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

1 ABL1 Dasatinib, Imatinib, Nilotinib

2 ACE Captopril, Lisinopril, Enalapril

Decamethonium, , Edrophonium, , , 3 ACES Succinylcholine 4 ADRB2 Pindolol,Timolol, Alprenolol, Dobutamine, Isoprenaline

5 ANDR , Cyproterone, , ,

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 , , Quinestrol, ,

11 FPPS Alendronate, Ibandronate, Pamidronate, Risedronate, Zoledronate

12 GCR , Propionate, (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, , , , Mifepristone

19 POL_P Darunavir, Indinavir, Lopinavir

20 POL_RT Delavirdine, , 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 transitions Thermodynamics, 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 IC 50 ) • Structural basis, molecular determinants of binding, role of entropy (compound flexibility analysis + hydrophobicity)

Organize info in a table for several drugs Sample Presentation Slide 1 Liothyronine and Levothyroxine

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 Nature reservoir for T3. T4 to T3 conversion is catalyzed by cellular iodothyronine deiodinases 2.5 days (free T3) 1 week (free T4) Half-life Cytomel , Tertroxin , etc. Levoxyl , Synthroid , etc. Marketing status + • Thyroid hormones (TH) and their synthetic analogs increase oxidative 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 2 Thyroid hormone carriers

• 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 Sample Presentation Slide 3 Role of TH carriers in drug-drug interaction

• 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 : TBG – pregnancy, -containing (TBG ↑) – infectious hepatitis (TBG ↑) – nephrosis, acromegaly (TBG ↓) – or therapy (TBG ↓) • T3/T4 binding by TTR is inhibited by salicylates.

TTR Sample Presentation Slide 4 Human Thyroid Hormone Receptors (target)

α β Transactivation DNA binding Ligand-binding • Two main types ( and ), domain domain domain ≥ each having 2 isoforms ααα 1 53 127 190 410 THR 1 • Length: 410-490 AA ααα 1 53 127 190 370 490 THR 2 βββ • MW ~ 46.8-54.8 kDa THR 1 1 107 181 244 461 βββ • Tissues: multiple THR 2 1 122 196 259 476

• Cellular localization: nucleus Hormone or agonist therapeutic Cell

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

Unbound Agonist-bound or antagonist-bound (initiates gene (inactive) transcription)

= Co-repressor Co-activator + RNA polymerase

DNA

HRE target gene HRE target gene βββ Ligand-binding domain of THR 1 in complex with T3, PDB 3gws Sample Presentation Slide 5 Liothyronine and Levothyroxine: ionization, dissolution, and phase partitioning

Graph from:

Acidic pKa = 2.13 Acidic pKa = 2.12, Basic pKa = 8.27 Basic pKa = 8.27 Zwitterion at pH = 7, base at pH = Zwitterion at pH = 7, base at pH = 2, 2, acid at pH = 9 acid at pH = 9 Solubility Free form: Free form: 3.96 mg/L ~ 6 uM, logS = -5.22 0.105 ml/L ~ 0.14 uM, logS = -6.87 Sodium salt: «poorly soluble» Sodium salt: 770 uM, logS = -3.11 – More soluble at pH < 2 and pH > 8 MβCD increases solubility MβCD increases solubility Phase partitioning LogP = 3.9 ( lipophilic , but within LogP = 4.7 ( more lipophilic ) reasonable range) LogD = 1.3 (ionization decreases LogD = 1.7 (ionization decreases lipophilicity) lipophilicity)

MβCD Sample Presentation Slide 6 Liothyronine and Levothyroxine: Target binding

Chemical structure(s)

Full agonist Not so potent agonist (~ 10% of potency Mode of action in cell culture) Thyroid hormone receptor A (THA) pKd = 9.6 - Equilibrium dissociation Kd = 0.25 nM constant for primary and ∆ secondary targets Gbind = -13.4 kcal/mol Thyroid hormone receptor B (THB) pKd = 10.39 Kd ~ 2 nM Equilibrium dissociation ∆ constant for primary and Kd = 0.04 nM Gbind ~ -12 kcal/mol ∆ secondary targets Gbind = -14.4 kcal/mol (30-50 fold lower affinity than T3)

•https://www.ebi.ac.uk/chembldb/ •Sandler B, et al. Thyroxine-thyroid hormone receptor interactions . J Biol Chem. 2004; 279(53):55801-8 Liothyronine and THR Sample Presentation Slide 7 Molecular basis of drug-target interaction Van der Waals interactions: Electrostatics: • Perfect steric fit between the • The three Arg in the bottom of ligand and the pocket ⇒ many the pocket create a strong VW contacts positive charge interacting with the negatively charged carboxyl group of the drug. Liothyronine and THR Sample Presentation Slide 8 Molecular basis of drug-target interaction 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 • Non-covalent interaction Sample Presentation Slide 9 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 3 4 • Estimated Gibbs energy contribution due to conformation entropy loss upon binding or crystallization is ∆ Gconf =0.6×4 = 2.4 kcal/mol Liothyronine: Sample Presentation Slide 10 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 A 2 of ASA. • There are 18 non-polar atoms, with the total of 441 A 2 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 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 Textual and quantitative DrugBank http://www.drugbank.ca/ Wikipedia http://en.wikipedia.org/ Universal Protein Resource http://www.uniprot.org/ Pharmacogenomics http://www.pharmgkb.org/index.jsp Knowledgebase ChEMBL medicinal https://www.ebi.ac.uk/chembldb/index.php chemistry database Micromedex http://www.thomsonhc.com/micromedex2/librarian/

Molecular 3D ICM molecular files http://xablab.ucsd.edu/12/p/ Software: ICM-Browser Course Lab Software: ActiveICM and ICM Browser

• Go to course web-page http://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