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CHEM E-120 Harvard University Extension School Spring 2011

CHEM E-120 Harvard University Extension School Spring 2011. Disorders of Mood and Behavior Bipolar Disorder. Bipolar Disorder. Formally known as manic-depressive disorder Consists of a manic episode and depressive episode Bipolar I - 0.5-1.3% of population

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CHEM E-120 Harvard University Extension School Spring 2011

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  1. CHEM E-120Harvard University Extension SchoolSpring 2011 Disorders of Mood and Behavior Bipolar Disorder CHEM E-120

  2. Bipolar Disorder Formally known as manic-depressive disorder Consists of a manic episode and depressive episode Bipolar I - 0.5-1.3% of population one or more manic or mixed episodes ( every day for 1 week) previous depressive episode not required Bipolar II - 0.25 of population one or more major depressive episodes and one hypomanic episode (> 4 days) No sex difference, life-long disease Identical twins (monozygotic) - 4-8 fold higher concordance than in nonidentical twins (dizygotic) Chromosomes 11, 18, X identified as associated with bipolar strong genetic factor - 60-85% Difficult to compare clinical phenotype with genotype - great variability in clinical phenotypes ~50% attempted suicide rate, 10-15% success rate CHEM E-120

  3. Bipolar Disorder Hypotheses Permissive Hypothesis Deficits in central serotonergic neurotransmission Both manic and depressive episode characterized by low central serotonergic transmission - hypofunctional serotonergic system Lower levels of norepinephrine in depressed state Some evidence of, noradrenergic receptor level changes? Neurotrophic Hypothesis Decreased expression of neural growth factors contributes to mood disorders CHEM E-120

  4. Summary of Neuropathology of Bipolar Disorder Limbic system serotonin norepinephrine CHEM E-120

  5. Bipolar Disorder – Medication Development 1. Lithium attenuates manic episode currently thought to exert its effects via inhibition of enzymes involved in second- messenger systems 2. Atypical Antipsychotic agents: Seroquel (Astra-Zeneca), Ziprasidone + Li (Pfizer) Antidepressants in combination with a mood stabilizer: olanzapine + fluoxetine Nuvigil (modafinal) + mood stabilizers – Phase 2 (to treat depressive stage) Future Therapeutic Approaches 1. Understand the therapeutically relevant biochemical targets of existing drugs and use that knowledge to design new drugs against the known targets and downstream targets. Monoaminergic system drugs - DA, DAT, NE, NET, 5-HT, SERT 2. Develop pathophysiological models of the illness and develop drugs to attenuate or prevent these processes. Intracellular pathways of signalling - control of expression GSK-3 inhibitors Neuropsychopharmacology 2008, 33, 2080-2092 CHEM E-120

  6. Lithium Mood Disorder Li+ ionic radius of 0.6 Å Does Li+ compete with Mg 2+? Mg2+ ionic radius of 0.65 Å Not naturally occurring in body, can replace Na+ (1.2 Å) in supporting a single action potential but cannot maintain action potentials Dosed as Li2CO3 For acute mania given 3x600 mg/day producing serum levels of 1.0 - 1.5 mEq/L (mM) Maintenance dosage of 900 - 1200mg/day producing 0.6 - 1.2 mM. >1.5 mM starts producing toxic effects Known to inhibit Mg2+-dependant enzymes inositol monophosphatase glycogen synthase kinase-3β adenylyl cyclase CHEM E-120 Acc Chem Res 2006, 39, 283

  7. Cell Surface Proteins G-protein Coupled Receptor – GPCR Membrane bound receptor bound to a G-protein that is in the cytoplasm 7 transmembrane domains (7TM) Receptor 3 extracellular domains 3 intracellular domains three distinct subunits , ,  G-protein bind to GTP and GDP Gs - stimulation of adenyl cyclase Gi - inhibition of adenyl cyclase ~ 60% of drugs act on GPCR’s CHEM E-120

  8. G-protein Coupled Receptor - GPCR agonist antagonist prevents binding of the agonist and stops the cycle Functional activity usually refers to some measurement of this cycle CHEM E-120

