THERAPEUTICSTRATEGIES

DRUG DISCOVERY

TODAY

Drug Discovery Today: Therapeutic Strategies Vol. 3, No. 4 2006

Editors-in-Chief

Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK

Eliot Ohlstein – GlaxoSmithKline, USA

Nervous system disorders

In-Target versus Off-Target allostericmodulators of GPCRsJuan Ballesteros1,*, John Ransom2

1OrphaMed, Parque Cientifico de Madrid, Polı́gono Industrial Zona Oeste, 28760 Tres Cantos, Madrid, Spain2Cytori Therapeutics, Inc., 3020 Callan Road, San Diego, CA 92121, USA

Allosteric modulators represent a new and possibly

safer mechanism of action for G protein coupled recep-

tor (GPCR) drugs, whose effect preserve the temporal

and local patterns of physiological activity of the endo-

genous ligand while binding at a different site. We

highlight the different molecular mechanisms that

could be responsible for this phenotype, distinguishing

between the commonly assumed In-Target and poten-

tial Off-Target modulators. In-Target modulators are

suitable for new chemical entities, while Off-Target

may be more suitable for new drug indications.

*Corresponding author: J. Ballesteros (jballesteros@orphamed.com)

1740-6773/$ � 2006 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddstr.2006.11.006

Section Editors:David Sibley – National Institute of Neurological Disordersand Stroke, National Institutes of Health, Bethesda, USAC. Anthony Altar – Psychiatric Genomics, Gaithersburg,USATheresa Branchek – Lundbeck Research, Paramus, USA

Introduction

Allosteric modulators represent a new and safer mechanism

of action for G protein coupled receptor (GPCR) drugs. These

are drugs that do not bind directly to the standard binding

site of a therapeutic target, but instead bind to a different site

modulating the function of the target [1–4]. Modulators

potentiate or diminish the effect of another drug or the

endogenous ligand on the therapeutic target of interest.

The key therapeutic strategies differ for In-Target and Off-

Target modulators. In-Target modulators bind to the same

target at a different site not exploited before, and thus offer

new chemical space for new chemical entities. Off-Target

modulators bind to a different target altogether that mod-

ulates the function of the therapeutic target, such as a GPCR

heterodimer or a distant protein, which depend on the

biological context representing a systems biology concept.

There are many potential targets for Off-Target modulators

and are thus best suited for new indications of existing drugs.

GPCR allosteric modulators: ‘To be or not to be?’

Although there are many discrepancies in the definition of

allosteric modulators, there is widespread agreement in their

expected advantages as drugs [1–4]. One, the allosteric effect

is saturable and this means that an excess of the drug cannot

have target related toxicity at excess concentrations. Two,

unless additional non-allosteric activities are present in the

drug, the drug is active only at tissue sites where the normal

physiologic release of the natural ligand occurs, and with the

same temporal pattern as the presence of the natural ligand.

This would mean that the drug would only enhance or

attenuate the tissue response on an as needed basis, a safer

therapeutic strategy than the direct action of a standard

orthosteric drug. Contrary to an agonist, this mechanism

prevents the potentially undesired activation of the same

receptor target expressed at other sites throughout the body

without a ligand present.

Pharmacological validation of an allosteric modulation

mechanism of action can become a difficult task, with

several definitions available and confusing claims for older

drugs. Most gold-standard measurements are based on bind-

ing experiments, while the real interest is in confirming

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Drug Discovery Today: Therapeutic Strategies | Nervous system disorders Vol. 3, No. 4 2006

physiologically relevant functional modulation of the recep-

tor. There is a risk to become trapped in the definition of what

a GPCR allosteric modulator is, and in the associated mea-

surements in the discovery process. The best solution may

require pharmacological tools such as labeled standard and

modulator ligands that are rarely available. The reality is that

standard discovery efforts are rarely sufficient to validate and

understand an allosteric mechanism for GPCRs, and novel

approaches are needed in this area.

