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Strategies for Antiviral Drug Discovery



Historical strategy trial and error Current strategies

target identification Substrate-based approaches Biostructural approach

lead development

Tools for Antiviral Drug Development



Genomics
Microarrays Determine host response to viral Infection

Proteomics
Crystal Structures Binding Sites Protein Data Bank

HCV NS3 protein (helicase) bound to DNA

Characteristics


A successful antiviral will

1. Interfere with a virus-specific function 2. Interfere with a cellular function so that the virus cannot replicate

An ideal drug will be

1. Water-soluble 2. Stable in the blood stream 3. Easily taken up by cells

An ideal drug should NOT be

1. Toxic 2. Carcinogenic 3. Allergenic 4. Mutagenic 5. Teratogenic

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Protease Inhibitors)

Anti-Herpes viruses


Nucleoside Analogues
       

Nucleotide Analogues
 

Acyclovir Ganciclovir Penciclovir Brivudine Sorivudine Idoxuridine Trifluridine Vidarabine PRODRUGS Valaciclovir Famciclovir Valganciclovir

Cidofovir Adefovir

Pyrophosphate Analogues


Foscarnet

Newer
 

   

Fomivirsen Docosanol

Under development
 

Maribavir Lobucavir Helicase Primase Inhibitors

  

BAY57-1293 BILS179 BS BILS45 BS

Anti-Retroviruses


Nucleoside RT Inhibitors
         

NNRTI
    

Zidovudine Didanosine Stavudine Zalcitabine Lamivudine Abacavir Tenofovir Dideoxycytidine Combivir Trizivir


Nevirapine Delaviridine Efavirenz TIBO HEPT Tipranivir BMS-232632

Protease Inhibitors
     

 

 

Saquinavir Ritonavir Indinavir Nelfinavir Amprenavir Lopinavir Fosamprenavir Atazanavir Caparavirine DPC-083

 

Fusion Inhibitors


 

Emivirine FTC

Enfuvirtide

Others


Anti-Hepatitis
     

rh-IFN peg-IFNs Ribavirin Lamivudine Adefovir dipivoxil Tenofovir Emricitabine Entecavir Clevudine -L Deoxythymidine

Anti-Orthomyxoviruses/ Paramyxoviruses
   

Papilloma v.


Afovirsin

Amantidine Rimantidine Zanamavir Oseltamivir RSV Inhibitors Ribavirin Palivizumab

Rhinoviruses
  

sICAM-1 Pleconaril AG7088

      

Influenza


RWJ 270701

Picornavirus Inhibitors
  

Enteroviruses


Pirodavir WIN-54954 Enviroxime

Pleconaril

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Assembly protein inhibitors)

Attachment Inhibitors


Neutralizing Antibodies
Passive Immunization Adoptive T-cell therapy

Receptor Antagonists
Sugar/ peptide analogues of cell receptor Chemokine receptor inhibitors

Enfuvirtide- the first fusion inhibitor

By 1993, antiretroviral activity Approved by FDA in March, 2003 36aa peptide, a peptido-mimetic of the HR2 domain in gp41 Bind to HR1 domain, prevent formation of 6-helix structure

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Assembly protein inhibitors)

Uncoating Inhibitors


Arildone and WIN compounds inhibit uncoating of picornaviruses



  

WIN inserts a canyon which fits into a hydrophobic pocket in VP I protein of virus and blocks ion transport Blocks conformational change that occurs when the virus and ICAM-1 interact stabilizes the structure of the virus Pore formation does not occur

Amantadine
approved in 1966


M2 ion channel inhibitor of Influenza virus

 

Prevents uncoating/nuclear import Prevents viral replication initiation



Inhibits acid mediated dissociation of RNP complex

Problems - not all Influenza viruses have the same ion channel Influenza B virus has BM2 - also part of the M gene but different ion channel properties


therefore no effect of amantadine on Influenza B

Side effects CNS ~ nervousness, insomnia, difficulty in concentrating Dosing

 

must be given within 24-48 h of initial infection reduce duration of illness

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Assembly protein inhibitors)

Transcription Inhibitors


IFNs


Potent cytokines
  

Antiviral Anti proliferative Immunomodulatory actions

IFN
 

I , , , II Mainly , have antiviral actions Stimulate cytotoxic activity of lymphocytes, NK cells, macrophages Upregulate MHC-I Ag

  

Activate JAK-STAT signal transduction pathway leading to nuclear translocation of a cellular protein complex


Binds to genes leading to synthesis of proteins contributing to viral resistance

IFN


Not effective by oral therapy



i/m, s/c

Short T1/2
  

IFN 40 min r-IFN 4 hr r-IFN 0.5 hr

Attachment to PEG molecules

  

Clearance Antigenicity Allows once weekly dosing

S/E Myelosuppression, neurotoxicity, ac. Influenza like syndrome, hypotension, tachycardia Uses
    

Chr. infections of HBV, HBV associated syndromes, chr. HDV with HBV Chr./ Ac. HCV infections Condyloma acuminata Genital/ mucocutaneous HSV, localized HZ CMV in renal transplants

Antisense Oligonucleotides
 

stearic hindrance induction of RNaseH

  

poor bioavailability degraded by nucleases very rapidly delivery systems ~ liposomes, nanoparticles

Fomivirsen
Antisense 21-mer phosphorothioate oligonucleotide Complementary to the immediate-early gene of hCMV Approved in 1998 treat CMV retinitis in AIDS.

