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DEPARTAMENTO DE

ARMACOLOGIA

BMF 0120
INTERAÇÃO FÁRMACO-RECEPTOR
E MECANISMOS DE TRANSDUÇÃO
Leticia Veras Costa Lotufo
Departamento de Farmacologia
Email: costalotufo@gmail.com

MECANISMOS DE TRANSDUÇÃO

MECANISMO
EFEITO
FÁRMACO RECEPTOR DE
BIOLÓGICO
TRANSDUÇÃO

Fármaco deve ligar-se a receptores ou alvos

farmacológicos, causar sua ativação para
enfim causar sua resposta biológica!!!

AFINIDADE è EFICÁCIA
ASPECTOS MOLECULARES DA AÇÃO DO FÁRMACOS

1
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ALVOS FARMACOLÓGICOS OU
RECEPTORES
“macromolécula (ou complexa macromolecular) com o
qual a molécula interage para produzir uma resposta
celular (i.e. uma alteração da função celular)”
Goodman & Gilman (12a. Edição - 2012)

• ALVOS PROTÉICOS OUTROS ALVOS:

– Enzimas Proteínas estruturais
– Transportadores Tubulina
– Canais iônicos DNA
– Receptores Parede Celular

LOCALIZAÇÃO DO RECEPTOR
s, fatty acid derivatives, and even dissolved
n monoxide. Most of these signal molecules
(A) CELL-SURFACE RECEPTORS
Forte relação com a natureza química do
plasma membrane
ace by exocytosis from the signaling cell, as cell-surface
receptor protein
fármaco:
wever, are emitted by diffusion through the
whereas others are displayed on the external
Hidrossolúveis – baixa
ed to it, providing a signal to other cells only permeabilidade celular
mbrane signal proteins may operate in this Lipossolúveis – alta permeabilidade
may be released from the signaling cell’s sur-
act at a distance.
hydrophilic signal
molecule target cell celular
gnal, the target cell responds by means of a
(B) INTRACELLULAR RECEPTORS
olecule and then initiates a response in the
eptor has a complex structure that is shaped small, hydrophobic Implicações:
signal molecule
h high specificity, helping to ensure that the Farmacocinética – absorção,
priate signal and not to the many other sig-
ll. Many signal molecules act at very low con- carrier protein
target cell transporte, eliminação,
their receptors usually bind them with high metabolismo
0–8 M; see Figure 3–44). Farmacodinâmica – Tipo de
smembrane proteins on the target-cell sur-
xtracellular signal molecule (a ligand), they receptores
ous intracellular signals that alter the behav- nucleus
eptor proteins are inside the target cell, and intracellular receptor protein

cell to bind to them: this requires that the

and hydrophobic to diffuse across the target
3). This chapter focuses primarily on signal-
ut we will briefly describe signaling through
apter.
espond to Specific Combinations4 of
sm is exposed to hundreds of different signal
olecules can be soluble, bound to the extra-
ce of a neighboring cell; they can be stimula-
numerable different combinations; and they
ell behavior. The cell responds to this blizzard
expressing only those receptors and intracel-
o the signals that are required for the regula-
erent signals in the environment, and some
sponse to other signals. One of the key chal-
Figure 15–3 The binding of extracellular
signal molecules to either cell-surface
Essential
or intracellular receptors. Cellsignal
(A) Most Biology. Alberts et al., 2013, 4th edition.
molecules are hydrophilic and are therefore
unable to cross the target cell’s plasma
membrane directly; instead, they bind to
cell-surface receptors, which in turn generate
signals inside the target cell (see Figure
15–1). (B) Some small signal molecules,
by contrast, diffuse across the plasma
membrane and bind to receptor proteins
inside the target cell—either in the cytosol
or in the nucleus (as shown here). Many of
these small signal molecules are hydrophobic
and poorly solublem15.03/15.03
MBoC6 in aqueous solutions; they
are therefore transported in the bloodstream 2
and other extracellular fluids bound to carrier
proteins, from which they dissociate before
entering the target cell.
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RECEPTORES DE MEMBRANA
TRNSDUÇÃO DO SINAL
814 Chapter 15: Cell Signaling

EXTRACELLULAR SIGNAL MOLECULE Figure 15–1 A simple intracellular

signaling pathway activated by an
extracellular signal molecule. The signal
RECEPTOR PROTEIN molecule usually binds to a receptor
protein that is embedded in the plasma
plasma membrane of membrane of the target cell. The receptor
target cell activates one or more intracellular signaling
CYTOSOL
pathways, involving a series of signaling
proteins. Finally, one or more of the
intracellular signaling proteins alters the
activity of effector proteins and thereby the
behavior of the cell.

INTRACELLULAR SIGNALING PROTEINS

EFFECTOR PROTEINS

metabolic transcription cytoskeletal

enzyme regulatory protein protein

altered altered gene altered cell

metabolism expression shape or
movement

Essential Cell Biology. Alberts et al., 2013, 4th edition.

