Orjala Lecture: Adrenergic Nervous System – Sites of Drug Action Adrenergic Actionable Sites (1) Biosynthesis of NE - NE Is Synthesized from Tyr Within the Neuron

(1/26) Orjala Lecture: Adrenergic Nervous System – Sites of Drug Action Adrenergic Actionable Sites (1) Biosynthesis of NE - NE is synthesized from Tyr within the neuron. Tyr is actively transported into the cell by dedicated AA-Na+ symporters - Tyr undergoes chemical modification, becoming DA as an intermediate, and NE as the final product (2) Storage of NE - DA is transported into intracellular vesicles, where is it modified into NE and stored until its release is stimulated - Drug: Reserpine inhibits DA transport into vesicles (3) Release of NE - Arrival of electrical potentials stimulate vesicular fusion with the presynaptic membrane resulting in NE exocytosis - Drug: Guanethidine inhibits vesicular fusion (4) Post- and Presynaptic Receptors (a,b) - Among the a and b adrenergic receptors, there are 9 subtypes - Presynaptic receptors are involved with negative feedback - Postsynaptic receptor activation triggers target tissue effects (5) Reuptake of NE - Majority of NE is taken up presynaptically and recycled - Drug: Cocaine and TCA inhibit reuptake, prolonging the signal (6) Metabolism: MAO - Monamine Oxidase (MAO) catabolizes NE - Drug: Target in PD and for antidepressants (7) Metabolism: COMT - Catechol-O-methyl transferase (COMT) inactivates NE via methylation. Drug: Target in PD Biosynthesis of NE and other Catecholamines - L-Phenylalanine à L-Tyrosine - L-Phe is an essential AA. It is hydroxylated by the para-directed enzyme L-phenylalanine hydroxylase utilizing Tetrahydrobiopterin as a cofactor, producing L-Tyr - L-Tyrosine à L-DOPA (cytosol) - Following absorption into the neuron, L-Tyr is hydroxylated at the meta position by the rate-limiting enzyme L-Tyrosine Hydroxylase utilizing Tetrahydrobiopterin as a cofactor, producing L-DOPA. - The production of L-DOPA is the committed step to the catecholamine biosynthetic pathway. - Drug: a-Methyltyrosine (Demser) is competitive inhibitor of L-Tyrosine Hydroxylase (Mentioned later) - L-DOPA à DA (cytosol) - In the cytoplasm, L-DOPA is decarboxylated by L-Aromatic Amino Acid Decarboxylase utilizing Pyridoxal Phosphate (Vit-B6) as a cofactor, producing Dopamine (DA). - The L-Aryl-AA-Decarboxylase enzyme is the first non-specific AA enzyme we have encountered. For instance, it can operate on His as well. Since it is not a rate-limiting enzyme, it is not a common target in therapy, albeit a few - Drug: Aldomet (a-methyldopa): A competitive inhibitor of L-Aryl-AA-Decarboxylase. Has antihypertensive activity due to its metabolite a-methyldopamine (Mentioned later in ‘Drugs and Catecholamine Synthesis’) - Drug: Sinemet (L-DOPA+Carbidopa): Carbidopa is a true inhibitor of L-aryl-AA-decarboxylase with a low ‘off’ rate. Carbidopa’s mechanism is modulated by its hydrazine group (-NH-NH2) o MoA: Taken alone, L-DOPA would be converted to DA peripherally, at which point it would be charged and unable to cross the BBB. Taken with Carbidopa, its conversion does not occur peripherally and there is enhanced uptake of L-DOPA in the CNS. This is an effective PD therapy. - DA à NE (vesicle) - DA is converted to NE via Dopamine b-hydroxylase (DBH) utilizing Vit-C as a cofactor, producing R(-)-NE. (Only the R form is active) - Drug: Antabuse (Disulfiram) and Nepicastat (SYN117) inhibit the radical reaction. The latter is currently in clinical trials for investigating its use in treating PSTD and cocaine dependence Biosynthesis of E - The adrenal medulla plays a significant