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Applications of computational drug design

Jan Kelder

Molecular Design & Informatics

N.V. Organon, part of MSD

CMBI Nijmegen:

International Computational Drug Discovery Course

13 June 2017

History

Organon 1923 / Diosynth 1971

Organon Biosciences 2005

Schering Plough 2007

MSD 2009

Aspen 2013

Drug targets

Nuclear hormone receptors

G-protein coupled receptors (GPCRs)

Ion channel receptors

Serine proteases

Kinases and Phosphatases

Phosphodiesterases

Progestin and AdipoQ receptors (PAQRs)

and many more

Drug target families

2%

2%

46%

4%

4%

15%

5%

22% Kinases

GPCRs

Ion channels

Ser proteases

Phosphatases

Cys proteases

Nuclear receptors

Others

A. L. Hopkins, Nature Rev. Drug Disc. 1, 727 - 730 (2002)

Drugs on the market by target

families

1%

4%

6%

1%

47%

30%

7%

4%

Transporters

GPCRs

Ion channels

Enzymes

DNA

Integrins

Nuclear receptors

Others

A. L. Hopkins, Nature Rev. Drug Disc. 1, 727 - 730 (2002)

Drug discovery

Molecular Modification

Screening (MTS and HTS)

Virtual Screening - 3D databases

Structure-Based Drug Design

Molecular modification towards

combined 5-HT2 and H1 antagonism

CH3

N

N

N

CH3

phenbenzamine (Antergan ®)

mianserin (Tolvon ®)

cyproheptadine (Periactin ®)

N

N

CH3

CH3

H1 antagonist

H1 + 5-HT2 antagonist antidepressant

no antidepressant activity


H1 + 5-HT2 antagonist



phenbenzamine (Antergan ®)

mianserin (Tolvon ®)


Org 3363

Molecular modification

mianserin (Tolvon ®) cyproheptadine (Periactin ®)



CH3

NN

N

N

CH3

tripelennamine (Azaron ®)

mirtazapine (Remeron ®)


N

N

CH3

N

CH3

H1 antagonist

H1 + 5-HT2 antagonist antidepressant



H1 + 5-HT2 antagonist


CH3

NN

Nmianserin mirtazapine

CH3

N

N

Noradrenalin (NA) Noradrenalin (NA)

NA uptake blocker --------

alpha-2 antagonist alpha-2 antagonist

alpha-1 antagonist ---------

Serotonin (5-HT) Serotonin (5-HT)

5-HT2A-2C antagonist 5-HT2A-2C antagonist

Histamine Histamine

H1 antagonist H1 antagonist


mianserin mirtazapine

5-HT GPCR subtypes

5-HT7 HUMAN

5-HT1A HUMAN

5-HT1B HUMAN

5-HT1D HUMAN

5-HT1E HUMAN

5-HT1F HUMAN

5-HT5A HUMAN

5-HT5B MOUSE

5-HT4 HUMAN

5-HT6 HUMAN 5-HT2B HUMAN

5-HT2A HUMAN

5-HT2C HUMAN


selective 5-HT2C antagonism

CH3

O

N

N

CH3

H

(S)-(+)-mianserin (R)-(+)-Org 3363 Org 37415

Org GC 94 SDZ SER-082 (+)

CH3

N

N

H

CH3

N

N H

CH3

N

N H

H

H

CH3

N

HH

N

(R)-(+) Org 3363 and (+) - SDZ SER-082 :

Two selective 5-HT2C antagonists

(R)-(+) Org 3363 SDZ SER-082 (+)

Fit of (R)-(+) Org 3363 and

(+) enantiomer of SDZ SER-82

Drug discovery





HTS Compound

library

HTS

Hit

Optimization

Lead

Optimization

Confirmed

Hit

• validated activity /

structure purified

sample

In vitro optimization

on potency & selectivity

Lead

• fulfill potency /

selectivity criteria

and show activity in

in vitro, ex vivo, or in

vivo proof of

principle model

Development

compound

A

D

M

E

T

High Throughput Screening

High throughput screening 200,000

Confirmed actives 100-500

Retesting solid (+ LC-MS) 50-200

Retesting

Purification/Resynthesis 10-50

Lead compounds 0-20

High Throughput Screening

320 compounds/plate

up to 150 plates/day

384-wells plate

Orally active LH agonist: Robot

screening for LH receptor agonists

Robot setup

Reader

Incubator Pipettor

Robot

Plate Hotel Dispensers

HTS on human luteinizing

hormone receptor agonists

N

NS S

NH2

O

O

Confirmed hit: EC50 = 1.4 M

N

NS S

NH2

N

O

O

H

Lead compound Org 41841: EC50 = 0.03 M (= 30 nM)

