‘Covalent Drugs’ ‘Covalent Drugs’ ––Covalent Drugs Covalent Drugs Drugs acting by forming a covalent Drugs acting by forming a covalent bond with their biological targetsbond with their biological targets

Lecture 6. 1st Year Graduate students.Lecture 6. 1 Year Graduate students.2016

PPictureictureof the of the daydaydayday

GGeneraleneral considerationsconsiderationsGGeneraleneral considerationsconsiderations• pharmaceutical companies are reluctant to apply covalent mechanisms of inhibition

• off-target reactivity

• idiosyncratic drug-related toxicity (e. g., by causing immune response)

• >40 FDA approved drugs bind covalently to their targets• >40 FDA-approved drugs bind covalently to their targets

• 1/3 of all enzyme drug targets have at least one example of approved covalent drugdrug

• a majority of successful covalent drugs were discovered in biological assays by chance and covalent mechanism of action was established later onchance and covalent mechanism of action was established later on

• recently, a concept of TARGETED COVALENT INHIBITORS (TCI) was developedp

TTargetedargeted covalent inhibitors (TCI)covalent inhibitors (TCI)TTargetedargeted covalent inhibitors (TCI)covalent inhibitors (TCI)BTK – Bruton’s tyrosine kinase, a target to treat B-cell malignanciesDiscovered in 1993.The only approved drug (13/11/2013) –Ibrutinib (Johnson & Johnson, marketed by AbbVie):

Hinge region

BTK IC50 = 5 nM

Hydrophobicpocket

First designed at Celera Genomics in 2007 Cys481

HS

IIbrutinibbrutinib selectivityselectivityIIbrutinibbrutinib selectivityselectivity

IIbrutinibbrutinib synthesissynthesisIIbrutinibbrutinib synthesissynthesis

AAcalabrutinibcalabrutinib –– next generation next generation BTK inhibitorBTK inhibitor

Phase III for chronic lymphocytic leukemia: > 95% reponse! Developed by Acerta Pharma

TTCIs vs. reversibleCIs vs. reversible (classical) (classical) inhibitorsinhibitors

Ki = koff / kon

Ki = koff / kon

AAdvantagesdvantages of covalent inhibitionof covalent inhibitionAAdvantagesdvantages of covalent inhibitionof covalent inhibition

Particularly difficult targets:

• protein-protein interactions (PPI) subject of a standalone lecture next semester!• kinase ATP binding site (due to high intracellular levels of ATP)

COVALENT MODIFICATION OF A BIOLOGICAL TARGET OFFERS OPPORTUNITY OF ESTABLISHING NON-EQUILIBRIUM BINDING KINETICS!

Covalent vs. reversible inhibitorsCovalent vs. reversible inhibitorsCovalent vs. reversible inhibitorsCovalent vs. reversible inhibitors Extremely useful graphs

Therapeutic window

Barf, T. & Kaptein, A. J. Med. Chem. 2012, 55, 6243 (posted in USEFUL)

Reactivity of TCIsReactivity of TCIsReactivity of TCIsReactivity of TCIs

‘warheads’ of TCIs are weakly electrophilic to non-selectively modify other biomolecules (off-targets)

this makes TCIs very different from reactive intermediates

Sulfotransferases(Liver, kidney, intestine)

DDesignesign and optimization of TCIsand optimization of TCIsDDesignesign and optimization of TCIsand optimization of TCIs1. Bioinformatic analysis to identify nucleophilic amino acid (e. g., kinase

i bi i f i )cysteinome bioinformatics)• Near functionally relevant binding site• More or less unique to that protein

2. Reversible inhibitor identified with a known binding mode (X-ray!)

3 Computer aided design to properly position the reactive functionality 3. Computer-aided design to properly position the reactive functionality

4. Iterative synthesis and testing

5. Counter-screen on closely related kinases

Afatinib(Boehringer-Ingelheim)

Inhibitor of EGFR, approved for NSCLC

RReversible covalent eversible covalent inhibitors (RCI)inhibitors (RCI)RReversible covalent eversible covalent inhibitors (RCI)inhibitors (RCI)

Reversible

RCEI di i f i f h h f i d d i RCEI dissociate from protein faster than the rate of its degradation

Similar potency/selectivity benefits as for true covalent inhibitors

minimi ed r d cti n f l n li ed m dified r teins ( imm ne res nse) minimized production of long-lived modified proteins ( immune response)

perfect example – serine hydrolases and proteases (see WARHEADS in

previous lectures)previous lectures)

SSerineerine proteaseprotease mechanismmechanism

WWhyhy are these RCIs of serine proteases?are these RCIs of serine proteases?yy pp

WITHDRAWNWITHDRAWN WITHDRAWNWITHDRAWN

T l iTelaprevir(Approved 2011,

HCV protease inhibitor, Vertex Pharma)

Boceprevir(Approved in 2011,

HCV protease inhibitor, S h Pl h)Vertex Pharma) Schering-Plough)

Telaprevir: $189,000 per treatment course Boceprevir: ~$200,000 per treatment course Both agents were voluntarily withdrawn ‘due to overwhelming superiority of newer direct-acting antiviral agents, such as ledipasvir/sofosbuvir.

