Transcript
Page 1: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Allosteric Modulation

David Hall GlaxoSmithKlineDavid Hall, GlaxoSmithKline

Page 2: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Topics

• What is an allosteric modulator?

• How do allosteric modulators behave?– Build up theory from known properties

Use theory to predict & qualify behaviours (illustrated with real world– Use theory to predict & qualify behaviours (illustrated with real world examples)

H h t i ll t i d l t ?• How can we characterise allosteric modulators?– To drive SAR– To understand mechanismTo understand mechanism– For PK/PD modelling or ‘dose prediction’

Page 3: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

What is an allosteric modulator?

• A ligand which binds to a receptor at a site distinct from that of the endogenous agonistendogenous agonist.

Competitiveorthosteric

sitep≡ Orthosteric

Orthosteric binding is mutually exclusive

s te

Allosteric

Orthosteric binding is mutually exclusive.An allosteric ligand can bind to the receptor at the same time as an allosteric

3

Allosteric orthosteric ligand. site

Page 4: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Immediate consequences of this mechanism

• The effects of an allosteric modulator are saturable – they have an upper ylimit.

• The effects of an allosteric modulator must be due to an effect onThe effects of an allosteric modulator must be due to an effect on receptor conformation (to which the orthosteric ligand is sensitive).

• Presumably then the orthosteric ligand induces a conformational change• Presumably then the orthosteric ligand induces a conformational change in the receptor to which the allosteric ligand is sensitive.

• Allosterism can be formally described in terms of ligand effects on• Allosterism can be formally described in terms of ligand effects on receptor conformation:

– Positive cooperativity the ligands have highest affinity for a common (set of) conformation(s) of the receptorthe receptor

– Negative cooperativity the ligands have highest affinity for distinct a (set of) conformation(s) of the receptor

Page 5: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Dihydrofolate ReductaseHydrogen deuterium exchange during allosteric ligand bindingHydrogen-deuterium exchange during allosteric ligand binding

Trimethoprim (TMP) & NADPH positively cooperativeFolinic acid & NADPH negatively cooperative Access of backbone

amide protons toamide protons to solvent

D d

Fig 4: Polshakov et al (2006) J Mol Biol 356 886-903

Decreased

Fig 4: Polshakov et al. (2006) J. Mol. Biol. 356, 886 903

Increased

5

15N-1H heteronuclear single quantum coherence spectroscopy

Page 6: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

More formally, in terms of binding

Competitive Allosteric

R ARKA

R ARKA

KB KB/KB

ARBRBKA/

BR ARBRBBRγ is the ‘allosteric constant’.

K & K di i ti t tKA & KB are dissociation constants,γ>1 indicates positive cooperativity

Page 7: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

A further property of allosteric modulation

• Reciprocity – the orthosteric ligand has the same effect on the allosteric y gligands affinity as the allosteric ligand has on the orthosteric ligand’saffinity.

• This is quantified by the allosteric constant.

• Reciprocity is a thermodynamic requirement of the system at equilibrium• Reciprocity is a thermodynamic requirement of the system at equilibrium (otherwise allosteric binding would provide a route to a perpetual motion machine).

Page 8: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

So what does allosterism look like: effect on bindingIn both cases [radioligand] = K

Competitive Allosteric

In both cases, [radioligand] = KA

80%

90%

100%

g

160%

180%

200%

g

50%

60%

70%

rol B

indi

ng

Increasing KB 100%

120%

140%

ol B

indi

ng

easi

ng γ

γ = 1

20%

30%

40%

% C

ontr

40%

60%

80%

% C

ontro

Incr

e

All curves

0%

10%

0.001 0.01 0.1 1 10 100 1000

C i C i

0%

20%

0.001 0.01 0.1 1 10 100 1000

M d l C i

have same KB

Competitor Concentration

Analysed by Cheng-Prusoff equation

Modulator Concentration

DO NOT use Cheng-Prusoff equation!