N

NS S

NH2

N

O

N

H

H

O

N

O

Optimized compound Org 42599: EC50 = 3.1 nM

Not orally active Orally active

Orally active

LMW LH agonists: Org 42599

selected for development

Drug discovery





Decision Tree program

Oss

Rotterdam

Amsterdam

Groningen

Arnhem

Utrecht

Oss

Rotterdam

Amsterdam

Utrecht

Groningen

Arnhem

above

below sea-level

Decision Tree program

Oss

Rotterdam

Amsterdam

Groningen

Arnhem

Utrecht

west of

Utrecht?

south of

Groningen?

south of

Amsterdam?

west of

Arnhem?

yes

no

no

no

no

yes

yes

yes

above

belowsea-level

N

N X R4

R7

R6

R3

R2

R1

Y

• Synthesize first set of compounds based on LH

agonist Lead Org 41841 (at least 50 compounds)

• Test LH receptor activity

• Calculate Molecular Descriptors:

(molecular weight, lipophilicity, polar surface)

• Build Decision Tree which separates active

from inactive compounds

Decision Tree program C5.0

Calculated

molecular

descriptors

active

inactive

Decision Tree pEC50 > 7.5

(n = 201)

23 actives (> 7.5) 23/23 correctly classified (100 %)

178 inactives (< 7.5) 173/178 correctly classified ( 97 %)

N

N X

R4

R7

R6

R3

R2

R1

Y

PSR6 >18.7

Inactive (14)

Inactive (3)

Inactive (7)

Inactive (12)

Inactive (137)

Active (26)

Active (2)

PSR6 >17.3

PSR4 >2.8

MWR7 >30.0

MWR6 >87.1

PSR2 >1.3

PSR6 > 18.7

PSR6 > 17.3

PSR4 > 2.8

MWR7 > 30.0

MWR6 > 87.1

PSR2 > 1.3

yes

yes

yes

yes

yes

yes

no

no

no

no

no

no

N

N X R4

R7

R6

R3

R2

R1

Y

• Select substitution site on molecular scaffold (R2 this time)

• Design virtual library of compounds

• Calculate Molecular Descriptors of all virtual compounds

• Apply Decision Tree and predict active and inactive

compounds

• Select, synthesize and test active compounds

Decision Tree program C5.0

Virtual library design

N

N

NH

S S

NH2

N

O

O

Br

Selection of amines based on availability (ACD database) and predicted potency

(pEC50 LH-CHO > 8.0; decision tree model derived from 250 analogues)

N

N

NH

S S

NH2

N

O

O

NR1

R2

HN

R1

R2 +

Virtual library design

ACD

Select

Reagents

N

N X

R1

R2

R3

R4

R6

R7

Y

Generate

Library

Predict

Actives

65

predicted

actives

1934

library

compounds

1934

amines

Virtual library for substitution at R2

derived from 1934 amines:

65 actives

1869 inactives

N

N X R4

R7

R6

R3

R2

R1

Y

Predicted LMW LH agonists

ClipArt

0

1

2

3

4

5

6

7

8

9

pEC50 CHO-LH

CMP01

CMP02

CMP03

CMP04

CMP05

CMP06

CMP07

CMP08

CMP09

CMP10

CMP11

CMP12

CMP13

CMP14

CMP1516/26 correctly predicted > 8.0 (62 %)

23/26 correctly predicted > 7.5 (88 %)

3D-database pharmacophore searches

5-HT2C and 5-HT2A antagonists

X-ray structure of mesulergine

3D query derived from mesulergine

6-Membered aromatic ring

at a distance of 5.18 Angstrom

of a basic N atom (type 14D)

with a tolerance of 1.0

Two aliphatic carbon atoms

connected to the basic N atom

Exclusion sphere placed in the

direction where the basic N atom

can be protonated at a distance

of 7.0 Angstrom with a radius of

5.3 Angstrom

A second exclusion sphere is placed

at a distance of 7.0 Angstrom of the

basic N atom in the direction of the

N-CH3 bond with a radius of 4.5

Angstrom

3D-database pharmacophore searches

5-HT2C and 5-HT2A antagonists

Chembase:

79716 3D structures

9229 hits (11.6 % of 79716)

1500 hits available for testing

979 hits used for testing after elimination of 521

compounds tested already on 5-HT2C

receptor binding

113 5-HT2C ligands found (11.5 % of 979)

211 5-HT2A ligands found (21.5 % of 979)

Comparison between MTS screen and

3D-database pharmacophore search

Chembase:

Mesulergine (5-HT2C) Ketanserin (5-HT2A)

# hits > 95 % competition # hits > 95 % comp.

MTS screening 49 (4.9 %) 83 (8.3 %)

(1000 compounds)

3D pharmacophore 113 (11.5 %) 211 (21.5 %)

screening

(979 compounds)

3D pharmacophore 283 (18.9 %) 470 (31.3 %)

screening

(1500 compounds) 3.9 x 3.8 x

Results 3D-database pharmacophore

searches 5-HT2C antagonists

NCH

3

HH

H

Org 9283

Two compounds were selected that showed

already interesting 5-HT2C antagonistic

potency and selectivity (Org 9283 and Org 20659)

