ANTIMICROBIALS AND VACCINES Flashcards
give an example of why we need antibiotics?
1941 mortality rate from S. aureus bacteraemia 82% and predominantly in young people
2009 mortality rate 21% and mostly old fucks
what do antibiotics do?
inhibit bacterial growth by targeting molecular targets
interfere with specific bacterial enzymes
different toxicity for bacterial cells as target not present or sufficiently different in eukaryotic cells
what are the three broad targets of antibiotics?
cell wall aka peptidoglycan synthesis
protein synthesis (ribosome)
nucleic acid synthesis
outline how antibiotics target peptidoglycan synthesis?
peptidoglycan layer gets cross linked to form lattice structure in both gram+ and gram- and it provides strength against osmotic lysis of bacteria
peptidoglycan synthesis has a number of steps which can be inhibited
e.g. bacitracin inhibits lipid carrier recycling, beta lactams and glycopeptides inhibit peptidoglycan subunit cross-linking
outline how beta lactams inhibit peptidoglycan cross-linking?
key molecule of beta lactams (e.g. penicillin) is beta-lactam ring as this responsible for its activity
cross linking of peptidoglycan key for providing its function; facilitated by transpeptidases called penicillin binding protein (PBP) and this has serine residue which crosslinks peptide side chains of peptidoglycan backbone
beta lactam ring reacts with serine residue on PBP forming covalent bond so PBP can no longer cross-link peptidoglycan chains
why is cross-linking so important for peptidoglycan function?
autolysis are enzymes present in peptidoglycan layer and are responsible for degrading it when activated
this is normal part of turnover of peptidoglycan, growth and division
so when no more cross linking (cause beta lactic activity) this leads to bacterial lysis to osmotic pressure (hence b-lactams are bactericidal)
autolysins most active during exponential cell growth so beta lactams most effective during this
compare and contrast the classes of beta-lactams?
there are multiple families of beta-lactams e.g penicillins, cephalosporins, carbapenem, monobactam
they have different spectra of activity and resistance to beta-lactamases which is influenced by side chain R group - can also affect pharmacokinetics (what body does to drug)
we can alter R-group to alter how drug works
all beta-lactams have beta-lactam ring tho and have all come from natural sources
how do glycopeptides work?
glycopeptides e.g. vancomycin are beta lactams
they are big molecules so only effective against gram positive as cannot penetrate gram negative outer membrane (also means have to be given intravenously unless luminal infection e.g. C. difficile)
glycopeptides recognise the D-alanine-D-alanine portion of muramylpentapeptide
inhibits ability of PBP to come in and cross-link peptidoglycan
outline how protein synthesis inhibiting antibiotics function?
bind either 16s rRNA in 30s subunit (e.g. aminoglycosides, tetracyclines) inhibiting mRNA from binding OR 23s rRNA in 50s subunit (macrolides, lincosamides, oxazolidinones) inhibiting tRNA from binding
all these drugs work by binding the ribosome thus inhibiting protein synthesis
reason they don’t inhibit our protein translation is difference between prokaryotic and eukaryotic ribosomes i.e. differential selectivity
what are the three main things inhibited by antibiotics that target nucleic synthesis?
inhibition of DNA synthesis
inhibition of RNA synthesis
inhibition of folate metabolism
how do antibiotics target folate synthesis and give examples?
folate synthesis is a bacterial pathway for production of tetrahydrofolic acid which is an essential co-factor for synthesis of nucleic acids
this pathway involves three key steps
sulphonamides (inhibits first step) are structurally similar to p-aminobenzoic acid (important precursor in folate synthesis) and thus is competitive inhibitor of dihydropteroate synthase
trimethoprim (inhibits final step) is structurally similar to dihydrofolic acid so is competitive inhibitor of dihydrofolate reductase
how does metronidazole target nucleic acid synthesis?
makes breaks in DNA
doesn’t damage our DNA cause is a prodrug i.e. requires activation
activation involves reduction by ferredoxin or flavodoxin - these are electron acceptors in anaerobes and microaerophiles
aerobes and mitochondria use pyruvate dehydrogenase so we allg
how do fluoroquinolones target nucleic acid synthesis?
