• $K_D$$K_D$ is the "equilibrium constant"

  • the lower the number ( concentration ) , the higher the affinity of a drug for the receptor

  • $K_D\frac{1}{\propto }\text{Affinity of a Drug for the Receptor}$$K_D\ \frac{1}{\propto}\ \text{Affinity of a Drug for the Receptor}$

  • $K_M$$K_{\color{red}{M}}$ is not the same as $K_D$$K_D$ because it reflects both binding and catalytic events.

• Let $A$$A$ = agonist

• Let $R$$R$ = receptor

• Let $AR$$AR$ = agonist-receptor complex

• Scatchard Plot :

  • graphical method of analyzing equilibrium ligand-binding data

  • used for determining :

    • the number of ligand binding sites on a receptor

    • whether the sites show cooperative interactions

    • whether more than one site exists

    • the respective affinities of each site

  • Y-Axis = $\frac{[bound]}{[\text{free}]}$$\frac{[bound]}{[\text{free}]}$

    • Y-Intercept = $\frac{B_{max}}{K_D}$$\frac{B_{max}}{K_D}$

  • X-axis = $[bound]$$[bound]$

    • X-Intercept = $B_{max}$$B_{max}$ = total number of receptors $(N)$$(\ N\ )$

• Scatchard Plot Equation for Single Binding System :

  $$\frac{[B]}{[F]}=(-\frac{1}{K_D}\ast [B])+\frac{B_{max}}{K_D}$$

  • $B_{max}$$B_{max}$ = total number of binding sites

    • density of receptors in the tissue

  • $[B]$$[B]$ = concentration of bound ligand

  • $[F]$$[F]$ = concentration of free ligand

  • $K_D$$K_D$ = dissociation constant , representing the ligand concentration at which half of the binding sites are occupied

  • Also note in this form , its the same as standard form : $y=(m\ast x)+b$$y = (\ m * x\ ) + b$

    • $y=\frac{[B]}{[F]}$$y = \frac{[B]}{[F]}$

    • $\text{Slope ( m )}=-\frac{1}{K_D}$$\text{Slope ( m )} = - \frac{1}{K_D}$

    • $x=[B]$$x = [B]$

    • $\text{y-intercept ( b )}=\frac{B_{max}}{K_D}$$\text{y-intercept ( b )} = \frac{B_{max}}{K_D}$

"Students will be able to interpret a Scatchard plot to assess the Kd of a compound"

• So essentially , you just need to know which term is the slope or y-intercept ( they both have a $K_D$$K_D$ term ) , and then solve for $K_D$$K_D$

$$K_D=-\frac{1}{m}$$

or

$$K_D=\frac{B_{max}}{b}$$

• Saturation Equations :

  • Affinity : How strong is the interaction? ( $K_D,K_M$$K_D\ ,\ K_M$ )

  • Capacity : How many sites are there? ( $B_{max}$$B_{max}$ , enzyme number )

  • Specificity : Can you distinguish binding of different ligands?

• Michaelis-Menten focuses on enzyme kinetics :

  • It relates substrate concentration to reaction rate and is used to find $K_M$$K_M$ ( substrate concentration at half-maximal velocity ) and $V_{max}$$V_{max}$ ( maximum reaction velocity )

• Scatchard focuses on binding , not catalysis :

  • It’s better for studying receptors, antibodies, or protein-ligand interactions where you're interested in how tightly a molecule binds ( affinity ) and how many binding sites are available

• Agonist = molecule that binds to a receptor and activates it , producing a biological response

  • it has both affinity ( binding to the receptor ) and efficacy ( it elicits a biological response )

• Antagonist = a molecule that binds to a receptor but does NOT activate it. Instead , it blocks or dampens the effect of an agonist

  • it has affinity ( it binds ) but no efficacy ( no intrinsic activity )

     

• p = potency

• A = antagonist

• The pA₂ value tells us how strong the fake ligand ( antagonist ) is at blocking the real ligand ( agonist )

  • a large pA₂ for the antagonist :

    • a small amount of antagonist doubles the dose of agonist needed to start the signal

  • a small pA₂ for the antagonist :

    • a large amount of the antagonist is needed to block the agonist

• Defined as negative logarithm of its molar concentration required to double the agonist dose needed to achieve the same effect.

