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principi attivi e medicinali

Dipartimento di Chimica

IX Ciclo di Laboratori Chimici di Aggiornamento per i Docenti delle Scuole Medie Superiori

Laura Belvisi, Ilenia Rossetti Commissione Orientamento del Collegio Didattico del Dipartimento di Chimica

16 Settembre 2014 Aula G10

via Golgi 19

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Farmaco

Sostanza che interagisce con un sistema biologico,

producendo una risposta biologica (effetto)

La maggior parte dei farmaci produce i propri effetti

biologici mediante interazione con una

macromolecola specifica (recettore o bersaglio).

Lo studio di come i farmaci interagiscono con i loro

bersagli macromolecolari attraverso le interazioni di

legame viene definito farmacodinamica.

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Fasi dell’azione di un farmaco

nei liquidi presenti nell’area di somministrazione

Effetto farmacologico ha avuto effetto terapeutico?

SOMMINISTRAZIONE

- Bersagli dei farmaci (drug targets) - Forze di legame intermolecolari legami ionici legami a idrogeno interazioni dipolo-dipolo e ione-dipolo interazioni dispersive (London) interazioni idrofobiche

G. L. Patrick Introduzione alla Chimica Farmaceutica, seconda edizione italiana (EdiSES, 2010) curata sulla quarta edizione inglese (Oxford University Press, 2009) An Introduction to Medicinal Chemistry 4e Capitolo 1

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Drug targets

Lipids Cell membrane lipids Proteins Receptors Enzymes Transport proteins Structural proteins (tubulin)

Nucleic acids

DNA RNA Carbohydrates Cell surface carbohydrates Antigens and recognition molecules

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Drug targets

• Drug targets are large molecules – macromolecules

• Drugs are generally much smaller than their targets

• Drugs interact with their targets by binding to binding sites

• Binding sites are typically hydrophobic clefts on the surface of macromolecules

• Binding interactions typically involve intermolecular bonds (non-covalent, weak)

• Functional groups on the drug are involved in binding interactions and are called binding groups

• Specific regions within the binding site that are involved in binding interactions are called binding regions

Drug targets

Most drugs are in equilibrium between being bound and unbound to their target

Macromolecular target

Drug

Bound drug

Induced fit Macromolecular target

Drug

Unbound drug

Binding site

Drug

Binding site

Binding regions

Binding groups

Intermolecular bonds

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L + R LR KA = 1/KD = [LR] / [L][R] ∆G° = - RT ln KA = RT ln KD

Equilibrium dissociation constant KD = [L] [R] / [LR] -thermodynamic parameter indicating the affinity of a drug for a particular receptor -KD is espressed in molar units and is an inverse measure of the receptor affinity -the smaller the KD value is, the higher is the affinity of a ligand Equilibrium association constant KA = [LR] / [L] [R] -reciprocal of the dissociation constant -the larger the KA value is, the higher is the affinity of a ligand

The drug-target or ligand-receptor interaction

Thermodynamic aspects

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The interaction is stabilized by the possibility to form a large number of weak non-covalent bonds.

Most drug-receptor interactions -reversible -weak chemical bonds Irreversible drug-receptor interactions -not common -strong chemical bonds (covalent, e.g. aspirin) -usually undesirable (toxicity)

The drug-receptor interaction

Interaction Energy (kcal/mol) Covalent bond 40-110 Ionic bond 5-10 Dipole-dipole bond 1-7 H-bonds 1-7 Dispersive 0.5-1 Hydrophobic 1

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Intermolecular bonding forces

Electrostatic or ionic bond • Strongest of the intermolecular bonds (20-40 kJ mol-1) • Takes place between groups of opposite charge • The strength of the ionic interaction is inversely proportional to the distance between the two charged groups • Stronger interactions occur in hydrophobic environments • The strength of interaction drops off less rapidly with distance than with other forms of intermolecular interactions • Ionic bonds are the most important initial interactions as a drug enters the binding site

DrugO

O H3N TargetDrug NH3

TargetO

O

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Intermolecular bonding forces