5 signaling to cells far away; others signal only to immediate neighbors. Most cells
in multicellular organisms both emit and receive signals. Reception of the signals
depends on receptor proteins, usually (but not always) at the cell surface, which
bind the signal molecule. The binding activates the receptor, which in turn acti-
vates one or more intracellular signaling pathways or systems. These systems
depend on intracellular signaling proteins, which process the signal inside the
MBoC6 m15.01/15.01
receiving cell and distribute it to the appropriate intracellular targets. The tar-
gets that lie at the end of signaling pathways are generally called effector proteins,
which are altered in some way by the incoming signal and implement the appro-
priate change in cell behavior. Depending on the signal and the type and state of
the receiving cell, these effectors can be transcription regulators, ion channels,

SINALIZAÇÃO
components of a metabolic pathway, or parts of the cytoskeleton (Figure 15–1).
The fundamental features of cell signaling have been conserved throughout the
evolution of the eukaryotes. In budding yeast, for example, the response to mating
factor depends on cell-surface receptor proteins, intracellular GTP-binding pro-
teins, and protein kinases that are clearly related to functionally similar proteins
UM TRANSMISSOR
in animal cells. Through – MÚLTIPLOS
gene duplication and EFEITOS
divergence, however, the signaling
systemsFatores a considerar:
in animals have become muchTipomore
de receptor, localozação,
elaborate than etc…
those in yeasts; the
human genome, for example, contains more than 1500 genes that encode recep-
tor proteins, and the number of different receptor proteins is further increased by
530 CHAPTER 16alternative RNA splicing and post-translational modifications.
Cell Signaling

(A) heart pacemaker cell

Extracellular Signals
(B) salivary Can Act Over
gland cell Short or Long Distances
(C) skeletal muscle cell (D) acetylcholine
Many
acetylcholine extracellular signal molecules remain bound to the surface of the signal-
O CH3
ing cell and influence only cells that contact it (Figure 15–2A). Such contact-
receptor
dependent signaling is especially important during development and H3in
C immune
C O CH2 CH2 N+ CH3
protein
responses. Contact-dependent signaling during development can sometimes
CH3
operate over relatively large distances if the communicating cells extend long thin
processes to make contact with one another.

DECREASED RATE SECRETION CONTRACTION

OF FIRING

Essential Cell Biology.

Figure 16–5 The same signal molecule
A typical
can induce different responses in
different target cells. Different cell
types are configured to respond to the
6 neurotransmitter acetylcholine in different
ways. Acetylcholine binds to similar receptor
proteins on heart pacemaker cells (A)
and salivary gland cells (B), but it evokes
different responses in each cell type.
Skeletal muscle cells (C) produce a different
type of receptor protein for the same
signal. (D) For such a versatile molecule,
acetylcholine has a fairly simple chemical
structure.
Alberts
et al., 2013,
cell possesses 4th edition.
many sorts of receptors—each present in tens
to hundreds of thousands of copies. Such variety makes the cell simul-
taneously sensitive to many different extracellular signals and allows
a relatively small number of signal molecules, used in different com-
binations, to exert subtle and complex control over cell behavior. A
combination of signals can evoke a response that is different from the
sum of the effects that each signal would trigger on its own. As we dis-
ECB4 e16.05/16.05
cuss later, this “tailoring” of a cell’s response occurs, in part, because the
intracellular relay systems activated by the different signals interact. Thus
the presence of one signal will often modify the effects of another. One
combination of signals might enable a cell to survive; another might drive
it to differentiate in some specialized way; and another might cause it to
divide. In the absence of any signals, most animal cells are programmed
3
to kill themselves (Figure 16–6).

A
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826 Chapter 15: Cell Signaling

extracellular signal molecule Figure 15–13 Slow and rapid res

to an extracellular signal. Certain
of signal-induced cellular response
as increased cell growth and divisio
intracellular signaling cell-surface involve changes in gene expression
pathway receptor protein and the synthesis of new proteins;
nucleus
therefore occur slowly, often startin
hour or more after the signal is rece
Other responses—such as change
movement, secretion, or metabolis
need not involve changes in gene
DNA transcription and therefore occur m
ALTERED more quickly, often starting in seco
FAST PROTEIN RNA SLOW or minutes; they may involve the ra
(< sec to mins) FUNCTION (mins to hrs)
phosphorylation of effector proteins
the cytoplasm, for example. Synap
ALTERED PROTEIN SYNTHESIS
responses mediated by changes in
membrane potential are even quick
can occur in milliseconds (not show
Some signaling systems generate b
ALTERED CYTOPLASMIC MACHINERY rapid and slow responses as show
allowing the cell to respond quickly
signal while simultaneously initiating
long-term, persistent response.
ALTERED CELL BEHAVIOR

Essential Cell Biology. Alberts et al., 2013, 4th edition.

7 because the signal exerts its effects by altering the concentrations of intracellular
molecules that are short-lived (unstable), undergoing continual turnover. Thus,
once the extracellular signal is gone, the degradation of the old molecules quickly
wipes out all traces of the signal’s action. It follows that the speed with which a cell
responds to signal removal depends on the rate of destruction, or turnover, of the
intracellular molecules that the signal affects.
It is also true, although much less obvious, that this turnover rate can deter-
536 PLASTICIDADE DA SINALIZAÇÃO
mine the promptness of the response when an extracellular signal arrives. Con-
sider, for example, CHAPTER 16 Cell Signaling
two intracellular MBoC6 m15.06/15.13
signaling molecules, X and Y, both of which
are normally maintained at
Figure 16–13 Intracellular signaling
proteins can relay, amplify, integrate, a steady-state concentration of 1000 molecules per extracellular signal molecule
receptor protein
cell. The cell synthesizes
example, a receptor protein and degrades molecule Y at a rate of 100 molecules per
and distribute the incoming signal. In this
A.S. Pupo et al. / Pharmacological Research 112 (2016) 49–57

located on
53
plasma membrane
PRIMARY
second, with each molecule having
the cell surface transduces an extracellular
signal into an intracellular signal, which an average lifetime of 10 seconds. Molecule X CYTOSOL
TRANSDUCTION

initiates one or more intracellular signaling

has a turnoverpathwaysrate that
that relay is 10
the signal times
into the cell slower than that of Y: it is both synthesized and RELAY
degraded atinterior.
a rate
signaling
Each pathway includes intracellular
ofthat
proteins 10canmolecules
function in per second, so that each molecule has an aver- scaffold

