Document

Transcript Document

Il sistema colinergico

Un sistema neuronale

THE AUTONOMIC NERVOUS SYSTEM

Autonomic Nervous System: parasympathetic division

THE AUTONOMIC NERVOUS SYSTEM Parasympathetic System

•Long preganglionic fibers •Somata located in brain and sacral regions of cord •Peripheral ganglia lie near target organs

Toraco-lombare

Spinal cord

Cranio-Sacrale

brain glands and organs ACh NA ACh Placca neuromuscolare

Ach and Dopamine

Central functions

Major groups of

cholinergic cells

are found in the

medial septal nucleus, the diagonal band nucleus

, and the

nucleus basalis

of the forebrain. The cholinergic cells project to the

hippocampus

( learning and memory ),

cerebellum

( movement regulation ) , and to the

cerebral cortex .

Modulation of peripheral functions

The parasympathetic system predominates when acute episodes of stress provide no obvious coping response. Under non-stressful conditions, each of the effector organs responds individually as necessary

Other peripheral functions

b -adrenergic agonists ( b 3 in particular) induce relaxation of the bladder body  -adrenergic agonists (  1a in particular) increase urethral tone and closure pressure Anthicholinergic agents (muscarinic) decrease the response to cholinergic stimulation

Il modulatore o

neurotrasmettitore

del sistema colinergico

Different Types of NT

1. Amine



Acetylcholine (Ach) 2. Monoamines

Catecholamines i.

Dopamine ii.

Norepinephrine Indoleamines i.

Serotonin



Acetylcholine (Ach) First identified and more abundant in the body.



First demonstrated by

Otto Loewi

in 1921, by electrically stimulating the vagus nerve of a frog, which he knew would

slow down

the heart rate.



He then took the fluid surrounding the stimulated heart and applied it to an unstimulated heart.



The second heart also slowed its beat, proving that the first heart had indeed released a chemical into the fluid.



He later proved that the chemical was

Ach .

cellula neuronale

+ N OH

colina

Neuronal biosynthesis of Ach

Colina acetiltransferasi

+ N

Acetil-CoA

AchE, acethyl cholinesterasi

Ach

N-methyl-metiltransferasi

HO H 2 N

etanolamina

HO H 2 N COOH

serina cellula epatica

aa-decarboxylasi

reuptake

Colina + acido acetico

Ach receptor system

Cartoon of the two types of acetylcholine receptors. Acetylcholine receptors are integral membrane proteins that occur as either nicotinic-sensitive ionotropic or muscarinic-sensitive metabotropic receptors. Nicotinic receptors are ion channels created by the assembly of five protein subunits.

Muscarinic receptors belong to the superfamily of GPCRs which are characterized as a single peptide which weaves in and out of the plasma membrane creating seven hydrophobic membrane spanning domains and three extracellular and cytoplasmic loops.

Family of five muscarinic acetylcholine receptors. The muscarinic receptor family has five known members designated M1-M5 based on the order in which they were cloned. The M1, M3, and M5 stimulatory receptors couple primarily to the mobilization of intracellular calcium. The increase in cytoplasmic calcium occurs following stimulation of a signaling cascade which begins with coupling to the heterotrimeric G protein, Gq, followed by stimulation of the effector enzyme, phospholipase C, which releases inositol-1,4,5-trisphosphate (IP3). The M2 and M4 of cAMP. inhibitory receptors negatively modulate adenylate cyclase to reduce cytoplasmic levels

