chemotherapy keeping viral infections in check. what are characteristics of an ideal drug? effective...
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Chemotherapy
Keeping Viral Infections in Check
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What are characteristics of an ideal drug?What are characteristics of an ideal drug?
• Effective – block spread quickly and not allow persistence
• No toxicity – chemotherapeutic index
• Manageable resistence
• Inexpensive
• Oral vs injection
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What are possible targets for interference?What are possible targets for interference?
• 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
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TargetsTargets
• 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)
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AttachmentAttachment
• Inhibitor of poliovirus
• Binds in canyon
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FusionFusion
• Fusion inhibitor approval
• Prevents conformational change in GP41 fusion peptide
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UncoatingUncoating
• Anti-influenza
• Affects uncoating step by interfering with virus M2 proton ion channel in membrane
• Must be given early after infection
• Resistance problems
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Nucleic acid synthesis:nucleoside analogsNucleic acid synthesis:nucleoside analogs
• Acyclovir – prodrug
• Chain termination• Derivatives – better oral
availability; different spectrum
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Dose response curve of acyclovir and herpes Dose response curve of acyclovir and herpes viruses and cellsviruses and cells
ACV um
% inhibition
HSV1HSV2 VZV Vero CMV
WI38
0.5 10 100 500
50
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• 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?
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• 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?
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Rev controls splicing and shift to late gene Rev controls splicing and shift to late gene expressionexpression
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siRNAssiRNAs
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• Protease critical for cleaving structural proteins to final configuration
• Works in particle maturation
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HIV after HAART - plasma RNA levels by PCRHIV after HAART - plasma RNA levels by PCR
• Highly active antiretroviral therapy
• Combination therapy to minimize resistance
• Can we cure HIV infection?
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Rational Drug DesignRational Drug Design
• Sialic acid analog
• Reduces symptoms about 1 to 3 days
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Fishing for antiviralsFishing 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
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•Phage libraries of peptides–Binding assay to target
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What is on the horizon?What is on the horizon?
• Inducible toxins
• Hiv LTR connected to toxin
– HSV TK (treat with acyclovir)
– diphtheria toxin
• What happens in body?
toxinHIVLTR
Cel
l con
c
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Inducing apoptosis is HIV infected cellsInducing 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 Tat-HIV protease
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Drugs that inhibit NefDrugs that inhibit Nef
• IKA inhibits surface molecule endocytosis
• Looked for its effect on CD4 presentation
• What would you expect for MHC presentation?
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Ribavirin - broad spectrumRibavirin - 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.
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Mutations per genome
# of
virions
normalMutagenized by ribavirin
“living”
“dead”
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InterferonsInterferons
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Treatment of chronic Hepatitis BTreatment 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
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Antiviral resistanceAntiviral 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
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Anti IF strategy of HCVAnti 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?
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Do PKR and E2 bind?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
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Does E2 interfere with PKR activity?Does E2 interfere with PKR activity?
• ATP- P32
• PKR +/- E2 and in presence of dsRNA activator and substrate H2a