Transcript Diabetes (type II) treatment, Dec. 7

Andrew Mumma, Shwetha Manjunath, and Asha Mahajan
Chemistry 315/515
What is Type II Diabetes (T2D)?
• Metabolic disorder involved in abnormally
high blood glucose levels caused by
insulin insensitivity
• Insulin insensitivity is caused by deficiency
of or unresponsiveness to insulin
• Risk Factors:
– High food intake
– Decreased exercise
– Genetics
Why Is Increased Blood Glucose Detrimental?
• Non-enzymatic glycation of proteins alter
their structure and function
• Measuring Blood Glucose:
D-Glucose + O2 glucose oxidase D-Glucono--lactone + H2O2
• This can lead to:
– Diabetic Nephropathy
– Neuropathy
– Retinopathy
– Heart complications
– Stroke
Glycation Mechanism
Schiff Base
Amadori
Product
AGE products
Pentosidine
Pyrraline Imine
Horvat, Š.; Jakas, A. J. Peptide Sci.
2004, 10, 119-137.
“Lipid Burden” Hypothesis for T2D
Cusi, K. Curr. Diab. Rep. 2010, 10, 306-315
How might chronic inflammation in fat tissue lead to
insulin resistance…
Lean fat cell (healthy condition)
Glucose
Guilherme et al. Nat Rev Molecular Cell Biol., 2008, 9, 367-377
…Potentially through inhibition of PPAR activity resulting in
increased Free Fatty Acids (FFA)
Macrophages
Insulinmediated
Adipocyte
(obese condition)
Glucose
Insulin
Resistance
FFA
http://www.aamdsglossary.co.uk/glossary/m
Guilherme et al. Nat Rev Molecular Cell Biol., 2008, 9, 367-377
Treatment
Metformin
Sulfonylureas
Insulin
http://en.wikipedia.org/wiki/Insulin
Thiazolidinediones (TZDs)
What is AMP-activated protein kinase
(AMPK) and its main role in the body?
• Balances catabolism (processes that produce ATP)
with ATP consumption to maintain high levels of ATP
• Expressed primarily in liver, skeletal muscle, and the
brain, which are all involved in energy intake,
consumption, and storage
• Heterotrimeric kinase (α, β, and γ subunits)
• Activated in two ways[1]
– Kinases that phosphorylate Thr172 on α subunit
– AMP binding of γ subunit that blocks dephosphorylation of
Thr172 on α subunit
[1] Zhang, BB. Cell Metab. 2009, 9, 407-416.
The master switch of AMPK and energy
homeostasis: the ratio of ATP to AMP
• ATP is depleted by
decreased
production or
increased
consumption
• AMP is a byproduct
of ATP consumption
• Decreased ATP and
increased AMP
activate AMPK
• AMPK triggers
mechanisms that
restore the balance
of ATP to AMP
Hardie, DG. Nature Rev. Mol. Cell. Biol. 2007, 8, 774-785.
What else regulates AMPK, and what
does AMPK do?
Cytokines
Natural products
Downstream mediators
Activation of ATPproducing processes
Hardie, DG. Nature Rev. Mol. Cell. Biol. 2007, 8, 774-785.
Inhibition of ATP-consuming
processes
A Potent and Selective AMPK
Activator That Inhibits de Novo
Lipogenesis
Gómez-Galeno JE et al ACS Med. Chem. Lett. 2010.
What about AMPK as a direct drug
target for treatment of Type II
Diabetes?
•
•
Endogenous
activator
•
Regulates
many
•
proteins
AMPK
AMP mimetic
Binds AMPK and
various proteins
regulated by AMP
[1] Cool, B. Cell Metab. 2006, 3, 403-416.
•
•
Binds specifically
to AMPK
Different binding
site from AMP[1]
Basic residues in the binding site of the
gamma subunit and phosphate interaction
Xiao, B. Nature Let. 2007, 449, 496-501.
How effective is compound 2 at activating
human AMPK?
