Chem 195 Drug Discovery Lecture 5

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Chem 195 Drug DiscoveryLecture 5

• Intellectual Property
• Molecular Pharmacology
• Quantitation of Modulation of Receptor and Enzyme
  Action

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Chem 195 - 3/1/2002

• Housekeeping
• Files on web page - Lecture5.ppt and hts2.pdf
• Possibility of change in day 3/22 to 3/20 for
  guest lecture by Dr. Dirksen Bussiere on
  structure based drug discovery
• Lectures by Ed Scolnick, President Merck Research
  Labs, 3/4 - Drugs for Mental Illness, 4 pm, 100
  Lewis Hall 3/5 Drug Discovery - Science and
  Cost, Sibley Auditorium 4 pm
• Visit to Chiron HTS facility 3/8 - meet at North
  Side of Stanley Hall at 130pm - need one more
  driver

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Intellectual Property

• Patents and Trade Secrets
• Time limited right to exclude others from
  making, using, or selling your invention
• Useful
• Novel
• Not Obvious
• Enablement - To one skilled in the art
• Cant patent something which exists in Nature
• Composition of Matter vs. Use vs. Tool patents
• Genes, genomes, and who owns what?

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Patents

• Description and Background
• Specific Embodiments
• Claims
• What you actually get
• Negotiated with the Patent Examiner

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Useful WEB Resources on IP

• http//www.uspto.gov/patft/index.html
• http//www.ajobonline.com/wol.php?taskviewarticl
  eID53

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Intellectual Property

• Patents on Biological Entities
• What is patentable? What claims to you get?
• A partial gene sequence?
• A complete gene sequence?
• A gene sequence with a function?
• A gene and protein sequence?
• A human gene if you have a rodent gene sequence?
• A 95 identical sequence to one you have
  described?
• A small molecule ligand that binds to the protein
  encoded by your gene?
• ..

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Intellectual Property

• Chemical Patents - what claims do you get?
• The molecules you made
• Molecules which are related?
• Molecules you can think of?
• Processes for making molecules
• Tools for making molecules
• Quantitative descriptions - specific stereoisomers

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Molecular Pharmacology

• Quantitative Analysis of Target Function
• How Target Activity is Measured
• Characterization of Drug Hits and Leads
• Molecules or Cells as Targets

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Enzymes as Targets

• Biological catalysts
• Almost always proteins but RNA can catalyze
  reactions as well
• Enzymes catalyze reactions in the intra or
  extracellular environments
• Tremendous rate accelerations are achieved - up
  to 17 orders of magnitude!!
• How? Take Chemistry 230 and find out.

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Enzymes as Targets

• Configuration of enzyme assays
• Detection of product formation or substrate
  depletion
• Which is better?
• Methodologies
• Spectroscopy is most common but also
  chromatography, scintillation or gamma counting,
  fluorescence methodologies have become the
  methods of choice (discussed further next week)

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Enzyme Targets

• How to determine enzymatic rates
• Steady state analysis depends on a determination
  of the dependence of the rate on substrate
  concentration.
• Once this is determined one can analyze the
  behavior of putative inhibitors
• Single turnover assays can be used but are rare
  in drug discovery

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Enzymes as Targets

• Michaelis-Menten Equation Derivation
• Assumptions
• E S ES E P (k-2 0)
• Etot E ES and Stot gtgt Etot
• Rate (v) k2ES and Vmax k2 Etot
• At steady state d(ES)/dt -d(ES)/dt
• The concentration of ES is a constant

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Enzymes as Targets

• Michaelis-Menten Equation Derivation

E S ES E P (k-2 0)
If ES constant then k1(Etot - ES)S (k-1
k2)ES S(Etot - ES) (k-1 k2) Km
Michaelis Constant ES k1
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Enzymes as Targets

• Michaelis-Menten Equation Derivation

SEtot - SES KmES EtotS ES(S
Km) ES EtotS

v k2ES
S Km
v k2 EtotS VmaxS
S Km
S Km
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Enzymes as Targets
v k2 EtotS VmaxS S Km
S Km
Rate depends on S as a rectangular hyperbola At
S Km, v Vmax/2
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Enzymes as Targets
Steady State Behavior with a Competitive
Inhibitor - What happens?
k1
k2
E S ES E P (k-2 0)
k-1
k-i -I
I ki
Ki EI k-i EI ki
EI
v k2 EtotS VmaxS
S Km(1 I/Ki) S Km (1 I/
Ki) IC50 Conc. of Inhibitor which causes 50
inhibition at a given S IC50 Ki(1 S/Km)
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Enzymes as Targets

• Practical Issues in Inhibitor Characterization
• Under what regime of S should one be to
  accurately measure Ki?
• If one has two enzymes and one wants to determine
  the relevant selectivity of an inhibitor what
  does one need to know?
• How would one determine if an inhibitor is
  irreversible?