M1,3,5

M2,4

Spinal cord brain glands and organs ACh NA ACh Placca neuromuscolare

Spinal cord brain glands and organs ACh NA ACh Placca neuromuscolare ganglia



are involved in cognition neurodegeneration, pain, anxiety and depression



are involved in D release and addition

The

4b4, 4b2, 3b2

receptors

The

7

receptor

Biological active conformer of Ach

O X N + O

Acetilcolina

2-(acetossi)-N,N,N-timetil-etanammina Cl

H + N(CH 3 ) 3 60

OCOCH 3 H H H

gauche o anticlinale from NMR and X-ray studies

180

H N(CH 3 ) 3 H H H OCOCH 3

trans o antiplanare from theorethical stidies

H H H + N(CH 3 ) 3 H 137° OCOCH 3 Sinclinale

Proposed biological active conformer

I VII Tyr-533 Tyr-529

X -

II VI Tyr-506

N + O

Thr-231

Thr-234 V Tyr-148 Asp-147 IV III

Modello di interazione dell’Ach con i recettori muscarinici

(la numerazione dei residui aa si riferusce al recettore m3 di ratto)

Structure activity relationship

O X N + O

Functional group accepting hydrogen bonding Protonated nitrogen

spacer

La sostituzione con atomi di H cosi come la sostituzione con radicali piu’ voluminosi del metile determina una progessiva diminuzione di attività agonista.

La sostituzione dell’N con altri atomi, S,P,As o Se è tollerata.

L’ammina quaternaria non è indispensabile per l’attività.

Il gruppo estereo puo’ essere sostituito con altri gruppi accettori di legami a H e piu’ resistenti alla degradazione enzimatica, (eteri, chetoni, carbamati, ecc.) Radicali acidi a catena piu’ lunga determinano diminuzione di attività mentre quelli aromatici in alcuni casi attività antagonista.

L’allungamento dello spacer è drasticamente non tollerato, in particolare dal recettore muscarinico, l’alchilazione con radicali piccoli (CH una diversa selettività recettoriale. 3 ) è tollerato e determina una maggiore resitenza alla degradazione enzimatica e in alcuni casi

Sostanze naturali che interagicono prevalentemente con i recettori

muscarinici con attività colinomimetica (agonisti).

OH X + N O

muscarina

[2S-(2



)]-N,N,N,-trimetil-4-idrossi-5-methyl-tetraidrofuran-2-il-metan amina Cl O O N O N + O

Acetilcolina

pilocarpina

(3S-cis)3-etil-4[(1-metil-1H-imidazol-5-il)-metil]-diidro-3H-furan-2-one

Sostanze naturali che interagicono prevalentemente con i recettori

muscarinici con attività colinomimetica (agonisti).

X O N + O

Acetilcolina

O N O

arecolina

1-methyl-1,2,5,6-tetrahydro-pyridine-3-carboxylic acid methyl ester

Sostanze di sintesi che interagicono prevalentemente con i recettori

muscarinici con attività colinomimetica (agonisti).

N + O O

metacolina

X O N + O

Acetilcolina

2-(acetossi)-N,N,N-trimetil-1-propan amina Cl —

N + O O NH 2

carbacolo

2-[(aminocarbonil)-oxi)-N,N,N-trimetil-etan amina Cl —

O N + J S P O O

ecotiopato

2-[(diethoxyphosphinyl)-thio)-N,N,N trimetil-etan amina J —

N + O O NH 2

betanacolo

2-[(aminocarbonil)-oxi)-N,N,N-trimetil-1-propan amina Cl-

Colinomimetici di sintesi derivati dall’arecolina

N O O

arecolina

Xanomeline analogue N N N N O N N S O

Xanomelina

5-(4-Hexyloxy-[1,2,5]thiadiazol-3-yl)-1-methyl-1,2,3,6-tetrahydro-pyridine

The selective

M 1 and M 4

agonist xanomeline was shown to significantly improve psychiatric symptoms in phase II clinical trials in Alzheimer's patients.

Unfortunately, xanomeline produced unwanted side effects associated with the activation of

M 3

compound.

receptors that severely limit the clinical utility of this

The implication of muscarinic receptors in patologies of brain

Muscarinic agonists such as xanomeline may exert an antipsychotic action by regulating the release of dopamine in the frontal cortex.

