Targeting S-Adenosylmethionine biosynthesis with a novel … · 2017-06-20 · Casey L. Quinlan*1,...

Targeting S-Adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

Supplementary information

Casey L. Quinlan*1, Stephen E. Kaiser*2, Ben Bolaños2, Dawn Nowlin1, Rita Grantner1, Shannon Karlicek-Bryant1, Jun Li Feng2, Stephen Jenkinson3, Kevin Freeman-Cook2, Stephen G. Dann1, Xiaoli Wang1, Peter A. Wells1, Valeria R. Fantin4, Al E. Stewart2, and Stephan K. Grant1

1 Pfizer; Oncology R&D; 10777 Science Center Dr. San Diego, CA; [email protected] Pfizer; Oncology Medicinal Chemistry; San Diego, CA3Pfizer; Drug Safety and Pharmacology; San Diego, CA4ORIC Pharmaceuticals; South San Francisco, CA*denotes joint first authorship

Nature Chemical Biology: doi:10.1038/nchembio.2384

2

Item Page

Supplementary Figure 1. Mat2A screening campaign. 3

Supplementary Figure 2. Analysis of PF-9366 off target inhibition. 4

Supplementary Figure 3. Analysis of Mat2A structure. 5

Supplementary Figure 4. Mat2A solution oligomerization state and crystallographically observed waters.

6

Supplementary Figure 5. Comparison of Mat1A with Mat2A. 7

Supplementary Figure 6. ITC and biochemical analysis of effects of titration of Mat2A with Mat2B. 8

Supplementary Figure 7. Mat2A and Mat2B quantification by western blotting 9

Supplementary Figure 8. SAM synthesis and proliferation in H520 cells 10

Supplementary Figure 9. Unprocessed immunoblot images with corresponding protein size markers 11

Supplementary Figure 10. SDS-PAGE and mass spectrometry characterization of Mat proteins 12

Supplementary Table 1. Small molecule screening data 13

Supplementary Table 2. Off-target mini-profiling panel 14

Supplementary Table 3. Mat2A kinetic parameters for effect of Mat2B and PF-9366 15

Supplementary Table 4. Data collection and refinement statistics (molecular replacement) 16

Supplementary results


3

b

a

Inhibition > 30% (20 µM)Mat2A – Mat2B

Hits: 0.05% Mat2A

Hits: 0.33%

IC50determination

Followup orthogonal assays

Kinase-targeted library 27,000

Methyltransferase-targeted library15,000

100

90

80

70

60

50

40

30Mat

2A %

inhi

bitio

n in

initi

al s

cree

n(N

orm

aliz

ed to

con

trol)

Mat2A pLog IC50 in followup screen≤4.0 4.5 5.0 5.5 6.0 6.5 7.0

Kinase-targeted libraryMethyltransferase-targeted library

PF-9366

110N

N N

N

Cl

Supplementary Figure 1. Mat2A screening campaign (a) The primary (% inhibition) and secondary (IC50) screens were executed using the luminescent Kinase-Glo platform from Promega that detects ATP depletion. Mat2A alone or in complex with Mat2B was screened against two targeted libraries, the kinase-targeted library and methyltransferase-targeted library containing 27K and 15K compounds, respectively. IC50s were determined for those compounds that met the criteria of >30% inhibition at the 20 µM test dose. Screen details in materials and methods. (b) The Mat2A hit profile plotted to show the relationship between compound percent inhibition in the primary screen and the positive log of the molar IC50 in the secondary Kinase-Glo screen. Some % inhibition values are >100% as a result of normalization to the control inhibitor, AMP-PNP (100 µM). The libraries are denoted by shape; square- Kinase targeted library, circle- Methyltransferase targeted library. Each unique chemical series is depicted in a different color. The chemical series containing PF-9366 is represented by pink. Many of the compounds were not advanced after further investigation in orthogonal screens. Orthogonal assays included kinetic assays for SAM detection, differential scanning fluorimetry, compound aggregation assays, and isothermal calorimetry.


