Construction

of

models

of

the

family

as

a

tool

to

predict

ligand

binding

characteristics

Aldosterone

is

synthesised

by

aldosterone

synthase

-LRB-

-RRB-

has

a

highly

homologous

isoform

steroid

-LRB-

-RRB-

which

is

responsible

for

the

biosynthesis

of

aldosterone

precursors

and

glucocorticoids

To

investigate

aldosterone

biosynthesis

and

facilitate

the

search

for

selective

inhibitors

we

constructed

three-dimensional

models

for

and

for

both

human

and

rat

The

models

were

constructed

based

on

the

crystal

structure

of

Pseudomonas

Putida

and

Oryctolagus

Cuniculus

Small

steric

active

site

differences

between

the

isoforms

were

found

to

be

the

most

important

determinants

for

the

regioselective

steroid

synthesis

A

possible

explanation

for

these

steric

differences

for

the

selective

synthesis

of

aldosterone

by

is

presented

The

activities

of

the

known

inhibitors

metyrapone

R-etomidate

R-fadrazole

and

S-fadrazole

were

determined

using

assays

of

cells

that

express

human

and

respectively

By

investigating

the

inhibitors

in

the

human

models

using

molecular

docking

and

molecular

dynamics

simulations

we

were

able

to

predict

a

similar

trend

in

potency

for

the

inhibitors

as

found

in

the

in

vitro

assays

Importantly

based

on

the

docking

and

dynamics

simulations

it

is

possible

to

understand

the

enantioselectivity

of

the

human

enzymes

for

the

inhibitor

fadrazole

the

R-enantiomer

being

selective

for

and

the

S-enantiomer

being

selective

for

Introduction

Aldosterone

is

a

member

of

the

renin

angiotensin

aldosterone

system

-LRB-

RAAS

-RRB-

that

plays

an

important

physiological

role

in

the

regulation

of

electrolyte

homeostasis

and

thereby

blood

pressure

This

endogenous

mineralocorticoid

exerts

its

function

by

binding

to

the

mineralocorticoid

receptor

Upon

ligand

binding

the

protein-ligand

complex

is

translocated

to

the

cell

nucleus

where

it

modulates

the

gene

expression

of

proteins

involved

in

electrolyte

homeostasis

-LSB-

-RSB-

Aldosterone

is

produced

predominantly

in

the

adrenal

cortex

and

is

derived

from

cholesterol

through

various

steps

involving

a

number

of

dehydrogenases

and

cytochrome

enzymes

-LRB-

CYPs

-RRB-

This

includes

aldosterone

synthase

-LRB-

-RRB-

which

catalyses

the

final

steps

of

aldosterone

biosynthesis

Recently

various

studies

on

the

pathophysiology

of

heart

failure

have

revealed

that

aldosterone

plays

a

role

in

the

formation

of

myocardial

hypertrophy

reactive

myocardial

fibrosis

vascular

remodelling

and

electrolyte

imbalance

This

may

contribute

to

the

development

of

arrhythmias

hypertension

and

congestive

heart

failure

-LSB-

-RSB-

Although

great

therapeutic

successes

have

been

achieved

by

treating

heart

failure

patients

with

blockers

of

the

RAAS

-LRB-

CONSENSUS

trial

-LSB-

-RSB-

SOLVD

trial

-LSB-

-RSB-

-RRB-

the

mortality

due

to

heart

failure

is

still

high

Therefore

exploring

new

therapeutic

possibilities

is

highly

desirable

Recently

it

became

clear

that

aldosterone

is

also

a

key

player

in

heart

failure

In

the

RALES

study

-LSB-

-RSB-

blocking

the

action

of

aldosterone

using

the

mineralocorticoid

receptor

antagonist

spironolactone

proved

to

greatly

reduce

mortality

hospitalisation

numbers

and

hospitalisation

time

in

patients

with

severe

heart

failure

Later

similar

encouraging

results

were

found

for

eplerenone

another

mineralocorticoid

receptor

antagonist

-LRB-

EPHESUS

trial

-LSB-

-RSB-

-RRB-

in

patients

with

post

myocardial

infarction

From

this

it

can

be

derived

that

reducing

aldosterone

action

seems

highly

beneficial

in

the

treatment

of

heart-failure

and

that

it

is

particularly

worthwhile

to

find

new

pharmacological

manners

to

interfere

with

this

hormone

An

alternative

manner

to

reduce

aldosterone

action

would

be

not

to

block

the

mineralocorticoid

receptor

but

to

prevent

formation

of

the

hormone

itself

by

inhibiting

its

biosynthesis

-LSB-

-RSB-

Although

the

net

outcome

of

aldosterone

receptor

inhibition

versus

aldosterone

synthesis

inhibition

apparently

is

identical

a

number

of

potential

advantages

for

the

latter

approach

exists

reduction

of

side

effects

as

particularly

observed

for

the

antagonist

spironolactone

-LSB-

-RSB-

prevention

of

compensatory

aldosterone

synthesis

inherent

to

mineralocorticoid

receptor

blockade

-LRB-

of

which

long

term

effects

are

unknown

-RRB-

-LSB-

-RSB-

and

possible

circumvention

of

inter-individual

variations

regarding

pharmacodynamics

and

pharmacokinetics

that

are

as

observed

for

mineralocorticoid

receptor

antagonists

-LSB-

-RSB-

The

clinical

use

of

inhibitors

of

aromatase

-LRB-

which

converts

androgens

into

estrogens

-RRB-

next

to

estrogen

receptor

antagonists

in

the

treatment

of

breast

cancer

is

one

of

the

examples

that

shows

that

the

approach

of

ligand

synthesis

inhibitors

clinically

can

be

of

great

use

despite

the

existence

of

receptor

blockers

for

that

ligand

-LSB-

-RSB-

The

last

steps

of

the

biosynthesis

of

aldosterone

are

mediated

by

the

mitochondrial

cytochrome

family

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

Members

of

this

protein

family

contain

a

heme

prosthetic

group

in

the

core

of

the

active

site

with

which

they

catalyse

-LRB-

subsequent

-RRB-

oxidation

reactions

on

and

on

the

β-side

of

the

steroid

skeleton

-LRB-

for

enumeration

see

Fig.

-RRB-

In

bovine

-LSB-

-RSB-

pig

-LSB-

-RSB-

and

frog

-LSB-

-RSB-

aldosterone

synthesis

is

performed

by

only

one

cytochrome

but

in

human

-LSB-

-RSB-

and

mouse

-LSB-

-RSB-

the

synthesis

involves

two

isoforms

-LRB-

steroid

-RRB-

and

-LRB-

aldosterone

synthase

-RRB-

Rat

possesses

four

isoforms

for

which

and

are

the

most

important

ones

is

only

expressed

in

neonatal

rat

and

carries

the

same

activity

as

and

encodes

a

pseudogene

-LSB-

-RSB-

Fig.

of

Aldosterone

by

the

family

Indicated

with

arrows

are

the

possible

substrate

conversions

performed

by

human

and

-LSB-

-RSB-

Rat

and

possess

the

same

activities

as

the

human

isoforms

except

that

rat

can

also

oxidise

on

-LSB-

-RSB-

Most

remarkable

is

the

substrate

specificity

of

the

different

isoforms

In

both

man

and

rat

only

the

isoform

can

perform

the

final

oxidation

of

to

produce

aldosterone

-LSB-

-RSB-

For

the

isoform

hydroxylation

has

been

reported

for

rat

but

not

yet

for

man

-LSB-

-RSB-

and

the

isoform

in

general

is

known

to

play

an

important

role

in

the

biosynthesis

of

glucocorticoids

If

an

inhibitor

of

aldosterone

synthesis

is

to

be

designed

the

biosynthesis

of

glucocorticoids

should

remain

unaffected

indicating

that

the

inhibition

must

be

selective

We

developed

three-dimensional

in

silico

models

as

a

decision

making

tool

to

facilitate

the

selection

of

potential

inhibitors

for

synthesis

and

in

vitro

testing

In

order

to

obtain

specific

inhibitors

we

also

investigated

its

highly

homologous

family

member

The

aim

of

this

report

is

-LRB-

-RRB-

to

deduce

by

homology/comparative

modelling

the

architecture

of

and

around

their

active

site

-LRB-

-RRB-

to

investigate

protein-substrate

interactions

and

propose

a

mechanism

for

substrate

regioselectivity

and

-LRB-

-RRB-

to

validate

the

homology

models

by

correlating

the

in

vitro

activity

of

four

known

inhibitors

to

in

silico

data

The

inhibitors

we

have

chosen

are

metyrapone

-LSB-

-RSB-

R-etomidate

-LSB-

-RSB-

R-fadrazole

and

S-fadrazole

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

The

in

silico

models

not

only

represent

an

important

tool

in

modern

drug

discovery

but

will

also

help

in

elucidating

molecular

mechanisms

and

-LRB-

substrate

binding

-RRB-

preferences

of

the

substrate

conversion

of

the

enzymes

of

interest

Fig.

