Physical Pharmacology Labs• Time: Fri 9-12

– Practice problems regularly posted online for independent study

• Goal: Develop better understanding of course materials via:– Problem solving– Semester-long group projects: Phys. Pharm.-characterization of drugs– Molecular visualization exercises & Data analysis

• Format:– 30-60 minute presentation by one of the instructors / review of self

tests, past midterms and quizzes– Online quiz (quizzes make 10% of the total grade)

• Course materials may be used during the quiz (but not during the exams)• Calculators are allowed during exams and quizzes• Week 1 only: the quiz will be available online till Mon morning

– Work on group projects

• Group project grade constitutes 15% of your total grade– Preparation: week 1-8 Fridays (time will be set aside during labs)– Presentations: weeks 9-10 Fridays (same time as labs)

Group Projects

Phys. Pharm.-characterization of drugs

Structural and Physical aspects of drug pharmacokinetics and phamacodynamics

Target Drugs: choose 3-4, preferably with different mechanisms* Name Name Name

1 ABL1 Dasatinib, Imatinib, Nilotinib

2 ACE Captopril, Lisinopril, Enalapril

3 ACESDecamethonium, Donepezil, Edrophonium, Galantamine, Tacrine, Succinylcholine

4 ADRB2 Pindolol,Timolol, Alprenolol, Dobutamine, Isoprenaline

5 ANDR Bicalutamide, Cyproterone, Fluoxymesterone, Flutamide, Testosterone

6 AOFB Moclobemide, Rasagiline, Selegiline

7 CAH2 Acetazolamide, Brinzolamide, Dorzolamide, Topiramate, Trichlormethiazide

8 D3DR Amisulpride, Remoxipride, Ropinirole, Rotigotine, Piribedil, Pramipexole

9 EGFR Erlotinib, Lapatinib, Gefitinib

10 ESR1 Diethylstilbestrol, Estradiol, Quinestrol, Raloxifene, Tamoxifen

11 FPPS Alendronate, Ibandronate, Pamidronate, Risedronate, Zoledronate

12 GCR Dexamethasone, Fluticasone Propionate, Mifepristone (off-target activity?)

13 HMDH Atorvastatin, Fluvastatin, Lovastatin, Rosuvastatin, Simvastatin

14 NRAM Oseltamivir, Zanamivir

15 PDE5A Sildenafil, Tadalafil, Vardenafil

16 PPARG Rosiglitazone, Pioglitazone, Troglitazone

17 PGH2 Celecoxib, Diclofenac, Flurbiprofen, Indomethacin

18 PRGR Norethindrone, Progesterone, Levonorgestrel, Norgestrel, Mifepristone

19 POL_P Darunavir, Indinavir, Lopinavir

20 POL_RT Delavirdine, Efavirenz, Nevirapine

21 RENI Aliskiren, Remikiren

22 THRB Argatroban, Ximelagatran

* For many targets in the list, drugs belong to two classes: agonists (activators) and antagonists (inhibitors). Then are often prescribed for opposite indications. When you choose drugs for the presentation, make sure to represent both classes.

PChem project chapter 1:Molecules

– Drug(s): names, chemical structures, MW– Other components of formulation(s) (e.g. Sodium salt? Hydrochloride?)– Drug target (DT) protein: name, length/MW, organism of origin,

subcellular location, domain organization, quaternary structure*. Role of the DT in the disease(s):• If DT is an enzyme: what reaction does it catalyze? • If DT is a receptor: what endogenous ligand stimulates its activation? • How is this reaction/activation related to the disease(s)?

– Name other important players: albumin and/or other plasma proteins, metabolizing enzymes, etc.

– Mention metabolic activation**, where applicable.

*Pick this information from online resources, follow the links if necessary. Use the “blobology”approach to represent the information on a single slide.

**Some compounds are administered as prodrugs, they undergo metabolic modification to become active drugs. If that is the case, please describe the metabolizing enzyme and the active metabolite.

PChem project chapter 2:Reactions and transitionsThermodynamics, kinetics, structural and molecular basis of:

– Dissolution/crystallization. Water solubility, for different crystal forms where available.