Hydrogen bonds

X HDrug

Y TargetDrug X

TargetHYδ+δ+

δ- δ-δ-δ-

HBD HBA HBA HBD

• Vary in strength • Weaker than electrostatic interactions but stronger than van der Waals interactions • A hydrogen bond takes place between an electron deficient hydrogen and an electron rich heteroatom (N or O) • The electron deficient hydrogen is usually attached to a heteroatom (O or N) • The electron deficient hydrogen is called a hydrogen bond donor • The electron rich heteroatom is called a hydrogen bond acceptor

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Intermolecular bonding forces

• The interaction involves orbitals and is directional

• Optimum orientation is where the X-H bond points directly to the lone pair on Y such that the angle between X, H and Y is 180o

Hydrogen bonds

YX H YX H

Hybridisedorbital

Hybridisedorbital

1sorbital

HBA HBD

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Intermolecular bonding forces

• Examples of strong hydrogen bond acceptors - carboxylate ion, phosphate ion, tertiary amine

• Examples of moderate hydrogen bond acceptors

- carboxylic acid, amide oxygen, ketone, ester, ether, alcohol

• Examples of poor hydrogen bond acceptors - sulfur, fluorine, chlorine, aromatic ring, amide nitrogen, aromatic amine

• Example of good hydrogen bond donors

- alkylammonium ion

Hydrogen bonds

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Intermolecular bonding forces

Dipole-dipole interactions • Can occur if the drug and the binding site have dipole moments • Dipoles align with each other as the drug enters the binding site • Dipole alignment orientates the molecule in the binding site • Orientation is beneficial if other binding groups are positioned correctly with respect to the corresponding binding regions • Orientation is detrimental if the binding groups are not positioned correctly • The strength of the interaction decreases with distance more quickly than with electrostatic interactions, but less quickly than with dispersive interactions

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Intermolecular bonding forces

Dipole-dipole interactions

Binding site

Localised d ipole moment

Dipole moment

R C

R

O

δ+

δ−

Binding site

R

C R O

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Intermolecular bonding forces

Ion-dipole interactions • Occur where the charge on one molecule interacts with the dipole moment of another • Stronger than a dipole-dipole interaction • Strength of interaction falls off less rapidly with distance than for a dipole-dipole interaction

CO

O

Binding site

δ+

δ−

R

C R O

H3N

Binding site

δ+

δ−

R

C R O

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Intermolecular bonding forces

Induced dipole interactions • Occur where the charge on one molecule induces a dipole on another • Occur between a quaternary ammonium ion and an aromatic ring

Binding site

R N R 3

+ δ−

δ+

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Intermolecular bonding forces

Dispersion interactions • Very weak interactions (2-4 kJ mol-1) • Occur between hydrophobic regions of the drug and the target • Transient areas of high and low electron densities cause temporary dipoles • Interactions drop off rapidly with distance • Drug must be close to the binding region for interactions to occur • The overall contribution of dispersive interactions can be crucial to binding

Hydrophobic regions

Transient dipole on drug δ+ δ-

London interaction

Binding site

DRUG

δ- δ+

δ+ δ-

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Desolvation penalties

• Polar regions of a drug and its target are solvated prior to interaction • Desolvation is necessary and requires energy • The energy gained by drug-target interactions must be greater than the energy required for desolvation

R C

R

O

O H

H H H

O

H

H

O

H

H

O

O H

Binding site Desolvation - Energy penalty Binding - Energy gain

O H

R C

R

O

Binding site

R C

R

O

O H

Binding site

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Intermolecular bonding forces

Hydrophobic interactions • Hydrophobic regions of a drug and its target are not solvated • Water molecules interact with each other and form an ordered layer next to hydrophobic regions - negative entropy • Interactions between the hydrophobic regions of a drug and its target ‘free up’ the ordered water molecules • Results in an increase in entropy • Beneficial to binding energy

Unstructured water Increase in entropy

Drug DRUG

Structured water layer round hydrophobic regions

Hydrophobic regions Water Binding site Binding site

Drug DRUG

Binding

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Receptors and drug action

When a drug interacts with a receptor, a number of chemical attractive forces are responsible for the initial interaction (affinity). AGONISTS ANTAGONISTS affinity for the receptor affinity for the receptor production of biological response but no activation as a result of the interaction and biological response

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L’interazione farmaco-recettore

La conoscenza della struttura, delle proprietà e della funzione dei bersagli macromolecolari è cruciale per la progettazione di nuovi farmaci. Alcuni esempi: Cicloossigenasi/farmaci antiinfiammatori Tubulina/farmaci antitumorali Chinasi/inibitori delle protein chinasi come antitumorali Il ruolo dei database

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Con il simbolo X sono stati indicati i bersagli molecolari dell'azione di alcuni farmaci antiinfiammatori.