Org 9283 has been chosen as the lead compound

for developing selective 5-HT2C antagonists as

potential antidepressants/anxiolytics

WO 98377

EP 98-201462

5

5.5

6

6.5

7

7.5

8

8.5

9

5-HT2C 5-HT2A 5-HT2B 5-HT1A

SDZ SER-082

mianserin

Org 3363

Org 37415

Org 9283

mesulergine

Drug discovery





Protein Data Bank

20946 structures (June 2003) – 100147 structures (14 May 2014)

130807 structures (6 June 2017)

Drug targets




Serine proteases


Phosphodiesterases


and many more

NR4A2-NOT

NR4A3-NOR1

NR4A1-NGFI

NR5A1-SF1

NR5A2-FTF

NR6A1-GCNF

NR2F1-COTF

NR2F2-ARP1

NR2F6-EAR2

NR2E3-PNR

NR2B1-RRXA NR2B2-RRXB

NR2A2-HN4G

NR2E1-TLX

NR2C1-TR2-11

NR2C2-TR4

NR2B3-RRXG

NR2A1-HNF4

NR0B1-DAX1

NR0B2-SHP

NR1C1-PPAR

NR1C2-PPAS

NR1C3-PPAT

NR1D1-EAR1

NR1D2-BD73

NR1I3-CAR

NR1H2-NER

NR1H3-LXR

NR1H4-FAR

NR1I1-VDR

NR1B3-RRG1

NR1F3-RORG

NR1F2-RORB

NR1F1-ROR1 NR1A2-THB1

NR1A1-THA1 NR1I2-PXR

NR1B2-RRB2 NR1B1-RRA1

NR3C1-GCR

NR3C4-ANDR

NR3C3-PRGR NR3A1-ESTR

NR3A2-ERBT

NR3B1-ERR1

NR3B2-ERR2

NR3C2-MCR

NR3B2-ERR3

Dimerisation

Lipid metabolism

Drug metabolism

Cholesterol

metabolism

Cell growth

Development

48 nuclear

receptors

NR4A2-NOT

NR5A2-FTF

NR2B1-RRXA NR2B2-RRXB NR2A1-HNF4

NR1C1-PPAR

NR1C2-PPAS

NR1C3-PPAT

NR1H2-NER

NR1H3-LXR

NR1H4-FAR

NR1I1-VDR

NR1B3-RRG1 NR1F2-RORB

NR1F1-ROR1 NR1A2-THB1

NR1I2-PXR

NR1B1-RRA1

NR3C1-GCR

NR3C4-ANDR

NR3C3-PRGR NR3A1-ESTR

NR3A2-ERBT

NR3B2-ERR3

25 X-rays

LBD

NR3C2-MCR

D C A/B F E

F E

F E

DAX1

Heterodimers:

CAR, RXR, RAR, TR,

PPAR, HNF4, ER

Heterodimers:

SF1

Drug targets: Nuclear hormone

receptors (typical and atypical)

LBD DBD

ERa nuclear receptor domains

AB

D

F

E

C

LBD

DBD

D C A/B F E

Ligand binding domains (LBD)

nuclear hormone receptors Progesterone receptor (PR) 1A28, 1E3K, 1SQN, 1SR7, 1E3K

Androgen receptor (AR) 1I37, 1I38, 1E3G, 2AM9, 2AMB

Estrogen receptor (ER) 1A52, 1ERE, 1ERR, 1QKM

1QKN, 1QKT, 1QKU, 3ERD

3ERT, 1G50, 1HJ1

Glucocorticoid receptor (GR) 1M2Z, 1NHZ, 1P93, 3CLD

Mineralocorticoid receptor (MR) 1Y9R, 1YA3, 2AA7

Vitamin D3 receptor (VDR) 1DB1, 1IE8, 1IE9, 2ZLC, 2HAR

Retinoic acid receptor (RAR) 1EXA, 1EXX, 1FCX, 1FCY

1FCZ, 2LBD, 3LBD, 4LBD

Retinoid X receptor (RXR) 1LBD, 1FBY, 1G1U, 1G5Y

1DKF, 1FM6, 1FM9

Peroxisome proliferator-activated rec. 1K74, 1K7L, 1KKQ, 1PRG (PPAR) 2PRG, 3PRG, 4PRG, 1GWX

2GWX, 3GWX

Steroid hormone receptors

O

H

H

H

O

Progesterone

Testosterone

Dihydrotestosterone

Estradiol

Aldosterone

Corticosterone

Calcitriol

etc

H-bond donor HD1 ----

---- H-bond donor HD2

Progesterone

LBD nuclear progesterone receptor

in complex with progesterone PDB code 1A28

P.B. Sigler and S.P. Williams , Nature 393, 392 - 396 (1998)

Q

R

T

Synthetic Steroidal Progestogens

O

O

progesterone (1933)

ethisterone (1938) O

OHCH

northisterone (1956) O

OHCH

O

OHCH

norethynodrel (1957)