– Ionization. Name ionizable groups with group-specific pKa where available. Describe ionization forms prevalent at different pH. How does ionization affect solubility? Any related recommendations for taking the drug?

– Partition between aqueous and lipid phases. LogP value and membrane permeation.

– Conformational transitions. Stereoisomers and their activity where available. Conformational entropy and its role in reactions of dissolution and target binding

– Binding/dissociation reaction. Describe interactions with the target and with other proteins, e.g. albumin and cytochromes where available• Thermodynamics of binding (Kd if available, Ki or IC50)• Structural basis, molecular determinants of binding, role of entropy

(compound flexibility analysis + hydrophobicity)

Organize info in a table for several 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 1

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 +

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

Role of TH carriers in drug-drug interaction

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

Human Thyroid Hormone Receptors (target)• Two main types (α and β),

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

Sample Presentation Slide 4

Transactivationdomain

DNA binding domain

Ligand-binding domain

THRαααα1

THRαααα2

THRββββ1

THRββββ2

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 THRββββ1in complex with T3, PDB 3gws

Liothyronine and Levothyroxine:ionization, dissolution, and phase partitioning

Sample Presentation Slide 5

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»–MβCD 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 MβCD 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:

MβCD

Liothyronine and Levothyroxine: Target binding

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 nM∆Gbind = -13.4 kcal/mol

- Equilibrium dissociation constant for primary and secondary targets

Thyroid hormone receptor B (THB)

pKd = 10.39Kd = 0.04 nM∆Gbind = -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: Estimating Conformational Entropy Contribution

• Liothyronine molecule has 4 freely rotatable bonds and 3 restricted rotatable bonds.

• Estimated number of drug conformers in solution is 34

• Estimated Gibbs energy contribution due to conformation entropy loss upon binding or crystallization is

∆Gconf =0.6×4 = 2.4 kcal/mol

Sample Presentation Slide 9

Liothyronine: Polar and Non-polar Surface Areas

• The surface of Liothyronine molecule is mostly non-polar • LogP = 3.9• There are 5 polar atoms, with the total of 151 A2 of ASA. • There are 18 non-polar atoms, with the total of 441 A2 of ASA. • Energy of transfer from water to non-polar solvents negatively

correlates with polar ASA: Liothyronine is lipophilic• Levothyroxine has an additional iodine atom: LogP = 4.7, more

lipophilic

Polar atoms Non-polar atoms

Sample Presentation Slide 10

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 lots of illustrations• Make it fun and special ☺

Sources of information

DrugBank http://www.drugbank.ca/Wikipedia http://en.wikipedia.org/Universal Protein Resource http://www.uniprot.org/PharmacogenomicsKnowledgebase

http://www.pharmgkb.org/index.jsp

ChEMBL medicinal chemistry database

https://www.ebi.ac.uk/chembldb/index.php

Micromedex http://www.thomsonhc.com/micromedex2/librarian/

ICM molecular files http://xablab.ucsd.edu/12/p/ Software: ICM-Browser

Textual and quantitative

Molecular 3D

Course Lab Software: ActiveICM and ICM Browser

• Go to course web-pagehttp://xablab.ucsd.edu/12/icmdw/

• Download and install ICM Browser– ICM Browser is a standalone software that manipulates ICM binary

(.icb) files

• Download and install ActiveICM plugin– ActiveICM is a plugin for a web-browser (Firefox, Safari, Chrome, IE,

etc.) – useful for viewing lab 3D materials

• Right-click (Windows) or Ctrl-click (Mac) and choose “Save”on your computer

• Run the file after the download is complete• Follow the steps of the installation procedure

ActiveICM and ICM browser Window Mouse Controls

• Center of the window: left mouse button = rotate; middle button = translate, right button = menu

• Left margin: left mouse button = zoom in/out• Top margin: left mouse button = Z-rotation• Bottom margin, two triangular arrows = switch to

next/previous slide.• Bottom right, Molsoft logo = Menu (switch slide, run script,

center view etc.)

Next/previous slide Menu