Agiscono inibendo la sintesi di importanti mediatori chimici dell’infiammazione, quali le prostaglandine. L’inibizione può avvenire a livello degli enzimi fosfolipasi (blocco del rilascio di acido arachidonico) oppure cicloossigenasi (blocco della conversione di acido arachidonico in prostaglandine).

Farmaci antiinfiammatori

COX = cicloossigenasi o prostaglandina G/H sintasi

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Farmaci antiinfiammatori non steroidei (FANS)

CLASSIFICAZIONE DEI FANS IN BASE ALLA SELETTIVITA’ PER COX-1 E COX-2

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Farmaco IC50 ratio COX-1/COX-2 Aspirina 0.01 COX-1 selettivo Ibuprofene 0.5

COX-1 inibitori non selettivi

Naprossene 0.56 Ketoprofene 0.61 Piroxicam 3.12 Nimesulide 17.69 COX-2 inibitori

relat. selettivi Diclofenac 18.90 Celecoxib 143.30 COX-2 inibitori

selettivi Rofecoxib 410

FANS – Inibizione COX-1 e COX-2 D

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Selectivity of NSAIDs expressed as log IC80 COX-2 IC80 COX-1 inhibitory concentration

human whole blood assay (WBA) human modified whole blood assay (WHMA)

FANS – Inibizione COX-1 e COX-2 D

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from Foye’s Principles of Medicinal Chemistry Seventh edition

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Farmaci antiinfiammatori non steroidei (FANS)

Ibuprofene principio attivo di Moment®, Brufen®, Nurofen®, Buscofen® Ketoprofene principio attivo di Oki®, Fastum gel®, Lasonil®, Orudis®, Ketodol®, Artrosilene® spesso commercializzato sotto forma di sale di lisina Flurbiprofene principio attivo di Froben®, Benactiv gola® Naprossene principio attivo di Momendol®, Synflex®, Aleve® Diclofenac principio attivo di Voltaren®, Voltfast®

classici non selettivi

SOLFONILIDICI Nimesulide inibisce entrambe le isoforme dell'enzima cicloossigenasi ma preferenzialmente, anche se non in modo esclusivo, inibisce la COX-2. Tale caratteristica lo colloca a metà strada tra i FANS classici non selettivi e i COX-2 selettivi (coxib).

COXIB Celecoxib principio attivo di Celebrex®, Pfizer Rofecoxib principio attivo di Vioxx, Merck ritirato nel 2004

Farmaci antiinfiammatori non steroidei (FANS)

principio attivo di Aulin®, Mesulid®, Nimedex®, Nimesulene®

COX-2 preferenziali

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COX-2 selettivi

I COX-2 inibitori (rofecoxib, celecoxib) sono stati sviluppati con l’idea di ottenere dei FANS meno gastrolesivi rispetto a quelli classici con maggiore attività sulla COX-1.

I dati degli studi pre-marketing sembravano confermare questa idea.

Gli studi osservazionali post-marketing e la segnalazione delle reazioni avverse da parte dei medici hanno evidenziato che anche i COX-2 provocano gravi effetti gastrointestinali, anche se probabilmente con una incidenza inferiore.

L’aspetto preoccupante di questi farmaci, che ha portato già al ritiro dal mercato del rofecoxib, valdecoxib e parecoxib, è la possibilità di incrementare eventi avversi cardiovascolari come l’infarto del miocardio.

