Chemotherapy

Keeping Viral Infections in Check

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?

• • • • • • •

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

• Anti-influenza • Affects uncoating step by interfering with virus M2 proton ion channel in membrane • Must be given early after infection • Resistance problems

Uncoating

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

CMV HSV2 VZV Vero WI38 HSV1 % 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

HIV LTR toxin

What is on the horizon?

• 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 Tat HIV 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.

# of virions normal Mutagenized by ribavirin “living” “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