The effects of an allosteric modulator on binding are described by 2 parameters

Page 9: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

A real world exampleAllosteric modulation of muscarinic receptorsAllosteric modulation of muscarinic receptors

Fig 3: Proška & Tuček (1995) Mol. Pharm. 48, 696-702g ( ) ,

Page 10: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Probe Dependence

• The allosteric constant characterises the interaction of a pair of ligands – the same ll t i li d d l t diff t th t i li d t diff t t tallosteric ligand can modulate different orthosteric ligands to different extents:

Ligand Cooperativity with:[3H]NMS ACh

St h i (M ) 2 2 ± 0 3 0 15 ± 0 02Strychnine (M2) 2.2 ± 0.3 0.15 ± 0.02Brucine (M2) 1.6 ± 0.06 0.28 ± 0.05Brucine (M1) 0.9 ± 0.04 1.6 ± 0.1

Lazareno et al (1998) Mol. Pharm. 53, 573 – 589.Lazareno & Birdall (1995) Mol. Pharm. 48, 362 - 378

Page 11: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

The properties of allosteric modulation

• Saturability – the effect of an allosteric modulator is inherently limited.y y

• Reciprocity – the orthosteric ligand affects the modulator’s properties to the same extent as the modulator affects those of the orthosteric ligandthe same extent as the modulator affects those of the orthosteric ligand.

• Probe dependence – the cooperativity constants describe the obe depe de ce t e coope at ty co sta ts desc be t einteraction between pairs of ligands – screen with the endogenous agonist, where ever possible!

Page 12: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

An advantage of positive allosteric modulationMaintains the temporal characteristics of signallingMaintains the temporal characteristics of signalling

• An agonist activates receptors continually when present and may well g p y p yinduce desensitisation.

• A positive allosteric modulator only activates receptors when theA positive allosteric modulator only activates receptors when the endogenous agonist is present.

• Particularly advantageous for neurotransmitter receptorsParticularly advantageous for neurotransmitter receptors

N lNormal

Agonist+ Positive modulator

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Page 13: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Further advantages & disadvantages

• Allosteric sites may be less well conserved between receptor subtypes y p ypthan the orthosteric site (which has evolved to bind to the same ligand) giving the potential for greater selectivity.

• The potential range of effects of allosteric modulators is more varied than that of orthosteric ligands.

• Demonstrating that a non-competitive ligand is actually binding to your target receptor requires more effort than for competing ligands.

• By definition a competing ligand binds to the same binding site on the receptor as the endogenous agonist.

• An allosteric ligand binds anywhere but the orthosteric site and may not displace an orthosteric radioligand

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Page 14: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Analogy with AgonismThe two ternary complex modelsThe two ternary complex models...

Allosteric GPCR Agonism commont

R ARKA R AR

KAstep

K / K K /αI i iKB KB/γ KG KG/αIntrinsicEfficacy

ARBRBKA/γ

ARGRGKA/α

• Ligand intrinsic efficacy in the GPCR TCM is an allosteric constant• Biased agonism is essentially a manifestation of the probe

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Biased agonism is essentially a manifestation of the probe dependence of allosteric modulation

Page 15: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Link the common reaction from the two models

K /αRG ARG

K

KA/α

K /gTCM

R ARKA

KG KG/αgTCM

R AR

KB KB/γaTCM

ARBRB K /γKA/γ

Completing the reaction scheme results in a model of allosterism in functional assays

15

Completing the reaction scheme results in a model of allosterism in functional assays ...

Page 16: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Functional effects of allosteric modulatorsExemplifies the complexity of the systemExemplifies the complexity of the system

RG ARGKA/α

RG ARGKG KG/αgTCM

R ARKA

KB KB/γKB/β KB/βγδaTCM

ARBRB KA/γActivation

Cooperativity

Depends on A, B and G

Intrinsic efficacy of the modulator

ARBGRBGKG/β KG/αβδ

K /αγδ

- biased modulation!

16Hall, 2006, In: Bowery NG (ed). Allosteric Receptor Modulation in Drug Targeting. Taylor & Francis: New York, pp 39–78.

KA/αγδ

Page 17: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Characterising a modulator requires 4 parameters!