one of the various ways shown; some,

age lifetimeforin theintegrate
example, cell of 100
signals from seconds.
other If a signal acting on the cell causes a tenfold TRANSDUCE AND
intracellular signaling pathways. Many of AMPLIFY
increase inthethe steps synthesis
in the process can berates
modulated of both X and Y with no change in the molecu-
by other molecules or events in the cell
lar lifetimes,(not at the
shown). Note end
that someof 1 second
proteins in the concentration of Y will have increased
the pathway may be held in close proximity
by nearly 900 molecules
by a scaffold perthem
protein, which allows cell (10 × 100 – 100), while the concentration of X
to be activated at a specific location in the
will have increased
cell and with greaterbyspeed,
only 90and
efficiency,
selectivity. We discuss the production and
molecules per cell. In fact, after a molecule’s syn- small intracellular
messenger molecules

thesis rate has

functionbeen either
of small intracellular
molecules later in the chapter.
increased
messenger or decreased abruptly, the time required for
the molecule to shift halfway from its old to its new equilibrium concentration is
equal to its half-life—that is, equal to the time that would be required for its con- INTEGRATE

centration to fall by half if all synthesis were stopped (Figure 15–14). DISTRIBUTE

The same principles apply to proteins and small molecules, whether the mol-
ecules are in the extracellular space or inside cells. Many intracellular proteins
have short half-lives, some surviving for less than 10 minutes. In most cases, these
are key regulatory proteins whose concentrations are rapidly controlled in the cell ALTERED
METABOLISM
ALTERED CELL
SHAPE OR
ALTERED
GENE
MOVEMENT EXPRESSION
by changes in their rates of synthesis.
As we have Essentialseen, many Cell Biology. Alberts ettoal.,extracellular
cell responses 2013, 4th edition. signals depend on the
conversion of intracellular signalingProteins proteins that actfrom an
as molecular inactive
switches fallto an into
mostly active
one of form,
two

8 rather than on their synthesis or degradation. classes. The first—and by far the largest—class consists of proteins that
Phosphorylation
are activated or inactivated by phosphorylation,or the binding
a chemical modification of
discussed in Chapter 4 (see Figure 4–42). For these molecules, the switch
GTP, for example, commonly activates signaling
is thrown in one directionproteins. Even
by a protein kinase, whichincovalently
theseattaches
cases,
Fig. 2. Schematic representation of differences of potency and efficacy in biased agonism. Agonist A has higher potency/efficacy than agonist B in pathway 1, but lower

however, the activation must be rapidly

potency/efficacy than agonist B in pathway 2.

(A)
and continuously reversed (by dephos-
a phosphate group onto the switch protein, and in the other direction
by a protein phosphatase, which takes the phosphate off again (Figure
ECB4 e16.13/16.13

phorylation or GTP hydrolysis to GDP, signaling

pathway 1
16–15A respectively, in these
). The activity of any protein
X examples)
that is regulated
Y to make
by phosphorylation
depends—moment by moment—on the balance between the activities of
+
rapid signaling possible. These inactivation processes
the protein kinases play ait crucial
that phosphorylate
positive feedback
part
and the protein in deter-
phosphatases

mining the magnitude, rapidity, andthat (B)

dephosphorylate it.
duration of the response.
Many of the switch proteins controlled by phosphorylation are themselves 4
signaling X Y
pathway 2 – protein kinases, and these are often organized into phosphorylation cas-
cades: one protein kinase, activated by phosphorylation, phosphorylates
negative feedback the next protein kinase in the sequence, and so on, transmitting the sig-
nal onward and, in the process, amplifying, distributing, and regulating
Figure 16–14 Feedback regulation within it. Two main types of protein kinases operate in intracellular signaling
an intracellular signaling pathway can pathways: the most common are serine/threonine kinases, which—as
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ENZIMAS

RANG et al., 2012. Farmacologia, 7a edição

Enzimas - Exemplos

10

5
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ASPIRINA – AÇÃO CLÁSSICA:

11

ASPIRINA - CICLOOXIGENASE

https://tmedweb.tulane.edu/pharmwiki/doku.
php/non-selective_cox_1_2_inhibitors

12

6
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ASPIRINA – MECANISMO DE AÇÃO:

13

INIBIDORES DA ANGIOTENSINA II
Inibidores da ECA
• captopril, enalapril, lisinopril, quinapril, ramipril, benzapril,
moexipril, fosipril, trandolapril, perindopril
• Maioria são pro-drogas, com exceção do captopril
• Ativação por enzimas hepáticas
• Importância da farmacologia brasileira

Enalapril

Captopril
lisinopril

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INIBIDORES DA ANGIOTENSINA II
Inibidores da ECA
• Bloqueiam a conversão da
angiotensina I em
angiotensina II
• Inibem a degradação da
bradicinina que é um
potente vasodilatador.
• Ação principal envolve
diminuição da resistência Ações da angiotensina II:
vascular periférica. • Vasoconstrição direta
• Aumento da descarga simpática
• Liberação de aldosterona
• Alterações renais

15

ESTATINAS
Mecanismo de ação:
• são inibidores da 3-hidroxi-3-
metilgluratil-coenzima A (HMG-CoA)
redutase (enzima da síntese de
colesterol)
• A diminuição da síntese de
colesterol causa up-regulation do
receptor de LDL
• Principal efeito terapêutico:
remoção do LDL do plasma.
• Reduzem triglicerídeos por redução
da secreção de VLDL e aumentando
seu clearance

16

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EXEMPLOS

17

MOLÉCULAS TRANSPORTADORAS

RANG et al., 2012. Farmacologia, 7a edição

18

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Transportadores – Exemplos:

