Disease: Hypothyroidis

mReasons:• Not enough iodine• Autoimmune (eg Hashimoto's

thyroiditis)• Radioactive Iodine• Surgery/injuryTreatment• liothyroxine (synthetic T3) as

hormonal replacement• There is no direct way of increasing

thyroid hormone secretion by the thyroid gland

Targets: thyroid hormone carriersSample Presentation Slide 2

• In human plasma, T3 & T4 are >99% bound to carrier proteins• Reference range in normal adult blood:

– Free (only free hormone is active)• T3: 3-8 pmol/L• T4: 10-20 pmol/L

– Total T3/T4: 1-2.5 nmol/L (~ 100-fold higher than free)• 70% bound to thyroxine-binding globulin, TBG• 10-15% to transthyretin, TTR (same as thyroxine-binding prealbumin, TBPA)• 15-20% to albumin

TBG TTR Albumin

Targets: Additional role of TH carriers in drug-drug interactions for other drugs

TTR

• Because >99% of T3/T4 are protein-bound in plasma, carrier protein disregulation may lead to large variations in free T3/T4

• Factors that may change TBG concentration:– pregnancy, estrogen-containing

medication (TBG)– infectious hepatitis (TBG)– nephrosis, acromegaly (TBG)– androgen or corticosteroid therapy (TBG)

• T3/T4 binding by TTR is inhibited by salicylates.

Sample Presentation Slide 3

TBG

Target: Human Thyroid Hormone Receptor• Two main types ( and ),

each having 2 isoforms• Length: 410-490 AA• MW ~ 46.8-54.8 kDa• Tissues: multiple• Cellular localization: nucleus

Sample Presentation Slide 4

Transactivation domain

DNA binding domain

Ligand-binding domain

THR1

THR2

THR1

THR2

1 53 127 190 370 490

1 53 127 190 410

1 107 181 244 461

1 122 196 259 476

Cell

Nucleus

HRE target gene HRE target gene

Type II Nuclear Hormone Receptor (e.g. THR)

Unbound or antagonist-bound

(inactive)

DNA

Co-activator+

RNApolymerase

Co-repressor

Agonist-bound (initiates gene transcription)

Hormone or agonist

therapeutic

=

Ligand-binding domain of THR1 in complex with T3, PDB 3gws

Drugs: Liothyronine and Levothyroxine

• Thyroid hormones (TH) and their synthetic analogs increase oxidative metabolism of carbohydrates, lipids and proteins by the mitochondria.

• They also increase the number of catecholamine (e.g. adrenaline) receptors on target cells, thereby increasing catecholamine sensitivity – may aggravate heart conditions; acute overdose may lead to heart failure

• Indications: hormone replacement therapy for hypothyroidism and myxedema

Sample Presentation Slide 5

Chemical structure(s)

Synthetic L-isomer of triiodothyronine (T3) Synthetic L-isomer of thyroxine (T4) Stereo-isomers

MW ~ 650 g/mol MW ~ 775 g/mol Molecular weight

Amino-acid: acidic pKa = 2.13, basic pKa = 8.27 Amino-acid: acidic pKa = 2.12, basic pKa = 8.27 Ionization states/species

Administered as sodium salt; orally available Administered as sodium salt; orally available Formulations

Most active, but short-lived form of TH Pro-hormone (3-4 times less active) and a reservoir for T3. T4 to T3 conversion is catalyzed by cellular iodothyronine deiodinases

Nature

2.5 days (free T3) 1 week (free T4) Half-life

Cytomel, Tertroxin, etc. Levoxyl, Synthroid, etc. Marketing status +

Liothyronine and Levothyroxine:ionization, dissolution, and phase partitioning

Sample Presentation Slide 5a

Acidic pKa = 2.13Basic pKa = 8.27Zwitterion at pH = 7, base at pH = 2, acid at pH = 9

Acidic pKa = 2.12, Basic pKa = 8.27Zwitterion at pH = 7, base at pH = 2, acid at pH = 9

Solubility

Free form: 3.96 mg/L ~ 6 uM, logS = -5.22Sodium salt: «poorly soluble»– MCD increases solubility

Free form: 0.105 ml/L ~ 0.14 uM, logS = -6.87Sodium salt: 770 uM, logS = -3.11More soluble at pH < 2 and pH > 8 MCD increases solubility

Phase partitioning

LogP = 3.9 (lipophilic, but within reasonable range) LogD = 1.3 (ionization decreases lipophilicity)

LogP = 4.7 (more lipophilic)

LogD = 1.7 (ionization decreases lipophilicity)

Graph from:

MCD

Liothyronine and Levothyroxine: Target binding Constants and Energies

Sample Presentation Slide 6

Chemical structure(s)

Full agonist Not so potent agonist (~ 10% of potency in cell culture)

Mode of action

Thyroid hormone receptor A (THA)pKd = 9.6Kd = 0.25 nMGbind = -13.4 kcal/mol

- Equilibrium dissociation constant for primary and secondary targets

Thyroid hormone receptor B (THB)pKd = 10.39Kd = 0.04 nMGbind = -14.4 kcal/mol

Kd ~ 2 nM Gbind ~ -12 kcal/mol(30-50 fold lower affinity than T3)

Equilibrium dissociation constant for primary and secondary targets

• https://www.ebi.ac.uk/chembldb/ • Sandler B, et al. Thyroxine-thyroid hormone receptor interactions. J Biol Chem. 2004; 279(53):55801-8

Van der Waals interactions:• Perfect steric fit between the

ligand and the pocket many VW contacts

Electrostatics:• The three Arg in the bottom of

the pocket create a strong positive charge interacting with the negatively charged carboxyl group of the drug.

Liothyronine and THRMolecular basis of drug-target interaction

Sample Presentation Slide 7

Hydrogen bonds:• Three residues make HBs with the

ligand: Arg-282, Asn-331, and His-435 near the pocket entrance. The ligand has no unsatisfied hydrogen bond donors/acceptors.

Other types of interactions are not observed:

• Two aromatic residues in the pocket, none of them makes a Pi-interaction.

• No metal ions• Non-covalent interaction

Sample Presentation Slide 8Liothyronine and THRMolecular basis of drug-target interaction

Liothyronine: LogP, PSA and lipophilicity

• The surface of Liothyronine molecule is mostly non-polar • LogP = 3.9 Liothyronine is lipophilic• Levothyroxine has an additional iodine atom: LogP = 4.7, more

lipophilic• PSA is 98A2 , good permeability ( < 140).• Has 4 freely rotatable and 3 restricted bonds

Polar atoms Non-polar atoms

Sample Presentation Slide 9

Introduction and conclusion• Interesting aspects of pharmacodynamics and

pharmacokinetics resulting from the drug properties described above. Bioavailability? Protein binding? Half life? Drug interactions?

• Past and future of the drug(s). Market and competition. Stage? OR: Discontinued? Why? OR: To become a generic in 1.5 yrs?

• Provide good illustrations, key references and acknowledgements.

• Make it fun and special