4

a

b

3 00

4 00AB

L1A

KT1

(PK

Bal

pha)

AU

RK

A(A

uror

aA

)B

TKC

AM

K2A

(CaM

KII

alph

a)C

DK

2/cy

clin

AC

HEK

1(C

HK

1)C

HEK

2(C

HK

2)C

SNK

1A1

(CK

1al

pha

1)C

SNK

2A2

(CK

2al

pha

2)EG

FR(E

rbB

1)EP

HA

2FG

FR1

GSK

3B(G

SK3

beta

)IN

SRIR

AK

4JA

K3

KD

R(V

EGFR

2)LC

KM

AP4

K4

(HG

K)

MA

PK1

(ER

K2)

MA

PK14

(p38

alph

a)M

APK

APK

2M

AR

K1

(MA

RK

)M

ET(c

Met

)M

ST4

MYL

K2

(skM

LCK

)N

TRK

1(T

RK

A)

PAK

4PD

K1

PIM

2PR

KA

CA

(PK

A)

PRK

CB

2(P

KC

beta

II)R

OC

K1

SGK

(SG

K1)

SRC

STK

3(M

ST2)

TAO

K2

(TA

O1)

TEK

(Tie

2)

-10

0

10

20

30

40

6080

100

%in

hibi

tion

ofki

nase

activ

ityat

10µM

PF-9

366

ATP at KM(app)

ATP at 1 mM

0.001 0.01 0.1 1 10 1000

20

40

60

compound (µM)

Mat

1A-g

ener

ated

SAM

(nm

ol)

PF-9366PF-9366+Mat2B

AMP-PNP

Kinase selectivity screen of PF-9366

PF-9366 inhibition profile of Mat1A

Supplementary Figure 2. (a) Percent inhibition of kinase activity by PF-9366. 10 µM PF-9366 was profiled against 39 kinases at two ATP concentrations (KM app and 1 mM) in the Thermo-Fisher Z´-LYTE™ biochemical assay. (b) Inhibition of Mat1A by PF-9366. A dose response of PF-9366 inhibition of Mat1A (50 nM) SAM synthesis (alone or in the presence of 50 nM Mat2B). AMP-PNP, a non-hydrolyzable ATP analogue that behaves as an ATP-competitive inhibitor of Mat1A, was used as a control to determine maximum inhibition.


5

Products

Substrates(Modeled)

d e

b c

Leu315

Leu315

Phe333 Tyr

335Arg313

Trp274

Phe20

Phe18

Glu342

Phe139

Gln317

Ser331

Ala276

Leu19

Thr17

Gln190

Val195

Phe333

Tyr335

Trp274

Phe20

Phe18

Glu342

Phe139

Gln317

Ser331

Ala276

Leu19

Thr17

Gln190

His323 Phe

322

Val321

Val195

PF-9366(1)

Mat2A Mat2A

Substrates(Modeled)

Substrates(Modeled) Products

Mat2B

Mat2A(2)

Mat2A(1)

Mat2B

PDB: 4NDN

PDB: 4NDN

PF-9366(2)

a

Shafquat et al (2003)human Mat2A

2P02.pdbI222

AU = monomer

Gonzalez et al (2000)rat Mat2A1QM4.pdb

P4122AU = dimer

This studyhuman Mat2A

xxxx.pdbC2

AU = tetramer

Supplementary Figure 3. (a) To date, crystal structures of Mat enzymes - including those shown here - from different organisms and in different crystal forms are observed in similar tetrameric configurations. (b) PF-9366 interactions with one monomer of the Mat2A dimer. Methionine (green carbon atoms) and Mg-AMP-PNP (yellow carbon atoms, Mg atoms grey spheres) were modeled in the Mat2A active site from PDB 1P7L. (c) Mat2B C-terminal tail interactions with one monomer of the Mat2A dimer from PDB 4NDN with substrate SAM (orange carbon atoms) and PPNP. (d) Mat2B interactions with the Mat2A dimer. (e) Superimposed Mat2A monomers bound to Mat2B and PF-9366 with coloring and representations as in panels c and d.