La vicenda dei COX-2 inibitori D

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Da alcuni anni è noto che Celecoxib a gli altri coxib sono in grado di sopprimere la formazione di PGI2 (prodotto predominante della cicloossigenasi a livello dell'endotelio). Mentre in un primo momento si riteneva che PGI2 derivasse prevalentemente dall’azione della COX-1, successive ricerche hanno permesso di determinare che è in realtà la COX-2 la principale fonte di produzione di tale sostanza. Gli effetti della PGI2 sono contrastati dal trombossano A2, il prodotto più importante della COX-1 a livello piastrinico. Poiché celecoxib e gli altri inibitori delle COX-2 non contrastano la produzione di trombossano ma solo quella di PGI2, i coxib potrebbero predisporre i pazienti che li assumono ad un aumentato rischio di infarto del miocardio e di ictus cerebrale.

prostaciclina (PGI2) -> determina vasodilatazione e inibisce l'aggregazione piastrinica trombossano (TxA2) –> determina vasocostrizione e stimola l’aggregazione piastrinica

Coxib e rischio cardiovascolare D

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Le due isoforme mostrano più del 60% di omologia e una simile affinità per l’acido arachidonico. Entrambe le COX sono configurate in modo tale che il sito attivo è posizionato al termine di un lungo canale idrofobico che i FANS non selettivi (es. ibuprofene) occupano, impedendo in tal modo all’acido arachidonico di raggiungere il sito catalitico cicloossigenasico, con conseguente inibizione della biosintesi delle prostaglandine.

(selettivo) (non selettivo) ibuprofene celecoxib

Confronto tra il sito catalitico di COX-1 e COX-2

Il gruppo sulfonamidico polare di Celecoxib interagisce con una tasca vicino al sito attivo di COX-2.

Confronto tra il sito catalitico di COX-1 e COX-2 D

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a sito attivo più ampio grazie alla sostituzione di Ile523 (COX-1) con Val523 (COX-2) Inibitori COX-2 selettivi progettati per sfruttare questa differenza

Aspirina - inibitore irreversibile di COX-1 - agisce come agente acilante (gruppo acetile si lega covalentemente al residuo Ser530 nel sito attivo) - tutti gli altri FANS sono inbitori reversibili (interazione enzima-inibitore non-covalente) ma mostrano diversi modi di legame

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L’interazione farmaco-recettore

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carboxylate of the fatty acid substrate interacts with Tyr-385 and Ser-530 at the apex of the channel nonproductive binding mode for AA

carboxylate lies near Arg-120 and Tyr-355 at the opening of the COX channel, similar to the productive binding mode of AA observed in the ovCOX-1:AA crystal structure

Arachidonic acid bound in the cyclooxygenase channel of the murine COX-2:AA complex

AA bind in different conformations in each monomer constituting the homodimer such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel.

3HS5

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Comparison of binding of AA in ov-COX-1 and mu-COX-2

AA in COX1 (1DIY) AA in COX2 (3HS5)

J. Biol. Chem. 2010, 285, 22152-22163

Molecular basis of interaction with COX enzymes D

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Investigation of the molecular determinants of inhibition by different classes of compounds reveals that the protein residues in the active site maintain similar orientations and that each chemical class forms distinct sets of interactions within the active site. Arylcarboxylic acid inhibitors bind in one of two orientations in the COX active site. Ibuprofen, flurbiprofen and naproxen bind in the canonical fashion with the carboxylate moiety ion-paired and hydrogen-bonded to the residues Arg-120 and Tyr-355. In contrast, diclofenac binds in an inverted orientation in which its carboxylate is hydrogen-bonded to the side chains of Tyr-385 and Ser-530.

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Crystal structures of flurbiprofen and diclofenac bound in muCOX-2 active site

3PGH 1PXX acidic group coordinated to residues Arg-120 and Tyr-355 at the base of the active site

acidic group coordinated to the catalytic Tyr-385 as well as Ser-530 at the top of the pocket

flurbiprofen diclofenac

Crystal structure of naproxen bound to muCOX-2

acidic group coordinated to residues Arg-120 and Tyr-355 at the base of the active site

naproxen

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3NT1

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Database

Protein Data Bank (PDB) An Information Portal to Biological Macromolecular Structures The PDB archive contains information about experimentally-determined structures of proteins, nucleic acids, and complex assemblies. http://www.pdb.org/pdb/ 100147 structures, May 13, 2014 (X-ray, NMR, homology modeling) Search PDB ID, molecule name, author 3D View Ligands – View interactions (View pocket in Jmol)

