andrew mumma, shwetha manjunath, and asha mahajan chemistry 315/515
TRANSCRIPT
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
OH
HO
OH
OH
CH2OH
H O
[Protein] NH2 +
OH
HO
OH
OH
CH2OH
OH
NH2
[Protein]
OH
HO
OH
OH
CH2OH
OHH
NH
[Protein]
H A
CH
OH
HO
OH
OH
CH2OH
N
[Protein]
O
OH
OH
OH
CH2
HN [Protein]
H2C
O
HO
OH
OH
CH2OH
[Protein]HN
CH3
C
O
OH
OH
CH2OH
C
CH2
O
OH
OH
CH2OH
OH
+ O
Glycation Mechanism
Schiff Base
Pyrraline Imine
AGE products
Amadori Product
Pentosidine
N
[Lys]
[Arg]
N
NH
HN
N
OH[Lys]
N
[Lys]
+
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
Transcription/
translation
FFA
Insulin-mediated
Insulin Resistance
GlucoseAdipocyte
(obese condition)
Guilherme et al. Nat Rev Molecular Cell Biol., 2008, 9, 367-377http://www.aamdsglossary.co.uk/glossary/m
Treatment
Insulin
Metformin
Sulfonylureas
N N
O
S
HN
O
OThiazolidinediones (TZDs)
Cl
S
NH
O O
NH
O
http://en.wikipedia.org/wiki/Insulin
N NH
NH2
NH NH
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
Activation of ATP-producing processes
Inhibition of ATP-consuming processes
Downstream mediators
Hardie, DG. Nature Rev. Mol. Cell. Biol. 2007, 8, 774-785.
A Potent and Selective AMPK Activator That Inhibits de Novo
LipogenesisGomez-Galeno JE et al ACS Med. Chem. Lett. 2010.
What about AMPK as a direct drug target for treatment of Type II
Diabetes?
AMPK• Endogenous
activator• Regulates
many proteins
• AMP mimetic• Binds AMPK and
various proteins regulated by AMP
• Binds specifically to AMPK
• Different binding site from AMP[1]
[1] Cool, B. Cell Metab. 2006, 3, 403-416.
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] http://chemistry2.csudh.edu/rpendarvis/aminrxn.html
[1]
Formal [3+2] cycloaddition
Compounds 12-18
How do various phosphonate prodrugs perform at inhibiting de novo lipogenesis in vitro and in
vivo?
12-188
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 permeability8 and 12: Did not activate isolated enzyme – phosphonic acid important for AMPK activation by 2.
Is AMPK activation by compound 13 responsible for DNL inhibition?
Control 1000uM 10uM 3uM 1uM AICAR compound 13
inhibitionAMPK ACCAcetyl-CoA carboxylase: Catalyzes fatty acid biosynthesisInhibits free fatty acid oxidation
Pi
ACC
Pi
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
N N
O
S
HN
O
OThiazolidinediones (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
A-ring
Cl
N
S
S
Cl
HN
S
CF3
Cl
O
O
T2384
B-ring
C-ring
N N
O
S
HN
O
O
Rosiglitazone
T2384 is chemically distinct from TZDs
Thiazolidinedione
Does T2384 bind with similar affinity to PPARγ like Rosiglitazone?
(+) (+)(+)(-)Measure radioactivity
(-)
PPARγ
Nitrocellulose Paper
3H-labeled Rosiglitazone
(+) (+) (+)
Unlabeled Rosiglitazone or T2384
Ki of T2384 = 200 nM
Results
Does T2384 activate PPARγ in cells like Rosiglitazone?
LBD = Ligand Binding DomainDBD = DNA Binding Domain
Gal4-UAS LuciferaseDNA
Gal4DBD
PPARγLBD
Rosiglitazone or T2384
GLOW
PPARγDBD
PPARγLBD
Gal4DBD
PPARγLBD
PPARγ
Meneely, P. Advanced Genetic Analysis. Oxford University Press, New York. 2009.
Results
Little lipid accumulation
T2384 inhibited Rosiglitazone’s effect
Does T2384 trigger lipid accumulation in preadipocytes like Rosiglitazone?
Log[Compound] (Log[Compound] (μμM)M)
T2384 inhibited Rosiglitazone’s effect
Partially activated PPARγ
NCoR/ SMRT
Sin3
HDACs
RX
R
PP
AR
PPRE
PPAR-RXR heterodimer when associated with Corepressor Complex NO Transcription
How does PPAR regulate transcription?
DRIP205
Coactivator complex
DRIP205
Coactivator complex
RNA Pol II
RX
R
PP
AR
Ac
AcAc
Ac TAFs/TBPTranscription!
PPRE
Transcription of PPAR gene targets when associated with coactivator complex
TR-FRET 665 nm
FRETEmission 620 nm
Excitation
GST
PPAR LBD
Ligand
No interaction
Interaction
APC
Peptide Corepressor/ Coactivator
biotin
strepavidin
Emission Intensity @ 665 nm Emission Intensity @ 620 nm
Quantification of Protein-Protein Binding
How does T2384 binding to PPAR LBD affect its interactions with transcriptional regulatory proteins?
APC
Peptide
Corepressor/
Coactivator
Em
issi
on I
nten
sity
@ 6
65 n
m
Em
issi
on I
nten
sity
@ 6
20 n
m
T2384 partial agonist profile
T2384 antagonist profile
How does T2384 binding affect PPAR LBD interactions with corepressor/coactivator derived peptides?
T2384 displays partial agonist activity at concentrations < 0.1M and antagonist activity at concentrations > 0.1M
Complex of PPARγ with T2384
Helix 3
“U” conformation
“S” conformation
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
Ile 341
Cys 285
Leu 353Met 364
His 449Leu 330
His 323
Tyr 473
Ser 289
Comparison: PPARγ with Rosiglitazone
No interaction with F363 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:
L228WA292WL333W
Disrupting U pocket:
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
T2384 (100mg/kg) + rosiglitazone (3mg/kg)
rosiglitazone
Does T2384 elicit PPAR-mediated side effects?
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 structure-
based 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?