptase inhibitors (NRTI) + 1 non-nucleoside reverse transcriptase inhibitors (NNRTI)

what are the protease inhibitors for influenza

• influenza → airborne virus causing respiratory disease

• mechanism of action

  • prevents docking, cellular uptake and spreading of the virus

  • blocks protease enzymes of the virus: neuraminidase (NA) and hemagglutinase (HA), which are responsible for the lysis of mucus, allowing the virus to reach epithelial cells (NA), access epithelial cells (HA) and budding (NA)

what are neuraminidase inhibitors (influenza)

• MOA:

  • they mimic the structure of the transition state (the protease-binding sialic acid unit on glycoproteins and glycolipids

  • before virus cuts the glycosidic bond connecting sialic acid to a glycoprotein or glycolipid, drugs bind very strongly to neuraminidase as they mimic that distorted transition-state structure → allowing the drug to bind very tightly to the enzyme and prevent it from cleaving the real sialic acid-glycoprotein bond.

• examples: Osletamavir, Zanamivir and Laninamivir

what are the protease inhibitors for hepatitis C

• hepatitis C → blood-borne virus that causes an asymptomatic infection

• MOA: block a protease that releases mature virus proteins from pro-peptides

• exampkes: Boceprevir and Telaprevir

what are other antiviral agents

• ion channel disrupters

• capsid-binding agents

• ribavirin

• interferone

what are ion channel disrupters

Adamantanes are a class of antiviral drugs that act as ion channel disruptors against influenza A viruses

• MOA for low dose: blocks an ion channel protein (M2 protein) on the virus → prevents exchange of electrolytes between virus

• MOA for high dose: lowers the pH of the endosome to delay uncoating (virus removes its protective coat (capsid) and release genetic material into host cell)

what are capsid-binding agents

• used against picornaviruses (polio, hepatitis A, FMD)

• MOA: bind to a pocked in the capsid of the virus to prevent release of viral RNA

• examples: pleconaril and disoxaril

what is ribavirin

• a broad-spectrum antiviral (works against RNA and DNA virusea)

• only licensed for hepatitis C

• MOA: induces mutations in the virus

what is interferone

• a family of peptides involved in cell response to viral infections

• examples of interferone: alpha, beta and gamma

• they are released by infected cells to alert neighbouring cells of infection