Antimicrobials 2 - Antivirals Flashcards
Fungi: what do they include, feed on, reproduction, pharma relevance?
- mold, mushrooms, yeast
- feed on decomposing organic matter
- reproduce sexually and asexually through spores
- source of antibiotics (ex. penecillins) and other drugs
- can symbiotic (lichen) or pathogenic (candidias)
What kind of cells are fungal cells? What are they similar to? What is in their cell wall?
- eukaryotic (have nucleus)
- similar to plant cells –> membrane, cell walll, nucleus, mitochondira, cytoplasm
- chitin (instead of cellulose) and glucan and manoproteins –> only organism to have chitin and glucan in cell wall
history of antifungals
- pre 1990s - home remeides of vinegar and gynpowder
- early 1990s - general antiseptic gentian violet that binds to thioredoxin (protein in redox cycle of fungi that results in death if malfunctions)
- first true antifungal: Amphotericin B (polyene) from soil bacteria in 1955
- flucytosines (1970), azoles (1990), echinocanids (2000)
What do antifungals target?
- cell wall/membranr
- fungal DNA
- protein synthesis
- essential cellular processes
- virulence factors
How do fungi acquire resistance? And how is this different from bacteria?
- block drug entry
- pump it out
- metabolize it
- mutate drug target
- avoid the pathway
but there is no transformation or conjugation because they dont have a mechanism for DNA transfer –> resistance grows slower than in bac
What are the 5 antifungals?
- amphotericin B
- nystatin
- fluconazole
- griseolfulvin
- flucysteine
Amphotericin B
What is it?
- Naturally occuring antifungal
Use
- blastomyces respiratory disease
- serious infections only
MOA
- binds directly to ergosterol in fungal cell wall and causes permeability –> leakage
- fungocidal
PK
- not orally bioavailable but is okay in the gut
- administered IV or lipid formulation
ADR
- toxi effects are dose dependent
- low TI (can bind cholesterol)
- hypotension
- anorexia
- kidney/liver damage
Nystatin
What is it?
- polyene antifungal made from streptomyces bacteria
Treatment
- candida, oral thrush
- broad spectrum
MOA
- directly binds to ergosterol (fungal specific sterol) in cell membrane and causes permeability by forming TM pore–> leakage
- fungicidal
PK
- not orally bioavailable (large polyene striucture)
- administered topically or for GI –> not absorbed
- good safety profile
fluconazole
What is it?
- synthetic azole antifungal - has azole rings
Treatment?
- oral trush and yeast infection
- broad spectrum
MOA?
- blocks ergosterol fluidity by blocking 14-demethylase (CYP450) that catalyzes lanosterol into ergosterol
- causes permeability and leakage
- fungicidal
PK?
- water soluble and very bioavailable
- administered orally and IV
ADR? Safety?
- very safe because we use HMG-CoA reductase –> selective
- minor GI issues
griseolfulvin
What is it?
- naturally produced antifungal
Treatment?
- nail and systemic infection
MOA?
- directly binds to tublin and leads to impaired microtubules –> blocks mitosis/replication
- fungistatic
PK?
- orally admin for superficial infections
ADR? Safety?
- sunlight sensitivity, yellowing of skin, liver damage, links to cancer –> can be used to treat cancer?
- slightly more selective for fungal tubulin but can also induce mutations in humans
flucytosine
What is it?
- synthetic base analogue
Treatment?
- systemic infections
- combination therapy for serious infections
MOA?
- prodrug converted by cytidine deaminase to 5-fluorouracil –> converted to NTP –> blocks DNA/RNA synthesis
- fungistatic
PK?
- orally bioavailable
ADR? Safety?
- blood count
- liver/kidney damage
- not safe in pregnancy
- possible carcigonen –> 5FU is toxic to human cells (used in chemo)
Side effects and contraindications of antifungals
- toxicity and DNA damage
- DNA damage from nucleotide analogues that can be incorporated into DNA of human cells (FLYCYTOSINE, GRISEOFULVIN)
- liver and kidney toxicity (AMPHOTERICIN B) - allergic reaction
- mild to serious
- specific to drug - GI issues - from nausea to diarrhea
- Pregnancy
- FLUCYTOSINE AND GRISEOFULVIN NOT OKAY
- FLUCONAZOLE was thought to be okay but new evidence suggests increased risk of miscarriage
What is a virus? What is its replication cycle? What genomes they that have?