19

Mecanismo de ação
aa

aa
E
hidroxilase
MAO
AAADC b
interm NT MET

VMAT
NT
NE, 5-HT
5-TH
T

ADT

Slide Profa. Rosana Camarini

ICB-USP

20

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CANAIS IÔNICOS

CANAIS IÔNICOS
BLOQUEIO DA
BLOQUEADORES PERMEAÇÃO

AUMENTO OU
DIMINUIÇÃO DA
MODULADORES PROBABILIDADE DE
ABERTURA

RANG et al., 2012. Farmacologia, 7a edição

21

CANAIS IÔNICOS - EXEMPLOS

22

11
 Structure Potency (Procaine = 1) Duration of Action
Esters
Cocaine O O CH3 2 Medium
C N CH3

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O

C O

Procaine (Novocain) O C2H5 1 Short

H2N C O CH2 CH2 N

C2H5

Tetracaine O CH3 16 Long

(Pontocaine)
HN C O CH2 CH2 N

C4H9 CH3

Benzocaine Surface use only

ANESTÉSICOS LOCAIS
O

H2N C O CH2 CH3

Amides
Lidocaine (Xylocaine) CH3 4 Medium
O C2H5

NH C CH2 N

C2H5
CH3

Mepivacaine CH3 2 Medium

(Carbocaine, O
Bloqueadores reversíveis
•Isocaine) NH C
do Canal de Na+-
dependente de voltagem N

• Bloqueam a condução
CH3 nervosa CH3
Bupivacaine CH3 16 Long
(Marcaine), O
Levobupivacaine
NH C
(Chirocaine)
N
CH3
C4H9

Ropivacaine CH3 16 Long

(Naropin) O

NH C

N
CH3
C3H7

Articaine CH3 nf2 Medium

O CH3

NH C CH NH C3H7
O
S
C OCH
1

23
Other chemical types are available including ethers (pramoxine), ketones (dyclonine), and phenetidin derivatives (phenacaine).
2
Data not found.

026-Katzung_Ch026_p449-464.indd 451 9/22/11 5:04:20 PM

RECEPTORES
Abertura e
RECEPTORES Direto fechamento de
canais iônicos
Ativação/Inibição
enzimática

Agonista Mecanismo de
Modulação de canais
transdução
iônicos

Transcrição do DNA

Sem efeito
Antagonista Ação farmacológicas: bloqueio dos
mediadores endógenos

RANG et al., 2012. Farmacologia, 7a edição

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RECEPTORES

RANG et al., 2012. Farmacologia, 7a edição

25

TIPO 1 – CANAIS ATIVADOS POR LIGANTES

• Localizados na membrana
• Formado por subunidades que delimitam um
poro
• Envolvidos na transmissão rápida
• Efetor – canal iônico
• Alteração na permeabilidade a íons
• Excitatórios/Inibitórios
• Exemplos: nAChR, GABAA, 5-HT, Glicina

26

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Exemplo: nACHR
Homomérico Heteromérico
• Formado por subunidades: α
(2 a 5), β (1-3), γ e δ
• Subunidade α – ligação da
Extracelular
acetilcolina
• Canal permeável a cátions
Intracelular • Influxo de Na+
• Despolarização na célula pós
sináptica

Número de canais abertos

Changeux, 2010. Nat. Rev. Neuroscience 11: 389-401.

27

RECEPTORES NICOTÍNICOS (nAChRs)

Receptor Musculares: Ganglionares: SNC: (α4)2(β2)3 SNC: (α7)5
(α1)2β1δε - adulto (α3)2(β2)3
Principais Junção Gânglios Muitas regiões do Muitas regiões do
localizações neuromuscular autonômicos SNC: pré e pós SNC: pré e pós
esquelética sináptica sináptica

Resposta da Excitatória Excitatória Excitatória Excitatória

membrana Aumento da Aumento da Aumento da Aumento da
permeabilidade a permeabilidade a permeabilidade a permeabilidade a
cátions (Ca+2, Na+) cátions (Ca+2, Na+) cátions (Ca+2, Na+) cátions (Ca+2)
Resposta Contração Estimulação do
funcional neurônio pós-
ganglionar simpático
e parassimpático

28

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Exemplos
Antagonistas nicotínicos
Fármaco Sítio principal Tipo de ação
Hexametônio Gânglios autonômicos Bloqueio da
transmissão
trimetafana Gânglios autonômicos Bloqueio da
transmissão
Tubocurarina Junção neuromuscular Bloqueio da
transmissão
Pancurônio Junção neuromuscular Bloqueio da
Atracurio transmissão
Vecurônio

Rang HP et al. Rang & Dale Farmacologia. 7a. Edição. Elsevier, 2012.

29

BENZODIAZEPÍNICOS
Sítio do • 1950: síntese CDZ
GABA Sternbach
Clordiazepóxido-
. 1960: introdução na
Introduzido em 1960
clínica
1977: descoberta dos
sítios BDZ – Mohler &
Okada

Sítio
BDZ

Córtex, hipocampo, amígdala

Adaptado de Stahl S M, Essential
Psychopharmacology (2000)
Slide Profa. Rosana Camarini
ICB-USP

30

15
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TIPO 2 – ACOPLADOS À PROTEÍNA G (GPCRs)

• Proteínas com 7 α-hélices
transmembrana, domínio N-terminal
extracelular e C-terminal intracelular.
• Acoplamento através da proteína G
• Efetor: canais ou enzimas
• Exemplos: Receptor muscarínicos da
ACh e adrenoceptores Cerca de 800 receptores
clonados

Domínios de ligação

Domínio de
ligação na
proteína G

31

PASSO A PASSO:
1. Ativação do receptor -
Agonista
agonista
2. GTP desloca GDP
3. Liga/ativa outra proteína
(enzima/canal iônico)
4. Ativa enzima e continua via
de sinalização
5. Atividade GTPásica