structures

of

the

known

inhibitors

metyrapone

R-etomidate

R-fadrazole

and

S-fadrazole

Modelling

considerations

Homology

modelling

has

been

widely

applied

to

the

family

of

cytochrome

enzymes

and

is

mainly

oriented

towards

enzyme

inhibition

Before

the

first

mammalian

cytochrome

structures

became

available

modelling

attempts

were

classically

performed

with

crystal

structures

possessing

sequence

identities

lower

than

because

no

realistic

alternatives

were

available

Homology

models

were

often

based

on

a

template

of

bacterial

-LSB-

-RSB-

but

the

introduction

of

bacterial

allowed

for

modelling

the

functional

properties

of

eukaryotic

class

II

-LSB-

-RSB-

Although

these

homology

models

contain

a

low

sequence

identity

with

their

template

structures

and

are

intuitively

suboptimal

it

has

been

shown

that

they

can

describe

key

features

of

protein-ligand

interactions

-LSB-

-RSB-

For

example

features

observed

for

inhibitor

binding

in

aromatase

models

have

provided

important

insights

for

the

development

of

drugs

-LSB-

-RSB-

Current

models

often

still

feature

bacterial

as

template

-LSB-

-RSB-

but

methods

involving

the

usage

of

multiple

crystal

structures

for

model

construction

may

prove

to

be

the

future

trend

-LSB-

-RSB-

A

model

based

on

the

structure

of

several

known

enzymes

would

be

more

accurate

since

every

additional

segment

will

improve

similarity

or

spatial

coordination

of

protein

regions

However

structural

flaws

can

be

expected

at

locations

where

the

template

structures

are

joined

and

if

these

regions

are

within

the

active

site

they

need

to

be

thoroughly

refined

Some

modelling

work

on

and

has

already

been

performed

earlier

by

Belkina

et

al.

-LSB-

-RSB-

and

Ulmschneider

et

al.

-LSB-

-RSB-

The

models

of

Belkina

et

al.

discuss

the

potential

spatial

arrangement

of

the

amino

acids

in

the

active

site

and

hypothesise

the

hydrogen-bonding

network

involved

in

heme

stabilisation

Furthermore

the

effect

of

several

amino

acid

mutations

have

been

detailed

The

models

of

Ulmschneider

et

al.

focus

on

describing

protein-inhibitor

interactions

and

structure

activity

relations

of

their

developed

inhibitors

The

so

far

published

models

were

thoroughly

characterised

for

those

specific

purposes

however

the

goal

of

our

model

construction

work

was

to

investigate

the

regioselectivity

of

the

natural

ligands

within

the

enzymes

and

to

detail

potential

protein-ligand

interactions

For

validation

of

our

own

three-dimensional

models

the

in

silico

data

are

presented

in

the

form

of

molecular

docking

and

molecular

dynamics

simulations

These

methods

are

regularly

used

to

investigate

protein-ligand

interactions

Because

the

only

difference

in

the

activity

of

the

two

isoforms

and

is

the

formation

of

aldosterone

by

the

latter

successful

modelling

of

the

isoforms

relies

on

a

careful

analysis

of

the

specific

substrate

conversion

activities

that

exists

between

these

two

isoforms

Because

of

this

we

reviewed

an

experimental

mutation

study

by

Bottner

et

al.

-LSB-

-RSB-

on

the

human

and

proteins

performed

in

a

similar

manner

as

by

Belkina

and

Ulmschneider

for

the

currently

published

models

-LSB-

-RSB-

The

study

by

Bottner

et

al.

showed

that

mutation

of

three

residues

outside

the

active

site

-LRB-

-RRB-

is

sufficient

to

convert

the

catalytic

activity

of

into

that

of

suggesting

that

remote

steric

aspects

play

a

more

important

role

in

the

substrate

binding

and

substrate

conversion

than

the

presence

of

different

amino

acids

in

the

active

sites

of

both

isoforms

This

led

us

to

postulate

that

the

difference

in

substrate

conversion

is

caused

by

a

difference

in

the

relative

positioning

of

the

substrate

above

the

heme

in

the

active

site

To

be

more

specific

we

postulate

that

there

is

a

correlation

between

substrate

selectivity

and

the

substrate

hydroxylation

distance

the

distance

between

the

heme

iron

and

the

substrate

carbon

In

other

words

the

binding

mode

of

the

natural

substrate

dictates

which

carbon

atom

is

oxidised

first

with

conversion

taking

place

on

the

carbon

atom

which

is

in

closest

proximity

to

the

iron-oxygen

complex

For

human

this

means

that

and

are

to

be

in

close

proximity

to

the

catalytic

iron

atom

with

closest

to

the

iron

Rat

possesses

a

similar

binding

mode

but

we

expect

that

it

also

presents

in

a

position

allowing

oxidation

Explaining

the

preference

for

human

and

rat

would

bind

with

closest

to

the

iron

atom

and

at

a

correct

distance

for

oxidation

To

substantiate

this

hypothesis

the

three

dimensional

architectures

of

the

human

and

rat

enzymes

were

constructed

using

comparative

modelling

For

reasons

of

relevance

only

the

and

isoforms

were

investigated

We

intend

to

show

how

knowledge

of

these

various

hydroxylation

patterns

of

aldosterone

precursors

can

result

in

working

models

for

the

substrate

selective

activity

of

the

two

isoforms

From

here

on

the

human

isoforms

will

be

noted

as

and

whereas

the

rat

isoforms

will

be

noted

as

and

As

stated

above

another

aim

was

to

validate

the

in

silico

models

with

in

vitro

activity

data

of

four

known

inhibitors

These

inhibitors

were

chosen

for

the

following

reasons

Metyrapone

is

a

known

inhibitor

of

and

is

clinically

used

in

the

diagnosis

of

Cushing

Syndrome

-LSB-

-RSB-

R-etomidate

is

clinically

used

as

anaesthetic

but

it

is

known

to

be

a

highly

potent

suppressor

of

the

family

-LSB-

-RSB-

Racemic

fadrazole

was

designed

for

the

selective

inhibition

of

aromatase

a

cytochrome

enzyme

which

is

closely

related

to

the

family

because

it

oxidises

steroids

on

Next

to

aromatase

inhibition

racemic

fadrazole

also

shows

considerable

inhibition

of

members

of

the

family

-LSB-

-RSB-

Methods

Homology

modelling

The

amino

acid

sequences

of

the

family

were

taken

from

Swissprot

-LSB-

-RSB-

-LRB-

accession

accession

accession

accession

accession

-RRB-

and

the

determination

of

the

secondary

structures

of

the

family

members

was

performed

using

the

secondary

structure

prediction

program

JPred

-LSB-

-RSB-

The

alignment

used

for

homology

modelling

was

obtained

with

MOE-Align

-LSB-

-RSB-

by

performing

a

topological

alignment

of

the

cytochrome

enzymes

for

which

a

crystal

structure

is

publicly

available

-LRB-

Fig.

-RRB-

The

three

dimensional

architecture

of

cytochrome

enzymes

consists

of

alpha-helices

annotated

from

A

to

L

as

well

as

six

beta-sheets

despite

having

a

sequence

identity

among

less

than

Additionally

several

short

helices

are

present

in

various

cytochrome

enzymes

-LRB-

annotated

B

F

G

J

K

-RRB-

whilst

being

absent

in

others

The

structural

core

of

all

cytochrome

enzymes

consists

of

a

four-helix

bundle

composed

of

alpha-helices

D

E

I

and

L

and

the

two

alpha-helices

J

and

K

The

variable

structural

features

involved

in

ligand

binding

and

substrate

specificity

are

situated

in

alpha-helices

A

B

B

F

G

and

their

adjacent

loops

-LSB-

-RSB-

Fig.

alignment

of

human

and

rat

isoforms

to

related

cytochrome

enzymes

of

which

a

three

dimensional

structure

has

been

elucidated

Indicated

with

a

are

the

in

alpha-helix

B

and

in

alpha-helix

I.

