Covalent drugs
Litelature seminar
2014.10.4
Takushi Araya
1

Recently approved targeted-covalent drugs

ONH2NNN

NNIbrutinib

O

EGFR inhibitor(Anti-NSCLC)Firstly approved

BTK inhibitor(Anti-mantle cell lymphoma)
Secondly approved

Solca, F. et al. J Pharmacol. Exp. Ther. 2012, 342, 342.

Pan, Z. et al. ChemMedChem 2007, 2, 58

Contents
1. Introduction
1-1. Covalent drug
1-2. History
2. Afatinib : first approved targeted covalent inhibitor
2-1. How to make Afatinib?
2-2. Learn from afatinib---design, benefit and risk
3. Structure in reacting groups
3-1. Target reactions and residues
3-2. Kinetic analysis of covalent inhibitors
4. Future application§1-1. Introduction

What is Covalent drug?
Compound to use as drug (medical use).
It has (or is going to have) chemical reacting groupand binds to target covalently.

Target (E) Inhibitor (I)

E・I

E-I

Non-covalent drug3§1-1. Introduction

Concept≡Inhibitor

Specificity group

Reacting group

Nu

Target protein

too low

Target protein

moderatereactivity

too strong4§1-2. Introduction

Aspirin(Side effects)(therapeutic effects)

COX-1
Funk, C. D. et al. The FASEB Journal 1991, 5, 2304.
DeWitt, D. L. et al. J. Biol. Chem. 1990, 265, 5192.
COX-2
Hla, T. et al. Proc. Natl. Acad. Sci. USA 1992, 89, 7384.
Figure: http://blog.metaome.com/use-case-2/different-strokes-common-painkillers-and-their-mechanisms/5§1-2. Introduction

Aspirin also acetylate other cellular proteins
Rat liver cell culture

Recombinant p53

Review : Alfonso, L. F. et al. Mol. Med. Reports, 2009, 2, 533.
Original (inaccessible from UT): Alfonso, L. F. et al. Int. J. Oncol. 2009, 34, 597.

6§1-2. Introduction

Penicillin

Levine, B. B. et al. Immunology 1964, 7, 542., 1964, 36, 527.
Figure: http://www.antibioticslist.com/images/design/penicillin_img.gif 7§1-2. Introduction

Omeprazole

Side effect is minimized
because Omeprazole is converted
active intermediate in low pH.

Olbe, L. et al. Nat. Rev. Drug Discov. 2003, 2, 132.

8

§1-2. Introduction

History of covalent drugs
OOOOH

AspirinFirst synthetic and covalent drug
Mechanism of action wasdiscovered in 1970s.
1899

Blockbuster PPIs
1940s

1980s

First-approved
Targeted covalent drug
1990s

2013

Development ofTargeted covalent drugs
for EGFR familyFirst antibiotics

Until recently, covalent drugs were discovered by serendipity.
Their mechanisms of action were reported after a long time later.
Can we design “Targeted” covalent drug?
Singh, J. et al. Nat. Rev. Drug Discov. 2011, 10, 307.
Warner, T. D. et al. Proc. Natl. Acad. Sci. USA 2002, 99, 13371.

9

Contents
1. Introduction
1-1. Covalent drug
1-2. History
2. Afatinib : first approved targeted covalent inhibitor
2-1. How to make Afatinib?
2-2. Learn from afatinib---design, benefit and risk
3. Structure in reacting groups
3-1. Target reactions and residues
3-2. Kinetic analysis of covalent inhibitors
4. Future application10§2-1. Afatinib

Afatinib
FNO

HNHN

ONO

ClN

Afatinib

EGFR inhibitor(Anti-NSCLC)Firstly approved targeted covalent drug

Afatinib covalently binds to Cys797
Lately, Cys 773 was corrected to Cys797.
Same Cys, but different numbering method.
In this material, they are described as “Cys797”.
Solca, F. et al. J Pharmacol. Exp. Ther. 2012, 342, 342.

NSCLC : Non-Small-Cell Lung Cancer§2-1. Afatinib

EGFR mutation induces NSCLC

Proliferation

Metastasis
Survival

Kerr, D. J. et al. Nat. Rev. Clin. Oncol. 2009, 6, 499.