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Receptors as Targets

• Receptors as Molecular Sensors and Transducers of
  Environmental Information
• Binding Events followed by Signal transduction
  events (usually catalytic)
• Cell surface - outside to inside
• 7 TMs (GPCRs)
• 1 TMs (Type 1 and 2), Receptor kinases, cytokine
  receptors, scavenger receptors, integrins
• Ion Channels (Multiple TMs)
• Intracellular - nuclear receptors - direct
  effects on transcription

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Receptors as Targets

• 7 TM receptors
• Ligands can be from small molecules to proteins
• Coupled to trimeric G-proteins
• Binding to exterior faces leads to G-protein
  dissociation inside the cell - a bc
• Subsequent signaling (functional response) via
  ion flux, cyclic AMP increase, protein kinase
  activation, other signaling events which are
  specific G-protein dependent ( there are many
  flavors of G proteins)

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Receptors as Targets

• Single TM Receptors
• One or more polypeptide chains
• Mostly protein ligands
• Signaling usually by dimerization dependent
  intramolecular phosphoryl transfer (enzymatic
  activity) followed by proteinprotein interaction
  cascades
• Growth factors - EGF
• Cytokines - Tumor necrosis factor
• Extracellular Matrix proteins - Integrins

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Receptors as Targets

• How to Measure Receptor Activity?
• Binding Assays
• Functional Assays
• These dont necessarily measure the same thing!

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Receptors as Targets
Receptor Binding Theory
Kd k-1 DR xd(Rtot-Rd) k1
DR Rd
k-1
Rtot DR Rd
rd Rd/Rtot receptor occupancy
Kd Rd xdRtot - Rd xd Kd Rd Rd xd xDRtot
Rd(Kd xd) xdRtot Rd /Rtot rd xd
Kd xd
Rearranging
Hill Equation - same form as Michaelis-Menten
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Receptors as Targets
Rearrangement of Hill Equation 1 - rd Kdn
xdn - xdn Kdn__
Kdn xdn Kdn xdn
Kdn xdn rd xdn
log( rd ) nlogxd - nlogKd 1 - rd
Kdn (1 - rd) Slope of
Hill Plot (loglog plot vs. xd) defines
cooperativity or stoichiometry of receptorligand
interaction and intercept defines Kd If n 1,
then no cooperativity and 11 stoichiometry
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Receptors as Targets
For Radio-Labelled Ligand Binding Studies on
Receptor Preparations At Equilibrium B (amount
bound) Bmaxxd where Bmax is total amount
bound
Kd xd Rearrangement yields the Scatchard
Equation B/xd Bmax/Kd - B/
Kd Where a plot of B/xd vs. B (Scatchard Plot)
has slope of -1/Kd and x intercept of Bmax
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Receptors as Targets
The Schild Equation - Competitive Antagonists
Kd I R D DR
Ki RI
rd xd/Kd/((xd/Kd) (xi/Ki) 1) r xd/xd
where x is the increased agonist which
compensates for xi r (xi/Ki) 1 log (r-1)
log xi - log Ki Schild plot - x intercept is
pA2, slope should 1 for a purely competitive
antagonist
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Receptors as Targets

• Functional Assays
• Can distinguish between occupancy and function
  (antagonists and agonists).
• Occupancy and signaling are usually monotonic in
  relationship (though not always)
• Maximal signaling by agonists often occurs at
  relatively low occupancy
• Due to coupling/amplication of initial binding
  events
• Thus, functional assays are often more sensitive
  than binding studies

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Receptors as Targets

• Quantitative Treatment of Functional Assays

k1
ka
D R
DR
DR
k-1
k-a
Binding
Activation
Kd k-1/k1 Kact k-a/ka
This is a very simple case where the functional
responseis one step ractive
xd/Kd(ka/k-a) 1 xd/Kd(1 ka/k-a)
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Summary

• Patents - novel, useful, not obvious
• Enabling disclosures for one skilled in the art
• Enzymes - Michaelis-Menten formalism for steady
  state kinetics
• Receptors - binding vs. functional assays
• Hill, Scatchard, and Schild Equations