Indicated

with

a

is

the

triple

mutant

in

alpha-helix

I.

Indicated

with

a

is

the

catalytic

in

alpha-helix

I.

Indicated

with

a

is

the

conserved

in

alpha-helix

L

The

predicted

secondary

structures

for

the

family

members

were

aligned

to

these

topological

features

of

the

crystal

structures

as

well

as

by

preserving

several

characteristic

CYP

features

-LSB-

-RSB-

W

-LRB-

R/K

-RRB-

XXR

-LRB-

X

indicates

any

amino

acid

-RRB-

in

helix

C

connecting

and

stabilising

the

heme

prosthetic

group

in

the

active

site

EXXR

in

helix

K

and

-LRB-

W/F

-RRB-

XXPXX

-LRB-

F/Y

-RRB-

XPX

-LRB-

H/R

-RRB-

-LRB-

W/F

-RRB-

following

helix

K

comprising

the

typical

meander

region

and

finally

XXF

-LRB-

G/S

-RRB-

XGX

-LRB-

H/R

-RRB-

XCXGXX

-LRB-

L/F

-RRB-

AXXE

before

helix

L

which

contains

the

cysteine

residue

by

which

the

heme

prosthetic

group

is

bound

to

the

enzyme

Furthermore

a

glutamic

acid

was

aligned

that

is

thought

to

be

involved

in

the

formation

of

a

catalytically

important

water

channel

-LSB-

-RSB-

-LRB-

in

in

the

family

-RRB-

and

the

entire

helix

I

was

aligned

for

its

catalytic

threonine

-LRB-

in

in

family

-RRB-

-LSB-

-RSB-

As

a

result

of

small

variations

in

loop

regions

some

insertions

and

deletions

were

introduced

in

segments

outside

the

active

site

These

segments

did

not

correspond

to

any

of

the

key

secondary

structures

Clearly

and

portray

a

high

degree

of

homology

possessing

a

pair

wise

sequence

identity

percentage

as

high

as

for

human

and

for

rat

-LRB-

Table

highlighted

-RRB-

This

emphasises

the

difficulty

of

modelling

the

difference

between

the

two

isoenzymes

and

the

challenge

of

reaching

the

level

of

modelling

accuracy

that

is

required

For

enzymes

for

which

a

crystal

structure

is

publicly

available

the

overall

pair

wise

sequence

identity

with

the

family

is

found

to

be

less

than

whereas

it

is

around

for

active

site

residues

This

low

degree

of

homology

indicates

that

of

the

reviewed

cytochromes

can

be

used

as

a

sole

representative

for

modelling

the

family

Table

pair

wise

sequence

identity

-LRB-

in

percentages

-RRB-

between

the

human

and

rat

isoforms

and

cytochrome

enzymes

for

which

a

three

dimensional

structure

has

been

17.330.237.739.638.523.521.223.130.823.111.519.626.424.524.526.426.42

24.528.339.619.229.426.934.630.832.714.847.126.426.424.524.524.51

28.350.936.521.619.221.221.225.014.321.620.818.920.818.918.91

35.828.829.425.026.928.825.017.929.426.426.428.328.328.31

34.627.528.828.832.728.825.029.426.426.428.326.426.41

19.617.319.221.223.118.519.618.917.017.017.017.01

23.128.825.025.011.135.320.820.817.020.820.81

57.765.459.633.333.332.132.132.130.230.21

.116.914.914.314.716.016.739.640.040.638.5

.122.019.614.517.214.816.522.822.022.921.917.9

.016.314.615.516.112.014.917.417.615.817.314.917.1

.316.913.614.815.812.014.517.417.616.217.715.517.593.6

.315.413.213.815.311.013.417.416.716.617.714.417.363.663.6

.315.814.414.516.613.014.715.916.215.817.113.516.868.268.882.6

.415.814.414.516.613.014.715.916.215.817.113.816.868.669.283.097.3

The

bottom

triangle

indicates

the

pair

wise

sequence

identity

of

the

whole

protein

the

top

triangle

indicates

the

pair

wise

sequence

identity

of

the

residues

within

Å

from

the

surface

of

the

active

site

cavity

of

Species

information

Pseudomonas-Putida

Bacillus

Megaterium

Saccharopolyspora-Erythreaea

Pseudomonas-SP

Archaeon

Sulfolobus

Solfataricus

Fusarium-Oxysporum

Mycobacterium

Tuberculosis

Oryctolagus

Cuniculus

and

Homo

Sapiens

and

Because

of

the

low

sequence

identity

of

the

family

we

have

chosen

to

create

a

hybrid

template

for

using

MOE-Homology

-LSB-

-RSB-

constructed

from

the

crystal

structures

of

-LRB-

pdb

code

-RRB-

and

-LRB-

pdb

code

-RRB-

Our

criteria

for

using

and

involve

similarity

in

functionality

of

both

the

cytochrome

reduction

system

and

ligand

characteristics

but

importantly

also

involves

the

spatial

positioning

of

active

site

regions

Thus

far

all

modelling

attempts

on

cytochrome

family

have

included

the

usage

of

microsomal

such

as

-LSB-

-RSB-

and

-LSB-

-RSB-

However

the

family

belongs

to

the

bacterial/mitochondrial

cytochrome

class

which

obtains

electrons

from

the

ferredoxin

reductase

family

in

the

electron

transfer

chain

-LSB-

-RSB-

Using

for

the

modelling

of

mitochondrial

is

therefore

more

intuitive

and

has

been

successfully

applied

to

other

mitochondrial

-LSB-

-RSB-

The

natural

ligands

of

the

family

are

steroids

and

steroids

can

be

substrates

for

hepatic

cytochromes

that

belong

to

the

microsomal

cytochrome

class

In

and

steroids

are

oxidised

on

the

β-side

of

the

steroid

skeleton

at

carbon

atoms

close

to

and

-LSB-

-RSB-

and

their

crystal

structures

may

possess

the

necessary

interaction

features

for

model

construction

However

investigation

of

the

crystal

structure

of

-LRB-

PDB

code

-RRB-

raised

doubt

on

the

appropriateness

for

its

use

as

a

template

structure

The

crystal

structure

is

resolved

at

low

resolution

-LRB-

Å

-RRB-

in

a

tetramer

complex

In

this

complex

numerous

amino

acids

participate

in

an

extensive

interaction

network

between

the

monomers

and

the

structure

was

therefore

excluded

from

our

modelling

work

Of

the

other

hepatic

enzymes

can

also

oxidise

steroids

but

its

oxidation

sites

are

on

the

opposite

side

of

the

steroid

skeleton

or

on

the

different

steroid

rings

This

means

that

the

steroids

bind

with

a

rotated

or

reversed

orientation

in

the

active

site

-LSB-

-RSB-

which

may

provide

different

active

site

conformation

than

the

family

Furthermore

many

substrates

are

characterised

by

a

basic

nitrogen

-LSB-

-RSB-

and

the

substrates

are

mostly

weakly

acidic

-LSB-

-RSB-

These

properties

are

found

to

be

less

desirable

for

modelling

of

the

family

than

the

functionally

similar

properties

of

the

cyclic

alkane

substrates

of

The

structures

of

also

do

not

provide

information

about

the

molecular

basis

of

regioselectivity

of

the

substrate

since

a

conformational

change

of

the

active

site

is

required

to

allow

the

substrate

hydroxylation

sites

to

contact

the

heme

-LSB-

-RSB-

Because

of

the

different

ligand

properties

the

structures

of

hepatic

and

were

found

to

be

less

suitable

as

template

structure

An

important

decision

criterion

to

use

as

the

template

is

also

its

spatial

properties

When

comparing

its

active

site

cavity

to

that

of

the

mammalian

we

found

that

the

steroidal

ligands

would

not

be

able

to

fit

into

the

active

site

cavity

of

the

mammalian

with

and

oriented

to

the

heme

unless

additional

space

is

introduced

near

helix

K.

Because

the

most

important

interactions

between

protein

and

ligand

take

place

near

the

heme

the

regions

lining

the

active

site

must

be

modelled

with

the

highest

accuracy

From

the

multiple

sequence

alignment

with

the

family

one

can

see

that

beta-sheet

following

helix

K

possesses

a

amino

acid

insertion

in

the

structure

and

a

amino

acid

deletion

in

the

mammalian

structures

-LRB-

Fig.