Komoto et al (2004)E. coli Mat1RG9.pdb

P212121AU = tetramer

Murray et al (2016)human Mat2A

5A1I.pdbI222

AU = monomer


6

Molar Mass vs. timeLJIC-2155 2 mg/ml LJIC-2155 4 mg/ml LJIC-2155 8 mg/ml

time (min)4.0 6.0 8.0 10.0 12.0 14.0

Mol

ar M

ass

(g/m

ol)

41.0x10

51.0x10

UV

Molar Mass vs. timeLJEC-1718 2 mg/ml LJEC1718 4 mg/ml LJEC1718 8 mg/ml

time (min)4.0 6.0 8.0 10.0 12.0 14.0

Mol

ar M

ass

(g/m

ol)

41.0x10

51.0x10

UV

~55 kD

~150 kD

~55 kD

~150 kD

Supplementary Figure 4. (a) Multi-angle laser light scattering (MALS) analysis of Mat2A oligomerization state. Human Mat2A expressed in and purified from baculovirus infected insect cells (LJIC-2155) and E. coli (LJEC-1718) cells were injected to a Phenomenex S3000 column equilibrated with 50 mM Tris 8, 100 mM NaCl, 100 mM KCl, 1 mM TCEP at 3 concentrations each (2, 4, 8 mg/ml) and analyzed by in line light scattering, refractive index, and UV absorbance detectors enabling determination of the molecular weight distribution across the eluting peaks. The observed molecular weight distribution and peak shifts suggest exchange between monomer, dimer, and tetramer with some bias toward dimer - as no discreet populations of tetramer (175 kD) or monomer (43.8 kD) were observed. (b,c) Water molecules at Mat2A dimer interface. (b) View of one monomer of Mat2A dimer with focus on water molecules (blue spheres) at dimer interface, allosteric site and active sites. (c) Side view of Mat2A dimer with focus on water molecules (blue spheres) at dimer interface, allosteric site and active sites.

a

b c


7

Supplementary Figure 5. Comparison of Mat1A with Mat2A. Mat1A and Mat2A are 84% identical and 94% similar over 395 residues. (a) Overlay of human Mat2A structure bound to PF-9366 with human Mat1A structure 2OBV.pdb with one monomer of the Mat2A or Mat1A dimer shown for clarity. The Mat2A surface is shown. PF-9366 molecules are shown with cyan carbons. Proteins are shown in stick representation with amino acids that are identical in Mat2A and Mat1A colored grey, amino acids that are different and similar are colored with pink carbon atoms (Mat2A) or with green carbon atoms (Mat1A): 1) hMat2A Arg192 (hMat1A Asn192) is separated from PF-9366(2) by Gln190. These side chains are > 9 Å from PF-9366 and point out of the allosteric site. 2) hMat2A Thr17 (hMat1A Val17) side chain faces away from the allosteric site. 3) hMat2A Leu19 (hMat1A Met19) points away from the allosteric site. 4) hMat2a His334 (hMat1a Thr334) is highlighted with a red circle and is 7 Å away from bound PF-9366 with Glu342 between hMat2A His334 and PF-9366. (b) Zoomed view of PF-9366(1) with hMat2a His334 and hMat1a Thr334 highlighted by a red circle. Coloring is as in panel A except that Phe333 (which flips to a new rotamer with PF-9366 binding) and Glu342 are colored with pink (Mat2A) or green (Mat1A) carbon atoms. The hMat2A Glu342 carboxyl group is 2.6 Å from PF-9366, 2.8 Å from the backbone amide of His334 and 3.5 Å from the side chain NH of His334. The presence of Thr334 in Mat1A may subtly alter hydrogen bonding networks supporting PF-9366 interaction.

a b

Phe333

Glu342

His334

PF-9366(1) PF-9366(2)1

2

3

44


8

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

-30.00

-25.00

-20.00

-15.00

-10.00

-5.00

0.00

-0.15

-0.10

-0.05

0.00

0 10 20 30 40 50 60 70 80 90 100

a1501303_RAW a1501303_BASE

35mM MAT2B into 10mM MAT2ATime (min)