OHCH

lynestrenol (1962)

O

CHOH

norgestrel (1966)

CHCH

2

OH

desogestrel (1981)

O

CHOH

gestodene (1987)

norgestimate (1986)

CH

N

OAc

OH

Synthetic Steroidal Progestogens

drospirenone (2000)

O

O

O

etonogestrel (1999) O

CHCH

2

OH

LBD nuclear progesterone receptor in

complex with etonogestrel (model) PDB code 1A28

Q

R

T

LBD nuclear androgen receptor in

complex with dihydrotestosterone PDB code 1I37

J.S. Sack et al. , Proc. Nat. Acad. Sci. USA 98, 4904 - 4909 (2001)

Q

R

T

Homology modelling

In case no experimental 3D structure of the LBD of a nuclear receptor is available homology modelling can be tried

Template selection

Sequence alignment between target and template

Model building

Optimization of the model

Validation

Ligand docking

Homology model LBD nuclear vitamin D3

receptor vs. experimental structure • Homology model (green)

LBD VDR based on LBD

PPARγ (gamma)

• X-ray structure VDR (blue)

• Alignment:

D. R. Boer et al. , Thesis University of Utrecht (2001)

33 % similarity for residues 131 - 427

53 % similarity for residues 226 - 427

LBDs nuclear vitamin D3 receptor and PR in

complex with calcitriol and progesterone

PDB code 1DB1

PDB code 1A28

Drug targets




Serine proteases


Phosphodiesterases


and many more

G-protein coupled receptors (GPCRs) •

• GPCRs trace back more than 1 billion years

• Ancestral nuclear steroid receptor (= estrogen receptor)

• back into evolution more than 500 million years

60

M. Barton et al., J. Steroid Biochem. Mol. Biol. 2017

Bovine rhodopsin X-ray model

K. Palczewski et al., Science 289, 739 - 745 (2000)

PDB code 1F88

X-ray models GPCR and G-protein

a

ß


G-protein coupled receptors(GPCRs)

Follicle Stimulating Hormone (FSH) receptor

Luteinizing Hormone (LH) receptor

Thyroid Stimulating Hormone (TSH) receptor

Serotonin (5-HT) receptors

and many more

Transmembrane region

hLH receptor + Org 41841 (MD)

Lead optimization LH agonist

LH receptor homology model

Mutation studies hLH receptor Org

41841 (Axel Themmen)

Thr 446 - Ala: 7 x less

Thr 446 - Val: 4 x less

Ser 450 - Ala: 2 x less

Glu 451 - Gln: 1 x

Thr 530 - Phe: 1 x

Thr 530 - Ser: 1 x

Asn 535 - Leu: 30 x less

Asn 535 - Ala: 6 x less

Ser 586 - Ala: 1 x

Tyr 612 - His: 1 x

Tyr 612 - Phe: 3 x less

Phe 515 - Leu: 17 x less

Thr 446

Tyr 612

Ser 450

Glu 451

Thr 530

Asn 535

Ser 586

CHO luciferase assay

Transmembrane region

hLH receptor + Org 42599 (MD)

Luteinizing hormone (LH) +

LMW LH agonist Org 41841

LH receptor activation

LHR TM

domain

EC domain LH/hCG

LMW LH agonist

LFR/FLR chimeric receptors transient transfection LFR/FLR.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

LHR-

LH

LHR-

FSH

FSHR-

LH

FSHR-

FSH

LFR-

LH

LFR-

FSH

FLR-

LH

FLR-

FSH

fold

in

cre

ase 0

0.1

1

10

100

Chimeric receptors respond as expected

mU/ml

LTR/TLR chimeric receptors

Org 42599 binds in TM domain of LH receptor

transient transfection LTR/TLR - ORG42599H.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

LHR TSHR LTR TLR

fold

in

cre

ase 0

-11

-9

-7

-6

M


G-protein coupled receptors(GPCRs

Follicle Stimulating Hormone receptor

Luteinizing Hormone receptor

Thyroid Stimulating Hormone receptor

Serotonin (5-HT) receptors

and many more

Serotonin GPCRs

OH

NH

NH3+

Acetylcholine

Noradrenalin

Adrenalin

Dopamin

Serotonin

Histamin

Opioid

etc

Acidic residue Asp ---- ---- H-bond acceptor Ser/Thr

Serotonin

5-HT2C GPCR transmembrane model

based on bacteriorhodopsin

Mutation studies 5-HT2C receptor

serotonin 8.0 6.5 5.3

Org 35018 7.6 6.4 5.5

OH

NH

NH2

NH

NH2

OH

S

NH2

Wildtype S219A F327A

5-HT2C mutant mutant

receptor receptor receptor

pKi pKi pKi

tryptamine 6.9 6.6 5.2

5-HT2C GPCR model based on

bovine rhodopsin

D

S

X-Ray 5-HT2B GPCR with

ergotamine

D

TM3

TM7

TM4

TM1

TM5

TM6

TM2

S

X-ray structures of 25 GPCR’s CLASS A: CLASS B:

Rhodopsin receptor Corticotropin R.F. CRHR1 recep.