  9. Receptors vs transporters CHEM E-120

  10. Receptors - Drugs Binding of a drug to a receptor may elicit a biological response The drug (D) will be competing with natural ligands (L) for the receptor An agonist will elicit a functional response Affinity - IC50, Ki (expressed in units of concentration, eg nM) Efficacy - EC50, Emax, ED50 (in vivo) An antagonist will bind to a receptor but will not elicit a functional response Affinity - IC50, Ki (expressed in units of concentration, eg nM) Efficacy - pA2 = -logKd L + D + R  LR + DR + L + D CHEM E-120

  11. Second-Messenger System agonist 7-TM receptor G-protein(α,β,γ) (agonist)(7-TM receptor)(G-protein) α + β,γ results in changes in second messenger concentrations cAMP cGMP Ca2+ IP (inositol phosphate) arachidonic acid NO CO • interact with effector proteins (enzymes) • inhibition or activation e.g. • adenylyl cyclase • phospholipase C • phospholipase A2 complex cascades of protein phosphorylations by protein kinases effecting protein activity via protein transcription CHEM E-120

  12. Second-Messenger System CHEM E-120

  13. Lithium – IP interaction IP3 (insoitol triphosphate) can regulate intracellular Ca2+ levels 5-HT2 and adrenergic 1 (norepinephrine) receptors use IP (PI) as the effector of the second-messenger system. The activity of these receptors will then be modulated in some fashion by lithium perturbing the inositol phosphorylation cycle. DAG Ca2+ regulatory domain catalytic domain Kandel p1220 Δ[Ca2+] CHEM E-120

  14. Lithium Mechanism in BPD CHEM E-120

  15. Second-Messenger Systems of interest in BPD 1. Brain-derived neurotropic factor (BDNF) involved in nueral plasticity (physical changes), decreased expression of BDNF likely contributes to mood disorders 2. Extracellular receptor-coupled kinase (ERK) mitogen-activated protein (MAP) kinase pathway - ERK MAP kinase MAP regulates expression of neurotropic (growth) protein Bcl-2 Li may upregulate Bcl-2 3. Phosphodiesterase (PDE) inhibition of PDE upregulates CREB (cAMP response element-binding protein) 4. Glycogen-Synthase Kinase-3 (GSK-3) constitutively active serine/threonine kinase, Li inhibits the enzyme 5. Protein Kinase C (PKC) Li attenuates PKC activity CHEM E-120

  16. GSK-3 (tau kinase) in mood disorders protein phosphatase protein + protein kinase/ATP phosphoprotein protein + PO43- (e.g. GSK-3) (- charge) GSK-3 modulates the expression of cytoskeletal proteins and synapse formation. Constitutively active, most 2nd messengers cause inactivation by phosphorylation. Inactivates transcription factors (nerve-growth factor, BDNF). Involved in apoptosis – interest in GSK-3 inhibitors GSK-3 phosphorylation increased by SSRI, TCA – possible role in 5-HT dysregulation GSK-3 & Li - role in regulation of circadian rhythms GSK-3 inhibitors act as antidepressants and antimanic in animal models CHEM E-120

  17. Bipolar Disorder - GSK-3 CHEM E-120

  18. Enzyme Kinetics Enzyme Subtrate Product d[product]/dt dependent on [enzyme], [substrate], pH and temperature. 4[E] 2[E] product [E] Time In vivo [substrate] >>> [enzyme] pseudo first-order reaction. CHEM E-120

  19. Enzyme Kinetics The interaction of enzyme and substrate to produce product can be expressed as: k1 k2 E + S ES P + E k-1 • To develop a rate equation three assumptions are made • Steady State Approximation: the concentration of ES complex is constant Km = (k-1+ k2) k1 2. Saturation conditions: the substrate is in an excess so all the enzyme is converted to ES, i.e no free enzyme is present. • When 2 is meet then the rate of the reaction will be maximal. Vmax = k2[ES] k2 is called kcat CHEM E-120

