Small Molecules, Big Expectations

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Small Molecules, Big Expectations
Dwayne L. Barber, PhD Room 9-410 Ontario Cancer
Institute dbarber_at_uhnresearch.ca
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ROLE OF ERYTHROPOIETIN IN HEMATOPOIESIS
BFU-E
CFU-E
Growth Differentiation Cell Survival
Proerythroblast
Erythrocyte
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ROLE OF ERYTHROPOIETIN IN HEMATOPOIESIS
Proerythroblast
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EPO SIGNAL TRANSDUCTION
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EPO-R EXISTS AS A PREFORMED DIMER
Livnah et al. Science 283 990, 1999
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BINDING OF EPO RESULTS IN CONFORMATIONAL CHANGES
Syed et al. Nature 395 511, 1998.
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STRATEGIES TO DEVELOP MIMETICS
Alter primary structure of ligand

• Glycosylation (Darboepoietin/ARANESP)

• Dimer (flexible peptide bridge)

• Dimer/Trimer (Chemical cross-linking

• Pegylation (CERA)

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STRATEGIES TO DEVELOP MIMETICS
Peptide mimetics

• Identical in primary sequence

• Phage display (distinct sequences)
• EMP1 (Agonist)
• EMP33 (Antagonist)

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EMP1 IS A PARTIAL AGONIST OF THE EPO-R
Livnah et al. Science 273 464, 1996
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SUMMARY OF EPO-R ACTIVATION
Jiang Hunter Curr Biol 9 R568, 1999.
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STRATEGIES TO DEVELOP MIMETICS
Small molecules
Merck compound Activates STAT-luciferase
gene Factor-dependent growth of erythroid cell
lines Erythroid differentiation of human
progenitors Qureshi et al PNAS 96 12156, 1999
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STRUCTURAL FEATURES OF KINASES
N-lobe C-lobe P-loop A-loop ?-C helix ?-G helix
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EGF-RECEPTOR ACTIVATION
Zhang et al Cell 125 1137, 2006
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ACTIVE VS. INACTIVE EGF RECEPTOR
(B) Active conformation of the EGF-R. L834 and
L837 are surface exposed and the K721/E738 ion
pair is intact C. Inactive conformation of the
EGF-R. L834 and L837 pack against helix aC,
preventing the formation of the K721/E738 ion pair
Zhang et al Cell 125 1137, 2006
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CHRONIC MYELOID LEUKEMIA
Clonal disorder 95 of patients have BCR-ABL
chromosomal translocation Median age of patients
is 67 Median survival is 4 to 6 yr Disease
proceeds in three phases Chronic
phase Accelerated phase Blast Crisis
Chronic phase Peripheral blood smear
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CLINICAL FEATURES OF CML
Peripheral blood
FISH
G-banding
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SIGNAL TRANSDUCTION PATHWAYS ACTIVATED BY BCR-ABL
CML is a stem cell disorder
CML cells have deregulated growth, survival,
adhesion migration
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IMATINIB AND PD180970 BINDING SITES ON ABL

• Imatinib binds to ABL when the kinase is in an
  inactive conformation. The activation loop is
  closed
• thereby preventing substrate binding. (B)
  PD180970 binds to a distinct region of ABL when
  the kinase
• is active. It contacts much less surface area of
  ABL.

Deininger et al Blood 105 2640, 2005.
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FREQUENCY OF BCR-ABL MUTATIONS
Deininger et al Blood 105 2640, 2005.
P-loop mutation
SH2 domain interaction
Imatinib binding
A-loop mutation
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SECOND GENERATION ABL INHIBITORS
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AMN107 IS EFFECTIVE AGAINST SEVERAL IM-RESISTANT
MUTANTS
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MODE OF BINDING OF VX-680 (MK-0457)

• Burial of VX-680 (left) and Imatinib (right). (B)
• Interactions between the P-loop of the kinase
  domain and VX-680 (left) and Imatinib (right). C.
  
• Residues that make side chain contact with
  VX-680. Residues that are substituted in Aurora-A
  are indicated by naming the Aurora-A residue in
  brackets. (D) Interaction between Vx-680 and
  Aurora-A, modelled on the Abl-VX680 structure.
  The position of VX-680 within Aurora was obtained
  by aligning the two kinase domains on the
  residues of the hinge region. L210 in Aurora-A,
  located at the gatekeeper position, is shown with
  spheres for the side-chain atoms. Interaction
  between VX-680 and Abl (T315I), modelled by
  substituting T315 in the crystal structure with
  isoleucine.

Young et al Cancer Res 66 1007, 2006
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ALTERNATIVE PATHWAYS FOR TARGETTING BCR-ABL
Nardi et al. Curr Opin Hematol. 11 35-43, 2003
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CML THERAPY IN THE POST-IMATINIB ERA
Rate of resistance to IM is 4/patient/year
Combination with other drugs -IM IFN, IM
cytarabine, IM homoharringtonine
Second generation tyrosine kinase inhibitors to
treat IM-resistant disease
Combination tyrosine kinase inhibitors
IM signal transduction inhibitors, IM
geldanamycin
IM allogeneic bone marrow transplantation-use
IM as induction therapy
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LUNG CANCER
Most lethal cancer diagnosis EGFR is found on
40-80 of non-small cell lung cancer Gefitinib
and erlotinib showed clinical response 10 of
European patients 30 of Japanese patients More
favoured women Adenocarcinoma Patients who had
never smoked
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EGFR MUTATIONS CONFERS DRUG SENSITIVITY
Responding patients have somatic mutations of the
EGFR Exon 19 mutations in frame deletions of
aa 746-750 G719C Exon 21 L858R L861Q
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EGF SIGNAL TRANSDUCTION
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EGF-RECEPTOR TYROSINE KINASE INHIBITORS
Yun et al. Cancer Cell 11 217, 2007
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STRUCTURE ACTIVITY OF MUTANT EGFR
(A) Structure of EGFR in complex with
AMP-PNP. Locations of the L858R and G719S
mutations in the A-loop and P-loop respectively
are shown. (B) The structure of the active site
region of the L858R mutant (green) superimposed
on the wt kinase (yellow). C The structure of
the active site region of the G719S mutant
(blue) superimposed on the wt kinase (yellow).
(D) Comparison of the activity of the Wt,
G719S and L858R kinases utilzing
poly-Glu-Tyr as substrate.
Yun et al. Cancer Cell 11 217, 2007
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MECHANISM OF ACTIVATION OF L858R EGF-R MUTANT
Lapatinib Wt EGF-R Inactive
Gefitinib EGF-R L858R Active
Yun et al. Cancer Cell 11 217, 2007
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ACTIVE VS. INACTIVE KINASE INHIBITORS
Inhibitors that target active kinases -more
rapid binding -faster dissociation
kinetics -bind to pre-existing cavity and compete
with ATP
Inhibitors that target inactive kinases -bind
through an induced fit -requires opening of the
kinase domain, displacement of ATP, competition
with ATP refolding of the kinase -inhibitors
should be more selective
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CHALLENGES IN USING TKI IN LUNG CANCER THERAPY
Etiology of EGFR mutations Potency
(oncogenicity) of various EGFR mutations Effects
of individual inhibitors on each EGFR
mutation Mechanisms of resistance in patients
who do not have a mutation in the T790
gatekeeper residue Combination therapies
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TARGETTED CHEMOTHERAPEUTICS
-Marker has to be activated (not
overexpressed) - Not all inhibitors are
efficacious on all targets I.e. Imatinib on GIST
vs. mastocytosis -Possibility of utilizing a
cocktail of inhibitors (FLT3) -Need for
partnering -Pursuit of niche markets