Beta1- and Beta2 adrenergic receptors Glucagon GCGR receptor

Adenosine A2A receptor CLASS C:

Muscarinic M1, M2, M3 receptors Glutamate GRM1 receptor

Histamine H1 receptor CLASS F:

Dopamine D3 receptor Frizzled SMOH receptor

Serotonin 5HT1B and 5HT2B receptors

Opioid OPRK1, OPRM1, OPRD1 recep.

Opioid-like ORL1 receptor

Sphingosine-1-phosphate EDG1 receptor

Neurotensin NTSR1 receptor

Protease activated PAR1 receptor

Adenosine diphosphate P2Y12 receptor

C-C Chemokine CCR5 receptor

CXC Chemokine CXCR4 receptor

http://gpcr.scripps.edu

Drug discovery





Target discovery

Steroid GPCR’s

Family Steroid receptor Ligand G-protein

1000 GPCR’s 3: GPER1(GPR30) E2 Gs

(GPRC6A T Gi)

(OXER1 T Gi)

11 PAQR’s 5: mPRα - mPRε P4 Gi/Golf

14 ZIP’s 1: ZIP9/mAR T Gs

GPCR: G-Protein Coupled Receptor

PAQR: Progestin - AdipoQ Receptor

ZIP : Zinc Influx Protein/Zrt-, Irt-like Protein

• Nuclear receptors (nPRs):

• Genomic effects

• Slow response time: minutes to hours

• Membrane bound receptors (mPRs, PGRMCs and nPRs):

Non-genomic effects

Fast response time: seconds to minutes

(e.g. sperm motility activation)

Progesterone receptors

Progesterone Hormone Action at

Target Cells

Membrane Receptor

1 2

slow activates genes

(genomic)

Nuclear Receptor

DNA

Nucleus

R

P

fast

activates 2nd messengers

(nongenomic)

Non-genomic steroidal signaling:

Controversies on 3 possible mechanisms

• Progesterone receptor membrane components (PGRMC1 & 2) or membrane-associated progesterone binding proteins (MAPR)

– Falkenstein/Wehling et al. (1996)

• mPRs coupled to G-proteins

– Zhu/Thomas et al. (2003)

• nPRs located (as dimers) in cytoplasm and/or cell membrane

– Bayaa/Tian et al. (2000)

Kyte & Doolittle Scale Mean Hydrophobicity ProfileScan-window size = 13

Position

19018518017517016516015515014514013513012512011511010510095908580757065605550454035302520151050

Mean H

ydro

phobic

ity

3,2

2,4

1,6

0,8

0

-0,8

-1,6

-2,4

-3,2

TM

Membrane Associated Progesterone

Receptor (MAPR) family : PGRMC1


Position

19018518017517016516015515014514013513012512011511010510095908580757065605550454035302520151050

Mean H

ydro

phobic

ity

3,2

2,4

1,6

0,8

0

-0,8

-1,6

-2,4

-3,2

TM

174


Position

19018518017517016516015515014514013513012512011511010510095908580757065605550454035302520151050

Mean H

ydro

phobic

ity

3,2

2,4

1,6

0,8

0

-0,8

-1,6

-2,4

-3,2

TM



Model of residues 71 – 171 of PGRMC1

H.J. Rohe et al. , Pharmacology & Therapeutics 121, 14 - 19 (2009)


Position

19018518017517016516015515014514013513012512011511010510095908580757065605550454035302520151050

Mean H

ydro

phobic

ity

3,2

2,4

1,6

0,8

0

-0,8

-1,6

-2,4

-3,2

TM



mPRα


Position

19018518017517016516015515014514013513012512011511010510095908580757065605550454035302520151050

Mean H

ydro

phobic

ity

3,2

2,4

1,6

0,8

0

-0,8

-1,6

-2,4

-3,2

TM



PGRMC1 is an adaptor

protein that activates

other proteins such as

enzymes and receptors.

It does not bind progesterone!

mPRα



PGRMC1 is an adaptor

protein that activates

other proteins such as

enzymes and receptors.

It does not bind progesterone!