- small particle containing RNA or DNA and capsid and sometimes envelops proteins
- not alive and reliant on host cell for replication and metabolism
replication cycle
attachement –> prenetration –> uncoating –> replication –> assembly –> release (lytic vs lysogenic)
genomes
- ssRNA, ssDNA, dsDNA
how do viruses interact with the host genome
- transiently - non permanent and non intergarting
- permanent but not integrating: episome - an extra chromosome piece that replicates independently
- permanently
how are viruses classified
- genetic component (DNA/RNA)
-morphology - mode of replication
- host: tropism - narrow means they can only go into one or two tissues, broad means they can go into many
- disease they cause
How do RNA viruses interact with the host genome?
- ssRNA and are non integrating ex SARS Cov 2
- retrovirus that uses reverse transcriptase to integrate into the host DNA ex. HIV 1
- dsRNA ex Reovirus
how does RT integrate into the genome?
- makes ssDNA from RNA
- degardes RNA strand and copies the DNA to form dsDNA
- goes into the nucleus and integrase helps with integration
- uses LTR to input DNA into host genome
Which viruses are DNA based? How are they categorized?
dsDNA: bacteriophage, adenovirus, HSV1/2
- dsDNA viruses can be non-permanent or permanent on episomes (ex. HSC1/2 can be latent or lytic)
ssDNA virsues: parovirus
- usually affects children and canines
- more mature immune systems dont get infected by ssDNA viruses
targets of antivirals
- membrane/envelope - prevent entry and fusion
- viral DNA/RNA - polymerase or RT
- proteases
- glycoenzymes
- increase the defences of human cells
What are the 5 antivirals?
docosanol
acyclovir
zivovudine
nirmatrelvir
amantidine
Docosanol
What is it?
- emulsifying agent (thickening) - very long lipid with alcohol group
Treatment?
HSV1 cold sore
MOA?
- inhibits the fusion of the viral envelope to the human membrane by directly inserting into the membranr and altering it in a way that blocks fusion
- no resistance!! –> new paradigm –> protecting YOU
PK?
- not orally bioavailable or systemic use
- only for topical infections HSV1 cold sore
ADR?
- low topical absorption
- very safe
acyclovir
What is it?
- synthetic purine analogue
Treatment?
- HSV1/2, varicella, EBV/CMV
MOA?
- thymidine kinase in virus converts prodrug to monophospahte –> tripphospahte NTP by host kinase –> integrates into DNA and causes chain termination
- our cells don’t have thymidine kinase so we are safe –> selective
PK?
- topical
- oral (prodrug increases absorption)
- IV
- good distribution
ADR?
- safe unless activated in non infected cells
zidovudine
What is it?
- thymidine nucleoside analogue
Treatment?
- against retroviruses, HIV
MOA?
- prodrug converted to triphosphate by thymidine kinase –> inhibits reverse transcriptase by competing with natural NTPs –> chain termination –> inabilityt o produce DNA and blocks integration
PK?
- orally bioavaiulable
ADR?
- complications in bone marrow
- act on mitochondrial DNA pol
nirmatrelvir
What is it?
- peptidomimetic
Treatment?
- covid 19
MOA?
- inhibits 3C like protease of SARS COV 2 –> competitive inhibitor that blocks viral replication (dummy substate for protease)
- (3C like protease is reposible for cleacing large pro-proteins into individual proteins)
PK?
- orally bioavailable
ADR?
- generallt well tolerated
- rebound effect
amantidine
What is it?
- tricyclic amine
Treatment?
- influenza A
MOA?
- blocks M2 ion channels in envelope imporatnt in viral uncoating envelope and insertion of protein into in host membrane for genetic delivery–> blocks replication
PK?
- orally bioavailable
ADR?