inhibit type II topoisomerase (e.g. DNA gyrase, topoisomerase IV) which are very important for bacterial replication
they are important for replication as they unwind positively supercoiled DNA into a relaxed state, negatively supercoil it to be packaged into cell and also decatenation of daughter chromosomes at cell division
how do rifamycins inhibit nucleic acid synthesis?
inhibit RNA synthesis by binding B subunit of DNA-dependent RNA polymerase inhibiting its function
resistance can occur due to a single point mutation - huge issue with rifampicin (important for TB) so almost always use in combination
what are some different approaches we could take to find new antibiotics?
improve existing antibiotics e.g. alter B-lactam ring
repurpose old drugs
discover untested new chemical diversity (either from natural or synthetic products; natural has proven most effective)
target based approach (finding novel targets then make drugs for them)
rediscover old antibiotics
why has most antibiotic discovery been from natural products?
many have come from bacteria and fungi which produce antibiotics in response to competition or stress
this is also an issue as it means producer species have intrinsic mechanism of resistance and so resistance exists for so many antibiotics
outline how teixobactin was discovered using iChip process?
potential new antibiotic for clinical use
discovered through growth in iChip in soil for a month, then move to agar (50% survive), bacterial extracts screened for activity against S. aureus
ones with anti-staph activity undergo purification into different compounds and are screened against a range of bacteria
assess compounds in vitro and work out mechanism (teixobactin related to cycling of lipids)
what is the research and development process for antibiotics?
drug discovery and pre-clinical (3-6 years) (teixobactin only just finished this)
clinical trials (6-7 years):
phase I - potential adverse side effects and safety
phase II - efficacy and safety
phase III - efficacy and safety (larger group)
seperate phase I and II trials for carried out each indication
only then can it be FDA approved and throughout this process fuck loads of compounds narrowed down to only a few
outline the financial issues with antibiotic research and development?
cost of bringing new drug to market >1 billion dollars and profits not made till a long way down the line (and not much)
so little incentive for pharmaceutical companies
this is fucked cause the antibiotics pipeline is running dry
how is the mortality rate with MRSA changing in New Zealand?
mortality rate for MRSA bacteraemia has doubled since 2009
methicillin first line drug for S. aureus bactaemia
outline the human and financial burden associated with increasing incidence of antimicrobial resistance?
increased cause of death - 10 million by 2050 at current rates
increased global loss of GDP - 100 trillion by 2050
outline the three broad mechanisms that AMR can come under?
restricted access to target (decreased permeability, increased efflux)
inactivation of antibiotic
modification of drug target
how do some bacteria restrict access to the antibiotic target by decreasing permeability?
beta lactams must cross outer membrane of gram negatives and many other antibiotics must access cytoplasm
gram negatives encode multiple porins in their outer membrane to allow selective diffusion of small molecules into periplasm - mutations can down regulate porin expression or restrict antibiotic access by narrowing channel (often affecting multiple antibiotics) - acquired resistance
gram negatives also intrinsically resistant to vancomycin cause it too big to cross outer membrane
how do bacteria restrict antibiotic access to target through increasing efflux?
efflux pumps actively pump small molecules out out of cytoplasm such as metabolites and toxic substances
bacteria contain multiple efflux pumps, some are highly specific (e.g. tetracycline efflux pump) and others more broad - can give resistance to multiple classes
this common resistance mechanism now described for almost every antibiotic class
outline how inactivation of antibiotics occurs via beta lactamases?
beta lactamases are enzymes secreted into periplasmic space (gram neg) or extracellularly (gram pos)
especially common in gram negs and can be distinguished into class A, B and C beta lactamases
class A and C have serine residue at active site which forms covalent bond with beta lactam ring and cleaves it and then undergoes hydrolysis freeing beta lactase from inactivated beta lactam - some of these more narrow spectrum (e.g. TEM) and some more broad (e.g. AmpC, ESBLs) destroying lots of beta lactams
AmpC are class C while ESBLs are class A
class B beta lactamases have different mechanism of action; are zinc dependent (have zinc ion at active site) and are broad spectrum
what are beta lactamase inhibitors and how effective are they?