-RRB-

Although

it

is

possible

to

model

this

site

through

a

deletion

in

the

mammalian

structures

the

resulting

active

site

cavity

would

become

strained

Since

the

structure

of

is

elongated

modelling

a

amino

acid

insertion

and

relaxing

the

fold

will

create

a

better

definition

of

this

active

site

region

Taking

into

account

that

its

substrate

and

reduction

system

are

similar

to

that

of

the

family

is

found

to

possess

the

best

structural

core

for

the

modelling

of

the

isoforms

Because

the

structural

core

of

the

cytochrome

enzyme

structure

is

the

four-helix

bundle

comprising

the

helices

D

E

I

and

L

and

the

two

helices

J

and

K

they

should

all

be

taken

from

one

and

the

same

template

structure

-LSB-

-RSB-

Hence

when

taking

the

helices

I

and

K

from

the

other

regions

must

be

used

as

well

Within

the

topological

alignment

of

these

regions

the

mammalian

contain

insertions

and

deletions

in

the

connectivity

between

helix

C

and

D

which

is

an

additional

reason

why

we

have

decided

to

construct

the

models

using

the

structure

Using

the

main

structural

features

of

the

remaining

variant

regions

of

the

fold

-LRB-

helices

B

F

G

J

and

the

meaner

region

-RRB-

were

modelled

with

was

chosen

because

after

aligning

the

desired

structures

it

was

found

to

possess

slightly

better

spatial

alignment

with

than

the

other

mammalian

crystal

structures

and

additionally

is

a

steroid

synthase

itself

Special

attention

was

given

to

the

modelling

of

helix

B

by

and

its

connection

to

the

helices

F

and

G

This

three

helical

complex

makes

up

the

entrance

of

the

active

site

cavity

and

closes

the

pocket

like

a

lid

The

helix

B

is

predicted

to

be

turns

long

in

the

family

and

there

is

an

apparent

insertion

of

residues

compared

to

that

of

the

other

cytochrome

structures

-LRB-

Fig.

-RRB-

In

the

structure

this

helix

is

positioned

too

high

in

the

active

site

cavity

which

is

probably

a

reason

why

it

is

regularly

seen

as

inappropriate

to

model

on

We

chose

the

structure

of

where

we

extended

its

helix

from

a

turn

length

to

a

turn

length

The

first

N-terminal

residues

corresponding

to

the

membrane

binding

region

of

the

human

cytochrome

family

were

omitted

from

the

models

because

no

complementary

sequence

is

present

in

the

crystal

structures

of

either

or

Some

manual

adjustments

were

made

to

the

model

to

compensate

for

large

steric

hindrances

and

the

model

was

refined

by

energy

minimisation

using

a

MOE

succession

method

of

steepest

descent

conjugate

gradient

and

truncated

newton

until

an

RMS

gradient

of

kcal

-LRB-

mol

Å

-RRB-

was

reached

The

forcefield

-LSB-

-RSB-

was

used

with

a

dielectric

constant

of

and

all

backbone

atoms

were

tethered

with

a

force

constant

of

kcal

-LRB-

mol

Å2

-RRB-

to

prevent

large

movements

To

investigate

the

influence

of

the

triple

mutant

introduced

in

the

protein

by

Bottner

et

al.

-LSB-

-RSB-

a

mutant

homology

model

-LRB-

-RRB-

was

created

The

three

dimensional

architecture

of

this

structure

should

turn

out

to

be

similar

to

the

active

site

of

Models

for

and

were

also

constructed

by

amino

acid

replacement

in

the

model

of

followed

by

a

structural

relaxation

of

the

fold

with

MOE

The

four

ligands

-LRB-

DOC

-RRB-

corticosterone

-LRB-

B

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

were

fit

in

the

active

sites

of

all

models

matching

all

three

hydroxylation

sites

-LRB-

-RRB-

to

the

oxygen

atom

occupying

the

ligating

position

of

the

heme

iron

First

the

steroids

were

docked

into

the

active

site

using

automated

docking

to

get

an

impression

of

the

relative

positioning

of

the

and

above

the

heme

Next

alternate

orientations

of

the

steroids

were

investigated

by

visual

inspection

taking

into

account

alternate

folding

of

amino

acid

side

chains

A

distance

of

Å

was

chosen

as

a

starting

distance

between

the

oxygen

and

carbon

atoms

which

is

a

near-optimal

distance

for

hydrogen

atom

abstraction

during

hydroxylation

-LSB-

-RSB-

A

threshold

of

Å

was

estimated

to

be

the

representative

maximal

distance

for

hydroxylation

The

models

were

then

equilibrated

with

MOE

without

tethering

the

backbone

atoms

allowing

the

model

active

sites

to

diverge

Ligand

docking

The

docking

of

all

ligands

was

performed

using

GOLD

-LSB-

-RSB-

in

order

to

investigate

the

protein-ligand

interactions

and

investigate

the

application

of

the

models

for

inhibitor

potency

prediction

The

docking

parameters

used

were

taken

from

the

default

GOLD

GA

settings

These

settings

were

used

for

all

docking

runs

presented

in

this

study

Each

ligand

was

docked

times

for

poses

each

for

which

the

GOLD

Fitness

score

was

obtained

using

the

standard

Goldscore

function

The

amount

of

docking

runs

and

poses

was

taken

to

ensure

an

appropriate

sampling

of

the

ligand

conformations

in

the

active

site

of

the

protein

For

the

inhibitors

the

fitness

score

was

averaged

for

the

top

ranking

poses

if

the

conformation

was

within

an

RMSD

of

Å

from

the

best

ranking

pose

-LRB-

results

Table

-RRB-

Subsequently

to

get

an

impression

of

the

correlation

of

the

docking

with

the

in

vitro

data

the

Goldscore

measure

of

binding

affinity

was

calculated

by

the

method

reported

by

Verdonk

et

al.

-LSB-

-RSB-

per

ΔGbinding

Goldscore

-LRB-

N

ΔGbinding

expressed

in

kcal/mol

-RRB-

The

average

poses

were

also

rescored

with

the

Chemscore

scoring

function

-LSB-

-RSB-

Docking

of

steroids

was

performed

in

the

presence

of

an

iron-bound

oxygen

atom

and

their

conformations

were

afterwards

checked

to

investigate

alternate

orientations

of

the

steroid

in

the

active

site

cavity

The

protein

structures

used

for

docking

the

substrates

were

the

unequilibrated

structures

whereas

for

docking

the

inhibitors

the

and

models

were

used

after

they

were

equilibrated

with

the

ligand

All

docking

runs

were

performed

in

the

absence

of

water

molecules

For

each

inhibitor

the

best

ranked

pose

was

used

as

input

for

the

molecular

dynamics

study

Molecular

dynamics

of

inhibitors

Several

molecular

dynamics

simulations

were

performed

to

investigate

the

stability

of

the

enzyme

models

of

and

and

the

dynamic

behaviour

of

the

three

inhibitors

inside

the

respective

active

sites

The

simulations

were

performed

with

the

NAMD

package

-LSB-

-RSB-

using

the

forcefield

-LSB-

-RSB-

The

protein

models

of

and

were

solvated

in

a

Å

equilibrated

water

box

removing

any

overlapping

water

Counter-ions

were

added

to

ensure

an

overall

net

charge

of

zero

The

protein-ligand

complexes

were

slightly

equilibrated

for

ps

at

a

temperature

of

K

and

were

subsequently

simulated

for

at

a

temperature

of

K

in

an

NPT

ensemble

All

simulations

were

carried

out

using

periodic

boundary

conditions

To

calculate

the

electrostatic

interactions

we

chose

the

implementation

of

NAMDs

Particle

Mesh

Ewald

-LSB-

-RSB-

To

accommodate

the

filling

of

the

active

site

cavity

volume

by

the

three

ligands

three

and

four

explicit

water

molecules

were

positioned

in

the

active

sites

of

and

respectively

-LRB-

volumes

are

Å3

Å3

etomidate

Å3

fadrazole

Å3

metyrapone

Å3

water

Å3

-RRB-

The

positioned

water

molecules

were

optimised

in

the

equilibration

step

Cellular

assay

for

measuring

inhibitor

in

vitro

activity

For

determining

inhibitor

potencies

for

and

an

assay

was

used

in

which

cells

overexpressing

or

convert

the

used

steroid

substrates

for

these

cytochromes

into

their

products

Product

formation

rates

were

assessed

by

HPLC

cells

overexpressing

-LRB-

stably

transfected

with

a

vector

carrying

a

hygromycin

resistance

box

-RRB-

were

constructed

at

NV

Organon

-LRB-

Oss

the

Netherlands

-RRB-

These

cells

were

constructed

in

the

following

manner

The

full

length

cDNA

for

human

was

obtained

by

PCR

from

human

adrenal

cDNA

as

described

by

Kawamoto

et

al

-LSB-

-RSB-

and

cloned

into

pPCR

SCRIPT

-LRB-

Stratagene

La

Jolla

USA

-RRB-

After

digestion

with

Xho

I

and

Not

I

the

obtained

cDNA

was

inserted

into

a

Xho

I/Not

I

digested

vector

-LRB-

InVitrogen

Breda

Netherlands

-RRB-

carrying

a

hygromycin

resistance

box

Following

transfection

to

the

cells

positive

cells

were

selected

based

on

their

hygromycin

resistance

The

presence

of

the

gene

was

confirmed

by

PCR

and

the

presence

of

activity

-LRB-

i.e.