• pharmacological term used to quantify the potency of a competitive antagonist

• used as an expression of antagonist potency

• where $K_B$$K_B$ is the equilibrium dissociation constant of the antagonist

  • B = used for antagonist / blocking agents

    • how tightly the antagonist binds to the receptor

  • D = used for ligands

  • $K_B$$K_B$ = the concentration of the agonist at which 50% of the receptors are bound

• Negative Log Approximation :

  $$-log(m\ast {10}^{-n})\approx n-0.m$$

  • where $m$$m$ is a number between $1$$1$ and $10$$10$

  • and $n$$n$ is an integer ( whole number )

  • Examples :

    • $-log(3\ast {10}^{-5})\approx 5-0.3=4.7$$-log\left(\ 3 * 10^{-5}\ \right) \approx 5 - 0.3 = 4.7$

    • $-log(7.1\ast {10}^{-9})\approx 9-0.71=8.29$$-log\left(\ 7.1 * 10^{-9}\ \right) \approx 9 - 0.71 = 8.29$

    • $-log(2.5\ast {10}^{-2})\approx 2-0.25=1.75$$-log\left(\ 2.5 * 10^{-2}\ \right) \approx 2 - 0.25 = 1.75$

• Thus , a $p{A}_2$$pA_2$ value of $9$$9$ means that $1\ast {10}^{-9}M$$1 * 10^{-9}\ M$ antagonist will cause the amount of agonist required to produce a given effect to be doubled

• A $p{A}_2$$pA_2$ value of $6$$6$ means that $1\ast {10}^{-6}M$$1 * 10^{-6}\ M$

• Thus , the larger the $p{A}_2$$pA_2$ value , the more potent the agonist

• Often calculated from dose-response curves in the presence of various antagonist concentrations

• It indicates the concentration at which a competitive antagonist shifts the agonist’s dose-response curve two-fold

  • i.e., the antagonist concentration that requires doubling of the agonist to achieve the same response

• pA2 helps compare relative potencies of different antagonists.

• A higher pA2 value indicates a more potent antagonist.

• Affinity = tendency of a drug ( or ligand ) to bind to its receptor

  • quantified by the $K_D$$K_D$

    • the lower the $K_D$$K_D$ , the higher the affinity

• Efficacy = the ability of a bound drug to initiate a response

  • a drug with high efficacy can produce a strong response once bound

Affinity = how well a ligand fits and binds

Efficacy = how effectively that ligand activates the receptor once bound

• What do we mean by "transduction" ?

  • refers to the process by which the binding of a ligand (e.g., a hormone, neurotransmitter, or drug) to its receptor initiates a series of intracellular events, ultimately leading to a cellular response.

  • This is a key step in signal transduction , where the extracellular signal (ligand binding) is converted into an intracellular signal that changes the cell's behavior or activity.

Ligand-Gated Ion Channels

• transduce effect in milliseconds

• critical for neuronal function

• Examples = GABA_A Receptor , Nicotinic ACh Receptor , 5HT₃ Receptor

• Mechanism: Ligand binding → opens ion channel → fast changes in membrane potential or ion flux

G-Protein Coupled Receptors ( GPCR )

• critical for neuronal function

• $G_i$$G_i$ = inhibitory

  1. Signal Molecule binds and Activates GPCR ( $G_i$$G_i$ )

  2. binds and inhibits adenylyl cyclase

  3. reduces PKA activation

  4. reduces cell response ( gene transcription )

• $G_s$$G_s$ = stimulatory

  1. Signal Molecule binds and Activates GPCR ( $G_s$$G_s$ )

  2. binds and stimulates adenylyl cyclase

  3. increases PKA activation

  4. increases cell response ( gene transcription )

• $G_q$$G_q$ = activates PLC via DAG and IP3

  1. Signal Molecule binds and Activates GPCR ( $G_q$$G_q$ )

  2. $PLC\beta$$PLC \beta$ becomes activated

  3. $\mathrm{PIP}{\phantom{A}}_2\underset{\mathrm{PLC}\beta }{\overset{}{\to }}DAG+I{P}_3$$\ce{PIP_2 ->[][PLC \beta]} DAG + IP_3$

     • this depletes levels of PIP2 in the membrane

     • but voltage gated M-channels ( potassium channels ) need PIP2 in order to open

       • so M-channels close

       • this is a SIGNALING event !