µcal

/sec

a1501303_NDH a1501303_Fit

Molar Ratio

kcal

mol

-1 o

f inj

ecta

nt

N = 0.46Kd = 4.5 nM

a

0.001 0.01 0.1 1 10 1000

10

20

30

40

50

PF-9366 (mM)

nmol

SAM

100 nM MAT2B50 nM MAT2B25 nM MAT2B12.5 nM MAT2B6.25 nM MAT2B3.125 nM MAT2Bno MAT2B

b

Mat2B binds to Mat2A in solution

Supplementary Figure 6. (a) Thermogram of 35 µM Mat2B titrated into 10 µM Mat2A to determine binding affinity and stoichiometry. The raw data are presented on top and the integrated peak areas are shown and fitted below. Kd = 4.5 ± 2 nM, N = 0.46 ± 0.01. Thermodynamic parameters: enthalpy (ΔH = -29 kcal mol-1), entropy (-TΔS = -17.5 kcal mol-1), and Gibbs energy (ΔG = -11.3 kcal mol-1) Representative of 2 independent experiments. (b) Data from Figure 1d before normalization to % activity. 12.5 nM Mat2A dimer titrated with increasing concentrations of Mat2B monomer. Increasing concentrations of Mat2B decrease the activity of Mat2A (saturates at 50% remaining activity) and decrease the efficacy of PF-9366 in a dose-dependent manner.

Both Mat2B and PF-9366 inhibit Mat2A activity


9

Supplementary Figure 7. Mat2A and Mat2B protein levels were quantified in cell lysates by protein standard curves run on every Western blot. Examples of experimental sets are shown here. Mat protein band intensity was normalized to Actin to account for small variations in protein loading and then further quantified using the relevant standard curve.

Mat standardcurve (ng)

Mat2A

Mat2B

Actin

cycloDMSO

60 30 15 7.5

PF-9366

10 5 2.5 1.25

a Huh-7 protein quantification


80 40 20 10

10 5 2.5 1.25

Mat2A

Mat2B

Actin

b H520-EV protein quantification


80 40 20 10

10 5 2.5 1.25

Mat2A

Mat2B

Actin

c H520-shMat2B protein quantification d Protein Standard Curves

0 20 40 600

100000

200000

300000

400000

500000

Mat2A (ng)

0 5 100

100000

200000

300000

LI-C

OR

fluo

resc

ence

sign

al(a

u)

Mat2B (ng)

LI-C

OR

fluo

resc

ence

sign

al(a

u)

cycloDMSO PF-9366

cycloDMSO PF-9366


10

0.0001 0.01 1 1000

200

400

600

dose (mM)

SAM

(fmol

/100

0ce

lls)

PF-9366 (EV)cycloleucine (EV)PF-9366 (shMat2B)cycloleucine (shMat2B)

0.0001 0.01 1 1000

25

50

75

100

dose (mM)

Inhi

bitio

nof

prol

ifera

tion

(%)

PF-9366 (EV)cycloleucine (EV)PF-9366 (shMat2B)cycloleucine (shMat2B)

A

3 00

4 00

B

IC50 (µM)

SAM levels (6 hrs)

SAM levels (72 hrs)

Proliferation (72 hrs)

H520 (EV)— PF-9366 1.2 5.2 15

H520 (shMat2B)—PF-9366 0.86 2.8 15

H520 (EV)— cycloleucine 5600 28000 27000

H520 (shMat2B)—cycloleucine 4988 15000 17000

C H520 IC50 table

Supplementary Figure 8. (a) SAM synthesis in H520 cells after 72 hours compound treatment. (b) The 72 hour antiproliferative effect of PF-9366 and cycloleucine. (c) Table comparing the IC50 values for inhibition of SAM synthesis at 6 and 72 hours, and inhibition of proliferation at 72 hours.

72 Hours: H520 inhibition of SAM synthesis 72 Hours: H520 inhibition of proliferation


11

Supplementary Figure 9. Unprocessed immunoblot images with corresponding protein size markers.

Fig 5b

1889862493828

Sup Fig 7a

18898

624938

Sup Fig 7b

18898

62493828

Sup Fig 7c

18898

62493828

α-Mat2A α-Mat2B α-actin


12

Supplementary Figure 10. SDS-PAGE and mass spectrometry analysis of purified proteins used in this study. For SDS-PAGE, 1, 5 and 20 mg protein was loaded.

