Chemotherapy Keeping Viral Infections in Check. What are characteristics of an ideal drug? Effective...
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Transcript of Chemotherapy Keeping Viral Infections in Check. What are characteristics of an ideal drug? Effective...
Chemotherapy
Keeping Viral Infections in Check
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
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
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)
AttachmentAttachment
• Inhibitor of poliovirus
• Binds in canyon
FusionFusion
• Fusion inhibitor approval
• Prevents conformational change in GP41 fusion peptide
UncoatingUncoating
• 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 analogsNucleic acid synthesis:nucleoside analogs
• Acyclovir – prodrug
• Chain termination• Derivatives – better oral
availability; different spectrum
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
• 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 Rev controls splicing and shift to late gene expressionexpression
siRNAssiRNAs
• Protease critical for cleaving structural proteins to final configuration
• Works in particle maturation
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?
Rational Drug DesignRational Drug Design
• Sialic acid analog
• Reduces symptoms about 1 to 3 days
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
•Phage libraries of peptides–Binding assay to target
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
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
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?
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.
Mutations per genome
# of
virions
normalMutagenized by ribavirin
“living”
“dead”
InterferonsInterferons
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
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
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?
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
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