lecture 6 - covalent drugs - krasavin group · • more or less unique to that protein 2....
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‘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
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PPictureictureof the of the daydaydayday
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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
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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
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IIbrutinibbrutinib selectivityselectivityIIbrutinibbrutinib selectivityselectivity
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IIbrutinibbrutinib synthesissynthesisIIbrutinibbrutinib synthesissynthesis
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AAcalabrutinibcalabrutinib –– next generation next generation BTK inhibitorBTK inhibitor
Phase III for chronic lymphocytic leukemia: > 95% reponse! Developed by Acerta Pharma
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TTCIs vs. reversibleCIs vs. reversible (classical) (classical) inhibitorsinhibitors
Ki = koff / kon
Ki = koff / kon
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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!
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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)
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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)
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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
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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)
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SSerineerine proteaseprotease mechanismmechanism
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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.
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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
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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
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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
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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
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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
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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
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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)
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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.
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Importance of the Importance of the pKapKa for for activation of activation of omeprazoleomeprazole
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WWhat happens next…hat happens next…pppp
Cys813 Cys813
C 813Cys813
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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
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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
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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 :
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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
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QUESTIONS?QUESTIONS?QUESTIONS?QUESTIONS?