the

ability

to

produce

corticosterone

or

cortisol

from

or

respectively

-RRB-

Although

no

electron-transporting

proteins

were

co-transfected

the

expressing

cells

showed

abundant

activity

as

was

previously

also

shown

by

Denner

et

al.

for

both

and

-LSB-

-RSB-

In

normal

-LRB-

non

transfected

-RRB-

cells

no

detectable

activity

or

PCR

signals

were

found

cells

stably

-LRB-

over

-RRB-

expressing

were

developed

in

the

laboratory

of

Prof.

R.

Bernhardt

Institute

of

Biochemistry

Saarland

University

Saarbrücken

Germany

-LSB-

-RSB-

Cells

were

cultured

under

standard

conditions

in

medium

-LRB-

Gibco

Gaitersburg

USA

-RRB-

supplemented

with

foetal

calf

serum

-LRB-

Hyclone

Logan

USA

-RRB-

penicillin/streptomycin

-LRB-

U/mL

and

μg/mL

respectively

Gibco

-RRB-

inside

a

humid

environment

of

°C

and

atmosphere

For

assessing

inhibitor

potencies

cells

were

transferred

to

well

plates

and

grown

until

they

were

confluent

Next

cells

were

incubated

for

h

in

serum-free

medium

with

cumulative

inhibitor

concentrations

followed

by

addition

of

nM

-LRB-

times

Km

-RRB-

-LRB-

Sigma-Aldrich

St

Louis

USA

-RRB-

as

substrate

Finally

cells

were

incubated

for

another

h

and

cells

for

another

h.

Medium

was

removed

and

the

-LRB-

produced

-RRB-

steroids

were

extracted

with

mL

diethylether

from

mL

medium

aliquots

mixed

with

mL

M

sodium-glycine

buffer

-LRB-

pH

-RRB-

containing

nM

methylprednisolone

-LRB-

Sigma-Aldrich

-RRB-

as

internal

standard

Extracts

were

dried

under

nitrogen

and

dissolved

in

mobile

phase

for

automated

HPLC

analysis

using

a

stationary

phase

consisting

of

an

MR

column

-LRB-

mm

particle

size

μm

-RRB-

and

a

mobile

phase

consisting

of

a

mixture

of

-LRB-

v/v/v

-RRB-

milliQ

water

acetonitrile

and

trifluoro-acetic

acid

Detection

of

UV

absorption

was

performed

at

nm

-LRB-

Shimadzu

Tokyo

Japan

-RRB-

Results

and

discussion

Model

quality

One

of

the

theorems

often

applied

to

the

quality

assessment

of

a

protein

model

is

the

Ramachandran

Plot

which

is

an

indicative

measure

for

the

correctness

of

the

residue

torsion

angles

The

plot

is

a

graphic

display

of

torsion

angle

φ

-LRB-

Cn

Nn

Cα

n

Cn

-RRB-

versus

torsion

angle

ψ

-LRB-

Nn

Cα

n

Cn

Nn

-RRB-

for

each

residue

of

the

protein

of

which

the

secondary

structure

character

of

the

residue

can

be

extracted

The

alpha-helix

character

of

a

protein

backbone

is

located

roughly

in

the

region

where

φ

and

ψ

and

the

beta-sheet

character

is

located

roughly

in

the

region

where

φ

and

ψ

-LSB-

-RSB-

In

Table

the

results

of

the

Ramachandran

Plot

are

summarised

for

both

and

models

after

they

were

equilibrated

with

the

ligand

Table

results

for

the

lowest

energy

models

of

and

and

the

crystal

structures

which

were

used

for

the

template

part

IRamachandran

Plot

-LRB-

core

regions

-RRB-

-LRB-

-RRB-

Ramachandran

Plot

-LRB-

favourable

regions

-RRB-

-LRB-

-RRB-

-LRB-

quality

factor

-RRB-

aVerify

-LRB-

total

score

-RRB-

bTemplate

-LRB-

PDB

resolution

-RRB-

-LRB-

Å

-RRB-

-LRB-

Å

-RRB-

a

Outliers

are

positioned

in

the

loop

regions

before

alpha-helix

F

where

the

two

template

structures

connect

and

at

the

end

of

alpha-helix

D

where

a

large

insertion

was

introducedb

Additional

outliers

are

positioned

at

the

end

of

alpha-helix

G

For

both

and

around

of

the

residues

are

positioned

in

the

favoured

and

core

regions

of

the

Ramachandran

Plot

indicating

that

for

hybrid

models

the

structures

are

of

acceptable

quality

Due

to

the

high

quantity

of

alpha-helices

and

beta-sheets

the

majority

of

residues

is

positioned

in

the

expected

regions

The

residues

which

are

situated

in

disallowed

and

unfavoured

regions

of

the

plot

are

located

in

loop

regions

outside

the

active

site

In

total

residues

in

the

model

are

situated

in

the

disallowed

regions

and

residues

in

the

unfavoured

regions

For

the

model

residues

are

situated

in

disallowed

regions

and

residues

in

the

unfavoured

regions

The

causes

for

these

disparities

are

several

insertions

or

deletions

introduced

in

the

models

for

which

the

structural

minimisation

was

not

sufficiently

adequate

to

correct

the

backbone

dihedrals

In

particular

these

regions

are

a

relatively

large

insertion

between

alpha-helix

D

and

beta-sheet

and

an

insertion

between

helix

G

and

H.

Additionally

the

amino

acid

environment

of

the

models

was

evaluated

with

Errat

-LSB-

-RSB-

and

-LSB-

-RSB-

-LRB-

Table

-RRB-

By

comparing

the

results

it

can

be

seen

that

the

models

all

score

equally

well

but

are

less

accurate

than

the

template

structures

An

Errat

quality

factor

of

is

expected

for

crystal

structures

resolved

at

a

resolution

of

Å

Using

the

Errat

score

per

amino

acid

we

were

able

to

locate

the

deviations

in

the

loop

regions

at

the

end

of

helix

E

where

our

two

template

structures

connect

and

at

the

end

of

helix

D

where

a

large

insertion

was

introduced

It

is

likely

that

in

the

minimisation

protocol

with

tethered

heavy

atoms

the

connectivity

between

template

structures

has

not

been

fully

optimised

Investigation

of

the

amino

acid

environment

with

resulted

in

similar

conclusions

as

found

with

Errat

and

the

Ramachandran

data

The

low

scoring

of

can

be

attributed

to

a

bad

folding

of

the

regions

around

the

helices

D

and

F

and

additional

potential

errors

were

located

at

the

end

of

helix

G

where

again

the

two

templates

have

been

connected

to

each

other

Finally

the

stereochemistry

of

the

models

was

analysed

with

MOE

-LRB-

Table

-LSB-

-RSB-

-RRB-

Except

for

the

already

determined

structural

flaws

in

the

regions

around

helices

D

F

and

G

no

additional

flaws

were

found

using

these

measurements

indicating

that

the

overall

fold

of

the

protein

is

of

acceptable

quality

Table

results

for

the

lowest

energy

models

of

and

and

the

crystal

structures

which

were

used

for

the

template

part

IIMOE-protein

reportObserved

-LRB-

-RRB-

Observed

-LRB-

-RRB-

Observed

modelObserved

modelReference

valuesaParameterMeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D.Trans-Omega176.62

C-alpha

chirality32.83.634.31.730.811.130.810.833.84.2Chi1-gauche

minus

plus55.420.756.316.451.526.753.028.364.115.7Chi1-trans185.313

Helix

phi

Helix

psi

S.D.