Katzung et al., 2011. Basic and Clinic Pharmacology. 11a. edição

32

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FUNÇÃO DA PROTEÍNA G

RANG et al., 2012. Farmacologia, 7a edição

33

PRINCIPAIS SUBTIPOS E FUNÇÕES

RECEPTOR 7TM

Canais iônicos
PI3K
Fosfolipase
Adenil ciclase
Receptores quinase

Canais iônicos Aumento AMPc Aumento DAG Ativação Rho

Inibição cAMP IP3
Fosfolipase

34

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PROTEÍNA G E SEUS EFETORES:

35

SEGUNDO MENSAGEIRO -AMPcíclico

1. Ativação da Adenil Agonista

ciclase
2. ATP é convertido em Membrana

AMPcíclico
3. Ativa proteína
quinase A (PKA)
4. Fosforilação de
substratos
5. Via inibitória – Gi –
diminui atividade da Resposta
Adenil ciclase
Katzung et al., 2011. Basic and Clinic Pharmacology. 11a. edição

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GPCR ACOPLADO A PROTEÍNA Gs

Efeitos rápidos
544 CHAPTER 16 Cell Signaling

plasma activated Figure 16–22 Enzymes activated by G proteins increase the

membrane enzyme concentrations of small intracellular signaling molecules. Because
EXTRACELLULAR
SPACE each activated enzyme generates many molecules of these second
messengers, the signal is greatly amplified at this step in the pathway
(see Figure 16–31). The signal is relayed onward by the second
CYTOSOL messenger molecules, which bind to specific signaling proteins
in the cell and influence their activity.
GTP

activated
α subunit intracellular signaling molecules inositol trisphosphate and diacylglycerol.
of G protein Inositol trisphosphate, in turn, promotes the accumulation of cytosolic
Ca2+—yet another small intracellular signaling molecule.
Adenylyl cyclase and phospholipase C are activated by different types of
SECOND MESSENGER MOLECULES G proteins, allowing cells to couple the production of these small intra-
DIFFUSE TO ACT ON INTRACELLULAR cellular signaling molecules to different extracellular signals. Although
SIGNALING PROTEINS
the coupling may be either stimulatory or inhibitory—as we saw in our
discussion of the actions of cholera toxin and pertussis toxin—we con-
centrate here on G proteins that stimulate enzyme activity.
The small intracellular signaling molecules generated by these enzymes
are often called small messengers, or second messengers—the “first mes-
sengers” being the extracellular signals that activated the enzymes in
the first place. Once activated, the enzymes generate large quantities of
small messengers, which rapidly diffuse away from their source, thereby
ECB4 e16.20/16.22 amplifying and spreading the intracellular signal (Figure 16–22).
Different small messenger molecules produce different responses. We
first examine the consequences of an increase in the cytosolic concen-
tration of cyclic AMP. This will take us along one of the main types of
signaling pathways that lead from the activation of GPCRs. We then
Essential Cell Biology. Alberts et al., 2013, 4th edition.
discuss the actions of three other small messenger molecules—inositol
trisphosphate, diacylglycerol, and Ca2+—which will lead us along a dif-
ferent signaling route.
37
NH2
The Cyclic AMP Signaling Pathway Can Activate Enzymes
N
N and Turn On Genes
O O O
_ N N Many extracellular signals acting via GPCRs affect the activity of the
O P O P O P O CH2 O
_ _ _ enzyme adenylyl cyclase and thus alter the intracellular concentration
O O O ATP of the small messenger molecule cyclic AMP. Most commonly, the acti-
vated G-protein subunit switches on the adenylyl cyclase, causing a
OH OH dramatic and sudden increase in the synthesis of cyclic AMP from ATP
adenylyl
NH2 (which is always present in the cell). Because it stimulates the cyclase, G-Protein-Coupled Receptors

GPCR ACOPLADO A PROTEÍNA Gs

cyclase
N this G protein is called Gs. To help terminate the signal, a second enzyme,
N
P Pi called cyclic AMP phosphodiesterase, rapidly converts cyclic AMP to ordi-
activated Figure 16–26 A rise in intracellular cyc
adenylyl cyclase
Efeitos lentos H2 O
N N nary AMP (Figure 16–23). One way that caffeine
inhibiting this phosphodiesteraseadrenaline
acts as
in the nervous system,
a stimulant is by
α
blocking
activated cyclic
AMP can activate gene transcription.
Binding of a signal molecule to its GPCR
C AMP degradation and thereby keeping the concentration ofofthis small
subunit can lead to the activation of adenylyl
stimulatory G plasma cyclase and a rise in the concentration o
O cyclic messenger high. protein (G ) membrane
O P O OH AMP
s cytosolic cyclic AMP. The increase in cyc
_ Cyclic AMP phosphodiesterase is continuously active inside the cell. AMP activates PKA, which then moves
O
Because it eliminates cyclic AMP so quickly, the cytosolic concentration into the nucleus and phosphorylates
H2O of this small messenger can change rapidly in response to extracellular specific transcription regulators. Once
cyclic AMP NH2
phosphodiesterase phosphorylated, these proteins stimulat
N GTP
N the transcription of a whole set of targe
Figure 16–23 Cyclic AMP is synthesizedactivated GPCR cyclase
by adenylyl
O (adrenergic receptor) genes (Movie 16.3). This type of signalin
N and degraded by cyclic AMP phosphodiesterase. Cyclic AMP
_
O P O CH2 O
N ATP pathway controls many processes in
(abbreviated cAMP) is formed from ATP by a cyclization reaction that
_ removes two phosphate groups from ATP and joins the “free” end of cells, ranging from hormone synthesis
O AMP the remaining phosphate group to the sugar part of the cyclic
AMP AMP
molecule in endocrine cells to the production of
(red bond). The degradation reaction breaks this new bond, forming proteins involved in long-term memory
OH OH AMP. in the brain. Activated PKA can also
inactive PKA active phosphorylate and thereby regulate oth
PKA proteins and enzymes in the cytosol (as
shown in Figure 16–25).
nuclear pore
CYTOSOL

