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Transcript
Chemotherapy
Keeping Viral Infections in Check
What are characteristics of an ideal drug?
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•
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•
•
Effective – block spread quickly and not allow persistence
No toxicity – chemotherapeutic index
Manageable resistence
Inexpensive
Oral vs injection
What are possible targets for interference?
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•
•
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Unique and essential - viral
encoded enzymes/proteins or NA
Attachment (soluble
receptors;small drugs)
Fusion – coreceptor blockers
Uncoating - vesicle acidification;
nuclear localization
Nucleic acid synthesis –RT,
polymerases (in others helicases,
primases)
Integration
Transcription - activator
interference
Targets
• Translation - antisense; splicing
inhibitors (Rev/RRE); mRNA
degradation
• No direct protein synthesis
inhibitors are known
• Maturation and release
• Proteases for cleavage
(common in viruses with
polyproteins)
• Packaging – herpes endonuclease to cleave
concatemer
• Release (flu – neuraminidase
inhibitors)
Attachment
• Inhibitor of poliovirus
• Binds in canyon
Fusion
• Fusion inhibitor approval
• Prevents conformational change
in GP41 fusion peptide
Uncoating
• Anti-influenza
• Affects uncoating step by
interfering with virus M2
proton ion channel in
membrane
• Must be given early after
infection
• Resistance problems
Nucleic acid synthesis:nucleoside analogs
• Acyclovir – prodrug
• Chain termination
• Derivatives – better oral
availability; different
spectrum
Dose response curve of acyclovir and herpes
viruses and cells
HSV1
HSV2
VZV
Vero
CMV
WI38
%
inhibition
50
0.5
ACV um
10
100
500
• TAT - transactivator of
transcription needed for
efficient transcription of HIV
• TAT binds to TAR in nascent
RNA and lets polymerase
elongate
• Initially low level of
transcription until TAT levels
rise
• What are possible targets?
• Two different cell lines
transfected with different
luciferase genes under control
of pHIV-LTR and pCMV with
pCMV Tat
• Added to some are an antiTAR
polyamide nucleotide analog
with/wo link to transportin that
gets it into cell
• Bottom row - scrambled
nucleotide sequence
• What do results show?
• Why might this approach have
an advantage over targeting
Tat?
• How would you show that it
prevents virus replication?
Rev controls splicing and shift to late gene
expression
siRNAs
• Protease critical for cleaving
structural proteins to final
configuration
• Works in particle maturation
HIV after HAART - plasma RNA levels by PCR
• Highly active antiretroviral
therapy
• Combination therapy to
minimize resistance
• Can we cure HIV infection?
Rational Drug Design
• Sialic acid analog
• Reduces symptoms about 1 to 3
days
Fishing for antivirals
• Combinatorial chemistry with good screen test – can do 50000
compounds a day
– Chemical libraries for big firms – any drug with some reaction can
be modified
– Screening assays
• Preventing replication
• Transcriptional regulation - luciferase expression
• Protease inhibitor - modify tetracycline efflux protein in
bacteria to have protease sensitive site
– transform with protease gene (makes cell sensitive to tet);
– then add putative inhibitor and see if tet-R or tet-S
•Phage libraries of peptides
–Binding assay to target
What is on the horizon?
Cell conc
HIV
LTR
toxin
• Inducible toxins
• Hiv LTR connected to toxin
– HSV TK (treat with
acyclovir)
– diphtheria toxin
• What happens in body?
Inducing apoptosis is HIV infected cells
• Tat protein linked to caspase
protein modified to have HIV
protease cleavage sites to
activate
• Tat transduces cells with
caspase protein or mutant
version of protein
• Also can transduce with TatHIV protease
Drugs that inhibit Nef
• IKA inhibits surface molecule
endocytosis
• Looked for its effect on CD4
presentation
• What would you expect for
MHC presentation?
Ribavirin - broad spectrum
• Used to treat hepatitis, RSV,
Hantavirus, Lassa fever
• Mode of action - RNA error
catastrophe
• Many viruses evolve rapidly
(particularly RNA) – a plus for
them to adapt; but if mutation
rate increases slightly the
population will no longer be
viable
• Hypothesis: Ribavirin is a
mutagen that works by shifting
viruses to error catastrophe.
normal
Mutagenized by
ribavirin
“living”
# of
virions
“dead”
Mutations per genome
Interferons
Treatment of chronic Hepatitis B
• HBV infection
• Partial dsDNA virus
• Infection - completes circle,
makes RNA copy and then with
RT makes partial dsDNA
• Incidence global - 50 million
• US - 140 - 320,000 cases
• Chronic rates - more common
in children with HBV
• Leads to cirrhosis, liver failure,
liver cancer
Antiviral resistance
•
•
•
•
Viral mutation frequency - error rate of replicase
Intrinsic mutability of the antiviral target site
Selective pressure exerted by the drug
Rate of virus replication
Anti IF strategy of HCV
• NS5a binds to PKR and
inactivates
• E2 gene has 12 aa homology to
autophosphorylation site of
PKR and eIF2a
• How do IFres and Ifsens differ?
• How might that help the virus?
Do PKR and E2 bind?
•
•
•
•
His tag binds to beads
Isolate and run on gel
Wt PKR
K296 = mutant in ATP binding
domain
• E2-C - no Phos site
• Hn - cell protein control
Does E2 interfere with PKR activity?
• ATP- P32
• PKR +/- E2 and in presence of
dsRNA activator and substrate
H2a