Proline

phi

Dihedral

angle

length

were

generated

with

the

MOE

module

protein

Eval

The

thresholds

were

chosen

to

be

for

the

Z-Score

and

for

the

vanderWaals

contactsa

Reference

values

were

published

in

a

statistical

survey

of

the

high-resolution

data

in

the

Protein

Data

Bank

-LSB-

-RSB-

of

the

structural

flaws

are

located

at

residues

in

the

active

site

or

at

residues

lining

the

active

site

The

occurrence

of

Ramachandran

errors

and

problems

with

modelling

external

loop

regions

seems

an

inevitable

circumstance

in

homology

modelling

-LSB-

-RSB-

and

will

probably

not

pose

a

problem

for

modelling

protein-ligand

interactions

All

the

abovementioned

errors

are

expected

to

be

alleviated

during

further

investigation

of

protein-ligand

interactions

using

molecular

dynamics

simulations

Therefore

the

current

models

have

been

selected

as

appropriate

starting

points

for

further

analysis

Protein-substrate

interactions

After

quality

assessment

of

the

created

protein

models

encompassing

the

various

ligands

we

investigated

the

steroid

binding

mode

All

docking

results

from

GOLD

favoured

the

β-side

of

the

steroid

oriented

to

the

heme

with

the

pointing

towards

alpha-helix

B

-LRB-

Fig.

-RRB-

Visual

inspection

revealed

that

in

case

of

reverse

orientation

of

the

steroid

unfavourable

clashes

of

the

in

the

opposite

side

of

the

pocket

would

occur

After

docking

the

steroids

into

the

active

site

the

protein

ligand

complexes

were

subjected

to

MOE

minimisation

as

described

in

the

homology

modelling

section

After

minimisation

the

distance

between

the

heme

iron

atom

and

the

different

substrate

hydroxylation

sites

-LRB-

and

-RRB-

was

measured

The

results

are

summarised

in

Table

Fig.

binding

of

-LRB-

-RRB-

inside

the

active

site

for

the

synthesis

of

aldosterone

The

heme

prosthetic

group

contains

a

bound

oxygen

atom

needed

for

catalytic

function

The

model

possesses

in

alpha-helix

B

which

is

stabilised

by

in

alpha-helix

I.

possesses

several

hydrogen

bonds

one

internal

hydrogen

bond

between

the

and

the

two

hydrogen

bonds

between

the

and

the

backbone

carbonyls

of

and

and

finally

a

hydrogen

bond

between

the

and

distance

table

-LRB-

iron

atom

carbon

atom

-RRB-

after

minimisation

with

MOE

-LRB-

distances

in

Angstrom

-RRB-

.305.614.374.655.324.304.565.484.304.754.834.704.245.5418

a4.314.515.21b4.314.605.19b4.304.685.17b4.324.315.39aB5.394.065.465.374.405.225.434.395.285.334.494.945.284.215.2018OH-B4.864

aa

Ligand

group

forms

a

hydrogen

bond

with

the

group

of

the

ligandb

Ligand

group

forms

a

hydrogen

bond

with

the

iron-oxygen

of

the

proteinc

Ligand

group

forms

a

hydrogen

bond

with

the

group

of

the

ligandd

Ligand

group

forms

a

hydrogen

bond

with

the

group

of

the

ligand

All

ligands

showed

two

very

distinct

interactions

in

the

modelled

active

site

cavities

Firstly

the

ligands

possess

a

steric

fit

for

the

and

the

in

a

small

cavity

created

between

helix

K

and

beta-sheet

Inside

this

cavity

the

group

possesses

two

hydrogen

bonding

interactions

with

the

protein

backbones

of

and

-LRB-

Fig.

-RRB-

The

presence

of

these

amino

acids

in

the

active

site

cavity

coincides

with

the

models

of

Belkina

et

al.

but

for

those

models

no

interactions

between

protein

and

ligand

were

discussed

-LSB-

-RSB-

Secondly

the

ligands

possess

an

interaction

between

the

and

active

site

residue

in

helix

B

is

stabilised

by

in

helix

I

which

has

further

stabilising

interactions

with

the

protein

backbone

coincides

with

an

aspartic

acid

in

the

family

which

from

visual

inspection

of

the

crystal

structures

of

-LRB-

pdb-code

-RRB-

and

-LRB-

pdb-code

-RRB-

seems

to

play

a

specific

stabilising

role

in

the

active

site

structure

it

is

expected

that

has

the

same

stabilising

role

The

difference

in

chain

length

between

an

aspartic

acid

and

a

glutamic

acid

also

determines

the

flexibility

of

helix

B

It

can

move

Å

further

out

of

the

active

site

cavity

in

all

models

allowing

the

steroid

to

fit

parallel

to

the

heme

The

ligands

also

possess

many

hydrophobic

interactions

in

this

region

particularly

with

-LRB-

Fig.

-RRB-

Due

to

the

close

interactions

with

the

A

and

B

rings

of

the

steroid

skeleton

this

amino

acid

might

play

an

important

role

in

substrate

stabilisation

The

presence

of

in

our

models

coincides

with

the

models

of

Ulmschneider

et

al.

-LSB-

-RSB-

where

it

is

seemingly

involved

in

ring

stacking

with

their

inhibitors

There

are

also

two

striking

differences

between

the

and

models

Firstly

the

active

site

cavity

near

beta-sheet

is

smaller

in

both

models

compared

to

the

models

with

being

the

largest

This

small

cavity

is

formed

by

the

loop

region

between

helix

K

and

beta-sheet

-LRB-

Fig.

-RRB-

Comparing

this

cavity

in

both

the

and

models

we

find

that

in

this

cavity

has

a

larger

volume

dependent

on

amino

acid

flexibility

as

well

as

the

probe

radius

chosen

for

calculation

of

the

volume

-LRB-

differing

from

to

Å

-RRB-

This

difference

in

size

is

caused

by

the

folding

of

which

induces

a

Å

outward

shift

of

the

loop

region

A

comparison

of

the

models

with

the

crystal

structures

of

the

family

reveals

that

the

loop

region

in

the

models

is

in

closer

proximity

to

helix

I

-LRB-

by

Å

-RRB-

This

contact

is

defined

by

two

amino

acids

having

relatively

smaller

sidechains

-LRB-

and

in

-RRB-

than

observed

in

the

family

-LRB-

generally

Thr/Val

and

Leu/Ile

-RRB-

Changes

in

helix

I

such

as

the

mutant

between

and

are

likely

to

have

a

direct

influence

on

the

folding

of

this

region

Fig.

site

volume

difference

between

the

-LRB-

white

-RRB-

and

models

-LRB-

orange

-RRB-

Shown

for

both

and

are

the

active

site

volume

and

the

backbone

trace

R-fadrazole

and

S-fadrazole

are

indicated

with

blue

and

purple

respectively

It

is

clear

to

see

that

contains

a

larger

active

site

between

helix

I

and

sheet

This

cavity

allows

S-fadrazole

to

fit

the

cavity

but

not

R-fadrazole

-LRB-

black

arrow

-RRB-

On

the

other

side

of

the

active

site

near

Helix

B

and

contains

the

larger

cavity

which

might

rationalise

the

better

fit

of

R-fadrazole

in

the

cavity

Secondly

helix

B

is

shifted

outward

by

Å

in

both

models

-LRB-

the

backbone

RMSD

of

helix

B

of

and

is

Å

-RRB-

The

change

in

spatial

positioning

of

this

helix

is

probably

caused

by

the

mutants

and

in

helix

I

but

because

this

region

is

on

the

surface

of

the

protein

the

exact

cause

is

less

clear

In

the

model

contacts

helix

B

at

which

forms

the

counter

charge

for

the

C-terminal

end

of

this

helix

Hasemann

et

al

have

posed

that

movement

of

helices

B

F

and

G

is

involved

in

the

opening

of

the

active

site

cavity

-LSB-

-RSB-

Changing

the

stabilising

environment

of

helix

B

has

a

likely

effect

on

the

structural

stability

of

the

helix

and

may

result

in

a

loss

of

activity

Introducing

the

triple

mutant

investigated

by

Bottner

et

al.

-LSB-

-RSB-

by

means

of

the

model

showed

that

the

hydroxylation

distance

pattern

of

the

substrates

shifts

as

expected

from

to

that

of

Both

and

display

similar

active

site

cavities

near

beta-sheet

-LRB-

backbone

RMSD

Å

-RRB-

with

only

slight

deviations

in

the

hydroxylation

distances

for

the

ligands

-LRB-

Table

-RRB-

This

confirms

the

behaviour

of

the

triple

mutant

in

enzymatic

activity

found

by

the

study

of

Bottner

et

al.