ECB4 e16.21/16.23
NUCLEUS
active inactive
PKA transcription
regulator
activated, phosphorylated
transcription regulator

P
activated target gene

Essential Cell Biology. Alberts et al., 2013, 4th edition. TRANSCRIPTION OF

TARGET GENE

38 Once activated, phospholipase C propagates the signal by cleaving a lipid

molecule that is a component of the plasma membrane. The molecule is
an inositol phospholipid (a phospholipid with the sugar inositol attached
to its head) that is present in small quantities in the cytosolic leaflet of the
membrane lipid bilayerECB4(seee16.24/15.26
Figure 11–18). Because of the involvement of
this phospholipid, the signaling pathway that begins with the activation
of phospholipase C is often referred to as the inositol phospholipid path-
way. It operates in almost all eukaryotic cells and can regulate a host of
different effector proteins. 19
The action of phospholipase C generates two small messenger molecules:
inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Both mol-
ecules play a crucial part in relaying the signal (Figure 16–27).
 maker cell, forcing the ion channel into an open conformation (Figure
16–21A and B). This channel opening slows the heart rate by increasing
the plasma membrane’s permeability to K+, making it more difficult to
electrically activate, as explained in Chapter 12. The original signal is ter-
minated—and the K+ channel recloses—when the subunit inactivates
itself by hydrolyzing its bound GTP, returning the G protein to its inactive
state (Figure 16–21C).
24/04/22
Many G Proteins Activate Membrane-bound Enzymes that
Produce Small Messenger Molecules
When G proteins interact with ion channels, they cause an immedi-
ate change in the state and behavior of the cell. Their interactions with
enzymes, in contrast, have consequences that are less rapid and more
complex, as they lead to the production of additional intracellular sig-
naling molecules. The two most frequent target enzymes for G proteins

GPCR ACOPLADO A PROTEÍNA Gi

are adenylyl cyclase, which produces the small intracellular signaling
molecule cyclic AMP, and phospholipase C, which generates the small

acetylcholine +
plasma membrane closed K channel

(A)

GTP activated
βγ complex
activated α subunit +
+
K CHANNEL open K channel
+
OPENING K
EXTRACELLULAR SPACE
(B)
Figure 16–21 A Gi protein directly
CYTOSOL
couples receptor activation to the
opening of K+ channels in the plasma
GTP
membrane of heart pacemaker cells.
(A) Binding of the neurotransmitter
acetylcholine to its GPCR on the heart
G-PROTEIN cells results in the activation of the
Pi INACTIVATION;
+ G protein, Gi. (B) The activated complex
K CHANNEL
CLOSING
+
closed K channel directly opens a K+ channel in the plasma
membrane, increasing its permeability to
K+ and thereby making the membrane
(C) harder to activate and slowing the heart
rate. (C) Inactivation of the subunit by
inactive hydrolysis of its bound GTP returns the
G protein GDP G protein to its inactive state, allowing the
K+ channel to close.

Essential Cell Biology. Alberts et al., 2013, 4th edition.

39
ECB4 E16.19/16.21

SEGUNDOS MENSAGEIROS - IP3 E DAG

Agonista

1. Ativação da
Fosfolipase C
Membrana
2. PIP2 é convertido
em IP3 e DAG
3. IP3 libera Ca+2 –
ativa Calmodulina
4. DAG estimula PKC
5. Fosforilação de
substratos
Resposta

Katzung et al., 2011. Basic and Clinic Pharmacology. 11a. edição

40

20
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GPCR ACOPLADO A PROTEÍNA Gq

548 CHAPTER 16 Cell Signaling

Figure 16–27 Phospholipase C activates signal molecule inositol

two signaling pathways. Two small phospholipid plasma
activated GPCR activated membrane
messenger molecules are produced when phospholipase C diacylglycerol
a membrane inositol phospholipid is
hydrolyzed by activated phospholipase C.
Inositol 1,4,5-trisphosphate (IP3) diffuses
through the cytosol and triggers the P
P
release of Ca2+ from the ER by binding to GTP
and opening special Ca2+ channels in the P activated
ER membrane. The large electrochemical P PKC
P
gradient for Ca2+ across this membrane
causes Ca2+ to rush out of the ER and into activated G protein (Gq) inositol
Ca 2+
1,4,5-trisphosphate P
the cytosol. Diacylglycerol remains in the (IP3)
plasma membrane and, together with Ca2+, open Ca2+
channel
helps activate the enzyme protein kinase C
(PKC), which is recruited from the cytosol to CYTOSOL
the cytosolic face of the plasma membrane. endoplasmic
PKC then phosphorylates its own set of reticulum
intracellular proteins, further propagating ER LUMEN
the signal. At the start of the pathway,
both the subunit and the subunit of
the G protein Gq are involved in activating
phospholipase C.