On the other hand, M 4 receptors regulate the release of dopamine and have been implicated in schizophrenia.

Xanomeline produces very few of the adverse side effects (e.g., catalepsy) associated with classical antipsychotics such as haloperidol, suggesting that selective muscarinic agonists might provide a useful alternative therapeutic approach to treating the symptoms of schizophrenia. Moreover, muscarinic agonists might be useful in improving cognitive function (including memory function, language use, and constructional praxis) in schizophrenic patients.

Similar to M 1 receptors, M 2 receptors also are expressed in the hippocampus and most other brain regions implicated in learning and memory processes. Since M 1 and M 2 receptors play a role in cognitive and memory function, agonists with M 1 and M 2 activity might be particularly useful in treating memory and cognitive deficits associated with schizophrenia.

Taken together, an

M 1 , M 2 , and M 4

agonists could provide efficacy in a broad range of symptomatic domains of schizophrenia, including enhancement of cognitive function.

Schematic representation of the nACh-R A : Heteropentameric complex of neuronal nicotinic



- and

-subunits (note the 2:3 ratio between the



and

). The ACh-binding site is localised at the interface of



and the adjacent subunit

Schematic binding of Ach to nicotinic receptors

In accordance with several different studies Tyr93 and Trp149 are oriented in close proximity to the quaternary ammonium group of Ach, with Tyr190, Tyr198 and the

cysteine

residues closer to the ester moiety of Ach.

Sostanze naturali che interagicono prevalentemente con i recettori

nicotinici con attività colinomimetica (agonisti).

Nicotina

(S) 3-(1-Methyl-pyrrolidin-2-yl)-pyridine X O N + O

Acetilcolina Altri composti naturali di interazione nicotinica

(-)-Cytisine

O N NH

Ach interacts to both receptors

N O + H 3 C CH 3 N CH 3 4.4A

O OH ------------------------ O CH 3 The distance between the protonated nitrogen and the chemical function accepting hydrogen bond of a cholinergic ligand is a crucial element determining interaction with muscarinic receptor (4.4A

) H + N H ------------------------ CH 3 + H 3 C CH 3 N CH 3 5.5 A

O N ------------------------ O CH 3 or nicotinic receptor (5.5A

) .

Ach is able to interact with both receptors.

Acetylcholinesterase, utilizes serine as the active site nucleophile (a common theme) and gets an assist from an histidine residue

X -

Ach

N + O O N H OH HN ----------------- N C O N H C O AChE

Prodotto di degradazione

Acetylcholinesterase,

is a

serine hydrolase

belongs to the esterase family within higher eukaryotes.

Acetylcholinesterase

that The active site is composed of two subsites: the

esteratic subsite

catalytic triad, and the

anionic subsite

acetylcholine.

which contains the The esteratic subsite contains the catalytic machinery of the enzyme: a catalytic triad of

Ser 200, His 440, and Glu 327 .

This catalytic triad is similar to other serine proteases.

X N + O

The anionic subsite is defined by Its role is to orient the charged part of the substrate that enters the active center, and this role is the main function of the

Trp Trp 84, Phe 330, and Phe 331

residue. .

Acetylcholinesterase,

is a serine hydrolase that belongs to the esterase family within higher eukaryotes. This family acts on different types of carboxylic esters.

The

anionic subsite

has another interesting characteristic, it is involved in a "

cross-talk

" mechanism with the

peripheral anionic site (PAS).

Site-directed labeling and mutagenesis studies place the location of the PAS at or near the rim of the aromatic gorge. This site has the ability to bind to many different types of ligands, and by doing so effects the conformation of the active center.

Six residues have established activity within this site: Trp 286, Tyr 72, Tyr 124, Glu 285, Asp 74 and Tyr 341, which are located on the opposite side of the gorge entrance. This array of residues exhibits flexibility which accommodates many distinct ligands, and also implies their conformational mobility.

