HIV as a model of rational drug design

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HIV as a model of rational drug design

• Objectives Rational drug design approaches now
  dominate the drug discovery process.  HIV
  infection and AIDS represent one of the first
  diseases for which the discovery of drugs was
  performed entirely via a rational drug design
  approach.  At the end of these lectures the
  student will have an overview of the components
  and life cycle of the HIV virus and of the steps
  involved in rational drug design, with emphasis
  on HIV and AIDS. 

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HIV early history

• 1981 5 cases of a rare type of pneumonia,
  Pneumocystis carinii
• Over next 30 months 26 cases of Kaposi's
  sarcoma   
• Increase in occurrences of chronic
  lymphadeonopathy and non-Hodgkin's lymphoma.
• 1982 Acquired immunodeficiency syndrome (AIDS).
• 1984 HIV isolated and serological test to
  identify virus developed (ELISA).
• Origins of HIV go back to 1959 or earlier (via
  primates)

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AIDS population distribution2006
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Steps to development of therapies for the
treatment for HIV infection and AIDS

• 1) Identification of cause (done in 1984)
• 2) Analysis of life cycle           Viral
  components           Biochemical pathways
• 3) Identification of sites of attack
• 4) Design of therapeutic agents          
  "traditional" antiviral agents (nucleoside
  analogs)           novel, rationally designed
  compounds

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Viral Components
Outer lipid bilayer gp120 (SU), gp41 (TM) Inner
side of bilayer p17 (MA) Inner conical
capsid p24 (CA) Capsid interior p6 and p7 (NC),
p11 (PR), p51 and p15 (RT), p34 (IN), Regulatory
proteins in the viral particle Nef, Vir, Vpr
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Non-expressed RNA

• 5LTR (long-terminal repeat)
• 3LTR
• Essential for initiation of transcription of RNA
  to DNA

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Structural proteins

• gp160 gp120 (SU) and gp41 (TM)
• p17 (MA)
• p24 (CA)
• p6 and p7two nucleocapsid proteins (NC)
• Located on the Gag and Env genes

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Catalytic proteins

• Reverse Transcriptase/RNase H (RT)
• Integrase (IN)
• Protease (PR)
• Located on the Pol gene

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Regulatory proteins/nucleic acids

• Regulatory proteins packaged in the virus
• Nef
• Vif (virion infectivity factor)
• Vpr     Regulatory proteins NOT packaged in the
  virus
• Tat activated by TAR (RNA trans-activation
  responsive region)
• Rev
• Vpu    
• DNA cis-acting elements     

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Frame shifting Same regions of the genome used
to encode different proteins

• Initiation of transcription (or translation) at a
  different base pair leads to a different amino
  acid sequence.  Allows for a maximum of three
  proteins to be obtained from a single DNA or RNA
  sequence.
•      CUC,ACG,CUU,A      Leu Thr Leu  frame
  shift by 1      C,UCA,CGC,UUA       Ser
  Arg Leu
• Gene splicing can also contribute via
  reorganization of introns.

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HIV 5 levels of attack
3 levels are the 3 possible proteins from 1
gene sequence Viral RNA (TAR) Viral DNA (DNA
cis acting element) Indicative of the efficiency
of HIV maximizes the use of its limited genetic
code.
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HIV-1 life cycle
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Infection/Early phase
1) HIV surface gp120 and transmembrane gp41 Env
proteins interact with target cell CD4 membrane
glycoprotein and then with a host "fusion"
(chemokine) co-receptor (cell to cell infection
can occur). 2) Viral fusion/internalization and
nucleocapsid uncoating 3) RNA genome is reverse
transcribed to dsDNA by RT (Vif facilitated,
5LTR and 3LTR essential) 4) Integrated as
provirus into host cell genome by IN in PIC
complex
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Initial attachment of HIV to a T cell
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Viral reproduction/late phase
1) Regulatory proteins Tat, Rev, Nef initially
synthesized in elevated levels. In combination
with cellular proteins and other HIV regulatory
proteins (Vif, Vpr and Vpu) activate expression
of remainder of viral genome. 2) Full length and
singly spliced HIV mRNA transcripts (Gag and
Gag-Pol genes) exported from nucleus to cytoplasm
mediated by Rev (cellular proteins assist this
process, which is facilitated by Tat) 3) gp160
synthesized and transported to cell membrane to
initiate viral synthesis 4) Host CD4 degraded
(facilitated by Vpu and Nef) 5) Gag and Gag-Pol
polyprotein synthesized and processed 6)
Rearrangement of structural proteins followed by
maturation of entire virus
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HIV cytopathic effects
Syncytium clusters of HIV particles and T
cells Apoptosis of T-helpers cells (CD4
cells) Other sites of attack Central nervous
system Gastrointestinal system Hematopoietic
system
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Potential sites for the rational design of
therapeutic agents

• CCR5 and CXCR4 (Maraviroc, entry inhibitor)
• gp41 (T-20 peptide, entry inhibitor)
• gp120 (vaccine target??)
• Reverse Transcriptase (nucleoside and
  non-nucleoside)
• Integrase (Raltegravir)
• TAT-TAR complex
• Protease (Saquinavir etc.)
• Zinc Finger proteins
• Maturation inhibitors (PA-457, Phase II trials)