Replication Inhibitors


DNA polymerase inhibitors

Reverse transcriptase inhibitors

DNA Polymerase Inhibitors



Acyclovir
One of the most successful antiviral drugs, 1983 HSV-1&2, VZV
nucleoside analog With an acyclic side chain lacks the 3-OH Blocks virus replication

a prodrug

converted to active form first by viral thymidine kinases, and then by cellular kinases

formation of the triphosphate form which leads to chain termination by the viral DNA polymerase

Basis for selectivity



Thymidine kinase (TK) is also a cellular enzyme,

Why is acyclovir such a good antiviral drug?

viral TK has a much broader substrate specificity than cellular TK ~ cellular TK does not recognize acyclovir ACV-TP is 50 more potent inhibitor of viral vs cellular DNA polymerase

Uses
   

Treatment and suppression of HSV infections in normal host Varicella and Herpes Zoster in normal hosts HSV and VZV infections in immunosuppressed patients Prophylaxis and suppression of HSV, VZV, CMV in transplant recipients

Resistance
HSV and VZV develop resistance due to three mechanisms
(a) Reduced/absent synthesis of viral TK (b) Altered substrate specificity of viral TK (c) Altered viral DNA polymerase

Derivatives of Acyclovir
   

addition of side chains oral delivery specificity decrease toxicity

Famciclovir
  

Disacetyl ester prodrug of Penciclovir Serve as a competitive inhibitor of viral DNA p Has a 3OH so not an obligate terminator of DNA synthesis but is a chain inhibitor

Ganciclovir
 

Special activity against CMV Phosphorylation achieved by

 

Viral TK Viral phosphotransferase

  

Eventually causes cessation of DNA chain elongation Myelosuppressive Approved for treatment & chr. suppression of CMV retinitis in immunocompromised and prevention of disease in Tx recipients. Resistance ~ mutation in UL 97 ~ viral DNA p

Idoxuridine
  

Iodinated thymidine analog Faulty DNA formed which breaks down easily Faulty transcription Uses


Only indication is HSV keratoconjunctivitis

Brivudine
 

Conversion of BVDU-MP & BVDU-DP by HSV TK Less active against HSV 2

Vidarabine
  

Adenosine analogue tri-P competitively inhibits deoxyadenosine tri-P Inhibits

  

Ribonucleoside reductase RNA polyadenylation S-adenosylhomosysteine hydrolase

Nucleotide Analogues


Cidofovir


Acyclic nucleotide phosphonate Does not require specific conversion to mono-P Targets the virus specific DNA p Very long tissue T1/2


 

Once weekly dose

Nephrotoxicity Uses


Sight threatening CMV retinitis in AIDS

Resistance


Mutations in viral DNA p

Pyrophosphate Analogues
Foscarnet


Organic analogue of inorganic pyrophosphate




Binds to DNA polymerase at its pyrophosphate binding site

Blocks DNA synthesis

Uses


CMV disease in immunocompromised patients, including disease caused by ganciclovir-resistant CMV Acyclovir-resistant HSV and VZV

Problems


Insoluble, only IV

Side Effects
 

Nephrotoxicity Symptomatic hypocalcemia

Resistance


Point mutation in DNA p

Ribavirin


Guanosine analog


Phosphorylated by cellular enzymes mono-P interferes with GTP synthesis tri-P competitively inhibits GTP dependent 5 capping of viral m-RNA

Immunomodulatory effects

Uses
 

Wide spectrum antiviral activity RNA viruses; paramyxoviruses, flaviviruses, picornaviruses, orthomyxoviruses.

Reverse transcriptase (RT) inhibitors

Azidothymidine(AZT)


One of the first anti-HIV drugs Also a nucleoside analog ~ chain terminator Targets reverse transcriptase (RT) Converted to triphosphate form


but only by cellular enzymes

HIV is 100 more susceptible to inhibition by AZT than the host cell DNA polymerase Less active in quiescent cells

Side Effects
Bone marrow suppression ~ granulocytopenia, anemia Myalgia Nausea Fatigue Headache Abnormal liver function

Uses
Patients with HIV infections in combination with at least two other antiretroviral agents Prevention of maternal/fetal HIV transmission as monotherapy.