Given the disagreements in the definition of an allosteric

modulator, and the agreement on how it should behave

physiologically as a drug, it seems logical we focus on the

latter. We propose that the ultimate test is functional mod-

ulation by a compound of the action of a known GPCR

ligand, such as the endogenous ligand or an exogenous drug,

measured in a physiologically relevant system.

In-Target GPCR allosteric modulators: the standard

When we read about GPCR allosteric modulators, it is

assumed that we refer to In-Target modulators. However, it

can be difficult to determine whether a modulator is an In-

Target or Off-Target allosteric modulator. Given the fact that

by definition it would bind at a different site, lack of dis-

placement by the endogenous ligand or a drug is not con-

clusive. Eventually, In-Target allosteric modulators could be

validated by labeling allosteric compounds that are displaced

by similar allosteric compounds in a relatively purified recep-

tor preparation, a very rare event.

All but one (Cinacalcet [5]) of the FDA approved drugs that

target G protein coupled receptors (GPCR) interact directly

with the orthosteric site of the GPCR, the site on the receptor

that is engaged by the naturally occurring agonist. Unlike a

standard agonist, the consequence of an allosteric interaction

is typically apparent only in the presence of an orthosteric

ligand, where the allosteric modulator modifies the func-

tional activity of the orthosteric ligand. Allosteric activity

is apparent as a right or left shift in the dose–response curve

for a given ligand in the presence of the modulator, a change

in the maximum efficacy, or both. A thorough and extensive

review of the theoretical mechanism underlying allosterism,

a comparison to orthosteric activities, experimental means

for detecting and identifying allosterism, its functional con-

sequences and the practical advantages of allosteric mole-

cules for therapeutic applications was presented several years

ago [2]. Subsequent reviews have since focused on practical

issues encountered in definitively ascribing GPCR allosteric

activity to a compound while highlighting the advantages of

such molecules [1,2,4,6–9] or surveyed the family of allosteric

ligands with activities at different GPCR family targets [3,10–

13], and other reports have described methods for quantify-

ing allosteric activities [8,14].

The range of GPCR targets suitable for allosteric modula-

tors could be very large since modulators have been described

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for at least 14 GPCR classes [2,3,8,15,16]. Structure–function

studies are beginning to pinpoint the molecular basis of

allosteric interaction with target GPCRs [17–25]. The emer-

ging pattern suggest interactions of the allosteric modulators

with extracellular loops for Family A receptors, and within

the transmembrane helical domain for Family C, with a less

clear pattern for Family B. This pattern is opposite to the

localization of their respective orthosteric sites, within the

transmembrane domain for Family A and in the extracellular

domain for Family C.

In-Target GPCR allosteric modulators: discovery

strategies

These compounds normally interact at a novel and pre-

viously undetected binding site within the receptor, and thus

represent primarily an opportunity for the discovery of new

chemical entities. Although it is possible that known drugs

interact as well on said site, this is expected to be an exception

in which case the compound would likely require further

chemical optimization for that new mechanism of action.

The strategies for the discovery of these compounds face two

major issues; the difficulty in screening approaches that can

detect these subtle indirect effects, and their pharmacological

validation as In-Target allosteric modulators.

An allosteric modulator has a subtle functional effect,

shifting the dose–response curve of the ligand by a maximum

of 10-fold, judged by published GPCR allosteric modulators.

GPCR allosteric modulators have been traditionally identified

by binding experiments measuring koff rates, which to our

understanding are not yet amenable to cost-efficient high

throughput screening approaches. There is a lot of interest in

developing the most commonly applied cell-based functional

screening formats to screen compound libraries systemati-

cally for GPCR allosteric modulators. The difficulty is the low

resolution of these screening approaches, where desirably

weak allosteric modulators, or initial weak hit compounds,

such as those inducing less than a two-fold shift, might be

difficult to detect. One solution to this problem has been the

adaptation of flow cytometers, whose intrinsic characteristic

is measuring single cell events at high resolution. These

systems can resolve subtle levels of GPCR activation, and

thus allosteric modulators, up to a small percentage of

individual cells activated in a whole cell population (http://

www.pharmacodiscovery2005.com, http://www.healthtech.

com/2006/gpc, and Ransom 2006, unpublished).