The common feature of these conformations is a core comprised of

Trp 286

and

Asp 74

On one hand, the increase of affinity for AChE has been pursued through a simultaneous interaction of the drug with both the active site of the enzyme and the

so-called peripheral site

, a less well-defined area located at the entrance of the catalytic gorge of AChE, approximately

14 Å

distant from the active site.

Sostanze naturali che inibiscono l’attività della AchE con attività parasimpaticomimetica indiretta.

Inibitori che trasferiscono sull’ enzima un gruppo isosterico della funzione esterea.

H N O O N H N

(3aS cis) (-)Fisostigmina

pKa(N1) = 8.46

pKa(N8) = 3.7

Methyl-carbamic acid 1,3a,8-trimethyl-1,2,3,3a,8,8a-hexahydro-pyrrolo[2,3-

]indol-5-yl ester X N + O O

Acetilcolina

Sostanze di sintesi che inibiscono l’attività della AchE con attività parasimpaticomimetica indiretta.

Inibitori che trasferiscono sull’ enzima un gruppo isosterico della funzione esterea.

R R' N O O N R 2 H N R 1 Phenyl-carbamic acid 1,3a,8-trimethyl-1,2,3,3a,8,8a-hexahydro-pyrrolo[2,3-

]indol-5-yl ester

Derivati di sintesi con semplificazione della struttura pirrolo-indolica R R' N O O N R 2 H N R 1

Pirodostigmina Neostigmina

N O + N Br N O N + Br O O

3  (dimetilamino)-carbonil  -ossi  -N,N,N-trimetilbenzenamina 3  (dimetilamino)-carbonil  -ossi  -1-metil-piridinio bromuro

N O N O

Rivastigmina

Ethyl-methyl-carbamic acid 3-(1-dimethylamino-ethyl)-phenyl ester

Inibitori reversibili della AchE che non trasferiscono sull’ enzima gruppi isosterici della funzione esterea.

The drug

Tacrine

has drawn considerable attention because of a report of its clinical efficacy for this Emzyme.

Its structure is,

1,2,3,4-Tetrahydro-acridin-9-yl-amine

The experimental structure of the AChE/tacrine complex show a strong interaction of the aromatic moieties bound solely to the hydrophobic sites. However, large-scale clinical trials show that the primary disadvantage of

Tacrine is its high toxicity

. This has led to investigations into possible variants of the basic Tacrine structure.

Some reported variations used the following structure,

Drug design of TACRINE analogues

The rationale for its use was related to the elevation of ACh levels that can compensate for the cholinergic deficiency associated with the brain lesions in AD. Nevertheless, the deficiency of tacrine in clinic has been related mainly to elevated liver transaminase levels and dose-related, low-selective peripheral cholinergic effects.

Tacrine (

) and Its Derivatives

In particular

6-chlorotacrine

(

) has been found to be more potent than other substituted analogues, the reduced electron density on the tacrine aromatic rings could favor p -interactions with nearby residues in active sites of the enzyme and strongly increase the inhibitory potency of tacrine.

J. Med. Chem., 45 (11), 2277 -2282, 2002

Polar interactions between donepezil and AChE as well as interactions of both aromatic rings of donepezil with aromatic amino acids of the AChE are similar to tacrine.

Galanthamine

Galanthamine

, Nivalin® a tertiary alkaloid extracted from several species of

Amaryllidacae

, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. Galantamine interacts with the

presinaptic nicotinic receptor

(autoreceptor) and the result is an improvement of release of Ach and other neurotrasmitters.

*** * ***

(-)-Galanthamine, 4a,5,9,10,11,12-esaidro-3-metossi-11-metil-6H-benzofuro-



3a, 3, 2-ef

 



-benzo azepin-6-olo

Drugs do not transferring chemical functions approved for AD treatment

The only drugs approved by the United States Food and Drug Administration (FDA) for the treatment of AD are reversible inhibitors of AChE. They are

Tacrine

, marketed as

Cognex

,®

Galantamin

, marketed as

Nivalin or Reminyl

, and, recently, the more potent

E2020

, marketed as

Aricept

®.