98

6249

38

28

14

986249

38

28

14

98

6249

38

28

14

986249

38

28

14

Mat2ALJEC-1718

Mat2ALJIC-2155 Mat2B Mat1A

MWcalc = 43804.8 MWcalc = 43804.8 MWcalc = 37695.9 MWcalc = 43792.0


13

Supplementary Table 1. Small molecule screening data Category Parameter Description

Assay Type of assay In-vitro, biochemical Target MAT2A

Primary measurement Detection of ATP substrate depletion by coupled luminescent assay

Key reagents Mat2A and Mat2B proteins were cloned, expressed and purified in-house. See Methods section “Mat2A and Mat2B cloning, expression, and purification”. ATP, methionine, KinaseGlo Plus™ (Promega Corp., cat. #V3772)

Assay protocol See Methods section “High-throughput Screen of Mat2A”

Additional comments

Library Library size KTL, 27000; MTTL,15120 compounds

Library composition KTL: established kinase pharmacophores, ATP analogs, kinase-like inhibitors. MTTL: mimetics of AdoMet, S-adenosyl homocysteine, adenosine, analogs of known methyl transferase inhibitors

Source Pfizer internal collection, de-novo synthesis, commercial

Additional comments Libraries are curated and maintained by Global Steering Committees

Screen Format 384-well, black flat bottom plate

Concentration(s) tested 20uM, 2% DMSO Plate controls Controls on each plate (n=8): 2% DMSO (zero

inhibition), 100uM AMP-PNP (100% inhibition), 2.6uM AMP-PNP (50% inhibition)

Reagent/ compound dispensing system Multidrop Combi liquid handler, acoustic or mosquito compound dispense

Detection instrument and software Envision multi-mode reader, luminescence. IDBS ActivityBase software for data analysis.

Assay validation/QC Z’ > 0.5 requirement, 0.7 mean Z’ for the HTS campaign.

Correction factors Calculation of %inhibition based on in-plate control wells to establish the assay window.

Normalization none

Additional comments

Post-HTS analysis Hit criteria Inhibition > 30% for n=1 at 20uM dose. Hit rate 0.33% hit rate for Mat2A, 0.04% for Mat2A/Mat2B

complex.

Additional assay(s) Hit follow-up by n=2 at 20uM dose and IC50 using KinaseGlo assay format. Orthogonal assays include RapidFire measure of SAM product, differential scanning fluorimetry, compound aggregation assay, and isothermal calorimetry

Confirmation of hit purity and structure QC analysis for hit purity and correct mass done on DMSO stock from HTS library sourcing. IC50 analysis from powder compound source. Lead compounds repurified or resynthesized.

Additional comments


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2 00

3 00

4 00

GPCR Targets Agonism Antagonism

Kinases Inhibition at KM(app) ATP

Inhibition at 1 mM

ATP

EC50 (nM) Kb (nM)

(%) (%) Adrenergic Alpha 1a >10,000 >10,000

ABL1 4.14 -1.42

Adrenergic Beta 2 >10,000 6,025

AKT1 (PKB alpha) 1.79 6.93 Cannabinoid 1 >10,000 >10,000

AURKA (Aurora A) 11.91 15.24

Dopamine 1 >10,000 >10,000

BTK -0.56 -3.66 Histamine 1 >10,000 >10,000

CAMK2A (CaMKII alpha) 7.77 11.51

Mu Opioid >10,000 >10,000

CDK2/cyclin A 2.21 3.85 Muscarinic 1 >10,000 >10,000

CHEK1 (CHK1) 10.88 2.94

Muscarinic 3 >10,000 2,879

CHEK2 (CHK2) 1.56 -1.82

Serotonin 2b >10,000 >10,000

CSNK1A1 (CK1 alpha 1) 2.31 5.01

CSNK2A2 (CK2 alpha 2) 3.93 1.32

Amine Transporters Inhibition

EGFR (ErbB1) 1.68 -0.64 IC50 (nM)

EPHA2 9.40 -0.02

Sertonin Transporter >10,000

FGFR1 8.82 -2.06 Norepinephrine Transporter >10,000

GSK3B (GSK3 beta) 2.94 1.86

Dopamine Transporter >10,000

INSR 2.50 -0.10

IRAK4 -4.13 4.08

Ion Channels Agonism Antagonism

JAK3 -1.37 -1.89 EC50 (nM) IC50 (nM)