1EQG THE 2.6 ANGSTROM MODEL OF OVINE COX-1 COMPLEXED WITH IBUPROFEN (2001) Biochemistry 40: 5172-5180 1CQE or 1EQH PROSTAGLANDIN H2 SYNTHASE-1 COMPLEX WITH FLURBIPROFEN (ovine COX-1 with flurbiprofen) (1994) Nature 367: 243-249 3PGH CYCLOOXYGENASE-2 COMPLEXED WITH A NON-SELECTIVE INHIBITOR, FLURBIPROFEN (murine COX-2 with flurbiprofen) (1996) Nature 384: 644-648 3LN1 Structure of celecoxib bound at the murine COX-2 active site (2010) Bioorg.Med.Chem.Lett. 20: 7159-7163

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Protein Data Bank (PDB)

Database

Drug Bank – Open Data Drug & Drug Target Database The DrugBank database is a unique bioinformatics and cheminformatics resource that combines detailed drug (i.e. chemical, pharmacological and pharmaceutical) data with comprehensive drug target (i.e. sequence, structure, and pathway) information. http://www.drugbank.ca/ 7739 drug entries Search for drugs Search results Pharmacodynamics Pharmacokinetics Properties and other information

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The binding database BindingDB is a public, web-accessible database of measured binding affinities, focusing chiefly on the interactions of protein considered to be drug-targets with small, drug-like molecules. BindingDB contains 1,058,945 binding data, for 6,997 protein targets and 453,657 small molecules. http://www.bindingdb.org/bind/index.jsp CREDO: A Structural Interactomics Database For Drug Discovery CREDO is a relational database storing all pairwise atomic interactions of inter- as well as intra-molecular contacts between small- and macromolecules found in experimentally-determined structures from the Protein Data Bank (PDB). http://www-cryst.bioc.cam.ac.uk/credo (search PDB file -> Ligands -> Interactions - Contacts)

Database D

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Given their central role in cell division, microtubules are the target of many important toxins and drugs. For instance, some anticancer drugs act this way: they are designed to block the malignantly rapid growth of the cancer cells by blocking the normal dynamics of microtubules. Three molecules that block microtubule action, all produced by plants, are: - colchicine and vinblastine block the assembly of

microtubules (PDB entry 1Z2B) - paclitaxel (Taxol) promotes the assembly, binding so

tightly that disassembly is virtually impossible (PDB entry 1JFF)

Anticancer drugs

The use of stathmin (an important regulatory protein of microtubule dynamics) has allowed the determination of the structures of many different inhibitors bound to tubulin. You can use the Ligand Explorer to explore the ways these inhibitors are bound.

1Z2B 1JFF

Anticancer drugs D

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Chapter 21 Anticancer agents G. L. Patrick, An introduction to Medicinal Chemistry Fourth edition

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Targeted cancer therapies Protein kinase inhibitors Protein Kinases

•Enzymes that catalyse phosphorylation reactions on protein substrates •500-2000 estimated protein kinases in a cell •Protein kinases are present in the cytoplasm •Protein kinase receptors - dual role as receptor and enzyme •Overexpression can result in cancer •Tyrosine kinases, serine-threonine kinases and histidine kinases •ATP used as enzyme cofactor - phosphorylating agent

N

N N

N

N

O

OH OH

H HH H

O P OO

OP OO

OPO

OO

H H

61N

N N

N

N

O

OH OH

H HH H

O P OO

OP OO

O

H H

61

O PO

OO

Tyrosine kinases HN

O

Protein Protein

H

OH

ATP ADP

HN

O

Protein Protein

H

OP

OHOH

O

Serine-threonine kinases

Serine

HN

O

OH

Protein Protein

H

ATP ADP

HN

O

O

Protein Protein

H

PO OH

OH

Threonine

HN

O

H3C OH

Protein Protein

H

ATP ADP

HN

O

H3C O

Protein Protein

H

PO OH

OH

Protein Kinases D

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Active Site •Contains the binding site for the protein substrate