role in producing the sympathetic stimulants for circulation. Upon stimulation, both NE and E are released (80% E!). This is unique to the medulla, compared to the CNS - NE à E, a methylation reaction (medulla) - R(-)-NE is converted the E via methylation at the Nitrogen group by Phenylethanolamine N-methyl Transferase (PNMT) utilizing S- adenosylmethionine (SAM) as a cofactor and methyl donor. - Conferring Selectivity: The addition of the methyl group: (1) Slows MAO Metabolism of E relative to NE (2) Increases E selectivity for b receptors - b-adrenergic receptors, as you’ll see in a moment, have more room in the active site to receive E and its methyl group. The addition of this methyl group also improves its circulation - FYI: In case you were sitting there wondering, ‘what was that other important endogenous methyl donor I learned about?’ – you’re in luck. It was Tetrahydrofolate, which methylates C1s during nucleic acid synthesis Metabolic Inactivation of NE (or DA) - Catabolism of NE, DA, or E involves a combination of modifications by MAO and COMT. The order of events does not matter, the final excreted product will be the same depending on the division of the nervous system o PNS: Aldehyde Dehydrogenase produces carboxylic acid products: Vanillyl Mandelic Acid (VMA) o CNS: Alcohol Dehydrogenase produces alcohol products: 3-Methoxy-4HydroxyPhenylGlycol (MHPG) - Method: “MAO first!”: Monoamine oxidase is present in various tissues and throughout the blood. It converts the primary amine to an aldehyde, producing DOPGAL. The aldehyde group is very reactive. o At this point, DOPGAL may be encountered by Aldehyde Dehydrogenase or COMT-SAM in any order. The aldehyde will be converted to a carboxylic acid group. o Aldehyde dehydrogenase will produce the major PNS metabolite, VMA to be excreted in the urine - Method: “COMT first!”: Catechol-O-Methyl Transferase, with the assistance of SAM, will methylate the catecholamine phenolic-OH, eliminating the activity altogether. This substituent on the catechol group is large enough that it ceases activity. MAO will convert the primary amine to an aldehyde. COMT Result= (1) Inactivation,(2) Detoxification o At this point, the final product depends on if metabolism is occurring in the CNS or PNS. If CNS- Alcohol dehydrogenase (ADH) will reduce the aldehyde to a hydroxyl, producing MHPG Adrenergic Receptors - Thus far, 9 subtypes of adrenergic receptors have been identified. Each of which are GPCR o 6 a: a1 (A, B, D) a2 (A, B, C) which, to be vague, are involved in vasoconstriction o 3 b: b1 (heart), b2 (lungs), b3 (adipose + bladder) - The receptor consists of 7-transmembrane domains, 3 of which are involved in substrate interaction. The right side, the polar side, has the majority of the interactions for substrate-receptor activity o Domain III: Anionic Binding Site: Asp-113 - + § Asp-COO engages in ionic interactions with catecholamine-NH3 o Domain V: Catechol Binding Site: Ser-204, Ser-207 § Ser-OH engages in H-bonding with the Catechol-OH groups o Domain VI: Aromatic Binding Site: Phe-290 § Phe-Aryl engages in Pi-stacking with the catechol-benzyl o Domain VI: H-Bonding Site: Asn-293 § Asn-Amide engages in H-bonding with the Catechol-b-OH § Note: DA does not have a b-OH, \ at [physiological] it has no effect - a vs b: Adrenergic Receptor Differences o Compared to the a-adrenergic receptors, the b receptor is capable of harboring larger hydrophobic moieties substituted on the Nitrogen. For example, the synthetic b-adrenergic agonist R-Isoproterenol has an isopropyl substitution on the ethanolamine. It is therefore b-selective.