To

speculate

further

Bottner

et

al

have

shown

that

the

mutant

alone

or

the

mutant

alone

is

not

enough

to

significantly

change

aldosterone

synthesis

-LSB-

-RSB-

Only

mutation

in

both

regions

results

in

an

almost

complete

loss

of

the

aldosterone

synthesis

capabilities

of

It

is

likely

that

the

subtle

changes

on

both

sides

of

the

active

site

go

hand-in-hand

to

-LRB-

nearly

-RRB-

completely

convert

the

activity

of

into

that

of

For

DOC

our

results

indicate

that

and

preferentially

catalyse

whereas

and

preferentially

catalyse

For

both

models

the

shifted

alpha-helix

B

causes

DOC

to

present

its

closest

to

the

iron

as

it

is

repositioned

by

the

strong

interaction

of

the

group

with

in

alpha-helix

B

Additionally

the

larger

active

site

of

around

beta-sheet

allows

DOC

to

fit

further

into

the

niche

presenting

into

hydroxylation

range

-LRB-

distance

less

than

Å

-RRB-

A

point

of

argument

against

a

preferred

of

DOC

by

is

that

in

vitro

measurements

indicate

higher

levels

of

the

product

B

-LSB-

-RSB-

However

both

and

products

of

DOC

can

be

promptly

consumed

as

a

substrate

for

the

production

of

and

subsequently

aldosterone

in

in

vitro

-LSB-

-RSB-

This

apparent

discrepancy

between

observed

and

predicted

regioselectivity

of

DOC

hydroxylation

by

may

possibly

indicate

that

other

factors

than

hydroxylation

distances

are

involved

in

the

formation

of

the

actual

products

For

the

group

forms

a

hydrogen

bond

with

the

iron

oxygen

limiting

hydroxylation

to

which

is

in

closest

proximity

to

the

iron

In

the

models

both

and

are

in

approximately

equal

distance

to

the

iron

and

an

internal

hydrogen

bond

is

formed

by

the

substrate

between

the

group

and

the

Although

both

and

are

shown

to

be

possible

in

such

a

complex

oxidation

on

the

unsubstituted

is

likely

to

form

a

more

stable

reaction

intermediate

In

all

models

the

two

ligands

B

and

only

portray

in

close

proximity

to

the

iron

as

the

group

blocks

access

of

to

the

heme

iron

The

positioning

of

the

group

of

in

the

active

site

cavity

appears

to

determine

conversion

into

aldosterone

In

both

models

the

natural

substrate

is

shifted

above

the

heme

which

creates

a

slightly

larger

active

site

cavity

near

beta-sheet

for

the

group

to

rotate

in

This

difference

in

size

allows

room

for

an

internal

hydrogen

bond

between

the

group

and

the

for

both

and

In

and

the

group

forms

a

hydrogen

bond

with

the

heme

oxygen

atom

which

blocks

for

hydroxylation

-LRB-

Fig.

-RRB-

Interestingly

in

the

the

group

forms

an

internal

hydrogen

bond

with

the

group

and

thereby

also

blocks

the

for

hydroxylation

In

conclusion

we

propose

that

the

immediate

folding

of

the

active

site

around

the

substrate

group

may

be

the

key

difference

between

the

two

isoforms

leading

to

the

production

of

aldosterone

by

and

not

by

To

speculate

further

on

the

mechanism

of

the

enzymatic

formation

of

aldosterone

from

we

hypothesise

that

a

stable

internal

hydrogen

bond

for

the

group

of

the

ligand

may

be

essential

to

stabilise

its

against

further

oxidation

A

second

oxygen

atom

can

then

be

inserted

between

and

one

of

its

hydrogen

atoms

forming

a

intermediate

which

forms

aldosterone

by

elimination

of

water

Although

the

formation

of

a

has

been

proposed

by

Johnston

et

al.

-LSB-

-RSB-

the

precise

mechanism

of

oxidation

is

still

unknown

This

proposed

method

of

substrate

stabilisation

may

explain

the

regioselectivity

of

the

two

isoforms

and

can

be

attributed

to

the

subtle

changes

observed

on

two

sides

of

the

active

site

cavity

the

cavity

near

helix

K

and

the

positioning

of

helix

B

Protein-inhibitor

interactions

The

non-steroidal

inhibitors

metyrapone

R-etomidate

R-fadrazole

and

S-fadrazole

were

docked

flexibly

into

the

active

site

of

the

human

models

where

their

aromatic

nitrogen

atom

forms

a

strong

interaction

with

the

sixth

ligating

position

of

the

heme

iron

atom

The

accessible

electron

lone

pair

of

the

heterocyclic

nitrogen

atom

is

required

for

heme

iron

complexation

a

well-known

interaction

for

non-steroidal

CYP

inhibitors

-LSB-

-RSB-

The

GOLD

docking

results

showed

all

ligands

to

bind

in

one

favourable

conformation

The

enantiomers

R-fadrazole

and

S-fadrazole

mainly

favoured

one

particular

orientation

in

the

active

site

cavities

of

and

respectively

The

higher

affinity

of

S-fadrazole

to

and

R-fadrazole

to

can

be

attributed

to

the

active

site

difference

near

beta-sheet

and

helix

I

-LRB-

Fig.

-RRB-

The

difference

in

affinity

is

determined

by

the

steric

aspects

of

the

active

site

cavity

allowing

only

S-fadrazole

to

fit

in

the

active

site

of

where

R-fadrazole

possesses

a

steric

clash

-LRB-

black

arrow

-RRB-

In

it

is

less

clear

why

R-fadrazole

fits

the

active

site

best

but

this

is

mainly

determined

by

steric

effects

near

helix

B

Due

to

the

presence

of

rotatable

bonds

R-etomidate

was

able

to

dock

in

different

orientations

but

favoured

one

particular

conformation

with

its

phenyl

ring

pointing

into

the

direction

of

helix

B

The

main

flexibility

observed

was

caused

by

the

ethyl-ester

group

and

the

docking

score

of

those

conformations

was

very

similar

Metyrapone

was

able

to

dock

with

both

pyridine

rings

to

the

iron

but

mainly

favoured

a

conformation

with

the

carbonyl

facing

Since

all

ligands

favoured

only

one

orientation

it

was

this

orientation

that

was

investigated

by

MD.