LyricalLyrical digression: Gileaddigression: Gilead SciencesSciencesyy gg

Ledipasvir/sofosbuvir (trade name Harvoni)

HCV replication

phosphoprotein

Nucleotide inhibitor of viral RNA phosphoprotein

NS5A inhibitorviral RNA

polymerase

a.k.a. Sovaldi

• Harvoni: $90,000 per treatment course• >95% cure rates for genotype 1 HCV

LLyricalyrical digression: Approved NS5Adigression: Approved NS5A inhibitorsinhibitors

Daclatasvir(Bristol-Myers-Squibb)\

Elbasvir(Merck)

Ombitasvir(AbbVie)

+ >10 other advanced compounds in development ll d f HCV all approved for HCV

BBackack to RCEI:to RCEI: FAAH inhibitorsFAAH inhibitorsBBackack to RCEI:to RCEI: FAAH inhibitorsFAAH inhibitorsEndocannabinoid system 2-AG = 2-arachidoyl glycerol

AEA = arachidoyl ethanolaminey AEA = arachidoyl ethanolamine(anadamide)MGL = monoglycerol lipaseFAAH = fatty acid amide hydrolase(serine protease)

PainMood

(serine protease)

Appetite

VVeryery simple simple ketoneketone FAAH inhibitorsFAAH inhibitors(RCEI type)(RCEI type)

FAAH IC50 = 4.7 nM FAAH IC50 = 22 MH+

N

O

ON

N

O

ON

OHOH

Ser241What about substituent effects?

H N ON

O

ON

H

Ser241

N

OR

O

SerR = COOMe, CF3: IC50 = 400-800 pM!

• > 300-fold selectivity over other Ser hydrolases• increses anandamide levels in vivo• potent analgesic activity in pain models

CCovalentovalent modification ofmodification of Serine by Serine by carbamoylationcarbamoylation

O

N

F3C

O

N NH

NN

NN

NN O

NMe2

hFAAH kinact/Ki 40,300hFAAH IC50 = 7.2 nM

hMGL IC50 = 54 nMhFAAH IC50 = 37 nM

LY 2183240

OR2 O

N NR

R1

HO S

Ser O N

OR1

R2

Ser

Other representative examples of reactive Other representative examples of reactive groups employed in TCI designgroups employed in TCI design

HO HNO

OO

Hinge region

NBr

GSK3 199 Cys

SH

R = HGSK3

(glycogen synthase kinase 3)IC50 = 5 nM

Cathepsin B (cystein protease, inflammation target):

IC50 = 13 nM

Carfilzomib (approved)Kinact/Ki = 34,000h20S proteasome 1h IC50 = 6 nM

LLyricalyrical digression: digression: proteosomeproteosome as a as a drug target (todrug target (to chase latest lecture)chase latest lecture)

•Protein complex in cells (often viewed as organelle)

•Responsible for degradation of proteinsRegulatory11S

• in particular, in various cancers, proteasome

degrades proteins responsible for apoptosis

(programmed cell death)

• therefore, proteasome (catalytic 20S subunit) was Catalytic considered a drug target for cancerCatalytic20S

T iti

Regulatory

Transition state analog

Regulatory11S

Bortozomib (Velcade®)First approved (2003) proteasome inhibitor

By Millennium Pharmaceuticals(for multiple myeloma)

RRecallingecalling: : omeprazoleomeprazole –– covalent covalent m difier f PROTONm difier f PROTON PUMPPUMPmodifier of PROTONmodifier of PROTON PUMPPUMP

Omeprazol

Lansoprazol

Pantoprazole

Esomeprazol These are inhibitors of gastric phydrogen potassium ATPase,

also known as H+/K+ ATPase, an enzyme whose purpose is to

d f h hDexlansoprasol acidify the stomach.

Importance of the Importance of the pKapKa for for activation of activation of omeprazoleomeprazole

WWhat happens next…hat happens next…pppp

Cys813 Cys813

C 813Cys813

CClopidogrellopidogrel ((PlavixPlavix) ) –– bestbest--selling selling antigoagulantantigoagulant drugdrug

Prevents platelet (thrombocyte) aggregation and is used to treat blood clotting Precribed to patients at risk of heart attack and strokeActs by blocking P2Y12 receptor on platelet

fsurface 2010 global sales >$9 bln! Only works in people with ‘good’ CYP 2C19 metabolism!metabolism! is a prodrug

HHowow does does clopidogrelclopidogrel work?work?HHowow does does clopidogrelclopidogrel work?work?

CYP 2C19

Only one stereoisomer is reactiveThe reactive thiol quickly forms a disulfide bond with P2Y12 Cys97

Pras rel (Effient Daiichi Sank ) eas ersi n f r r CYP2C19 metab li ers: Prasugrel (Effient, Daiichi Sankyo) – easy version for poor CYP2C19 metabolizers:

Metabolized by i i intestine esterases

FFinallyinally,, the aspirin storythe aspirin story

OH

O

OH

Oprodrug

OHCOX inhibitory

Too acidic, can causeStomach bleeding

OAcmetabolism?

However, aspirin itself is COX inhibitory, in vitro g

where metabolic activation is not

possible…

Unlike salicylic acid aspirin acts as a covalent modifier of COX-1/2 Ser530:Unlike salicylic acid, aspirin acts as a covalent modifier of COX-1/2 Ser :

CConclusionsonclusionsCConclusionsonclusions

covalent drugs are important

many of the known covalent drugs were discovered serendipitously

chances of discovering new covalent inhibitors via screening nowadays are nil

targeted covalent inhibitor (TCIs) are designed via bioinformatics and in silico

d li modeling

TCIs can act irreversibly by forming covalent bond with unique nucleophilic

residuesresidues

TCIs can act as reversible covalent inhibitors (RCI) which appears a better

alternative to covalent inhibitorsalternative to covalent inhibitors

QUESTIONS?QUESTIONS?QUESTIONS?QUESTIONS?