1. The covalent targeting of cysteine residues in drug discovery and its application to the discovery of osimertinib (TagrissoTM)
Richard A. Ward
Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
BigChem Training School, Gothenburg
May 2019

2. Targeted covalent inhibition
A covalent inhibitor forms a reversible or irreversible bond to the target
Potential gains in:
Potency
Selectivity
Duration of action
Cysteine targeting:
Relatively rare and often poorly conserved
Nucleophilic
Two important steps:
Examples of covalent warheads: acrylamide, vinyl sulphonamides etc.
Popular within Oncology for targeting kinases, mutant kinases
Reduce side-effects
The has been an explosion of interest around covalent inhibitors in recent years

3. Targeted covalent inhibition strategies
Phenotypic screening of covalent inhibitors
Covalent fragment approach
Screening of reversible covalent libraries
Addition of covalent warhead to reversible lead
Addition of covalent warhead to reversible inhibitor
EARLY
ADDITION OF COVALENT GROUP
LATE

4. Designing covalent inhibitors: optimizing k2
The right cysteine
Are there cysteines in the binding pocket that can react with a warhead?
The right warhead
Too reactive – toxicity issues
Not reactive enough – potency issues
The right geometry
Can the warhead get close enough to the target cysteine for reaction to take place?
Assess local environment of cysteine
Tune reactivity by varying substituents near warhead
Conformation of cysteine
Warhead vector
H2O? C S S S H C C S
B-?
Cys
Cys- S C C

5. Could cysteine pKa be an indicator of ease of activation?
Cysteine activation
Thiolate form of cysteine is a strong nucleophile
Deprotonation occurs before reaction with warhead
Cysteines in proteins generally less easily activated than free cysteine in solution
Thiolate form favoured in polar environment
Activation can be assisted by solvent or by interaction with neighbouring residues
e.g. deprotonation by histidine in cysteine proteases
Acrylamide warhead
D800
C797
C797 in EGFR:
Solvent exposed
Nearby aspartic acid
Could cysteine pKa be an indicator of ease of activation?

6. pKa analysis of cysteines in EGFR
pKa of cysteine sensitive to change in local environment
More solvent exposure -> lower pKa
Large fluctuations in pKa
pKa of cysteines – calculated1 over all PDB EGFR X-ray structures
C797
C818
C950
C939
C781
C775
C781
C775
C797
C818
C950
C939
pKa of cysteines – calculated* from MD simulation
Conformational sampling of cysteines is important
C797
C818
C950
C939
C781
C775
Reported experimental pKa of 5.5
1 Finite difference Poisson-Boltzmann/Debye-Huckel (FD/DH) method – Jim Warwicker (Manchester) 2 Truong et al. Cell Chemical Biology (2016), 23,

7. Predicted pKa alone can be challenging to use prospectively

8. Designing covalent inhibitors: optimizing k2
The right cysteine
Are there cysteines in the binding pocket that can react with a warhead?
The right warhead
Too reactive – toxicity issues
Not reactive enough – potency issues
The right geometry
Can the warhead get close enough to the target cysteine for reaction to take place?
Assess local environment of cysteine
Tune reactivity by varying substituents near warhead
Conformation of cysteine
Warhead vector
H2O? C S S S H C C S
B-?
Cys
Cys- S C C

9. Glutathione half-life [GSH t1/2]
Half-life to glutathione conjugation is an indicator of relative warhead reactivity
Similar reactivity towards Michael acceptors as cysteine
Useful assay, requires compound to be synthesized
SAR from Hammett parameters useful for rational modification of reactivity of model aromatic acrylamides1
Cannot be used for saturated compounds, e.g.
GSH t1/2 (mins)
R2=0.92
σind parameter
ARS-8532
Can we predict warhead reactivity towards GSH for a diverse range of compounds?
Ward et al., J. Med. Chem. 2013, 56,
Rosen et al., Science 2016, 351,

