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