• $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

1. Perform Total Binding ( Curve 1 )

   • Use only radiolabeled ligand ( "hot" ligand )

   • This includes both specific and non-specific binding.

   • Incubate receptor preparation with increasing concentrations of radioligand

   • Measure total bound radioactivity at each concentration

   • Total Binding $B_{\text{total}}$$B_{\text{total}}$ is a combination of :

     • Specific binding ( to actual receptors )

       • Non-specific binding ( to membranes , non-receptor sites )

2. Measure Non-Specific Binding ( Curve 2 ) :

   • Add an excess of non-radioactive ligand ( "cold" ligand )

   • This competes with the radioligand for receptor sites , preventing it from binding specifically

   • What remains bound is only non-specific binding

   • Measure bound radioactivity at each radioligand concentration

   • This curve represents non-specific binding ( $B_{\text{NSB}}$$B_{\text{NSB}}$ )

3. Calculate Specific Binding ( Curve 3 ) :

   • Subtract non-specific binding from total binding

     • $B_{\text{specific}}=B_{\text{total}}-B_{\text{NSB}}$$B_{\text{specific}} = B_{\text{total}} - B_{\text{NSB}}$

   • This isolates only the radioligand bound to receptors.

   • curve represents specific binding , which follows saturation kinetics and is used for Scatchard analysis.

   

4. Generate a Scatchard Plot :

   • Use specific binding ( $B_{\text{specific}}$$B_{\text{specific}}$ ) and free ligand concentration ( $[L]$$[L]$ ) to construct the Scatchard plot

   • Plot:

     $$\frac{B_{\text{specific}}}{[L]}\text{ (y-axis) vs. }{B}_{\text{specific}}\text{ (x-axis)}$$
     $$\frac{B}{[L]}=\frac{B_{\text{max}}-B}{K_d}$$

   • Where:

     • $B$$B$ = bound ligand (pmol/mg protein)

     • $[L]$$[L]$ = free ligand concentration (nM)

     • $B_{\text{max}}$$B_{\text{max}}$ = maximum number of binding sites

     • $K_d$$K_d$ = dissociation constant (affinity, nM)

   • Rearrange into a linear equation :

    

   • Scatchard Plot Equation for Single Binding System :

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

   • The slope gives $-1/K_d$$-1/K_d$ ( binding affinity ) , and the x-intercept gives $B_{\text{max}}$$B_{\text{max}}$ ( total receptor density )

• The binding of drugs/transmitters/hormones to receptors can be measured directly by the use of radioactive drug molecules

• Used to determine affinity of drug for receptor and receptor density of tissue.

• Radioligand Binding Methods :

  • Centrifugation

  • Filtration Assay :

    1. Homogenise tissue

    2. Incubate with ‘hot’ compound with without saturating [] cold compound.

    3. Rapidly filter.

    4. Count bound and free.

• 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

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

1. Oral : Drugs are taken through the mouth and absorbed via the gastrointestinal tract

2. Rectal : Administered through the rectum , useful when oral administration is not feasible

3. Topical : Applied to the skin or mucous membranes , such as creams or eye drops

4. Injection : Includes intravenous ( IV ) , intramuscular ( IM ) , and subcutaneous ( SC ) methods for direct delivery into blood or tissue

5. Respiratory : Inhaled drugs ( e.g., nasal sprays, inhalers ) absorbed through the respiratory tract

O - R - T - I -R

1. Intervascular = blood plasma within blood vessels

   • 3 Liters = 4%

2. Interstitial = fluid surrounding the cells in tissues

   • 9 Liters = 13%

3. Intracellular = fluid inside cells ( cytosol )

   • 28 Liters = 41%

1. Absorption : Passage through membranes , impacted by drug solubility and ionization

2. First-Pass Metabolism : Degradation in the gut wall or liver before reaching systemic circulation

3. Chemical Stability : Destruction of the drug in the gastrointestinal tract ( e.g., stomach acid )

1. Phase I :

   • Convert parent compound into a more polar / hydrophilic metabolite

   • Involves oxidation, reduction, or hydrolysis to add or unmask functional groups ( e.g., -OH , -NH2 ) , often preparing the drug for Phase II

2. Phase II : Conjugation reactions with endogenous substrates to further increase water solubility for excretion.

   • conjugation with glucoronide , sulfate , acetate , amino acid

   • try to make them even more polar , to get them out of the body and into the urine

   • Phase II is the true “detoxification” step in the metabolism process

1. Renal Excretion : Through urine , involving filtration , secretion , and reabsorption in the kidneys

2. Biliary Excretion : Through bile , often leading to fecal elimination

3. Respiratory Excretion : Drugs exhaled through the lungs , especially volatile compounds

• TNF :

  • induces apoptosis , which is damaging , but limits the spread of more damage in the long-term

  • increases inflammation , and BBB permeability

• IL-1 :

  • Key driver of sickness behavior: fatigue, loss of appetite, fever, and social withdrawal.

  • Enhances HPA axis activation by stimulating CRH release → leads to higher cortisol levels.

  • In stroke and inflammation, IL-1 stimulates neurotrophic factors (like NGF), helping neurons recover.

  • Role in Stress Adaptation

    • Short-term : Enhances alertness , mobilizes energy, and promotes fever, which can help fight infections.

    • Long-term : Chronic IL-1 elevation disrupts cognition and mood ( linked to depression and anxiety in chronic stress conditions

• IL-6 :

  • Activates Janus Kinase ( JAK ) and STAT signaling, which turn on genes related to immune response and neuronal plasticity.

  • Increases astrocyte proliferation (helps with neuroprotection but can also contribute to neuroinflammation).

  • Induces fever, sleep regulation, and food intake changes.

  • Helps modulate glutamate-induced neurotoxicity, protecting neurons.

  • Role in Stress Adaptation

    • Promotes neuronal survival, especially in glutamate toxicity scenarios.

    • Enhances resilience to stress by regulating the HPA axis.

    • However, excessive IL-6 can increase pain sensitivity (allodynia, hyperalgesia) and worsen neuroinflammation.

• know timing differences for the 4 different receptor types