KDR (VEGFR2) 7.61 5.62

L- Type Calcium Channel >20,000 >10,000

LCK -1.37 -4.61

Soadium Channel (Nav 1.5) >10,000 4,085

MAP4K4 (HGK) 14.71 3.99

MAPK1 (ERK2) 5.34 2.97

PDE Targets Inhibition

MAPK14 (p38 alpha) -1.63 -5.36 IC50 (nM)

MAPKAPK2 1.61 4.95

PDE1B1 115,081

MARK1 (MARK) 7.42 9.20 PDE2A1 >200,000

MET (cMet) 1.36 -1.44

PDE3A1 >200,000

MST4 5.68 0.04 PDE4D3 138,989

MYLK2 (skMLCK) 3.74 2.88

PDE5A1 91,570

NTRK1 (TRKA) 26.27 15.27 PDE6 (Bovine) >200,000

PAK4 2.30 2.55

PDE7B 115,090

PDK1 -3.16 3.50 PDE8B >200,000

PIM2 1.32 12.90

PDE9A1 >200,000

PRKACA (PKA) 2.67 1.78 PDE10A1 41,873

PRKCB2 (PKC beta II) 4.27 6.11

PDE11A4 29,186

ROCK1 3.19 1.26

SGK (SGK1) 4.32 3.28

SRC 11.32 0.90

STK3 (MST2) 1.19 -0.74

TAOK2 (TAO1) -11.49 -5.79

TEK (Tie2) 9.55 5.73

Supplementary Table 2. Mini-profiling panel examining off-target inhibition by PF-9366.


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Ligand (into 12.5 nM Mat2A dimer)

ATP KM (µM)

Methionine KM (µM)

Vmax (pmol SAM/min)

DMSO 52.7 ± 7.6 14.1 ± 2 5.2 ± 0.2

6.25 nM MAT2B 54.8 ± 7.8 7.1 ± 1.2* 3.5 ± 0.2*

12.5 nM MAT2B 56.7 ± 7.2 6.1 ± 0.8* 2.9 ± 0.2*

25 nM MAT2B 50.2 ± 5.3 3.1 ± 0.6* 2.2 ± 0.2*

50 nM MAT2B 41.6 ± 5.5 2.5 ± 0.5* 1.82 ± 0.2*

0.3 µM PF-9366 51.6 ± 7.8 15.2 ± 0.8 3.9 ± 0.2*

1 µM PF-9366 51.9 ± 11.3 14.9 ± 3.1 1.9 ± 0.2*

3 µM PF-9366 38.1 ± 7.8 2.9 ± 0.8* 0.7 ± 0.1*

Supplementary Table 3. Mat2A kinetic parameters for effect of Mat2B and PF-9366

* Statistically significant (p<0.05) difference from DMSO control; determined by One-way ANOVA and Dunnett’s multiple comparison test. Data are mean ± SEM (n=5).


16

Supplementary Table 4. Data collection and refinement statistics (molecular replacement)

Crystal 1, PDB 5UGH Data collection Space group C2 Cell dimensions a, b, c (Å) 99.73 109.10 149.01 α, β, γ (°) 90 100.04 90 Resolution (Å) 38.14 - 2.062 (2.135 - 2.062)* Rmerge 0.03252 (0.3049) I / σI 13.92 (2.22) Completeness (%) 99.70 (99.94) Redundancy 1.9 (2.0) Refinement Resolution (Å) 38.14 - 2.062 No. reflections 96483 (9603) Rwork / Rfree 17.6 / 20.3 No. atoms Protein 11165 Ligand/ion 100 Water 385 B-factors Protein 48.50 Ligand/ion 32.90 Water 43.40 R.m.s. deviations Bond lengths (Å) 0.004 Bond angles (°) 0.80 1


Targeting S-Adenosylmethionine biosynthesis with a novel … · 2017-06-20 · Casey L. Quinlan*1,...

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Transcript of Targeting S-Adenosylmethionine biosynthesis with a novel … · 2017-06-20 · Casey L. Quinlan*1,...