Chemotherapy

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Chemotherapy

• Keeping Viral Infections in Check

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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?

• Unique and essential - viral encoded
  enzymes/proteins or NA
• Attachment (soluble receptorssmall 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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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)

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Attachment

• Inhibitor of poliovirus
• Binds in canyon

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Fusion

• Fusion inhibitor approval
• Prevents conformational change in GP41 fusion
  peptide

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Uncoating

• 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 synthesisnucleoside analogs

• Acyclovir prodrug
• Chain termination
• Derivatives better oral availability different
  spectrum

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Dose response curve of acyclovir and herpes
viruses and cells
CMV
Vero
VZV
HSV2
WI38
HSV1
inhibition
50
0.5
10
100
500
ACV um
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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
expression
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siRNAs
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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 PCR

• Highly active antiretroviral therapy
• Combination therapy to minimize resistance
• Can we cure HIV infection?

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Rational Drug Design

• Sialic acid analog
• Reduces symptoms about 1 to 3 days

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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

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• Phage libraries of peptides
• Binding assay to target

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What is on the horizon?

• Inducible toxins
• Hiv LTR connected to toxin
• HSV TK (treat with acyclovir)
• diphtheria toxin
• What happens in body?

HIV LTR
toxin
Cell conc
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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 Tat-HIV protease

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Drugs 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 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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Mutagenized by ribavirin
normal
living
of virions
dead
Mutations per genome
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Affects of ribavirin

• Infected cells /- drug
• Over time measured PFU
• loss increases with drug conc 3 to 2000 fold
  reduction
• Therefore direct effect on genome viability

untreated
pfu
100um
400um
Viral RNA
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Interferons
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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

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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

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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?

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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?

• ATP- P32
• PKR /- E2 and in presence of dsRNA activator and
  substrate H2a