Although it is not clear what the desired level of modula-

tion is, and how the in vitro measurements relate to in vivo

activities, few principles are starting to emerge from in vivo

studies. It may depend on several properties in ways we do

not yet understand, such as basal levels of activation, barriers

for activation, speed and strength of GPCR inactivation and

downregulation, and the effective concentration at the recep-

tor site. Some GPCRs may require just a small modulation

Vol. 3, No. 4 2006 Drug Discovery Today: Therapeutic Strategies | Nervous system disorders

where a 10-fold shift may become toxic, such as those

involved in synapses with a high and quickly transient effec-

tive concentration where the modulatory effect may be most

sensitive. Other GPCRs may require a more robust modula-

tion, such as those activated by systematically diffusible

ligands with low effective concentrations whose effect might

be less drastic.

In-Target GPCR allosteric modulators: therapeutic

strategies

We consider three key therapeutic strategies related to their

novel mechanism of action, independent of the therapeutic

area; potentiators versus attenuators, undruggable GPCR tar-

gets, and druggable GPCR targets.

Potentiators versus attenuators

Typically GPCR allosteric modulators represent potentiators

of the GPCR response, rather than attenuators. This is true

historically for modulators of endogenous ligands, where the

potential for a safer mechanism of action relative to agonist

drugs by sharing the temporal and spatial patterns of activa-

tion of the endogenous agonists are more clear and intuitive.

There have been some reports of allosteric attenuators of

GPCRs [6], whose therapeutic benefit is not obvious. In the

absence of high basal activity of the GPCR target, an allosteric

attenuator may behave just like an antagonist, inhibiting the

response of the endogenous agonist only when said agonist is

present. The lack of a maximal effect of allosteric modulators

means that an attenuator could behave as a partial antago-

nist, partially inhibiting the agonist response. However, for

GPCRs with high basal activity, an antagonist that behaves as

an inverse agonist would block basal levels, while an allosteric

attenuator may not, a pattern which might be beneficial in

some exceptional cases. A more obvious benefit of an attenua-

tor versus an antagonist is when antagonists cannot achieve

the desired selectivity, as described below for undruggable

targets.

Undruggable GPCR targets

One of the most promising opportunities for GPCR allosteric

modulators is to enable the discovery of small molecule drugs

to ‘undruggable’ GPCR targets. These represent valuable

GPCR targets often validated biologically where it has not

been possible to discover drug candidates based on small

molecule synthetic compounds. We distinguish two cases,

large peptide receptors and highly homologous GPCRs. Many

of these ‘undruggable’ GPCR targets are large peptide GPCRs

such as Family B, glycohormone and some peptide receptors.

In these cases a small molecule may not be able to displace or

compete with the endogenous ligand, but could very well

modulate its effectiveness. This is especially true for small

molecule agonists, which do not seem able to have enough

contacts to mimic the activating effect of the large peptide

ligand, such as the glucagon-like peptide 1 (GLP1) and the

luteinizing hormone (LH) GPCRs. The idea is that a small

molecule could still behave as a modulator, lowering the

activation barrier for the endogenous ligand by acting on a

key activation motif. A different therapeutic benefit may be

when allosteric modulators enable the selectivity profile that

have prevented discovery of small molecule drugs for some

GPCR targets. By binding at a site different than the endo-

genous ligand, which might be highly conserved across GPCR

subtypes, modulators may find more divergent binding sites

enabling the desired selectivity profile. Muscarinic GPCRs

represent a good example, where allosteric modulators are

enabling selectivity among these five subtypes not possible

before with standard agonists and antagonists. A case of

special interest might also be for GPCRs drugs that suffer

from off-target toxic activities difficult to avoid, such as the

known preference of histaminergic, aminergic, or some pep-

tide GPCR drugs to interact with the HERG cardiac potassium

channel inducing cardiac toxicity [1].