Synthesis of compound 2 prodrugs
[1]
[1] http://chemistry2.csudh.edu/rpendarvis/aminrxn.html
Formal [3+2] cycloaddition
Compounds 12-18
How do various phosphonate prodrugs perform at
inhibiting de novo lipogenesis in vitro and in vivo?
EC50: inhibition of de novo
lipogenesis (DNL) in rat and
mouse hepatocytes
in vivo DNL inhibition:
inhibition of DNL in mice
livers after intraperitoneal
injection
Compounds:
2: anionic, poor cellular
permeability
8 and 12: Did not activate
isolated enzyme –
phosphonic acid important
for AMPK activation by 2.
8
12-18
Is AMPK activation by compound 13
responsible for DNL inhibition?
Pi
AMPK
inhibition
ACC
Acetyl-CoA carboxylase:
Catalyzes fatty acid biosynthesis
Inhibits free fatty acid oxidation
Pi
ACC
Control 1000uM 10uM
AICAR
3uM
1uM
compound 13
Results and future direction
• Evaluated compound 2,
the phosphonic acid
derivative that potently
activates AMPK
• Synthesized a line of
compound 2 prodrugs
that are esterase
sensitive, bioavailable,
and activators of AMPK
• Future use of these
AMPK-specific drugs
can help clarify the
exact role that AMPK
has in modulating
energy homestasis
• Test the potential of
these compounds as a
therepeutic treatment
for Type II diabetes
Paper #2:
http://diabetescure.hct.ac.ae/speaker-profiles/
Quest to Optimize T2D Treatment
Thiazolidinediones (TZDs)
•TZDs
-Bind to PPARγ
•Negative Side Effects:
-Weight gain
-Anemia
Rationale
• We know: Inhibition of PPARγ leads to T2D
• A paradox exists: Reduction of PPARγ can
lead to improvement of insulin sensitivity
– A mutation in PPARγ resulting in partial
loss of normal function reduced risk for
T2D
• Goal: Search for a partial agonist of PPARγ
that increases insulin responsiveness without
negative side effects
Chemical Structures
Thiazolidinedione
T2384
A-ring
Rosiglitazone
B-ring
C-ring
T2384 is chemically distinct from TZDs
Does T2384 bind with similar affinity to PPARγ
like Rosiglitazone?
Unlabeled Rosiglitazone
or T2384
3H-labeled
Results
Rosiglitazone
PPARγ
(-)
(+)
(-)
(+)
(+)
(+)
Nitrocellulose
Paper
(+)
(+)
Measure
radioactivity
Ki of T2384 = 200 nM
Does T2384 activate PPARγ in cells like
Rosiglitazone?
LBD = Ligand Binding Domain
DBD = DNA Binding Domain
PPARγ
LBD
PPARγ
PPARγ
LBD
PPARγ
DBD
Gal4
DBD
Rosiglitazone
or T2384
PPARγ
LBD
Gal4
DBD
DNA
Gal4-UAS
Luciferase
Meneely, P. Advanced Genetic Analysis. Oxford University Press, New York. 2009.
GLOW
Results
Partially activated PPARγ
T2384 inhibited
Rosiglitazone’s effect
Log[Compound] (μM)
Does T2384 trigger lipid accumulation in preadipocytes like
Rosiglitazone?
Little lipid accumulation
T2384 inhibited
Rosiglitazone’s effect
How does PPAR regulate transcription?
Coactivator
complex
DRIP205
Coactivator
complex
Sin3
DRIP205 NCoR/
HDACs
Ac
RXR
PPAR
Ac
RXR
Ac
PPAR
SMRT
Transcription!
PPRE
Ac
PPRE
PPAR
-RXR heterodimer
when
associated
with
Transcription
of PPAR gene
targets
when
Corepressor
 NOcomplex
Trans cription
associatedComplex
with coactivator
How does T2384 binding to PPAR LBD affect its interactions
with transcriptional regulatory proteins?
