Andrew Mumma, Shwetha Manjunath, and Asha Mahajan Chemistry 315/515.

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?