Drugs, and the protective role of COMT: COMT is present in the adrenergic/dopaminergic nerve terminals to degrade NT. At higher concentrations, it is located in the Liver and Kidneys for the detoxification of catechol metabolites. - à Catechols are VERY reactive, capable of oxidizing within cells to ortho-Quinone. O-Quinone is a neurotoxin that can alkylate proteins and neuronal content, leading to mutations, cancer, and cell death. - High [COMT] dominates catechol metabolism to produce the (1) inactivated and (2) non-toxic metabolites - Entacapone and Tolcapone are inhibitors of COMT used as adjuncts to L-DOPA in PD patients o MoA: Levodopa therapy requires the L-DOPA enter the brain to help restore/increase DA concentrations. To prevent peripheral metabolism, similar to carbidopa’s mechanism, these compounds inhibit COMT – effectively increasing L-DOPA passage into the CNS. Remember, peripheral conversion of L-DOPA to DA severely limits the opportunity of transport across the BBB. o While Entacapone is peripherally-acting only, Tolcapone is more lipophilic and can cross the BBB § Though Black box warning; can cause life-threatening Liver damage. Drugs and Catecholamine Synthesis - Featured to the right is a comparison between the natural activity Normal of catecholamine synthesis and the drug-manipulated activity

- a-Methyl-L-Tyrosine (Metyrosine/Demser): is a competitive inhibitor of Step 1: L-Tyr hydroxylase, mimicking L-Tyr. This is the rate-limiting enzyme, a-methyl-L-DOPA, and the other Demser and Aldomet Activity downstream products will be produced slowly. o CNS effects of this drug are mediated by a-methyl-DA and a-methyl NE. They are a-2 agonists o Aldomet (a-methyl-L-dopa), another option. - COMT-I Potentiation: COMT inhibitors, such as Entacapone, will potentiate the CNS effects of a-methyl-L-Tyr (Metyrosine) and a-methyl-L-DOPA (Aldomet) because, by preventing their metabolism, they have increased opportunity to cross the BBB via active transport, through their AA-like structure (carboxylate, amine). This is NOT the case for a-methyl-DA or a-methyl-NE, as these do not have AA-like structures. Drugs and Signal Termination - There are 2 mechanisms by which the signaling response of NE activity is terminated: - (1) Reuptake via NE Transporter: Uptake-1/NET o Cocaine and TCA: These compounds potentiate the sympathetic effects of adrenergic activity by inhibiting the reuptake of NE in sympathetic nerves by Uptake-1. § This is considered an “AE” – Recall, Cocaine is indicated as an anesthetic, not for recreation ;) - (2) Repackaging by Vesicular Monoamine Transporter (VMAT) o Reserpine blocks VMAT activity, depleting the storage of NE over time. Due to NE being pre-packaged in most cases, it will take time for this compound to elicit an effect until those reserves are depleted. This is an anti-hypertensive drug popularly used in India Drugs and Catecholamine Release - Guanethidine and Guanadrel will ‘replace’ NE in the vesicles, and prevent vesicular fusion with the membrane. However, due to their charged guanidinyl groups, they cannot cross the BBB thereby preventing CNS effects. Overall, they limit the release of NE into the synapse. Drugs: a-Adrenergic Receptor Agonists - When discussing the a-adrenergic agonists, the phenylethanolamine derivatives are the key players. These are substituted phenethylamines, which is a diverse class of drugs certainly worth looking into – in your ‘free’ time. An excellent unbiased resource is Shulgin’s PiHKAL o Phenylethanolamine: Phenyl group, Ethane chain – substituted with a hydroxyl, that is aminated. - Phenylephrine: a1 selective adrenergic agonist. Since it does not have the catechol moiety, it cannot act at a2 - Methylepinephrine: a2 selective adrenergic agonist. Methylation at the a-carbon produces steric hindrance, prohibiting Methylepinephrine from activating the a1 receptors. a2 receptors can accommodate the methyl because they have an additional ‘methyl binding pocket.’ - (S)-NE vs (R)-NE: Endogenous NE is the (R) configuration because the (S) is not as active. The (S) configuration prohibits the b-OH from H-bomding - Midodrine: Midodrine is a prodrug that is cleaved by CYP in the Liver to its active metabolite. It is a1-selective due to its lack of a catechol moiety. This drug is used to ÝBP, indicated for conditions like orthostatic HypoT.