Druggable GPCR targets

Druggable GPCR targets are by definition those for which it is

possible to discover small molecule synthetic drug candi-

dates, such as standard agonists and antagonists. Allosteric

modulators to druggable GPCRs with drug candidates early in

the pipeline may offer a new and potentially safer mechanism

of action [1,3,4], but they also imply higher risks due to the

higher complexity and uncertainty in their discovery. Allos-

teric modulators to GPCR targets with drugs already in the

market or close to it [1,3,4,11] may offer an opportunity to

discover next generation drugs for biologically and market

validated GPCR targets with a novel and safer mechanism of

action. This could actually apply to novel multi-target selec-

tivity profiles, where the new binding site of modulators may

result in novel multi-target profiles of therapeutic benefit. An

example would be the neurotransmitter GPCRs, where many

receptors share binding motifs in their orthostatic site often

leading to multi-target activities, where modulators could

have a very different multi-target profile of therapeutic inter-

est. There might even be endogenous allosteric compounds

whose binding sites are conserved across very different GPCR

subtypes than the standard orthosteric binding sites. Albeit

not a binding site for drugs, the effect of sodium in inhibiting

activation by abolishing the high affinity site of receptors is a

clear example of a conserved allosteric site across GPCRs, with

many peptides having a preference for divalent cations such

as calcium rather than sodium for monoamine GPCRs.

Off-Target GPCR allosteric modulators: reprofiling

strategies

Although almost all programs for discovery of GPCR

allosteric modulators are based on searching for In-Target

modulators, there are many ways for compounds to modulate

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Figure 1. In-Target vs. Off-Target GPCR allosteric modulators. In-Target vs. Off-Target GPCR allosteric modulators (green arrows), whose functional

modulation of the effect of the ligand (yellow arrow) for the desired GPCR therapeutic target (upper left red rectangle) may be equivalent and

undistinguishable in a physiologically relevant system. In-Target modulators (upper left text box) bind to the same GPCR target at a different site. Off-Target

modulators may bind to a GPCR that forms a heterodimer (upper right red rectangle) with the GPCR target (top right text box), or to another protein

within the receptor complex (dark blue rectangles under red rectangles) physically associated with the GPCR Target (right second text box from top), or to

other proteins without physical contact with the receptor complex of the GPCR target (orange objects). In-Target modulators represent new unexploited

binding sites suitable for new chemical entities (NCEs). For every In-Target modulator, there might be many potential Off-Target modulators, suitable for

new drug indications (NDIs) of existing drugs.

the function of a GPCR target without interacting directly

with it. We could distinguish physically associated proteins

and other Off-Targets without direct physical contact, as

shown in Fig. 1.

Off-Target modulators may act on a protein physically

associated with the GPCR target that forms the receptor

complex [16,26–28], such as G Proteins or GPCR heterodi-

mers [29–32] discussed also in this volume by L. Devi. In fact,

G proteins are indeed allosteric modulators of GPCR func-

tion, and their activity has been described using allosteric

models such as the ternary complex model. The potential of

GPCR heterodimers as new targets for drug discovery has

received much attention, as reviewed recently [29–32]. How-

ever, proving their ability to generate pharmacologically

distinct entities has been possible only in few cases [29,32],

which ultimately requires a physiologically relevant context.

Recently, a new example has been published of an antagonist

of the cannabinoid CB1 receptor, an analog of the

recently approved anticraving drug Rimonabant, acting as

448 www.drugdiscoverytoday.com

an Off-Target GPCR heterodimer allosteric modulator of the

Orexin-1 receptor [33]. Given the known role of the Orexin-1

GPCR in obesity, this potential Off-Target modulation of

Rimonabant may be related to its known antiobesity proper-

ties. There is a need to derive new strategies to search for

pharmacologically relevant and distinct entities formed by

GPCR heterodimers, or other forms of oligomerization.

It is possible that compounds behaving as GPCR allosteric

modulators may do so by interacting with a target not directly

associated with the GPCR that modulates the GPCR signal.