10. QM-based predictions of GSH t1/2
Energy barrier ΔE‡ to reaction with model compound (MeSH) previously shown to correlate with GSH t1/2 for model compounds1
Approach employed against our test set (a)
Correlation also observed for ΔE against GSH t1/2 (b)
Quicker to calculate than ΔE‡
Can be more routinely applied to larger molecules
ΔE‡ ΔE
Accurate but slow and resource intensive
Flanagan et al., J. Med. Chem. 2014, 57,
Ward et al., J. Med. Chem. 2013, 56,

11. Relationship between GSH t1/2 and pKa of parent base
Acrylamide reactivity can be increased by making terminal alkene carbon more electrophilic
Internal compound collection searched for matched pairs of acrylamides and amine precursors
Calculated (and experimental) pKa of parent base in good agreement with experiment in majority of cases
Can predict GSH t1/2 for saturated acrylamides – ‘warhead corrections’ can be applied
ibrutinib
Can tune warhead reactivity rationally using same techniques as for varying pKa
Lonsdale, Ward. JCIM, 2017, 57 (12), 3124.

12. Designing covalent inhibitors: optimizing k2
The right cysteine
Are there cysteines in the binding pocket that can react with a warhead?
The right warhead
Too reactive – toxicity issues
Not reactive enough – potency issues
The right geometry
Can the warhead get close enough to the target cysteine for reaction to take place?
Assess local environment of cysteine
Tune reactivity by varying substituents near warhead
Conformation of cysteine
Warhead vector
H2O? C S S S H C C S
B-?
Cys
Cys- S C C

13. The right geometry: cysteine sidechain conformation
Example project with difficult to target cysteine on edge of binding pocket
Slow covalent binding observed
MD simulations reveal two distinct conformations of targeted cysteine
Cysteine spends the majority of time in an inaccessible conformation
Dihedral angle (degrees)
out in
Time (ps)
Ligand
% in accessible conformation
None
9.7 1
5.2 2
0.5
Inaccessible conformation
Accessible conformation
Residue conformation may help explain lack of residue targetability

14. The discovery of osimertinib (TagrissoTM) The rational design of an irreversible EGFR T790M mutant specific inhibitor from project start to FDA approval in 6 years

15. EGFR mutant-selective inhibitor hypothesis (2009)
Exploitation of methionine gatekeeper residue to leverage a mutant-selective profile
Desired profile with activity in DM (and AM) and selectivity over WT
Set of EGFR inhibitors screened using a biochemical assay (DM & WT)
Selection of compounds from AZ Projects targeting methionine gatekeepers1
EGFR DM pIC50
EGFR WT pIC50
‘Methionine-GK’ Selection
Irreversible AQZ
Reversible AQZ
Generation
Activating Mutants (AM)
Double Mutant (DM)
Wild-Type
(WT)
1st
Active
Inactive
2nd
3rd
Margin
AM = L858R or Exon 19 Del (EGFR+)
DM = L858R/T790M or Exon 19 Del/T790M

16. An EGFR Mutant-Selective Template
Mutant-selective examples identified based on a pyrimidine scaffold
R1=OMe
PDB Code: 4LI5
EGFR-WT Structure 2
SBDD
Cellular activity observed in DM and AM
Compound DM
(µM, enzyme) WT
DM/WT Selectivity
(µM, cell) 1
0.009
0.79 88
0.77 1 1
Large enzyme-cell drop-off observed
Binding mode of Compound 1 modelled into EGFR-T790M structure
Molecular modelling deployed to target Cys-797 by covalent mechanism
1. Ward, R. A et al, J. Med. Chem. 2013, 56, 7025.

17. Piperazine modifications: selected examples
Cell potent, wild-type selective inhibitors with improved LLE and excellent oral exposure 3 4 5
Compound DM
(µM, cell) AM WT
DM/WT Selectivity
Log D7.4
hERG IC50 (µM)
IGF IC50 (µM) 3
0.005
0.002
0.363 72
3.1
5.6
0.007 4
0.0006
0.228
380
2.8
6.7
0.006 5
0.003
0.02
0.823
274
3.3
4.0
0.026 3 4 5 6
Excellent double mutant and activating mutant efficacy with margin over WT
Very low oral dose required (10 mpk)
Examples had issues with off-target activities (insulin receptor and/or hERG)
Reversible and irreversible off-targets both important for optimisation
Finlay et al. AACR-EORTC-NCI Molecular Targets & Cancer Therapeutics Conference, October 2013.