However, both have drawbacks such as hepatotoxicity and, in the case of tacrine,

low selectivity for AChE

vs. butyrylcholinesterase (BChE); this may be important, as it has been suggested that inhibition of BChE, which is abundant in human plasma, may cause increased side effects.

Enzyme selectivity of different AchE inhibitors

Alzheimer's disease

Alzheimer's disease

(AD) is the most common progressive dementia associated with aging, with

-amyloid plaques, neurofibrillary tangles, and synaptic loss being the

major neuropathological hallmarks of the disease.

Alzheimer's disease

The pathological hallmarks observed in the brains of AD patients are the: a) extracellular amyloid plaques, mainly composed of an amyloid β peptide with 42 amino acids in length (Aβ1 -42), b) the intracellular neurofibrillary tangles of hyperphosphorylated τ -protein. According to the amyloid hypothesis, the prevailing theory in the field, the underlying cause of AD is the aggregation and deposition of Aβ1 -42 in the brain due to its overproduction and/or diminished clearance.

This hypothesis is supported by strong genetic, histopathological, and clinical evidence.

Biological steps in the Alzheimer’s desease

Amyloid-beta formation The highly hydrophobic A

b

can nucleate and form fibrils that are neurotoxic and are capable of activating inflammatory processes in the brain.

Among AD-causing genetic mutations are those in the amyloid precursor protein (

APP

), an integral membrane protein of unknown function that is proteolytically processed to A

b.

Cleavage of APP just outside the single transmembrane region by



and

-secretases

forms soluble versions of APP (



- and

-APPs) that display neurotrophic activities and membrane-associated 83- and 99-amino acid C-terminal fragments of APP (C83 and C99), respectively.

-Secretase then cleaves these fragments in the middle of the transmembrane region to form A

(from C99) and p3, a 3-kDa N-terminally truncated A-

peptide (from C83). The cleavage of

-secretase is heterogeneous but principally leads to 40- and 42-amino acid peptides, A-

40 and A

42 , in roughly 9:1 proportions.

Another rappresentation of Amyloid-beta formation

Amyloid precursor protein (APP) and its metabolites

aa Sequence of Amyloid

-Protein (1-42)

H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys Leu-Val-Phe-Phe-Ala Glu-Asp-Val Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met -Val-Gly-Gly-Val-Val-Ile-Ala-OH The peptide evolves in well defined structure, the core of fibril formation “Leu 17 -Val-Phe-Phe-Ala 21 ”, critical sequence for fibril formation “Gly 25 -Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met 35 ” , minimal sequence for fibril formation

Hypothetical mechanisms of conformational transitions for Aß peptides (A).

The peptides are proteolytically cleaved from APP, precipitate as ß-structures in equilibrium with oligomers which may eventually redissolve into the membrane.

Structural models of aggregates

AD treatment

To date,

AChE inhibitors

are the major drugs approved for the symptomatic treatment of Alzheimer's disease.

It has also been demonstrated that

AChE

could play a key role during the early stage in the development of the senile plaques by

accelerating

-amyloid peptide deposition.

It is observed that inhibition of the

peripheral binding site (PBS)

prevent the deposition of b -amyloid peptide induced by AChE. of AChE might

MEMANTINE Brief Profile Memantine – Innovation for Alzheimer Patients Memantine is the first and only representative of a new class of Alzheimer drugs – a moderate affinity NMDA-receptor antagonist. Memantine has been developed by Merz Pharmaceuticals and is approved in Europe and the USA for the treatment of moderate to severe Alzheimer's disease. Efficacy of Memantine Clinical data on memantine show * Benefit in cognitive and psychomotor functioning * Benefit in activities of daily living * Reduction of care dependence * Excellent tolerability H 2 N