Resistance


Point mutation in RT codons ~ 41, 67, 70, 215 & 219

Non-nucleoside reverse transcriptase inhibitor (NNRTI)



Nevirapine (NVP) ~ 1996

   

Binds adjacent to the catalytic site Induces conformational changes ~ inactivation of the enzyme S/E ~ rashes, elevated liver enzymes Used in combination for HIV

Delaviridine (DLV) ~ 1997

 

Binds to a hydrophobic pocket in p66 subunit of RT Rashes, severe dermatitis

Efavirenz (EFV) ~ 1998

 

Binds adjacent to the catalytic site Headache, dizziness, abnormal dreams, avoided in pregnancy

Basic properties of NNRTIs



Highly potent ( effective at nanomolar concentrations) Resistance can develops through single mutations in RT codons


Can develop after a single dose in monotherapy

Delavirdine and Nevirapine are antagonistic in action on the HIV RT activity ~ not used together Interaction with some drugs occurs due to induction and/or inhibition of cytochrome P450 enzymes Rash is common especially in women

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Assembly protein inhibitors)

Translation Inhibitors


IFN therapy Ribozymes

Enzymes containing only RNA Cut at a specific sequence

siRNA (small interfering RNA)

RNAi against virus

RNAi against viruses



RNAi been successfully used against viruses in mammalian cells



RSV, HIV, Dengue virus, Rhesus rotavirus (RRV), Semliki forest virus (SFV), Influenza virus, HBV, HCV, etc.

Major challenge in turning RNA interference into an effective therapeutic strategy is the delivery of the RNA interference agents

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Protease Inhibitors)

Protease Inhibitors


Aspartic acid protease



99 AA monomers, homodimer

 

1000 higher affinity Binds to the active site of protease



Prevents cleaving

Protease Inhibitors
Peptidomimetic PIs


Moderate or poor oral bioavailability Short T1/2 Highly bound to serum proteins (compromises activity in vivo) Frequent, high doses (several grams) ~ diminished patient compliance Cross-resistance to against mutant HIV strains


Step wise mutations of protease gene ~ suboptimal doses predisposes

Significant side effects



Paresthesias, hypertriglyceridemia, glucose intolerance, visceral fat, buffalo hump

Protease Inhibitors
Non-Peptidomimetic PIs


Tipranavir In late Phase III trials

90% of HIV isolates highly resistant (more than ten-fold) to approved PIs indinavir, nelfinavir, ritonavir, and saquinavir highly sensitive to tipranavir

Show little cross-resistance with peptidomimetic PIs

(Fusion protein inhibitors)

(Ion channel blockers Capsid stabilizers)

7. Release
(NA inhibitors Abs) (Inhibitors of viral DNA p RNA p RT helicase primase integrase)

& PT Modifications
(IFNs Assembly protein inhibitors)

Release inhibitors


Zanamivir and Oseltamivir

Neuraminidase inhibitors Target- active site of enzyme Sialic acid analog replacement of the 4 OH with an amino or guanidinyl group Blocks virus release Both influenza A and B blocked Taken very early No effect on non-influenza neuraminidases

Scientific American Jan 1999:78-87

Antiviral Drug Susceptibility Testing



Essential
   

Defining mechanisms of resistance Determining the frequency testing cross resistance Evaluating new antiviral drugs

Helpful in
  

Persistent/worsening HSV or VZV in pts taking ACV Persistent/worsening CMV retinitis, pneumonitis, colitis Monitoring for antiretroviral resistance

Resistance testing
 

Phenotyping and genotyping Expressed as an altered IC50 or IC90

Antiviral Drug Susceptibility Testing



Phenotypic assays
        

PRA DU DNA Hybridization EIAs Flowcytometry NI Assay YRA PBMC cocultivation Recombinant virus assays

Genotypic assays
  

DNA sequencing PCR-RFLP Microarray hybridization

Antiviral Phytochemicals


Several hundred plant and herb species that have potential as novel antiviral agents have been studied

A wide variety of active phytochemicals



Flavonoids, terpenoids, lignans, sulphides, polyphenolics, coumarins, saponins, furyl compounds, alkaloids, polyines, thiophenes, proteins and peptides have been identified.

Some volatile essential oils of commonly used culinary herbs, spices and herbal teas have also exhibited a high level of antiviral activity Extracts of fruits, vegetables, grains, bark, roots, stems, leaves and flowers.

Antiviral Phytochemicals


Several of these phytochemicals have complementary and overlapping mechanisms of action

 

Antiviral effects by either inhibiting the formation of viral DNA or RNA Enhancing immunity

To be evaluated against diverse viruses families such as Retroviridae, Hepadnaviridae and Herpesviridae.

Antiviral Phytochemicals


Phyllanthus amarus ~ Hepatitis B



3 substance isolated ~ tablets

  

Dr. S.P. Tyagarajan

Hepavirin ~ Virohep Successfully cleared HBsAg from carriers Efficacy 30-40%

Neem ~ HIV, Herpes, Chicken Pox



NIH ~ in-vitro studies



Extract kills HIV

Leaves and bark ~ clinical trials

Melalenca alternifolia (tea tree)

 

Terpinen-4-ol ~ high antiviral activity against HSV 1&2 Black tea flavinoid ~ theaflavin


Antioxidant/ free radical scavanging activity

Thank You