Cytochrome b5



X-ray of residues 68 – 179 of PGRMC1

Y. Kabe et al. , Nature Communications 7, 11030 - 11042 (2016) (PDB: 4X8Y)

Y113 Y107 Y164

K163

PGRMC1

Non-genomic progesterone signaling:

Controversies on 3 possible mechanisms

• Progesterone receptor membrane components (PGRMC1 & 2) or membrane-associated progesterone binding proteins (MAPR)

– Falkenstein/Wehling et al. (1996)

• mPRs coupled to G-proteins

– Zhu/Thomas et al. (2003)

• nPRs located (as dimers) in cytoplasm and/or cell membrane

– Bayaa/Tian et al. (2000)

An emerging receptor family:

PAQR

Progestin – AdipoQ Receptor

mPR receptors Y. Zhu, C.D. Rice, Y. Pang, M. Pace, and P. Thomas

Cloning, expression, and characterization of a membrane

progestin receptor and evidence it is an intermediary in meiotic

maturation of fish oocytes

PNAS 100, 2231 – 2236 (2003)

Y. Zhu, J. Bond, and P. Thomas

Identification, classification, and partial characterization of

genes in humans and other vertebrates homologous to a fish

membrane progestin receptor

PNAS 100, 2237 - 2242 (2003)

Peter Thomas: Marine Science

Institute, University of Texas

• Progesterone GPCR:

• Cloned from spotted seatrout ovaries

• 40 kDa protein (352 AA)

• High affinity for 20β-S and progesterone

• Steroid specificity different from nuclear PR

• Coupled to Gi protein

• Up regulated by gonadotropins (hCG)

• Involved in oocyte maturation

• Tissue specificity: oocytes, testis, brain

Peter Thomas: Marine Science

Institute, University of Texas

• Mammalian Progesterone GPCR:

• 3 Human genes in testis, brain, and kidney

– mPRa :gonads: testis and ovary (weak)

– mPR :brain: all over the brain

– mPR :kidney

• Involved in oocyte maturation (pig?)

• Involved in (human) sperm motility

R.norvegicus PAQR4

M.musculus PAQR4

H.sapiens PAQR4

P.Troglodytes PAQR4

P.Troglodytes MPRA

H.sapiens MPRA

R.norvegicus MPRA

M.musculus MPRA

P.Troglodytes MPRB H.sapiens MPRB

P.pygmaeus MPRG

H.sapiens MPRG

R.norvegicus MPRG

M.musculus MPRG

P.Troglodytes PAQR9

H.sapiens PAQR9

R.norvegicus PAQR9

M.musculus PAQR9

P.Troglodytes PAQR11

H.Sapiens PAQR11

R.norvegicus PAQR11

M.musculus PAQR11

P.Troglodytes PAQR10

H.sapiens PAQR10

R.norvegicus PAQR10

M.musculus PAQR10

R.norvegicus ADR2

M.musculus ADR2

R.norvegicus ADR1

M.musculus ADR1

P.Troglodytes ADR1

H.sapiens ADR1

PAQR9 -

mPRε

PAQR5 -

mPRγ

PAQR6- mPRδ PAQR8-mPRβ

PAQR7-

mPRα

PAQR11-

MMD

PAQR10-

MMD2

PAQR3

PAQR2

-ADR2

PAQR1

-ADR1

PAQR4

PAQR Family

Progesterone and adiponectin receptors • PAQR I AdipoR1 ADIPOR1 3WXV

• PAQR II AdipoR2 ADIPOR2 3WXW

• PAQR III AdipoR3 ADIPOR3

• PAQR IV PAQR4

• PAQR V mPR PAQR5

• PAQR VI mPRδ PAQR6

• PAQR VII mPRa PAQR7

• PAQR VIII mPR PAQR8

• PAQR IX mPRε PAQR9

• PAQR X MMD2

• PAQR XI MMD

Progesterone and adiponectin receptors • PAQR I AdipoR1 ADIPOR1 5LXG

• PAQR II AdipoR2 ADIPOR2 5LWY

• PAQR III AdipoR3 ADIPOR3

• PAQR IV PAQR4

• PAQR V mPR PAQR5

• PAQR VI mPRδ PAQR6

• PAQR VII mPRa PAQR7

• PAQR VIII mPR PAQR8

• PAQR IX mPRε PAQR9

• PAQR X MMD2

• PAQR XI MMD

Seatrout mSR

Picture of seatrout mPR (352 AA)

EL2

Seatrout mSR

1 2 3 4 5 6 7

Hydrophobicity plot for seatrout

mPR

EL-2

1 2 3 4 5 6 7

Hydrophobicity plot for seatrout

mPR

No large hydrophobic EL-2 anymore!

Recombinant seatrout mPR binding is specific for

natural progestins but not for the nPR ligands RU486

and R5020 (human cell expression)

-10 -9 -8 -7 -6 -5 -40

25

50

75

100

12520 -S

E2

T

P4

Cort

R5020

RU486

Concentration (M)

3H

-20

-S B

ou

nd

(%

)

Down-regulation of adenylyl cyclase activity by 20β-S in

transfected cells blocked by pertussis toxin (PTX)

0

5

10

15

cDNA inserted vector Empty vector Reversed insert

Buffer 20-S 20-S 20-S

+ PTX + inPTX

Buffer Buffer20-S 20-S

*

cA

MP

(p

mo

l/m

l)

*

• suggests mPR coupled to inhibitory G-protein

Thomas et al., Endocrinology, 148(2):705–718 (2007)

Characterization of recombinant human mPRα in

MDA-231 cells

• mPRα is most abundantly

expressed mPR and is

widely distributed in tissues

and cell lines, suggesting

that it is the major mPR

mediating progestin

signaling in most cell types.