IP3 is a water-soluble sugar phosphate that is released into the cytosol;

thereEssential
it binds toCell
and Biology. Alberts
opens Ca2+ et al.,
channels that2013, 4th edition.
are embedded in the endo-
plasmic reticulum (ER) membrane. Ca2+ stored inside the ER rushes out
into the cytosol through these open channels, causing a sharp rise in the
41 cytosolic concentration of free Ca2+, which is normally kept very low.
ECB4 proteins,
This Ca2+ in turn signals to other e16.25/16.27
as discussed below.
Diacylglycerol is a lipid that remains embedded in the plasma membrane
after it is produced by phospholipase C; there, it helps recruit and acti-
vate a protein kinase, which translocates from the cytosol to the plasma
membrane. This enzyme is called protein kinase C (PKC) because it also
needs to bind Ca2+ to become active (see Figure 16–27). Once activated,
PKC phosphorylates a set of intracellular proteins that varies depending

CONTROLE DOS SISTEMAS EFETORES

on the cell type. PKC operates on the same principle as PKA, although the
proteins it phosphorylates are different.

A Ca2+ Signal Triggers Many Biological Processes

Ca2+ has such an important and widespread role as an intracellular mes-
senger that we will digress to consider its functions more generally. A
surge in the cytosolic concentration of free Ca2+ is triggered by many kinds
of cell stimuli, not only those that act through GPCRs. When a sperm ferti-
lizes an egg cell, for example, Ca2+ channels open, and the resulting rise
in cytosolic Ca2+ triggers the egg to start development (Figure 16–28); for
muscle cells, a signal from a nerve triggers a rise in cytosolic Ca2+ that
initiates muscle contraction; and in many secretory cells, including nerve
cells, Ca2+ triggers secretion. Ca2+ stimulates all these responses by bind-
ing to and influencing the activity of various Ca2+-responsive proteins.
The concentration of free Ca2+ in the cytosol of an unstimulated cell is
extremely low (10–7 M) compared with its concentration in the extracel-
lular fluid (about 10–3 M) and in the ER. These differences are maintained
by membrane-embedded Ca2+ pumps that actively remove Ca2+ from the
cytosol—sending it either into the ER or across the plasma membrane and
out of the cell. As a result, a steep electrochemical gradient of Ca2+ exists
across both the ER membrane and the plasma membrane (discussed in
Chapter 12). When a signal transiently opens Ca2+ channels in either of
these membranes, Ca2+ rushes down its electrochemical gradient into
the cytosol, where it triggers changes in Ca2+-responsive proteins in the

RANG et al., 2012. Farmacologia, 7a edição

42

21
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General Principles of Cell Signaling

Figure 16–15 Many intracellular sign

SIGNAL
OFF
SIGNAL OFF proteins act as molecular switches. T
IN IN proteins can be activated—or in some
GDP Pi inhibited—by the addition or removal
Pi
GTP
phosphate group. (A) In one class of sw
protein protein GDP GTP
ATP
kinase phosphatase hydrolysis protein, the phosphate is added coval
binding
GTP by a protein kinase, which transfers the
ADP
terminal phosphate group from ATP to
ON the signaling protein; the phosphate is
ON then removed by a protein phosphata
GTP (B) In the other class of switch protein,
P
SIGNAL SIGNAL GTP-binding protein is activated when
OUT OUT exchanges its bound GDP for GTP (wh
a sense, adds a phosphate to the prot
the protein then switches itself off by
(A) SIGNALING BY (B) SIGNALING BY GTP-BINDING PROTEINS hydrolyzing its bound GTP to GDP.
PROTEIN PHOSPHORYLATION

them, respectively (Figure

Essential Cell 16–15B
Biology. ). Once
Alberts et al.,activated
2013, 4th by GTP binding, these
edition.
proteins have intrinsic GTP-hydrolyzing (GTPase) activity, and they shut
themselves off by hydrolyzing their bound GTP to GDP.
43 ECB4 e16.14/16.15
Two main types of GTP-binding proteins participate in intracellular
signaling pathways. Large, trimeric GTP-binding proteins (also called G
proteins) relay messages from G-protein-coupled receptors; we discuss
this major class of GTP-binding proteins in detail shortly. Other cell-
surface receptors rely on small, monomeric GTPases to help relay their
signals. These monomeric GTP-binding proteins are aided by two sets
of regulatory proteins. Guanine nucleotide exchange factors (GEFs) acti-
vate the switch proteins by promoting the exchange of GDP for GTP, and
GTPase-activating proteins (GAPs) turn them off by promoting GTP hydrol-
ysis (Figure 16–16).

Cell-Surface Receptors Fall into Three Main Classes

All cell-surface receptor proteins bind to an extracellular signal molecule
and transduce its message into one or more intracellular signaling mol-
ecules that alter the cell’s behavior. Most of these receptors belong to INACTIVE
one of three large classes, which differ in the transduction mechanism MONOMERIC GTPase

they use.
OFF
1. Ion-channel-coupled receptors change the permeability of the
GEF
plasma membrane to selected ions, thereby altering the membrane GDP Pi
potential and, if the conditions are right, producing an electrical
current (Figure 16–17A). GDP
GTP GAP
2. G-protein-coupled receptors activate membrane-bound, trimeric
GTP-binding proteins (G proteins), which then activate (or inhibit) ON
an enzyme or an ion channel in the plasma membrane, initiating GTP
an intracellular signaling cascade (Figure 16–17B).
3. Enzyme-coupled receptors either act as enzymes or associate ACTIVE
MONOMERIC GTPase
with enzymes inside the cell (Figure 16–17C); when stimulated,
the enzymes can activate a wide variety of intracellular signaling
pathways. Figure 16–16 The activity of monom
GTP-binding proteins is controlled b
44 The number of different types of receptors in each of these three classes two types of regulatory proteins. Gu
is even greater than the number of extracellular signals that act on them. nucleotide exchange factors (GEFs)
ECB4
promote the m15.19/16.16
exchange of GDP for GTP
This is because for many extracellular signal molecules there is more
thereby switching the GTP-binding pro
than one type of receptor, and these may belong to different receptor
on. GTPase-activating proteins (GAPs)
classes. The neurotransmitter acetylcholine, for example, acts on skel- stimulate the hydrolysis of GTP to GDP
etal muscle cells via an ion-channel-coupled receptor, whereas in heart 22
thereby switching the GTP-binding
cells it acts through a G-protein-coupled receptor. These two types of protein off.
 24/04/22

Agonismo tendencioso

A.S. Pupo et al. / Pharmacological Research 112 (2016) 49–57 53

45

Agonismo tendencioso
50 A.S. Pupo et al. / Pharmacological Research 112 (2016) 49–57

Fig. 2. Schematic representation of differences of potency and efficacy in biased agonism. Agonist A has higher potency/efficacy than agonist B in pathway 1, but lower
potency/efficacy than agonist B in pathway 2.