e.g. clavulanic acid
can inhibit class A beta lactamases (e.g. ESBL) by binding it and remaining bound thus removing it from action
does not inhibit class C beta-lactamases (AmpC) produced by ESCAPPM group organisms which express class C beta-lactamase via inducible production or have mutation which can allow constitutive production - so can become resistant to beta lactams during treatment
metallic-beta-lactamases (aka class B) are not inhibited by clavulanic acid but are by EDTA (can’t use this clinically tho)
how do bacteria gain antibiotic resistance through modification or protection of antibiotic target- give some fuckiogn exampels?
mutations in target: such as gyrA/B and parC/E mutations for fluoroquinolones, rpoB point mutation for rifampicin, PBP mutations for beta lactams
enzymatic modification of target: stops antibiotic function but still allows target function e.g. glycopeptides; d-alanine-d-alanine to d-alanine-d-lactate
bypassing target: for example MRSA acquire different PBP (PBP2A encoded by mecA) which beta lactams don’t work on
where has altered penicillin binding proteins via mutation conferred resistance?
common in gram-positives
mutation in chromosomal copies of PBPs can then be acquired by horizontal gene transfer
particularly a problem in viridans group streptococci e.g. S. pneumoniae
how does resistance to glycopeptides (e.g. vancomycin) occur through enzymatic modification of target?
vancomycin binds D-alanine-D-alanine at end of peptide to inhibit PBP from coming in and cross linking it
bacteria can become vancomycin resistant through acquiring a new group/operon of genes which changes peptide to D-alanine-D-lactate
operon of genes has a two component regulatory system: VanS recognises presence of vancomycin and phosphorylates VanR which is transcriptional regulator of the system (i.e. turns on all the other genes) leading to synthesis of D-alanine-D-lactate via VanH and VanA and then destruction of D-alanine-D-alanine via VanX and VanY
how can altered-penicillin binding proteins (PBPs) allow bypassing of antibiotic target leading to resistance?
mecA in MRSA encodes PBP2A which isn’t inhibited by methicillin
this replaces the normal transpeptidase allowing peptidoglycan cross-linking in presence of antibiotic
what are the two main drivers of antibiotic resistance?
antibiotic-mediated selection
horizontal gene transfer
what are the different ways resistance genes can undergo horizontal gene transfer?
conjugation - most common, especially between different species, occurs via pillus
transformation - uptake of DNA from external environment, integrates by homologous recombination so has to be closely related or same species
bacteriophage transduction - usually between same species or closely related cause need the same phage receptor
HGT much easier way to acquire resistance genes than through mutation, frequent exchanges of genetic material e.g. within animal guts
describe multiple antibiotic resistance?
most resistance mechanisms against single class of antibiotics; exceptions include multi-drug efflux pumps and macrolide-lincosamide-streptogramin B (MLSb) resistance (binds overlapping sites on ribosome)
HOWEVER multi drug resistance due to genetic linkage on MGEs (where resistance genes often accumulate) is more common
selection by one class of antibiotics can maintain resistance genes to other unrelated antibiotics (cross-selection) cause they genetically linked
antibiotic resistance genes can also be linked to disinfectant resistance genes i.e. disinfectants may also be selecting antibiotic resistant bacteria
what are integrons?
often encoded by mobile genetic elements
integrons encode integrase which integrates circular DNA gene cassettes (e.g. resistance gene) into the att site and creates operons by sequential integration of gene cassettes (they contain a promoter so they get expressed)
what is a transposon?
a mobile genetic element flanked by insertion sequences which encode transposase and are recognised by transposase meaning they can cut themselves out of DNA along with their cargo and insert somewhere else (e.g. plasmid, chromosome)
can be conjugative (chromosome to chromosome) and have a broader host range than most plasmids (e.g. can pass from gram positive to gram negative)
why do we need to think of the flux of resistance through the environment?