• Activates and is coupled to

an inhibitory G protein.

• Regulates nPR

transactivation in human

myometrial cells

[3H]progesterone binding increased in transfected cells.

High affinity progesterone binding, Kd 4.2 nM .

Immunoprecipitation

results show activation

of an inhibitory G

protein

Competition of human mPR a -transfected cells membrane preparation

-11 -10 -9 -8 -7 -6 -5 -4 -3

0

20

40

60

80

100

120

P4

R5020

Ru486

Org2058

[ 3 H]P4: 1 nM final;

Mem protein: 0.15mg/ml final

Competitor conc. (log M)

% S

pec

ific

Bin

din

g

1 2 3 4 5 6 7

Human Testis/ovary specific mSR

Hydrophobicity plot for human

testis/ovary specific mPRa

EL-2

1 2 3 4 5 6 7

Human Testis/ovary specific mSR

Hydrophobicity plot for human

testis/ovary specific mPRa

No large hydrophobic EL-2 anymore!

PAQR5-9 + Adiponectin

111

adiponectin PAQR5-9

progesterone

Competition experiments with

mPRα

112 P. Thomas et al., Neuroendocrinology 96, 162-171 (2012)

Adiponectin receptors:

AdipoR1 + AdipoR2

113

7TM model: N-terminus inside and C-terminus outside!

T. Kadowaki et al. , Endocr. Rev. 26, 439-451 (2005)

Progesterone and adiponectin receptors • AdipoR1 ADIPOR1 7TM N-term inside Zn2+

• AdipoR2 ADIPOR2 7TM N-term inside Zn2+

• AdipoR3 ADIPOR3 7TM N-term inside Zn2+

• PAQR4

• mPR PAQR5 7TM N-term outside GPCR

• mPRδ PAQR6 7TM N-term outside GPCR

• mPRa PAQR7 7TM N-term outside GPCR

• mPR PAQR8 7TM N-term outside GPCR

• mPRε PAQR9 7TM N-term outside GPCR

• MMD2

• MMD

114

Progesterone and adiponectin receptors

• AdipoRs: mPRs:

• 7TM with N-term inside 7TM with N-term outside

• Helix 0 Helix 0 absent?

Ligands: Adiponectin & Ligands: Progesterone &

AdipoRon Org OD 02-0

No GPCR activities GPCR activities

• Zn2+ in the 7TM pocket no Zn2+ in the 7TM pocket

• Ceramidase activity No ceramidase activity

115

X-ray

structure

Homology

model

N-terminus

N-terminus

7TM 7TM

Helix 0

Helix 0

Zn2+

AdipoRs 1 & 2 structures suggest

ceramidase activity

116

AdipoRs 1 & 2 suggest

ceramidase activity

117

FFA:

R represents the alkyl

portion of a fatty acid.

Ceramide:

Sphingosine:

AdipoRs 1 & 2 structures suggest

ceramidase activity

118

X-ray human adipoR2 receptor

(3WXW)

119

TM3 TM4

TM5

TM2

TM1

TM6 TM7

Helix0

H. Tanabe et al. , Nature 520, 312-316 (2015)

X-ray human AdipoR2 receptor

(3WXW)

120

His348 His352

His202 Zn2+

Asp219

H2O

TM3

Tyr220

TM7

TM1

TM2

TM4

TM5

TM6

Homology model mPRa receptor

based on X-ray AdipoR2 (3WXW)

121

TM3 TM4

TM5 TM2

TM1 TM6

TM7

Helix0

122

His275

His279

His125

Asp142 TM3

TM7

TM1

TM2

TM4

TM5

TM6

Homology model mPRa receptor without Zn2+

and with P4 (docked with MOE2012 pose 6)

Val146 Ser210

Ser121 Val211

Ala252

Four new X-rays AdipoRs 1 & 2

123

AdipoR1 5LXG in an open conformation supersedes 3WXV

AdipoR2 5LX9 in complex with C18 (S1)

AdipoR2 5LWY in complex with C18 (S2) supersedes 3WXW

AdipoR2 5LXA in complex with C18 (S3)

I. Vasiliauskaité-Brooks et al. , Nature 544, 120-123 (2017)

New X-ray 5LWY AdipoR2 with cis-oleic acid

124 29-04-2017

CO2- of oleic acid interacts with Y220 in TM3 and R278 in TM5,

H2O and Y328 in TM6

Arg278

Asp219

His202

TM1

TM2

TM3

TM5

TM6 TM7

Tyr220

TM4

Helix 0

His348 His352

Ser198

Tyr328

Zn2+

New X-ray 5LWY AdipoR2 with cis-oleic acid

125 29-04-2017

CO2- of oleic acid interacts with Y220 in TM3 and R278 in TM5,

H2O and Y328 in TM6

Ile223 Arg278 Asp219

His202

TM1

TM2

TM3

TM5

TM6 TM7

Tyr220

TM4

Ser198

His352

His348

Tyr328

Zn2+

H2O

New homology model MPRα (PAQR7) with

Progesterone (pose 01)