Memantine

Memantine produces symptomatic improvements in learning under conditions of tonic NMDA receptor activation in Alzheimer’s disease. In contrast to first generation therapies, memantine is likely to show neuroprotective effects at concentrations used in the treatment of Alzheimer’s disease and to slow down disease progression. |

Physiological condition NMDA receptors are transietly activated by mM concentrations of glutamate. In order to prevent excessive influx, the ion channel is blocked by a Mg ++ under resting conditions. If the NMDA is activated by glutamate and the postsynaptic neuron is depolarized at the same time, Mg ++ leaves the ion channel and the Ca ++ can flow in, which is important for learning processes.

Pathological condition

NMDA protection with “Memantine”

Protease inhibitors,

a real therapy?

and

- secretases inhibitors

- and

- secretase inhibitors could be a more appropriate therapy of AD.

BACE ( beta amyloid claving enzyme) is a more studied enzyme and more inhibitors are reported

g 

Secretase has not been definitively identified nor well-characterized, and even the issue of whether the formation of A-

b

g

-secretases is unclear. and A-

b

42 is due to different Inhibitor studies demonstrated that while A-

b

40 and A-

b

42 formation is pharmacologically distinct, panels of

g

-secretase inhibitors show the same rank order of potency for blocking the formation of the two peptides suggesting that the active site topologies for the responsible enzymes are similar, if not identical.

Fine presentazione

Nervous System

Central Nervous System (CNS)

(Brain and Spinal Cord) Peripheral Nervous System (PNS)

Efferent (motor) Nerves

( Transmit information to the perifery of the body)

Afferent (sensory) Nerves

(Carry sensory information to the CNS from the perifery of the body)

Autonomic Sympathetic Somatic Parasympathetic



Peripheral Nervous System (PNS)

•

Efferent (motor) Nerves

– Transmit information to muscles or glands

.

Somatic Nervous System

Stimulates Skeletal muscles



Autonomic Nervous System

•

Stimulates Glands and Organs (e.g. heart)

•

Sympathetic - Adrenergic – stress response

•

Parasympathetic - Cholinergic – basic functions

Autonomic Nervous System: sympathetic division

Struttura delle cellule neuronali

Esempi di comunicazione neuronale

The arrival of the action potential causes the release of chemical messengers from the terminal

endings of the axon.

THE AUTONOMIC NERVOUS SYSTEM Parasympathetic System Long preganglionic fibers Peripheral ganglia lie near target organs BRAIN Ach and Dopamine BRAIN glands and organs NA ACh ACh Placca neuromuscolare BRAIN

THE AUTONOMIC NERVOUS SYSTEM

Sympathetic System Short preganglionic fibers Peripheral ganglia lie distant target organs Ach and Dopamine BRAIN glands and organs NA ACh ACh Placca neuromuscolare Peripheral activities of sympathetic nervous system involve many organs: heart, bladder,pupils, bronchi, vessels, glands

Principal traget tissues: 1. Brain 2. Eye 3. Heart 4. Broncus/Lung 5. Gastrointestinal

The autonomic nervous system uses a combination of different anatomical organizations and different chemical mediators to cause different (usually opposing) effects in the same target organs.

Biological active conformer of Ach

O X N + O

Acetilcolina

2-(acetossi)-N,N,N-timetil-etanammina Cl

H + N(CH 3 ) 3 OCOCH 3 H H H 60

° gauche o anticlinale from NMR and X-ray studies

+ (H 3 C) 3 N H H N(CH 3 ) 3 H H 180

H OCOCH 3

trans o antiplanare from theorethical stidies

H H H + N(CH 3 ) 3 H 137° OCOCH 3 Sinclinale Derivati ciclopropanici rigidi O-CO-CH H 3 + (H 3 C) 3 N H H O-CO-CH 3 cis-ACTM trans-ACTM

1S,2S