Angiogenesis12§2-1. Afatinib

1st generation EGFR inhibitors (non-covalent)
O

Me O
SONH

HNFClONNHN
MeO
MeOBr

Lapatinib (Tykerb )FNNO

HNNONPD153035
MeO

EGFR inhibitor
IC50=29 pM, Ki=5.2 pM

ClN

Gefitinib (Iressa )

HN
MeO
MeOO
ON
N

Erlotinib (Tarceva )Fry, D. W. et al. Science 1994, 265, 1093.

etc…

§2-1. Afatinib

X-ray structure of EGFR-Erlotinib complex

Cys797

Eigenbrot, C. et al. J. Biol. Chem. 2002, 277, 46265.

PDB code:1M17 14§2-1. Afatinib

Afatinib : 2nd generation EGFR inhibitor (covalent)
X-ray structure of Afatinib-EGFR complex

F

PDB code:4G5J
NO

HN

HNO

NO

ClN

AfatinibAfatinib covalently binds to Cys797
Also confirmed by MS/MSSolca, F. et al. J Pharmacol. Exp. Ther. 2012, 342, 342.

15

§2-1. Afatinib

Afatinib inhibition continues after washout
FNO

H
NHN

O

NO

Cl
N

BI37781
FNO

H
NHN

O

NO

Cl
N

afatinib

Solca, F. et al. J Pharmacol. Exp. Ther. 2012, 342, 342.

16

§2-1. Afatinib

Afatinib (BIBW2992) effect in vivo

NCI-H1975 cell

Human NSCLC cell lines (mutant proteins)(EGFR)(HER2)(EGFR)

Solca, F. et al. Oncogene 2008, 27, 4702.

(Kras)Median Tumor Volume [mm3]Mice xenograft model17§2-1. Afatinib

Phase III Study : Afatinib or chemotherapy

Direct comparisons between Afatinib and Gefitinib (or Erlotinib)
in clinical trial are ongoing (Lux-Lung 7 (or 8)).
Yang, J. C.-H. et al. J. Clin. Oncol. 2013, 31, 3327.

18

§2-1. Afatinib

Potential risk : alkylate Bmx kinase

Cys797 (EGFR) is highly conserved in TK family

No data about Afatinib, but there may be potential risk.
Non-conserved residue is desired for target.
Gray, N. S. et al. Bioorg. Med. Chem. Lett. 2008, 18, 5916.

19§2-2. Afatinib

Points to obtain targeted covalent inhibitor (1)1) Inhibitor known, Target known→target-based HTS ⇔ phenotypic screening
HN
MeO
MeO

Br
N

EGFR

NPD153035

2) High selectivity
minimized-interaction with off-target

HNNO

N
H

MeO
NHMe

Zarrinkar, P. P. et al. Nat. Biotech. 2008, 26, 127.

20

§2-2. Afatinib

Points to obtain targeted covalent inhibitor (2)3) Introduce reactive warhead to inhibitor
Target-inhibitor structure, interaction known
or homology modeling available
Computational
chemistry
Warhead of moderate reactivity, at appropriate positionFNO

HNHNONOClN

Bioinformatics

Afatinib

Target residue is preferred to non-conserved
using protein database (PDB, UniProt, genomatix etc…)

This step might be bottle neck.
21§2-2. Afatinib

Points to obtain targeted covalent inhibitor (3)4) Check affinity and covalent bond formation
by MS/MS, X-ray crystal, wash-out experiment etc…

5) Therapeutic effects vs adverse effectin vitro (enzyme), in vivo (cell culture), and clinical stage (patients)22§2-2. Afatinib

Characteristics (1)Median Tumor Volume [mm3]・Strong and prolonged pharmacodynamic activity
1) More complete target inhibition

2) Lower dose
Afatinib: 50 mg/day (Mw:486)
Lapatinib: 1250 mg/day (Mw:943)
Gefitinib: 250 mg/day (Mw:447)
Erlotinib: 150 mg/day (Mw:486)
23

§2-2. Afatinib

Characteristics (2)・Toxicity due to off-target・・・Current target is limited to severe (fatal) disease.
Does covalent type give more severe toxicity?
→Necessity of comprehensive study
Targeting non-conserved residue, using moderate reacting group.

・Can be Best-in-class, but can not be First-in-class(common issue in target-based drugs)

Distribution of new drugs (1999-2008)

Swinney, D. C. et al.
Nat. Rev. Drug Discov. 2011, 10, 507.

24§2-2. Afatinib

http://www.sulsa.ac.uk/research-facilities/uk-npsc/phenotypic-screening

Increase in knowledge

Drug discovery: Phenotipic and Target-orientede.g) Phenotipic screening using Zebrafish larvae in 96-well plate
http://www.ddw-online.com/chemistry/p102797-zebrafish:-a-versatile-in-vivo-model-for-drug-safety-assessmentfall-06.html

Swinney, D. C. Clin. Pharmacol. Ther. 2013, 93, 299.