126 17-05-2017

20-keto of P4 interacts with Y143 in TM3 and Q206 in TM5

(optimize further)

Val146 Gln206 Asp142

His125

TM1

TM2

TM3

TM5

TM6 TM7

Tyr143

TM4

His275

His279

H L/P D

Y

SAR human mPRa vs human nPR-B

R1

R2R3

R4

O

Et > Me > H

Et > Me C=OMe > OH

H >> CΞCH

H > Me

Et > Me C=OMe >> OH

CΞCH >> H

Human nPR-B

Human mPRa

CoMFA steric and electrostatic field

contour plots for the mPRα model

(right) and the nPR model (left)

n = 60 n = 48

129

J. Kelder et al., Steroids 75: 314-322 (2010)

Progesterone (RBA mPRα: 100 %)

Org OD 02-0 (RBA mPRα: 258 %)

Org OD 13-0 (RBA mPRα: 93 %) Org OE 64-0 (RBA mPRα: 2009 %)

Org 33663-0 (RBA mPRα: 168 %)

Selective most potent mPRα agonists: Non Selective:

Transactivation results of

P4(low dose), OD 02-0 and OD 13-0 in MCF-7 Luc reporter system

* * * *

0.0

0.5

1.0

1.5

2.0 R

ela

tive

luci

fera

se

act

ivit

y (

FF

/Ven

)

Conclusion: Org OD 02-0 and OD 13-0 are nPR antagonists

OD 02-0

OD 13-0

131

O.V. Lisanova et al., Biochemistry (Moscow) 78: 236-243 (2013)

Progesterone (RBA mPRα: 100 %)

Compound XX (RBA mPRα: 11 %) Compound XXI (RBA mPRα: 10 %)

Compound IV (RBA mPRα: 4.5 %)

3-keto is essential for nPR binding

20-keto is essential for mPRα binding

Progesterone (RBA nPR: 100 %)

Compound IV (RBA nPR: 95 %)

Compound XX (RBA nPR: 7 %)

Compound XXI (RBA nPR: 0.4 %)

mPRs: 20-keto is essential for binding via H-bonds (Y and Q/R)

• nPRs: 3-keto is essential for binding via H-bonds (R and Q)

X-ray nPR Homology model mPRα

Tissue distribution mPR receptors

A. van Heijst, P.M.A. Groenen, J. Kelder, Steroids 70, 482 – 483 (2005)

mPR receptors P. Thomas, Y. Pang, J. Dong, P. Groenen, J. Kelder, J. de Vlieg,

Y. Zhu, and C. Tubbs

Steroid and G protein binding characteristics of the seatrout and

human progestin membrane receptor alpha subtypes and their

evolutionary origins

Endocrinology 148, 705 – 718 (2007)

J. Kelder, R. Azevedo, Y. Pang, J. de Vlieg, J. Dong, and P.

Thomas

Comparison between steroid binding to membrane progesterone

α (mPRα ) and to nuclear progesterone receptor: Correlations

with physicochemical properties assessed by comparative

molecular field analysis and identification of mPRα-specific

agonists

Steroids 75, 314 - 322 (2010)

Sperm hmPRα protein levels are

decreased in men with low sperm motility

Low High0.00

0.25

0.50

0.75

Sperm Motility

Rela

tive B

an

d I

nte

nsit

y

mPRα expression

• mPRα is present on human sperm

• Sperm mPRa levels are decreased in men with low sperm motility

Low High0.00

0.25

0.50

0.75

Sperm Motility

Rela

tive B

an

d I

nte

nsit

y

Potential role of mPRs in human male

fertility

Thomas , Tubbs & Gary, Steroids 2009

mPRα expression

Membrane progesterone receptor

mPRα

137

Sex steroids regulate skin pigmentation

through GPER and mPRα (PAQR7)

C.A. Natale, et al., eLife 5, e15104 (2016)

Sex steroids regulate skin pigmentation

through GPER and mPRα (PAQR7)

C.A. Natale, et al., eLife 5, e15104 (2016)

= Org OD02

Conclusions

Org OD 02-0 and Org OD 13-0 have been identified as potent and selective mPRa ligands

Both compounds are weak antagonists for the nPR

Therefore they are useful reference compounds for demonstrating the role and function of mPRs

Still lacking are antagonists for mPRa

The PAQRs form an emerging receptor family

Conclusions (2)

The PDB forms a rich source of experimental structures that expands rapidly

Homology modeling is useful in cases where experimental structures are not yet available and good templates exist

Knowledge of how ligands bind to proteins can be utilised to suggest useful mutation studies

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