TR-FRET
665 nm
Emission
620 nm
FRET
APC
Excitation
strepavidin
GST
PPAR LBD
biotin
Ligand
Peptide
Corepressor/
Coactivator
No interaction
Interaction
Quantification of
Protein-Protein Binding
Emission Intensity @ 665 nm
Emission Intensity @ 620 nm
How does T2384 binding affect PPAR LBD interactions
with corepressor/coactivator derived peptides?
T2384 partial agonist profile
Emission Intensity @ 665 nm
Emission Intensity @ 620 nm
T2384 antagonist profile
T2384 displays partial
agonist activity at
concentrations < 0.1M
and antagonist activity at
concentrations > 0.1M
Complex of PPARγ with T2384
“S”
conformation
“U”
conformation
Helix 3
PPARγ LBD as homodimer
No direct
binding to
T2384
Complex (cont’d)
“U” conformation
“S” conformation
Pink dashed lines = H bonds
Black dashed lines= dipole-dipole
Grey dashed lines = van der Waals
Aromatic Stacking
Comparison:
PPARγ with
Rosiglitazone
His 323 His 449
Leu 330
Met 364
Leu 353
Cys 285
No interaction
with F363
Tyr 473
Ser 289
Ile 341
Rosiglitazone
Chandra, V. et al. Nature 2008, 456,
350-356.
Does T2384 binding to U vs. S pockets differentially
affect PPARγ activity?
Disrupting S pocket:
Disrupting U pocket:
L228W
A292W
L333W
G284I
•
Tested these mutant proteins with Rosiglitazone and T2384 ligand in coregulator
recruitment assays:
-If ligand binding induced PPARγ to recruit DRIP205 coactivator  agonist
-If ligand binding induced PPARγ to recruit NCoR corepressor  antagonist
Mutation in U pocket disrupts rosiglitazone’s
agonist affect on PPARγ activity
• Mutations in S pocket
do not hinder activity of
Rosiglitazone (data not
shown)
• Mutations in U pocket
disrupted agonist activity
of Rosiglitazone
T2384’s interactions with U and S binding sites
trigger different PPARγ responses
•
Mutations in S pocket
disrupt T2384’s antagonist
activity
•
Mutations in U pocket
disrupt T2384’s agonist
activity
• Biphasic phase was disrupted
• Different binding conformations of T2384 can elicit different PPAR
activity.
T2384 lowers plasma glucose and
insulin concentration in KKA
obese/diabetic mice
T2384
T2384 + rosiglitazone
rosiglitazone
T2384 lowers plasma glucose and insulin levels in a dose-dependent
manner.
Co-administration of T2384+rosiglitazone shows no significant
additive effect in improvement of insulin sensitivity.
T2384
Does T2384 elicit PPAR-mediated
side effects?
T2384 (100mg/kg) +
rosiglitazone (3mg/kg)
rosiglitazone
Unlike rosiglitazone, T2384 did not increase body weight or cause anemia.
Coadministration of T2384+rosiglitazone ameliorates body weight gain and
reduction in red blood cells caused by rosiglitazone treatment alone.
Conclusions and Future Directions
• Conclusion
– S pocket occupancy without interaction with AF2
helix may result in optimal PPARγ activity without
side effects
• Future Directions
– Investigate how T2384 reduces fat accumulation
and increases insulin sensitivity
– Create and explore other drugs through structurebased drug design that bind to S pocket and note
effects on PPARγ
Quiz!!!
• Which pocket (U or S) is associated with T2384’s
antagonistic activity?
• What additional binding interaction forms between
PPARγ and T2384 that is not present between
PPARγ and rosiglitazone?
• Why is the phosphonic acid compound 2 not active
when given to rat hepatocytes or injected in mice?
• The dynamic between which two molecules directly
modulates the activity of AMPK?
• Multi-part question (BONUS for getting more than
one!):
– What two structures react in the formal [3+2] cycloaddition?
– What is the name of the resulting ring structure?
Quiz!!!
• Is pursuing T2D drugs condoning personal
irresponsibility to one’s own health?