25

Contents
1. Introduction
1-1. Covalent drug
1-2. History
2. Afatinib : first approved targeted covalent inhibitor
2-1. How to make Afatinib?
2-2. Learn from afatinib---design, benefit and risk
3. Structure in reacting groups
3-1. Target reactions and residues
3-2. Kinetic analysis of covalent inhibitors
4. Future application26

§3-1. Structure

Target reactions and residues
Cys-SHAlkylation-S-S (Se)- formation
Thio-Carbamate
formation*DNA-targeted drugs are excepted

Thr-OH
Ser-OH

Others(coenzyme)
Iminiumformation

Carbamate formation
OthersHemiketalformation

Michaeladdition

Lys-NH2

Pinner reaction

Other nucleophiles(Asp-COO-, Glu-COO-,
His-imidazole-H, Tyr-OH)

Esterification(In all cases, compound after phase ll are picked up and derivatives are omitted.)
Potashman, M. H. et al. J. Med. Chem. 2009, 52, 1231.; Barf, T. et al. J. Med. Chem. 2012, 55, 6243.27

§3-1. Structure

1. Michael addition

Neratinib
EGFR inhibitor
Phase lllFNO

HNHNONOCl
N

Afatinib
EGFR inhibitor(Anti-NSCLC)

Tsou, H.-R. et al. J. Med. Chem. 2005, 48, 1107.

Wortmannin
PI3K inhibitor(antidiabetic)

Gemcitaine (prodrug)
DNA synthetase inhibitor(Anti-cancer)
Stubbe, J. et al. J. Med. Chem. 1991, 34, 1879.

Furan-ring opening
Wymann, M. P. et al. Mol. Cell. Biol. 1996, 16, 1722.
28

§3-1. Structure

2. -S-S(Se)- formation

Omeprazole (prodrug)
Proton Pump Inhibitor
Olbe, L. et al. Nat. Rev. Drug Discov. 2003, 2, 132.

Clopidogrel (prodrug)
ADP receptor inhibitor(Anti-platelet aggregation)

Propylthiouracil
thyroxine 5’-deionase inhibitor(Anti-hyperthyroidism)

Herbert, J. M. et al. Thromb. Haemostasis 2000, 84, 891.

(Target amino acid is Seleno-cystein)Sies, H. et al. Org. Biomol. Chem. 2003, 1, 2848. 29

§3-1. Structure

3. Alkylation

Carfizomib
Proteasome inhibitor(anti-cancer)
Orlowski, R. Z. et al.
Blood. 2007, 110, 3281.

Fosfomycin
MurA inhibitor(antibiotic)

Epoxomicin

Macheroux, P. et al.
Eur. J. Biochem. 2004, 271, 2682.
(by X-ray structure)

Crews, C. M. et al. J. Am. Chem. Soc. 2000, 122, 1237.

4. Hemiketal formation (reversible)

Telaprevir
HCV NS3・4A inhibitor

Lin, C. et al. J. Biol. Chem. 2004, 279, 17508.

30§3-1. Structure

5. (Thio)Carbamate formation
Rivastigmine
AChE inhibitor(reversible)
Silman, I. et al. Biochemistry, 2002, 41, 3555.

Dislfiram
Aldehyde dehydrogenase inhibitor
Naylor, S. et al. Biochem. Pharmacol. 2001, 61, 537.

6. Pinner reaction (reversible)

Vildagliptin
DPP-4 inhibitor
Peters, J.-U. Curr. Top. Med. Chem. 2007, 7, 579.

31§3-1. Structure

7. Esterification (reversible)
HNO

NO

Aspirin
COX-2 inhibitor

SCO2H

Penicillin
Peptidoglycan synthetase inhibitor

Warfarin
V.K. reductase inhibitor(Anticoagulant)

Fasco, M. J. et al. J. Biol. Chem. 1982, 257, 4894.

Orlistat
Lipase inhibitor
Hadvary, P. et al. J. Biol. Chem. 1991, 266, 2021.