Our

in

vitro

test

results

show

a

striking

enantioselectivity

of

fadrazole

binding

for

the

family

We

find

R-fadrazole

to

be

the

most

active

enantiomer

for

as

well

as

being

selective

-LRB-

Table

-RRB-

In

contrast

for

it

is

S-fadrazole

which

appears

to

be

the

most

potent

enantiomer

A

similar

stereoselectivity

as

for

has

also

been

described

for

aromatase

wwith

the

S-enantiomer

being

the

better

aromatase

inhibitor

-LSB-

-RSB-

The

docking

results

from

both

Goldscore

and

Chemscore

predict

the

same

enantioselectivity

for

fadrazole

in

the

family

as

observed

in

vitro

indicating

that

the

three

dimensional

models

contain

promising

accuracy

for

the

valid

prediction

of

enantiomer

selectivity

Docking

results

of

the

two

other

inhibitors

metyrapone

and

R-etomidate

confirm

the

trend

of

inhibitor

potency

as

determined

by

the

in

vitro

experiments

-LRB-

Table

-RRB-

Table

of

docking

and

molecular

dynamics

results

to

in

vitro

data

for

both

human

and

-LRB-

nM

-RRB-

GoldscoreΔG

Goldscore

-LRB-

kcal/mol

-RRB-

ΔG

Chemscore

-LRB-

kcal/mol

-RRB-

Unon-bonded

-LRB-

kcal/mol

-RRB-

Indicated

are

the

Goldscore

and

the

extracted

Goldscore

binding

free

energy

as

well

as

the

Chemscore

binding

free

energy

Unon-bonded

indicates

the

total

non-bonded

energies

between

the

protein

and

the

ligand

for

the

molecular

dynamics

simulations

Our

human

and

models

are

able

to

rationalise

the

-LRB-

inverse

-RRB-

enantioselectivity

of

and

inhibition

by

fadrazole

as

observed

in

the

in

vitro

tests

The

enantioselectivity

can

be

attributed

to

the

small

cavity

size

differences

of

the

models

Both

enantiomers

bind

with

a

steric

fit

comparable

to

the

A

B

and

C

rings

of

the

endogenous

substrates

where

the

cyanobenzyl

moiety

forms

a

π

π

stacking

interaction

with

In

addition

to

the

steric

interactions

both

enantiomers

possess

a

strong

polar

interaction

between

their

cyanide

moiety

and

mimicking

the

interaction

that

the

of

the

natural

substrates

possesses

with

this

amino

acid

Metyrapone

was

not

found

to

possess

any

hydrogen

bonding

or

polar

interaction

with

the

active

site

other

than

the

aromatic

nitrogen

Instead

it

possesses

a

pronounced

steric

fit

with

its

second

aromatic

ring

overlapping

the

space

occupied

by

the

A-ring

in

case

of

the

steroidal

substrate

R-etomidate

which

was

not

designed

as

a

specific

inhibitor

does

possess

a

hydrogen

bonding

interaction

The

hydrogen

bond

is

made

between

its

ester

moiety

and

the

catalytic

and

is

present

in

both

and

This

may

explain

the

non-selective

inhibitory

action

of

R-etomidate

on

both

isoforms

Its

aromatic

phenyl

ring

also

possesses

strong

steric

interactions

overlapping

the

same

space

of

the

steroidal

A-ring

like

metyrapone

Molecular

dynamics

Given

the

promising

prediction

of

the

models

for

substrate

conversion

substrate

binding

inhibitor

binding

and

the

satisfying

Ramachandran

Plots

we

conducted

molecular

dynamics

simulations

to

obtain

a

better

understanding

of

the

dynamic

behaviour

of

the

inhibitors

in

the

active

site

of

the

enzyme

models

To

investigate

the

structural

integrity

change

during

the

simulation

we

calculated

the

relative

root

mean

square

deviation

-LRB-

RMSD

-RRB-

over

all

heavy

atoms

-LRB-

Fig.

-RRB-

Fig.

for

the

molecular

dynamics

simulations

of

the

different

protein-ligand

complexes

of

the

and

models

Metyrapone

is

coloured

black

R-etomidate

green

R-fadrazole

red

and

S-fadrazole

blue

During

the

first

ps

the

RMSD

increased

and

the

protein

still

adapted

towards

its

optimal

conformation

After

this

point

in

time

hardly

any

change

in

the

three

dimensional

structures

of

the

proteins

was

observed

The

largest

fluctuations

of

the

protein

were

found

in

the

flexible

regions

with

peak

values

located

in

the

structures

around

alpha-helix

D

-LRB-

not

shown

-RRB-

In

the

random

coil

following

alpha-helix

D

we

introduced

a

large

insertion

of

seven

amino

acids

which

elongates

the

alpha-helix

by

one

turn

before

it

connects

to

the

following

beta-sheet

Inside

the

water

box

this

region

is

found

to

protrude

into

the

water

without

any

stabilising

protein

interactions

and

unfolds

due

to

interaction

with

water

In

all

the

simulations

we

observed

an

opening

of

the

active

site

and

the

continuous

flow

of

water

molecules

in

and

out

of

the

active

site

cavity

Several

water

molecules

retained

key

positions

such

as

the

water

molecules

that

make

up

the

channel

towards

the

conserved

-LRB-

not

shown

-RRB-

All

the

inhibitors

maintained

the

same

interactions

as

observed

in

the

docking

study

In

Fig.

the

resulting

poses

of

the

inhibitors

metyrapone

and

R-etomidate

are

displayed

Both

possess

a

ring

stacking

with

in

the

active

site

and

R-etomidate

also

possesses

a

hydrogen

bond

with

stabilises

the

heme

in

the

active

site

In

Fig.

the

poses

of

R-fadrazole

in

the

active

site

and

S-fadrazole

in

the

active

site

are

compared

Here

it

can

be

seen

that

in

the

model

alpha-helix

B

is

moved

further

out

of

the

active

site

In

the

model

possesses

a

polar

interaction

with

whereas

provides

a

horizontal

ring

stacking

for

R-fadrazole

In

the

model

S-fadrazole

possesses

the

same

interactions

with

the

protein

although

the

ring

stacking

with

is

vertical

rather

than

horizontal

Importantly

the

interaction

points

of

the

fadrazole

enantiomers

and

those

of

the

natural

substrate

coincide

very

well

Fig.

active

site

model

with

metyrapone

-LRB-

purple

-RRB-

and

R-etomidate

-LRB-

blue

-RRB-

in

the

active

site

Indicated

are

the

stabilising

interaction

between

and

the

heme

as

well

as

which

accommodates

a

ring

stacking

with

the

ligands

The

ester

group

of

R-etomidate

possesses

a

hydrogen

bond

with

model

-LRB-

orange

-RRB-

containing

S-fadrazole

-LRB-

purple

-RRB-

in

the

active

site

and

model

-LRB-

white

-RRB-

containing

R-fadrazole

-LRB-

blue

-RRB-

in

the

active

site

Indicated

the

stabilising

interaction

between

and

the

heme

as

well

as

the

stabilising

hydrogen

bonds

between

and

accommodates

a

ring

stacking

with

the

fadrazole

enantiomers

horizontally

for

the

R-enantiomer

and

vertically

for

the

S-enantiomer

R-fadrazole

and

S-fadrazole

displayed

different

behaviour

in

the

other

protein

models

R-fadrazole

lost

its

direct

interaction

with

the

heme

iron

atom

in

the

model

exchanged

it

for

a

water

molecule

and

drifted

to

the

top

of

the

active

site

S-fadrazole

displayed

a

similar

behaviour

in

the

model

Repeated

simulations

did

not

change

either

scenario

indicating

that

unlike

the

eutomers

the

distomers

do

not

comfortably

fit

our

starting

models

Because

the

complex

was

stable

over

the

last

ps

we

sampled

the

non-bonded

interactions

between

the

protein

and

the

ligand

for

this

time

period

-LRB-

Table

-RRB-

These

interactions

contribute

to

the

binding

free

energy

although

the

solvation

effects

of

free

ligand

still

need

to

be

subtracted

-LSB-

-RSB-

These

energies

follow

the

same

generic

trend

which

emphasise

the

strong

interactions

for

R-etomidate

in

both

models

and

the

strong

interactions

of

S-fadrazole

in

and

R-fadrazole

in

respectively

Conclusion

We

have

constructed

homology

models

of

the

two

isoforms

of

the

human

family

as

well

as

promising

starting

models

for

the

rat

isoforms

These

models

are

based

on

the

knowledge

of

substrate

specificity

which

were

defined

as

the

differences

in

hydroxylation

distances

between

the

active

site

and

the

and

of

the

steroid

skeleton

As

both

and

possess

virtually

similar

active

sites

the

steric

fit

of

the

different

steroidal

ligands

seems

to

be

one

of

the

strongest

determinants

for

substrate

specificity

We

have

found

that

within

the

active

site

of

the

model

the

endogenous

ligand

forms

a

stabilising

internal

hydrogen

bond

This

is

not

observed

for

the

model

which

might

rationalise

why

is

solely

a

substrate

for

to

yield

aldosterone

In

addition

the

interactions

of

the

with

in

alpha-helix

B

the

interaction

of

the

with

the

backbones

of

and

and

the

presence

of

are

also

important

for

the

stabilisation

of

the

ligand

in

the

protein

active

site

Determinant

for

the

interaction

of

is

the

presence

of

in

alpha-helix

I

which

stabilises

and

alpha-helix

B

in

the

active

site

The

known

non-steroidal

inhibitors

metyrapone

R-etomidate

R-fadrazole

and

S-fadrazole

were

postulated

to

occupy

the

same

space

in

the

active

site

as

the

endogenous

substrates

Conclusions

from

both

molecular

docking

and

molecular

dynamics

simulations

corroborate

the

measured

activity

data

from

in

vitro

experiments

supporting

the

validity

of

the

constructed

models

for

these

ligands

Importantly

these

models

rationalise

the

enantioselectivity

of

fadrazole

with

the

R-enantiomer

being

most

potent

on

and

the

S-enantiomer

being

most

potent

in

The

constructed

models

are

useful

tools

in

trying

to

understand

some

of

the

molecular

mechanisms

involved

in

ligand

binding

and

substrate

conversion

for

the

family

As

such

these

models

might

also

be

appropriate

tools

for

more

detailed

protein-inhibitor

modelling

studies

as

well

as

for

ligand

design

or

database

screening

following

further

model

optimisation

and

model

tuning

aldosterone synthesis
cyp11b1
cyp11b2
homology modelling
molecular docking
molecular dynamics
fadrazole