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

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Presentation on theme: "Andrew Mumma, Shwetha Manjunath, and Asha Mahajan Chemistry 315/515."— Presentation transcript:

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

2 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

3 Why Is Increased Blood Glucose Detrimental? Non-enzymatic glycation of proteins alter their structure and function Measuring Blood Glucose: D-Glucose + O 2 glucose oxidase D-Glucono-  -lactone + H 2 O 2 This can lead to: –Diabetic Nephropathy –Neuropathy –Retinopathy –Heart complications –Stroke

4 Glycation Mechanism Schiff Base Pyrraline Imine AGE products Amadori Product Pentosidine Horvat, Š.; Jakas, A. J. Peptide Sci. 2004, 10, 119-137.

5 “Lipid Burden” Hypothesis for T2D Cusi, K. Curr. Diab. Rep. 2010, 10, 306-315

6 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

7 …Potentially through inhibition of PPAR  activity resulting in increased Free Fatty Acids (FFA) Macrophages Transcription/ translation  FFA Insulin- mediated Insulin Resistance Glucose Adipocyte (obese condition) Guilherme et al. Nat Rev Molecular Cell Biol., 2008, 9, 367-377http://www.aamdsglossary.co.uk/glossary/m

8 Treatment Insulin Metformin Sulfonylureas Thiazolidinediones (TZDs) http://en.wikipedia.org/wiki/Insulin

9 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.

10 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.

11 What else regulates AMPK, and what does AMPK do? CytokinesNatural products Activation of ATP- producing processes Inhibition of ATP-consuming processes Downstream mediators Hardie, DG. Nature Rev. Mol. Cell. Biol. 2007, 8, 774-785.

12 A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis Gómez-Galeno JE et al ACS Med. Chem. Lett. 2010.

13 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.

14 Basic residues in the binding site of the gamma subunit and phosphate interaction Xiao, B. Nature Let. 2007, 449, 496-501.

15 How effective is compound 2 at activating human AMPK?

16 Synthesis of compound 2 prodrugs [1] http://chemistry2.csudh.edu/rpendarvis/aminrxn.html [1]

17 Formal [3+2] cycloaddition Compounds 12-18

18 How do various phosphonate prodrugs perform at inhibiting de novo lipogenesis in vitro and in vivo? 12-18 8 EC 50 : 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.

19 Is AMPK activation by compound 13 responsible for DNL inhibition? Control 1000uM 10uM 3uM 1uM AICAR compound 13 inhibition AMPK ACC Acetyl-CoA carboxylase: Catalyzes fatty acid biosynthesis Inhibits free fatty acid oxidation PiPi ACC PiPi

20 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

21 Paper #2: http://diabetescure.hct.ac.ae/speaker-profiles/

22 Quest to Optimize T2D Treatment Thiazolidinediones (TZDs) TZDs -Bind to PPARγ Negative Side Effects: -Weight gain -Anemia

23 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

24 Chemical Structures A-ring T2384 B-ring C-ring Rosiglitazone T2384 is chemically distinct from TZDs Thiazolidinedione

25 Does T2384 bind with similar affinity to PPARγ like Rosiglitazone? (+)(+)(+)(+) (+)(+) (-)(-) Measure radioactivity (-)(-) PPARγ Nitrocellulose Paper 3 H-labeled Rosiglitazone (+)(+)(+)(+)(+)(+) Unlabeled Rosiglitazone or T2384 K i of T2384 = 200 nM Results

26 Does T2384 activate PPARγ in cells like Rosiglitazone? LBD = Ligand Binding Domain DBD = DNA Binding Domain Gal4-UASLuciferase DNA Gal4 DBD PPARγ LBD Rosiglitazone or T2384 GLOW PPARγ DBD PPARγ LBD Gal4 DBD PPARγ LBD PPARγ Meneely, P. Advanced Genetic Analysis. Oxford University Press, New York. 2009.

27 Results Little lipid accumulation T2384 inhibited Rosiglitazone’s effect Does T2384 trigger lipid accumulation in preadipocytes like Rosiglitazone? Log[Compound] (μM) T2384 inhibited Rosiglitazone’s effect Partially activated PPARγ

28 NCoR/ SMRT Sin3 HDACs RXR  PPAR  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 RXR  PPAR  Ac TAFs/ TBP Transcription! PPRE Transcription of PPAR  gene targets when associated with coactivator complex

29 TR-FRET 665 nm FRET Emission 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

30 Emission Intensity @ 665 nm Emission Intensity @ 620 nm 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 0.1  M

31 Complex of PPARγ with T2384 Helix 3 “U” conformation “S” conformation PPARγ LBD as homodimer No direct binding to T2384

32 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 353 Met 364 His 449 Leu 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.

33 Does T2384 binding to U vs. S pockets differentially affect PPARγ activity? Disrupting S pocket: L228W A292W L333W 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

34 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

35 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.

36 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.

37 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.

38 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

39 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?

40 Quiz!!! Is pursuing T2D drugs condoning personal irresponsibility to one’s own health?


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