32§3-1. Structure

8. Iminium formation
PLPVigabatrin
GABA-AT inhibitor(anti-epilepsy)

John, R. A. et al. J. Biol. Chem. 1991, 266, 20056.
D-cycloserine

N O
HONH2Alanine racemase inhibitor
N O(antibiotic)

N OHOEnz

O

LysN

N

HN
H

OPO32NHO

OPO32N+OPO32N

OPO32N

PLP

Ringe, D. et al. Biochemistry 2003, 42, 5775.
33§3-1. Structure

9. Others(reversible)

Finasteride
5-α-reductase inhibitor(Anti-AGA)

Bull, H. G. et al. J. Am. Chem. Soc. 1996, 118, 2359.
Mn2+
KatG

N

H2N

O

MAO

MAO

F

MAO

F-・SET

H

N

N・+・

MAO
MAO

RN

S

N

NH2H

NH2O

O

O

NADH

NRNAD radicalONH

NO

SETO

O

NH2+FH-N
H

N

Isoniazid (prodrug)
InhA inhibitor
Anti-tuberculous+N

Selegiline
MAO-B inhibitor(Anti-Alzheimer)

FH・Mn2+
KatG

NN
R

N
R
NAD++・

N

N+

Mariano, P. S. et al. J. Am. Chem. Soc. 1998, 120, 5864-5872.

NR

O
NH2NO

Sacchettini, J. C. et al. Science, 1998, 279, 98.

Kinetic analysis of MA-covalent inhibitors§3-2. Structure

Murraya, B. W. et al.
Proc. Natl. Acad. Sci. USA 2014, 111, 173.

Kinetic analysis of MA-covalent inhibitors§3-2. Structure

Murraya, B. W. et al.
Proc. Natl. Acad. Sci. USA
2014, 111, 173.

1) Ki correlates to cellular IC50 (R2=0.89)
2) kinact correlates to cellular IC50 (R2=0.60 )3) Ki / kinact strongly correlates to cellular IC50 (R2=0.95)

Low KiHigh kinact
Low Ki / kinact 36

Kinetic analysis of MA-covalent inhibitors§3-2. Structure1) Low Ki

Reactivity(Toxicity)

Moderate
2) High kintact●●

3) Low Ki / kinact

●
●

●

●
●

□ WT, ● Mutant● Effective, ● DropAffinity
Murraya, B. W. et al. Proc. Natl. Acad. Sci. USA 2014, 111, 173.

Cl-1033 (Canertinib) drop (phase2)2 (PD168393), CL-387785 drop
Dacomitinib (Phase2)
Neratinib (FDA application pending)37

Contents
1. Introduction
1-1. Covalent drug
1-2. History
2. Afatinib : first approved targeted covalent inhibitor
2-1. How to make Afatinib?
2-2. Learn from afatinib---design, benefit and risk
3. Structure in reacting groups
3-1. Target reactions and residues
3-2. Kinetic analysis of covalent inhibitors4. Future application

38§4. Future

PPIs are important drug target cf. Literature Seminar, Shimizu, 2014

There are 375,000 PPIs (estimated), 32,000 PPIs (reported)
39

Mooney, E. M. et al. Genome Biology 2005, 6, R40.; Fu, H. et al. Trends Pharmacol. Sci. 2013, 34, 393.

§4. Future

PPIs + covalent inhibitor

Shallow, large binding surface (600-1300Å2)
Wilson, A. J. Chem. Soc. Rev. 2009, 38, 3289.

Nuweak interaction

strong interaction

Covalent modifying approach may increase potency.
May be a breakthrough of PPI inhibitor?
Way, J. C. Curr. Opin. Chem. Biol. 2000, 4, 40

40

Summary
Section 1・Historically, many covalent drugs give us lots of benefit, in spite of serendipity.
Their mechanisms of action were determined at later stage.
Section 2・Afatinib is first approved Targeted covalent inhibitor.
“Targeted covalent inhibitor can be designed by medicinal chemists”
・Targeted covalent inhibitor is irreversible, the strongest inhibitor class.
It may be a best-in-class method, although there are some limitations.
・Especially, potential off-target toxicity is remained, but there are few data.
Necessity to compare with conventional drugs. Currently under investigation.
Section 3・Covalent drugs can be classified into some target and reaction types
・Drug candidate should pursue the low Ki, moderate kinact and low Ki / kinact value.
Section 4
・Covalent modifying method will bring a new perspective into PPI inhibition.
41

That’s all,thank you for your kind attention!

Appendix

BTK relates to cancer

Hendriks, R. W. et al. Nat. Rev. Cancer 2014, 14, 219.

Appendix

PPIShin, J. M. et al. J. Am. Chem. Soc. 2004, 126, 7800.

Revision

In previous version (and in my presentation)
Wortomannin was Amidation type,but actually, Michael addition type.
There are no Amidation type.
Sorry to tell you mistakes.