       • the removal of PIP2 closed potassium channels , depolarizes the membrane ,

         • makes it very easy to fire action potential now

         • only a little bit of sodium is need to reach the action potential threshold

  4. DAG then activates PKC

  5. IP3 causes calcium to be released from endoplasmic reticulum

     • the calcium can also reinforce / enhance signaling of PKC

  • 3 Different Ways $G_q$$G_q$ can signal :

    1. Depletion of $PI{P}_2$$PIP_2$ in the membrane

    2. Liberation of $I{P}_3$$IP_3$ and hence intracellular calcium release

    3. Libration of DAG and hence activation of PKC

• Describe one alternative pathway that G-protein activation may signal via

  • Conventional Way = producing signaling molecules

  • Alternative Way = reduction ! of $PI{P}_2$$PIP_2$

• Example: β-adrenergic receptors , GABA_B , muscarinic ACh , histamine receptors

• Mechanism: Ligand binding → G-protein activation ( Gs , Gi , Gq ) → second messenger cascades (e.g., cAMP, IP3/DAG).

Kinase Coupled Receptors

• much slower , hours

• critical for hormonal function

• Example: Insulin receptor ( tyrosine kinase ) , epidermal growth factor receptors , nerve growth factor receptors

• Mechanism: Ligand binding → receptor dimerization/activation of intrinsic enzyme activity ( often tyrosine kinase ) → phosphorylation cascades.

  1. Binding of ligand activates intracellular tyrosine kinase domain.

  2. Phosphate from ATP transferred to selected tyrosine residues on side Chains, on receptor and on intracellular signalling proteins.

  3. 2 or more receptor oligomers then come together to form dimers or higher multimers.

• secondary messenger systems and intermediary products can cross activate with other secondary messenger systems

Nuclear Receptors

• very slow , transducer effect in hours

• critical for steroid hormone function

• Example: Steroid hormone receptors , thyroid hormones , retinoids , and vitamin D

• receptors are NOT in the membrane , ONLY in the cytosol

• Mechanism: Lipid-soluble ligand enters cell → binds cytosolic or nuclear receptor → alters gene transcription and protein synthesis

  1. receptor generally held in in-active state by inhibitory protein

  2. ligand binds , activating it

  3. receptor moves into nucleus and acts as transcription factor

• An important example of hormonal activation of nuclear receptors Is the action of the steroid hormone Cortisol

• Specific signal transduction mechanisms determine the effect that receptor activation has on tissue.

• Receptor activation can engage more than one signal transduction mechanism.

• Thus the same receptor activated in different tissues may mediate different effects depending on the transduction mechanism it is coupled to.

• Thus histamine receptors expressed on a nerve will mediate different effects than the same Histamine receptors on smooth muscle.

• The signal transduction pathway may be different (Histamine receptors are terribly promiscuous) and the effectors are almost certainly different.

• Which receptors does the excitatory neurotransmitter glutamate act upon?

  • Ionotorpic Glutamate Receptors = NMDA , AMPA , Kainate

    • ligand gated

  • Metabotropic Glutamate Receptors = GPCRs , modulate secondary messanger pathways and synaptic plasticity

• Are there selective agonists / antagonists for these receptors? Name some examples

  • selective agonists = NMDA , Glutamate

  • selective antagonists = ketamine , memantine

• Are any of these drugs used clinically? What for?

  • Ketamine (NMDA Receptor Antagonist) :

    • Uses: Dissociative anesthetic, analgesic, and (in lower doses) treatment for treatment-resistant depression.

    • Mechanism: Non-competitive NMDA receptor blockade, reducing excitatory neurotransmission.

  • Memantine (NMDA Receptor Antagonist)

    • Uses: Approved for moderate to severe Alzheimer’s disease.

    • Mechanism: Uncompetitive (voltage-dependent) NMDA receptor antagonist, helps prevent excessive glutamate-induced excitotoxicity.

  • Perampanel (AMPA Receptor Antagonist)

    • Uses: Adjunctive therapy for partial-onset seizures in epilepsy.

    • Mechanism: Non-competitive AMPA receptor antagonism, reducing excitatory synaptic transmission.

  • Riluzole (though not a direct, selective receptor antagonist, it modulates glutamate release among other actions)

    • Uses: Amyotrophic Lateral Sclerosis (ALS) to prolong survival and reduce excitotoxic damage.

  • Topiramate (broad-spectrum anti-epileptic)

    • Has multiple mechanisms, including some inhibition of AMPA/kainate receptors.