resistance genes in bacteria can travel through multiple hosts and locations due to their tendency to accumulate on MGEs
resistance bacteria can be transmitted between people, excreted into environment, accumulate in shellfish, the genes can get amplified in livestock and then back into people
how does New Zealand use antibiotics?
compared to other countries NZ has relatively low rates of antibiotic use in agriculture, but relatively high rates of antibiotic usage in medicine
broadly how did we come to the antibiotic resistance crisis?
inappropriate use of antibiotics (e.g. wrong use, wrong duration, over-prescription) applying selective pressure on bacteria with high genetic plasticity
this combined with increased spread as a result of socioeconomic issues, hospitals and increasing international travel and increasing susceptibility to infection and antibiotic usage as a result of poor nutrition, immunosuppression and invasive medical procedures
what are antiviral drugs?
inhibit viral replication by inhibiting specific viral proteins
they are quite specific (specific antivirals for specific viruses)
resistance develops by mutation of target protein
outline HIV virology?
retrovirus (subgroup lentivirus)
enveloped; important envelope glycoproteins gp120 and gp41
nucleocapsid (encoded by gag) main structural protein is p24
essential enzymes (encoded by pol) include integrate, protease and reverse transcriptase
how the fuck does HIV enter the host cell?
gp120 binds CD4 with high affinity
conformational change allows binding of gp120 to co-receptor which is CCR5 (or CCR4)
this exposes gp41 allowing membrane fusion and entry of nucleocapsid
how does maraviroc inhibit HIV attachment to host cell?
maraviroc is a CCR5 inhibitor
it binds CCR5 thus preventing gp120 binding
how does enfuvirtide inhibit HIV fusion host cell?
enfuvirtide is a peptide analogue of gp41 fusion domain
binds gp41 preventing fusion with host cell membrane
how can we inhibit HIV fusion and attachment to host cells?
maraviroc (attachment)
enfuvirtide (fusion)
how can monoclonal neutralising antibodies work as attachment inhibitors?
can be isolated from B cells encoding antibodies that neutralise SARS-CoV-2 and then be cloned and administered to patients
prevents virus binding to entry receptor (e.g. SARS-CoV-2 spike to ACE2)
already some covid drugs out there using monoclonal antibodies
how has viral resistance to monoclonal antibodies began to emerge?
immune escape variants can escape monoclonal antibodies due to surface proteins (e.g. of SARS-CoV-2) rapidly mutating causing resistant strains
in vitro assessment of the ability of monoclonal antibodies to neutralise original and omicron variants of SARS-CoV-2 show increasingly high doses of antibody required
what does amantadine do?
binds influenza A M2 protein inhibiting uncoating
mutation in the M2 protein however confers resistance
what antivirals inhibit HIV reverse transcriptase?
once HIV enters the cytoplasm it needs to start reverse transcription to convert RNA genome to cDNA which is catalysed by viral enzyme reverse transcriptase
two drugs inhibiting this (reverse transcriptase inhibitors or RTI)
- nucleoside analogues (NRTI)
- non-nucleoside RT inhibitors (NNRTI)
how do nucleoside analogues (NRTI) function to inhibit reverse transcriptase?
e.g. zidovudine (thymidine analogue; 3’ hydroxyl group replaced with nitrogens) - competes with the natural substrates (dNTPs) for HIV reverse transcriptase; gets incorporated into DNA leading to chain termination (as no 3’ OH)
e.g. acyclovir is an analogue of the nucleoside deoxyguanasine and has activity against HSV-1 and 2 and also VSV. Requires activation by virally encoded thymidine kinase which phosphorylates it allowing its incorporation by DNA polymerase leading to chain termination - resistance can develop through mutations in thymidine kinase or DNA pol
how do non-nucleoside reverse transcriptase inhibitors (NNRTI) function?
bind hydrophobic pocket near the catalytic site of HIV reverse transcriptase leading to a structural change in RT so it can no longer function