• The affinity (KB) and (intrinsic) efficacy (β) of the allosteric modulator

• i.e., characterise the modulator as a ligand in its own right

• The binding (γ) and activation (δ) cooperativity

• The characteristics of the allosteric interaction

• Each signalling pathway needs to be characterised separately!Each signalling pathway needs to be characterised separately!

• If there is more than one endogenous agonist, the cooperativity constants need to be measured for each one!constants need to be measured for each one!

• However, this means it is (theoretically) possible to design an allosteric ligand that selectively affects only the biological process that you wantligand that selectively affects only the biological process that you want to and has no effect on any other response via that receptor.

• This is impossible for an orthosteric ligand

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Page 18: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Example of biased positive allosteric modulatorGLP 1 receptor biased allosteric agonistsGLP-1 receptor biased allosteric agonists

cAMP ERK Phosph

Fi E K l l (2010) M l Ph 8 4 6 46Fig 5E: Koole et al (2010) Mol. Pharm. 78, 456-465.Fig 3D: Koole et al (2010) Mol. Pharm. 78, 456-465.

Potentiates cAMP production but has no effect of on ERK phosphorylation

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Page 19: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Example of a biased negative allosteric modulator.LPI805 at NK2 receptorsLPI805 at NK2 receptors

cAMP Ca2+

Fi 4C M ill t t l (2007) FASEB J 21 2124 2134Fig4C: Maillet et al (2007) FASEB J. 21, 2124-2134. Fig4A: Maillet et al (2007) FASEB J. 21, 2124-2134.

Negatively modulates cAMP production but (very weakly) positively modulates Ca2+

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Page 20: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

The properties of allosteric modulation

• Saturability – the effect of an allosteric modulator is inherently limited.y y

• Reciprocity – the orthosteric ligand affects the modulator’s properties to the same extent as the modulator affects those of the orthostericto the same extent as the modulator affects those of the orthostericligand.

• Probe dependence – the cooperativity constants describe theProbe dependence the cooperativity constants describe the interaction between pairs of ligands – screen with the endogenous agonist, where ever possible!

• Transducer dependence – allosteric effects may depend on the signaling pathway that you measure.

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Page 21: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

How can we quantify allosteric effects?

What can we actually measure or calculate?

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Page 22: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

First we need a theoretical model The previous model is a little too mechanistic and specificThe previous model is a little too mechanistic and specific

KA SAR ARKA SA

KB KB/γ

KA/γ βSARBRB

KA/γ βSA

SB Pragmatic solution for experimental systems which lack constitutive activity – doesn’t permit inverse agonism

22Leach et al. (2007) Trends Pharmacol. Sci. 28, 382 – 389.

Page 23: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

A more complete modelIncludes constitutive activity and the possibility of inverse agonismIncludes constitutive activity and the possibility of inverse agonism

χ

R ARKA

χ εAχ

KB KB/γ

ARBRBKA/γ

ARBRBεBχ

δεAεBχ

23Hall (2013) Prog. Mol. Biol. Translational Sci. (Kenakin T, ed) 115, 217 – 290

Page 24: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Behaviour of the modelInteraction of intrinsic efficacy: γ = δ = 1 vary εInteraction of intrinsic efficacy: γ = δ = 1, vary εB

1 1

εB = 10

0 5 asin

g ε B

εB = 1max 0.5

0 1

B

0.5

Incr

ea

B

E/E

0ax

εB = 0.1

00.0001 0.01 1 100

Modulator conc.

00.0001 0.01 1 100

E/E

ma

1

0.5

εB = 10

εB = 0.1The Leach et al model doesn’t account for this aspect of an allosteric interaction

24Agonist conc.

00.0001 0.01 1 100

Page 25: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Real World Example?Allosteric GLP1 agonistAllosteric GLP1 agonist

x

1βγ = 25

odul

inm

a

0 5max

% o

xyto

mo 0.5

E/E

m

εB = 100

%

00.0001 0.001 0.01 0.1 1 100.0001 0.001 0.01 0.1 1 10

Agonist conc.

Note the GLP1 receptor data requires some negative cooperativity to cause the relatively small level of leftward shift seen in this case

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small level of leftward shift seen in this case.