46

23
 24/04/22

Agonismo tendencioso

Fig. 2. Schematic representation of differences of potency and efficacy in biased agonism. Agonist A has higher potency/efficacy than agonist B in pathway 1, but lower
potency/efficacy than agonist B in pathway 2.

47

Exemplo
54 A.S. Pupo et al. / Pharmacological Research 112 (2016) 49–57

Fig. 3. Assessment of noradrenaline and oxymetazoline G protein-dependent signaling in !1A adrenergic receptor. Concentration-response curves for intracellular calcium
mobilization (A) and Gq activation (B) on HEK293T cells transiently transfected with !1A adrenergic receptor and stimulated with increasing amounts of noradrenaline or
oxymetazoline.

nist is noradrenaline [1]. If concentration-response curves for the
internalization induced by oxymetazoline and noradrenaline are
performed at 30 min of agonist incubation, oxymetazoline is con-
sidered a full agonist for human !1A-adrenoceptor internalization,
whereas noradrenaline is inactive at this specific time point [1],
giving the false impression that noradrenaline is absolutely biased
toward Gq signaling versus receptor internalization.
Recently, at least three useful pharmacological parameters were
48 proposed for the quantification of biased agonism, the Intrinsic Rel-
ative Activity (RAi , [29]) ratios, the bias factor " [70] and the log Bias
or !!log #/KA [46]. Because of space constraints, the reader will
be referred to the specific literature above for the mathematical
grounds of each of them. A common feature of all three parame-
ters is that they relate functional estimates of ligands efficacy and
affinity and assume that changes in its ratios in two or more signal-
ing pathways result from agonist directed stabilization of different
receptor conformations.
The most straightforward in terms of calculation is the Intrinsic
Relative Activity (RAi ) ratio described by [29] that only requires the
knowledge of Maximal Effect (Emax) and pEC50 as combined sur-
rogates of efficacy and affinity of agonists in two of the triggered
signaling pathways. However, when the Hill coefficients of the log
concentration-response curves differ from 1.0, RAi ratios will be
largely affected by cell dependent interferences such as receptor
densities and coupling efficiencies (reviewed in Refs. [29,46]) . For
4. Perspectives for biased agonism application
Despite the increasing complexity of what can be considered
biased agonism as above discussed, discovery of ligand bias, as
a general concept of a broader capability of signaling, has raised
speculations about the possibility of achieving pharmacological
profiles distinct from those classically related to agonists or antago-
nists [44,83]. Biased agonists so far described have shown ability to
emphasize a beneficial cellular signal, to de-emphasize a debilitat-
ing signal, or to de-emphasize a debilitating signal while blocking
the capacity of the endogenous agonist to produce the signal [45].
The development of novel GPCR drugs with potentially
increased efficacy and/or fewer on-target adverse effects has
already prompted a few compounds into clinical trials. The first
one being tested in humans against an unbiased ligand is TRV130,
a G protein biased agonist (versus "-arrestin-2) that binds to the
$ opioid receptor (MOR) and produces analgesia comparable to
that of morphine [16]. Preclinical studies have suggested that opi-
oid analgesia is associated with G!i coupling at the MOR, whereas 24
gastrointestinal dysfunction, respiratory depression and tolerance
to the analgesic effect are all linked to "-arrestin-2 recruitment
[13,12,39,69,56,89,26].
On the other side, a potent "-arrestin biased ligand of the
angiotensin II type 1 receptor (AT1 R) [82], referred to as TRV027, is
currently being investigated in Phase 2b as an intravenous drug
 24/04/22

DESSENSIBILIZAÇÃO Agonista

NOS GPCRs: Receptor

• Homóloga (agonista Receptor

ativado
Ativação de
PKA, PKC, etc.

específica)
Fosforilação de
– Papel da GRK Receptor
fosforilado
receptores
(Não-específica)

– Arrestina
• Heteróloga (cruzada)
– Papel de quinases Complexo
Receptor-arrestina

com PKA e PKC Arrestina

Desacopla Endocitose Redução do

mento acoplamento à
proteína G proteína G

HOMÓLOGA HETERÓLOGA
RANG et al., 2012. Farmacologia, 7a edição

49

RECICLAGEM
Agonista no espaço extracelular

Lisossomos

Endossomos

Katzung et al., 2011. Basic and Clinic Pharmacology. 11a. edição

50

25
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Essential Cell Biology. Alberts et al., 2013, 4th edition.

51

FÁRMACOS QUE AGEM VIA GPCRs

• ∼40% de todos os fármacos em uso clínico

52

26
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FÁRMACOS QUE AGEM VIA GPCRs

53

REFERÊNCIAS
• Bibliografia básica:
– Rang HP et al. Rang & Dale Farmacologia. 7a.
Edição. Elsevier, 2012.
– Katzung BG et al. Farmacologia Básica e Clínica.
11a. edição. MgGraw Hill – Artmed, 2013.
– Alberts, B. et al. Essential Cell Biology. 4th edition.
Garland Science, 2013.

54

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