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

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 ACV um % inhibition HSV1 HSV2 VZV Vero CMV WI

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

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.

Mutations per genome # of virions normal Mutagenized by ribavirin “living” “dead”

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 Viral RNA pfu untreated 100um 400um

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 ,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