Koole et al (2010) Mol. Pharm. 78, 456-465.

Page 26: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Behaviour of the modelBinding cooperativity: ε = δ = 1 vary γBinding cooperativity: εB = δ = 1, vary γ

1 1

γ = 10

0 5 asin

g γ

γ = 1max 0.5

0 1

γ

0.5

Incr

ea

E/E

0ax

γ = 0.1

00.0001 0.01 1 100

Modulator conc.

00.0001 0.01 1 100

E/E

ma

1

0.5

γ = 10

γ = 0.1

2626Agonist conc.

00.0001 0.01 1 100

Page 27: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Real World Examples?DFB at mGluR5; CCR4 antagonistDFB at mGluR5; CCR4 antagonist

Difluorobenzaldazine: PAM at mGluR5 NAM at CCR4N

S

NH

1 9

2.1Control+ 3M

N

tin c

onte

nt

1.7

1.9 + 3M+ 10M+ 30M

Fig 4: O’Brien et al (2003) Mol. Pharm. 64, 731-740

Rel

ativ

e F-

act

1.3

1.5

R

0.9

1.1

10-12 10-10 10-910-11 10-8 10-7

CCL17 concentration / M

27Weston & Hall (2008) P066 BPS Winter Meeting

Page 28: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Behaviour of the modelActivation cooperativity: ε = γ = 1 vary δActivation cooperativity: εB = γ = 1, vary δ

1 1

δ = 10

0 5 asin

g δ

δ = 1max 0.5

δ 0 10.5

Incr

ea

E/E

0ax

δ = 0.1

00.0001 0.01 1 100

Modulator conc.

00.0001 0.01 1 100

E/E

ma

1

0.5 δ = 0 1

δ = 10

δ = 0.1

282828Agonist conc.

00.0001 0.01 1 100

Page 29: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Real World Examples?CCR4 antagonistsCCR4 antagonists

For this profile see next slide:+ve activation coop with –ve binding coop

CCL17 concentration / M

We have seen little evidence of inverse agonism with these compounds in any system

29Slack et al. (2013) Pharm. Res. Persp. 1, e00019

We have seen little evidence of inverse agonism with these compounds in any system

Page 30: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

The product γδεB(≈βγ in the Leach et al model)(≈βγ in the Leach et al. model)

• Can be used to characterise the overall effect of an allosteric modulator• But DOES NOT represent a unique profile of effect• But DOES NOT represent a unique profile of effect

γδεB = 0.1

1

γ = 10, δ = 0.0003, εB = 30

C t l1

γ = 10, δ = 0.1, εB = 0.1

1

γ = 0.01, δ = 10, εB = 1.

0 5

Control

0 5 0 5

εA = 1000 εA = 30

Controlmax

0.50.5 0.5

ε = 1000

Control

ControlE/E

m

00.0001 0.01 1

00.0001 0.01 1

00.0001 0.01 1

εA = 1000

Agonist conc. cf. cpd 7 on

30

previous slide

The overall effect of a compound is the summation of its properties

Page 31: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

What can we measure? XC50How far can XC take us?How far can XC50 take us?

A

1

εB

BAB

AA

B

AKAK

XC

11

11

50

0.5

ncre

asin

g

εB = 1

E/E

max

BAA

B K

This is a complicated function of the

0

Inaffinity, intrinsic efficacy and the cooperativity constants.

O ti i i t DOES NOT0.0001 0.01 1 100Modulator conc.

Optimising potency DOES NOToptimise any specific property.

The maximal effect of a modulator is a similarly composite parameter.

NB: XC50 does NOT translate between experimental systems – it

31

C50 does O t a s ate bet ee e pe e ta syste s tCAN’T be used to predict effects in one system based on another

Page 32: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

What can we measure? Use of concentration-ratiosWhen the curves are ‘sufficiently parallel’When the curves are sufficiently parallel

1Assuming εA >> (1, εB),

10.0

o

KB = 3δ = 0.1

0 5max

δ = 10

1

max BDR

Assuming εA (1, εB),

1.0ratio

n-ra

tio

A2 = 3.75

0.5

E/E

δ = 0.1 1

max2 21 DRAKB

21max50 DRAKBor,

0

Con

cent

r

A0.5 = 0.375

δ 1000.0001 0.01 1 100

Agonist conc.

max5.0B,0.1

0.01 0.1 1 10 100 1000

Modulator conc. BpKpA 2

δ = 10

Thus, under some generally reasonable (and testable) assumptions, a classical null analysis of the curve shifts can provide an estimate of affinity and y ythe overall allosteric effect of a modulator.