  20. Enzyme Kinetics - Michaelis-Menten equation From these assumptions one can derive the Michaelis-Menten equation Vmax [S] Km + [S] v = V = moles substrate converted to product/min At Vmax the active site is saturated. Increasing S has no effect on the rate. Vmax if v = 1/2Vmax Then KM = [S]0.5Vmax velocity Substrate Saturation Curve initial velocity area, v0, where kinetic measurements are made initial rate measurements Km [substrate] CHEM E-120

  21. Lineweaver-Burk Plot To determine the values of Km and Vmax the rate of product formation is measured at different concentrations of substrate and at constant enzyme concentration. The values obtained are plotted in a double reciprocal plot called a Lineweaver-Burk plot. from v = Vmax[S]/(Km + [S]) one can obtain the following equation of a line: 1/v = (Km/Vmax)(1/[S]) + 1/VmaxLineweaver-Burk equation 1/v m = Km/Vmax 1/Vmax -1/Km 1/[S] CHEM E-120

  22. Significance of Km and kcat k1 k2 E + S ES P k-1 Km can be regarded as an apparent dissociation constant (KD) of ES (or all enzyme-bound species. KD = [E][S]/[ES] = k-1/k1 Km = (k-1 + k2)/k1 Only when k-1 >> k2 does Km = KD In general the concentration of substrates in cells is near the Km. Smaller Km, the greater the affinity of the substrate for the enzyme (k1 >>k-1) kcat (k2) first-order rate constant that includes the rate of conversion of all ES/EP complexes to P. kcat is called the turnover number: mole product/min/mole enzyme greater kcat the greater the activity of the enzyme kcat/Km is called the specificity constant. It is an apparent second-order rate constant that refers to the properties of the free enzyme and free substrate CHEM E-120

  23. Enzyme Inhibition Enzymes are normally inhibited by another substrate (inhibitor) binding to the active site and preventing binding of the normal substrate or by an inhibitor binding to an allosteric site. 1. Reversible inhibitors (noncovalent interaction) Competitive (inhibitor binds at active site and competes with the substrate) Noncompetitive (inhibitor binds at another site on the enzyme, allosteric site) Uncompetitive (inhibitor binds to ES complex) 2. Irreversible inhibitors (covalent modification of active site) affinity labels (structure similar to substrate but contains reactive functional groups) Mechanism-based inactivators (suicide inhibitors) structure similar to substrate and contains a functional group that can be transformed in the active site to a reactive functional group 3. Slow, tight-Binding inhibitors (can be reversible or irreversible) 4. Transition State Analogs (similar to competitive or mechanism-based) CHEM E-120

  24. Measurement of Enzyme Inhibition - Ki The activity of enzyme inhibitors: Ki (M) 1 Km(1 + [I]/Ki) - -1/Kmapp = 100 0 IC50 = Ki (1 + [S]/Km) % inhibition [I] CHEM E-120

  25. GSK-3 IC50 = 5 nM water solubility < 0.005 g/L drug competes with ATP for the ATP binding site increase water solubility while maintaining enzyme inhibition? J Med. Chem. 2008, 51, 6421 CHEM E-120

  26. Synthesis of GSK-3 Inhibitors – Diversity from a Common Scaffold CHEM E-120

  27. Molecular Modeling of 11 in ATP binding pocket of GSK-3 Molecular Modeling of 13 in ATP binding pocket of GSK-3 IC50 = 5 nM Log D ~ 1.5 IC50 = 3.3 nM Log D (7.4) = -0.87 CHEM E-120

  28. Increased water solubility while maintaining enzyme inhibition CHEM E-120

  29. Pyrazolone GSK-3 Inhibitors – Vertex Pharmaceuticals Ki = 1490 nM 1.5 μM CHEM E-120

  30. Conformational Analysis & Molecular Modeling of 1 intramolecular steric hindrance? steric hindrance with enzyme? Ki = 1490 nM 2 -3 points of contact CHEM E-120

  31. Asp133 Val135 Asp200 backbone NH Lys85 NH2 made 52 compounds Ki = 1490 nM (1) to < 2 nM (46) 2 points contact to 5 points of contact CHEM E-120

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