- CNS effects
- teratogenic
- resistance due to mutations of M2 channel
Antiviral resistance mechanisms
- high mutation rates and are produced in high numbers –> prone to evolution
- resistance arises from
- mutations in target proteins that block drug action
-mutations in membrane envelope proteins
side effects and contradindications of antivirals
- toxicity and DNA damage - nucleotide analogues release and incoroprated into DNA of health human cells, liver toxicity for AZT
- allergic reactions
- GI issues
- pregnancy - most safe, but drug dependent
How do traditional vaccines work
- contain attenuated or inactiated virus or a purified recombinant protein
- viral peptide fragments are captured and expressed on antigen presenting cells (APCs)
- APCs activate T and B cells –> cytotxic T cells and plasma cells –> antibodies and memory cells
How do next-generation nucleic acid vaccines work
- contain DNA or mRNA encoding for a specific protein from the virus and transfered into a plasmid
- delivered in a safe virus or chemical reagent
- when inside the cell DNA –> mRNA –> protein
- goal is to directly target APC cells so that the protein is expressed on the surface (don’t need phagocytosis anymore)
What is SARS COV 2
- severe acute respratory syndrome coronavirus 2
- ssRNA vaccine
- uses RNA-dependent RNA-polymerase for replication
- has 29 proteins
- uses spike protein to bind to ACE2 receptors on surface of cells - lung tissue has a lot of these
how do the COVID vaccines work?
- use a lipid nanoparticle to package modified RNA (pseudouridine) which encodes teh spike protein
- BioNTech uses RNA/LPX that targets dendritic cells
- dendritic cells present the protein to B and T cells
resistance to COVID-19 vaccines
- evolutionary pressure –> selection of mutations in the spike protein
- if the protein changes by 6-8 AA then teh antibody can’t clear it –> resistance
Concerns and contraindications with vaccines
- allergic reaction - rare and to components of vaccine
- off target immune reaction/autoimmune reaction - antibodies generated form vaccine cross react with targets on human cells
- infection - only an issue with attenuated or partially inactived vaccines, if immune system compromised it might lead to infection
- pregnancy - only an issue with attenuated and live vaccines causing risk to fetus
What are antibodies?
- match between APC and B cell receptor stimulates proliferation of B cell –> matures into plasma cell –> secretes antibodies
- antibody antigen complexes are specific
- contain fixed structural region and variable region for targeting antigens
How are recombinant antibodies produced?
- identify antibody from library –> immunize rabits and collect serum (natural diversity)/or perfrom an in vitro phage display/or in silico –> this gives monoclonal antibody
- scale up production - can be performed using expression systems inclusing mammalian or insect cells, yeast, etc
How do monoclonal antibodies function as antivirals and what is their PK?
bind to
- host viral receptors to block entry
- protein in free virus
- viral epitopes expressed on host cell –> stimulates secondary effector cells to activate B/T cells
PK - not orally bioavailable (stomach pH and proteases) - must be injected
- half life is days to weeks
Examples of antiviral monoclonal antibodies
- palivizumab: binds to virus F protein on RSV envelope to prevent fusion
- ibalizumab: binds to CD4 T cell receptors on HIV to block entry
- REGEN-COV: made of 2 mAb and binds to 2 epitopes of the COV spike to block binding to ACE2 Rs, admin subcutaenously after exposure
Hurdles and contraindications with antibody therapies (5)
- difficult to produce and purify: have to use expression systems and purify the cellular contaminants
- can only administer with injection
- can only be used as a prophylatic or in early stage of infection
- immune reaction/allergy
- pregnancy - can pass pregnancy barrier
CRISPR/CAS9 as treatment for HIV
goal: target sequence of viral DNA inside cell and leave it to block replication
CAS9
- 20pb programmable guide RNA and cleaves dsDNA
- excellent specificity, only cuts in cis
HIV: integrated retrovirus
Cas9 can
- disrupt replication genees (1 guide RNA)
- cut out the HIV provirus from host genome (cure) (2 guide RNAS)
- edit host cell so they lack the receptor for HIV to bind
strategy: delivered using safe virus or lipid nanoparticle
CRISPR/Cas13 therapy for COV
COV: ssRNA virus
Cas13
- 30bp programmable guide RNA that cleaves ssRNA
- trans collateral RNA cleavage after cis cleavage (only in bacteria or viruses)
- can use it to cleave viral RNA and block replication –> no collateral cleavage is observed in mammalian cells
strategy: Cas13 as protein or NA with RNA into cell using safe virus or lipid nanoparticle
Contraindications with CRISPR/Cas antiviral therapies
- viral escape/evolution stimulation
- resistance due to mutations in spacer sequence that blocks Cas from cutting –> viruses that have this can be selected and amplified
- cleavage can stimulate this too) - delivery - difficult to get system into all virally infected cells
- immune reactions to Cas if introduced into systemic circualtion
- human genome editing: off target DNA and RNA cleavage of similar human sequences if the enzyme escaped into human cells