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Reverse Transcriptase Inhibitors
Multiple nucleoside and non-nucleoside RT
inhibitors including AZT, ddI, ddC,
2-deoxy-3-thiacytidine and more.
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General Problems with HIV Therapies

• Persistence (latency)
• Resistance due to mutations
• HAART Highly active antiretroviral therapy
  (combination therapy)

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Requirements for an HIV targeted therapeutic agent

• 1) Ki values in nanomolar to subnanomolar range
• 2) Adequate specificity
• 3) Adequate bioavailability

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Approach to rational design of antiHIV agents

• 1) Base initial structure on physiological
  peptide substrate
• 2) transition state analog of peptide bond
  hydrolysis
• 3) replace amide bonds
• 4) minimize molecular weight
• 5) enhance binding and specificity based on 3D
  structure of the enzyme active-site

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1) Base initial structure on physiological
peptide substrate
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2) Transition state analog of peptide bond
hydrolysis
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3) Replace amide bonds, 4) minimize molecular
weight, 5) enhance binding
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Structure of Saquinavir
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Additional assays on best compound

• i)  less then 50 inhibition of human aspartic
  proteases at 10 mM           renin, pepsin,
  cathepsin D and cathepsin E (check of
  specificity)
• ii) no effect on serine, cysteine and metallo
  proteases (check of specificity)
• iii) antiviral activity         IC50 of 2 nM
  versus HIV infected C8166 cells           
  compared to  3 to 30 nM for AZT         IC50 of
  2.5 nM versus HIV infected JM cells           
  compared to ca. 30 nM for ddC            AZT,
  inactive up to 100 mM due to poor phosphorylation
  
•         IC50 of 1 nM versus HIV infected CEM
  cells
• iv) Toxicity studies           TD50 from 5 to
  100 mM in JM and C8166 cells

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Clinical applications

• Synergestic with zidovudine, didanosine or
  zalcitabine
• drug resistance develops if used alone
• poor bioavailability
• well tolerated (diarrhea, nausea and abdominal
  pain)
• induction of cytochrome P450 by certain drugs may
  lower serum levels and may interfere with
  metabolism of other drugs

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Agouron (now Pfizer!) development of new HIV
protease based agent(lead optimization)

• Goals Improve poor bioavailability     
  Improve binding constant      Decrease molecular
  weight

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Iterative approach applied to develop the
compound AG1343 by Agouron
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Structures of Saquinavir and Viracept
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Clinical information on Viracept

• 1) Ki of 2 nM for inhibition of HIV protease 2)
  EC95 from 7 to 196 nM versus several clinic
  isolates of HIV-1 and HIV-2 3) EC95 values
  between 0.005 to 0.08 mg/ml 4) LD50 values
  between 23 and 28 mM 5) Therapeutic index of 18
  mg/ml (related to EC and LD values) 6) additive
  with didanosine or stavudine 7) synergestic with
  zidovudine, lamivudine or zalcitabine       
  combination therapy is clinically more effective
  because it        requires mutation of two
  enzymes (protease and RT) to become resistant 8)
  single and double mutants in HIV protease lead to
  resistance       Ile 84 to Val             
  imparts cross resistance to other protease
  inhibitors      Arg 8 to Lys and Met 46 to Ile
       Ile 84 to Val and Met 46 to Ile

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Clinical information on Viracept

• 10) mutants with resistance to other protease
  inhibitors are often still susceptible to
  Viracept 11) Protease as well as viral growth
  shown to still be significantly inhibited 36 hrs
  after discontinued administration of the drug
        Time of dose not as important       may
  make it more difficult for resistance to develop
  12) Formulation aspects         very stable
          favorable solubility of mesylate salt
  allows for oral administration         tmax of 2
  to 4 hours         volume distribution of 2 - 7
  L/Kg         highly protein bound (gt 98 )
          care must be taken in patients with
  varying plasma protein levels due to hepatic
  problems         plasma levels remain above ED95
  for over 6 hours         concentrations
  substantially above ED95 in lymph nodes and
  spleen                   sites were HIV is
  particularly active         oxidized by
  cytochrome P450 system (as with Saquinavir)
          avoid coadministration with certain
  compounds         87 of drug recovered in feces
          22 of parent drug excreted
  unmetabolized         78 oxidative metabolites
  13) one compound with activity equal to parent
  drug identified (new lead???)

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Successes and Failures of antiHIV therapy

• 1) 60 - 90 decrease in deaths from HIV infection
• 2) viremia not controlled in 40-50 of treated
  cases
• 3) Long term side effects increased risk of
  diabetes mellitus increased risk of
  cardiovascular disease avascular necrosis of
  the hip     lipoatrophy via mitochondrial
  toxicity bloated abdomens with thin limbs
• 4) Limited compliance due to large and frequent
  dosages
• 5) Cross-reaction of protease inhibitors with
  other drugs
• 6) High cost of treatment (7,000 to 12,000/year)
        

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Alternatives for prevention/treatment HIV
infection and AIDS

• Vaccines
• Gene therapy
• Topical Control of HIV Transmission
• Mutagens