18. 5-Position SAR leading to the discovery of ‘AZD9291’
Combinations explored with indole head groups and pyrimidine 5-groups
Removal of 5-Cl beneficial for properties and off-target activities
Possible to achieve good DM potency and WT selectivity
AZD9291 selected due to target potency/selectivity, mouse exposure, hERG potency, IGF profile and in vivo tolerability
5-Position SAR leading to the discovery of AZD9291
EGFR DM (Cell) Potency (log µM)
IGF1R Enzyme Potency (log µM)
hERG Ionworks (log µM)
R2=Cl R2=H
R2=Cl R2=H
R2=Cl R2=H
Compound R1 R2 DM
(µM, cell) AM WT
DM/WT Selectivity
Log D7.4
IGF IC50 (µM)
6 (AZD9291) Me H
0.015
0.017
0.48 32
3.4
2.9
Divergent SAR identified between primary target(s) and off-targets

19. The profile of AZD9291 (Osimertinib/TagrissoTM)
AZD9291 delivers profound regression in H1975 (DM) and PC9 (AM) xenograft models
AZD9291
Cellular Activity (DM / AM / WT)
(DM vs WT Selectivity)
0.015 / / 0.48 mM
(32-fold)
IGF1R (biochemical)
2.9 mM
hERG IC50 (Ionworks)
16 mM
Aq. Solubility (mesylate salt, pH=6.8, mg/mL)
>490
Predicted dose to Man
17-90mg (PC9) 7-38mg (H1975)
Predicted maximum absorbable dose (MAD)
207 mg
Finlay & Cross et al, AACR-EORTC-NCI Molecular Targets & Cancer Therapeutics Conference, October 2013.

20. The clinical phases of osimertinib/TAGRISSOTM
Rapid clinical development plan in T790M mutation positive non-small cell lung cancer (NSCLC)
Summer 2013: Clinical activity observed at the first dose
March 2013: First time in man (FTIM) achieved for TAGRISSOTM
(AURA, NCT )
Adverse events consistent with EGFR pharmacology
Prof M Ranson at The European Cancer Congress 2013; European Journal of Cancer Vol 49, supplement 3: LBA 33
2013-4: Emerging evidence of activity in brain metastases
Brain MRI
A) Baseline Aug 2013 B) Oct 2013
Kim et al. European Society for Medical Oncology 2014 Congress,
26–30 September, Madrid, Spain; Abstract #6771
Anti-tumour study
Nov 2015: TAGRISSOTM receives U.S. FDA approval as 2L therapy
April 2014: Breakthrough designation granted by the U.S. FDA
April 2018: TAGRISSOTM receives U.S. FDA approval as 1L therapy
N Engl J Med 2017; 376: , 2017

21. Summary and Acknowledgements
Cysteine
Are there cysteines in the binding pocket that can react with a warhead?
Warhead
Too reactive – toxicity issues
Not reactive enough – potency issues
Geometry
Can the warhead get close enough to the target cysteine for reaction to take place?
Use MD and pKa calculations to assess local environment of cysteine
Solvent accessibility
pKa
GSH t1/2 predictions – relative reactivities
Hammett parameters (aromatics)
QM calculations of ΔE and ΔE‡
pKa of parent base
Matched molecular pairs analysis for warhead swap
Use MD simulations to explore conformational flexibility of cysteines close to binding pocket
Thanks to Richard Lonsdale, numerous AZ colleagues and collaborators and the AZ Postdoc Programme for the cysteine targeting work presented
I would like to acknowledge all our AstraZeneca colleagues involved in the discovery of osimertinib, of which there are many
In addition to our collaborators, clinicians and above all the patients and their families