This does rely on us being able to define a meaningful concentration-ratio, so the behaviour can’t be too exotic (e g γ = 10 δ = 0 0003 ε = 30)

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the behaviour can t be too exotic (e.g. γ = 10, δ = 0.0003, εB = 30).

Hall (2013) Prog. Mol. Biol. Translational Sci. (Kenakin T, ed) 115, 217 – 290

Page 33: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

What can we measure? Model FittingCan we actually fit the Leach et al or Hall Models?Can we actually fit the Leach et al or Hall Models?

• Yes, but the experiments are very labour intensive.

• To fit the Leach et al model requires a ‘complete’ family of concentration-response curves at two different receptor densities.

• There must be no evidence of constitutive activity in the system• There must be no evidence of constitutive activity in the system• The orthosteric agonist must become partial at one of the receptor

densities

• To fit the Hall model requires a complete family of concentration-response curves at two different receptor densities in a system with constitutive activity.

• Again, the orthosteric agonist must become partial at one of the receptor densities and the basal activity must changereceptor densities and the basal activity must change

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Page 34: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

An illustration – allosteric inverse agonistSimulated data from Hall (2013)Simulated data from Hall (2013)

34

(εAB = δεAεB)

(α = 1/γ)

Page 35: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Is this level of analysis really necessary?

• For screening work NO

• Tracking XC50 and maximal effect is probably enough to drive routine SAR decisionsI hift id tit ti i f ti ffi it• In many cases curve shifts can provide quantitative information on affinity and overall cooperativity and qualitative information on underlying mechanisms

• Very strong negative cooperativity can be treated as competitive antagonism

• For dose prediction and PK/PD modelling work concentration ratios or model• For dose prediction and PK/PD modelling work concentration-ratios or model fitting approaches are the only ways to provide system independent parameters which can be translated into complex physiological systems.

Th l th ti h th i i th lik l• The more complex your therapeutic hypothesis is, the more likely you are to need to use the fitting approaches.

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Page 36: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

One final illustrationTranslation between systems: negatively cooperative agonistTranslation between systems: negatively cooperative agonist

0 911

Weakly Coupled System Highly Coupled System

0 50.60.70.80.9

0 50.60.70.80.9

Em

ax

0.10.20.30.40.5

0.10.20.30.40.5

E/E DRs approx.

constant

00.001 0.01 0.1 1 10 100

00.001 0.01 0.1 1 10 100

Agonist conc.1

0.6

0.8

1

max

Kb 10alpha 1eA 10000eB 10

These allow me to specify what will happen

0.2

0.4E/E

m

Which of these curves predicts this change in behaviour?

delta 0.01

36

01 100 10000

Modulator conc.

Page 37: Allosteric Modulation - Royal Society of Chemistry · The properties of allosteric modulation • Saturability – the effect of an allosteric modulator is inherently limited. •

Summary

• Characteristics of allosteric modulation

• Saturability – the effect of an allosteric modulator is inherently limited.

• Reciprocity – the orthosteric ligand affects the modulator’s properties to the same extent as the modulator affects those of the orthosteric ligand.g

• Probe dependence – the cooperativity constants describe the interaction between pairs of ligands – screen with the endogenous agonist, where ever possible!

• Transducer dependence – allosteric effects may depend on the signaling pathway that you measure.

• The effects of allosteric modulators on binding do not necessarily translate directly into functional systems

• XC and maximal effect are of limited value in the characterisation of allosteric• XC50 and maximal effect are of limited value in the characterisation of allosteric modulators

• At a minimum curve shift analysis (if not model fitting) is required to predict behaviour

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across experimental or physiological systems


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