•Contains the binding site for the ATP cofactor

•Clinically useful inhibitors target the ATP binding site

•ATP binding site is similar but not identical for all protein kinases

•Allows selectivity of inhibitor action

Protein Kinases

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ATP binding site

Binding of ATP to the kinase active site of EGF-R

Protein Kinases

N

N

O

H

H

O

H N

H 2 N O C

H 3 C

H 3 C

O

S H 3 C

Gln-767

Met-769

Leu-768

Ribose pocket

Hydrophobic pocket

Cleft

N

N N

N

N

O

OH OH

H HH H

O P OO

OP OO

OPO

OO

H HHBD

HBA N

N N

N

N

O

OH OH

H HH H

O P OO

OP OO

OPO

OO

H H

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•Purine base is buried deep into the binding site •Purine forms two hydrogen bonding interactions to the binding site •Ribose sugar binds to a ‘ribose binding pocket’ •Triphosphate chain lies along a cleft towards the enzyme surface •Triphosphate interacts with two metal ions and amino acids •Specificity surface is an area of unoccupied binding site •An empty hydrophobic pocket lies opposite the ribose binding pocket •The gatekeeper residue is an amino acid situated at the entrance to the hydrophobic pocket •The size of the gatekeeper residue is important in drug design •The nature of aa in the binding pockets is important to drug design

ATP binding site

Protein Kinases

EGF-R kinase inhibitors Gefitinib (Iressa)

Notes •Developed by Astra Zeneca (first EGF-R inhibitor to reach the clinic) •Inhibits the kinase active site of the epidermal growth factor receptor •The EGF-receptor is a tyrosine kinase receptor •Gefitinib is a 4-anilinoquinazoline structure

N

N

HN

OMe

O N

O

F

Cl4 6

7

31 Morpholine

Quinazoline

Aniline

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Binding interactions •Binds to the ATP binding site (ATP mimic) •Aniline ring occupies the normally vacant hydrophobic pocket opposite the ribose binding pocket •Quinazoline binds to the same region as the purine ring of ATP

N

N

N

OMe

O

F

ClH

N

O

Hydrophobic pocket

Cleft

Met-769

OH2

Thr-830

N

N

N

OMe

O

F

ClH

N

OHBA

HBA

Hydrophobic pocket

Cleft

Met-769

OH2

Thr-830

N

N

N

OMe

O

F

ClH

N

OHBA

HBA

Hydrophobic pocket

Cleft

Met-769

OH2

Thr-830

N

N

N

OMe

O

F

ClH

N

O

EGF-R kinase inhibitors Gefitinib (Iressa)

PDB entry 2ITY CRYSTAL STRUCTURE OF EGFR KINASE DOMAIN IN COMPLEX WITH IRESSA

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Imatinib (Glivec or Gleevec)

Notes •First protein kinase inhibitor to reach the market (2001) •Selective inhibitor for a hybrid tyrosine kinase (Bcr-Abl) •Bcr-Abl is active in certain tumour cells

N

N

N

HN

Me

NH

O

N

NMe

The first FDA approved ATP-competitive kinase inhibitor (imatinib, Gleevec) was designed against BCR-Abl kinase for chronic myeloid leukemia but was also FDA-approved for use in cKit kinase driven diseases such as gastrointestinal stromal tumors.

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Imatinib (Glivec or Gleevec)

Binding interactions •Identified from the crystal structure of imatinib-Abl kinase complex •Amide serves as an ‘anchoring group’ and orientates the molecule •Amide binds to Glu and Asp (important to the catalytic mechanism) •A hydrogen bond to the gatekeeper Thr is essential to activity

PDB entry 2HYY

NNN

NH

Me

NH

O

N N MeH

O NH

O

MeS

Met

Gatekeeperresidue

Hydrophobic pocketSelectivity region 1

O

Thr

O

OGlu

H

N

O

O2C Asp

H

Hydrophobic regionSelectivity region 2

Piperazinyl group

Glu

Ionic bond

Conformational blocker Mutation to Isoleucine

introduces resistance