According to our definition of functional behavior in a phy-

siologically relevant system, those compounds would con-

stitute also GPCR allosteric modulators, and may offer similar

therapeutic benefits than standard In-Target GPCR modula-

tors. There are actually many targets within a cell that may

qualify as an Off-Target GPCR modulator; by being a member

of the GPCR signaling network, or of other signaling net-

works that modulate the GPCR signaling network, by affect-

ing the expression of any said member of these signaling

Vol. 3, No. 4 2006 Drug Discovery Today: Therapeutic Strategies | Nervous system disorders

Table 1. Comparison summary table

Strategy Pro Cons Who is working on this strategy

In-Target GPCR

allosteric modulators

New chemical

entities (NCEs)

Challenging

screening assays

http://www.addexpharma.com

http://www.novasite.com

http://www.orphamed.com

Off-Target GPCR

allosteric modulators

New drug indications (NDIs) Require physiologically

relevant system

http://www.orphamed.com

http://www.combinatorx.com

http://www.caratherapeutics.com

http://www.cadenbiosciences.com

networks, among others. . . There are known examples such as

dipeptidyl peptidase IV (DPP-IV) inhibitors that block degra-

dation of GLP-1, behaving as allosteric potentiators of the

GLP-1 receptor, or lithium and valproate, drugs for bipolar

disorder that act on the phosphoinositol signaling pathway

attenuating the signaling of many GPCRs coupled to Gq. The

emphasis on target-based drug discovery based on screening

in vitro artificial cell expression systems has hindered the

discovery of these compounds.

For a new chemical entity, directed at a specific GPCR

target, the preferred strategy is clearly an In-Target allosteric

modulator. However, the emphasis of drug candidates in

clinical phases that has lowered considerably the value of

preclinical compounds have generated a renewed interest in

finding new indications for existing drugs with proven lack

of toxicity in humans. This effort is called reprofiling, or

repurposing. Known drugs may be unlikely to bind with

sufficient affinity to newly identified allosteric sites in

GPCRs, but they represent a suitable option to search for

Off-Target modulators. That is because known drugs interact

already in sufficient efficacy with different targets, and for

every GPCR target there is likely to be tens of other targets

that would modulate its function. A reprofiling strategy to

identify Off-Target GPCR allosteric modulators based on

known drugs may represent a shorter-term complementary

strategy to the discovery of In-Target GPCR allosteric mod-

ulators based on new chemical entities. Off-Target modula-

tors represent a systems biology concept and are dependent

on their native biological context. New approaches are

necessary to screen compound libraries such as collections

of known drugs against physiologically relevant human

freshly isolated samples for the discovery of Off’-Target

GPCR Allosteric Modulators, a process called ex vivo screen-

ing. For example, we in OrphaMed are applying flow cyto-

metry instruments converted into medium throughput

screening platforms, described above to be suitable for the

discovery of allosteric modulators, to evaluate known drugs

and their combinations against human patients ex vivo sam-

ples. Several alternative approaches using microscopy or

microfluidics are also becoming available (http://www.

cellomics.com, http://www.genoptix.com, http://www.

vitrabio.com, http://www.ttplabtech.com).

Conclusion

We have considered two strategies to discover GPCR allosteric

modulator drug candidates, In-Target and Off-Target mod-

ulators (see Table 1). As shown schematically in Fig. 1, In-

Target modulators are the most commonly studied modula-

tors that represent novel unexplored binding sites and are

thus best suited for new chemical entities (NCEs). Off-Target

modulators may interact with a large variety of targets that in

turn modulate the function of the GPCR target of interest,

and are best suited to discover new indications of existing

drugs (reprofiling). There are Off-Target modulators that act

on a protein that is in direct contact with the GPCR target

forming the receptor complex, the most common being

GPCR heterodimers (Fig. 1). There are also Off-Target mod-

ulators that act on proteins or other targets not directly in

contact with the GPCR target of interest (Fig. 1) but which can

modulate its function.

Acknowledgements

We are thankful to Boban Thomas, M.D., for valuable com-

ments and for proposing DPP-IV inhibitors as Off-Target

allosteric modulators of the GLP-1 GPCR.

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