Download finding new tricks for old drugs: an efficient route

REVIEWS
FINDING NEW TRICKS FOR OLD
DRUGS: AN EFFICIENT ROUTE FOR
PUBLICSECTOR DRUG DISCOVERY
Kerry A. O’Connor and Bryan L. Roth
Abstract | With the annotation of the human genome approaching completion, public-sector
researchers — spurred in part by various National Institutes of Health Roadmap Initiatives —
have become increasingly engaged in drug discovery and development efforts. Although large
and diverse chemical libraries of ‘drug-like’ compounds can be readily screened to yield
chemically novel scaffolds, transforming these ‘chemical probes’ into drugs is a daunting
endeavour. A more efficient approach involves screening libraries of approved and off-patent
medications; both phenotypic- and molecular target-based screening of ‘old drugs’ can readily
yield compounds that could be immediately used in clinical trials. Using case studies, we
describe how this approach has rapidly identified candidate medications suitable for clinical trials
in disorders such as progressive multifocal leukoencephalopathy and amyotrophic lateral
sclerosis. This approach has also led to the discovery of the molecular targets responsible for
serious drug side effects, thereby allowing efficient ‘counter-screening’ to avoid these side effects.
Departments of
Biochemistry, Psychiatry,
Neurosciences,
Comprehensive Cancer
Center and National
Institute of Mental Health
Psychoactive Drug Screening
Program, 2109 Adelbert
Road, Case Western Reserve
University Medical School,
Cleveland, Ohio 44106,
USA.
Correspondence to B.L.R.
e-mail:
[email protected]
doi:10.1038/nrd1900
Now that the human genome has been sequenced and
its annotation is approaching completion1, publicsector researchers have been urged to focus their attention on exploiting this information for the purposes of
drug discovery2,3. The National Institutes of Health’s
(NIH) Molecular Libraries Initiative, for example, proposes “to expand the availability, flexibility, and use of
small-molecule chemical probes for basic research”3— an
effort that has become the subject of intense debate within
academia and the private sector4–6. Although efforts such
as the NIH Molecular Libraries Initiative do not propose
to provide new drugs for human disease — except in
“exceptional circumstances”3 — it is likely that many
‘drug-like’ molecules will be discovered. It is implied that
some of the ‘chemical probes’ discovered and validated by
these emerging public efforts will eventually be optimized
by commercial partners for therapeutic uses.
In large measure, the reticence for entering fullfledged drug discovery and development efforts in the
public sector stems mainly from the recognition of the
NATURE REVIEWS | DRUG DISCOVERY
enormous costs and risks associated with therapeutic
drug discovery. Indeed, current estimates for successful launch of a single new medication are in excess
of US$800 million7 — nearly an order of magnitude
higher than that for the NIH Molecular Libraries
Initiative. Given the extraordinarily high costs and
risks associated with therapeutic drug discovery, it
is exceedingly unlikely that any single public-sector
research group will successfully see a novel chemical
‘probe’ become a ‘drug’.
The main approach of the NIH Molecular Libraries
Initiative and similar public-sector small-moleculebased screening centres is to screen vast libraries
of chemically diverse scaffolds for novel actions3,6.
Typically, either PHENOTYPIC SCREENS or MOLECULAR TAR
GETBASED SCREENS are performed in a high-throughput
screening (HTS)-like fashion with more than 100,000
chemically diverse compounds screened and ‘hits’ subsequently identified and validated (FIG. 1). Currently, a
large number of public-sector groups TABLE 1 seem
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a
b Multiple drug plates containing chemically diverse
Plate cells and incubate with various compounds
compounds
Analyse phenotypic changes using microscopy
No effect
Phenotype 1:
cell death
Screen against
a single
molecular target
Find ‘hits’,
optimize and
validate data
Phenotype 2:
cell growth
Custom
2
1
3
4
5
Plate 1
6
7
8
9
10
11
12
Deconvolute data to
determine molecular
target producing
phenotypic change
Phenotype 3:
change in
cellular
morphology
m1 receptor partial agonists
A
B
C
D
E
F
Find ‘hits’,
optimize
and
validate
data
G
H
Figure 1 | The road to drug discovery: phenotypic versus single-target approaches. a | The phenotypic approach begins
with the hypothesis-based selection of compounds to test. This selection process can be applied to drugs currently on the
market. Compounds can be selected for their binding profiles with certain proteins of interest (for example, transporters and
receptors), known therapeutic uses, or structural similarities to known therapeutic compounds. Once selected, these compounds
are then tested against a cellular read-out to identify ‘hits’. A ‘hit’ is a compound that has activity at the particular phenotypic readout examined. The compounds can be further optimized if the molecular target can be identified. Alternatively, if the compounds
used already represent approved medications, investigators can directly attempt in vivo validation studies in suitable animal
models and/or small-scale proof-of-concept clinical trials. b | The single-molecular-target approach is the typical approach in which
a library of approved medications is simply screened for interactions at a single molecular target. ‘Hits’ can be further optimized or
used directly for in vivo studies. Shown are typical screening results from a campaign we undertook to identify muscarinic M1
receptor partial agonists for the treatment of cognitive impairment that can occur in schizophrenia. N-desmethyl-clozapine — a
major clozapine metabolite — was identified as a potent muscarinic M1 receptor agonist50.
PHENOTYPIC SCREENS
A type of chemical screen in
which cell responses, such as
motility or membrane ruffling,
are monitored as an index of
drug action. Frequently,
phenotypic screens are carried
out using high-throughput
microscopy and automated
image capture and analysis.
MOLECULAR TARGETBASED
SCREENS
A type of chemical screen in
which a single molecular target,
such as a receptor or enzyme,
is screened using a simplified
readout such as fluorescence.
1006 | DECEMBER 2005
to have the capacity to carry out near-industrial scale
HTS campaigns. An outgrowth of these efforts has
been the emerging field of chemical genomics in which
chemical probes are used “for activating or inactivating protein functions, thereby providing resources that
help discern the functions of gene products in normal
and disease cells, as well as in tissues”6,8.
An alternative approach probes multiple targets
simultaneously using smaller libraries of chemically
diverse compounds. One approach that we have
pioneered has been dubbed RECEPTOROMICS, in which,
ultimately, the entire complement of ‘receptors’ in the
genome is screened in a massively parallel fashion9,10.
As will be discussed below, this approach has yielded
several important discoveries that have accelerated
therapeutic drug discovery efforts. In a similar vein,
KINOMICS has emerged as an approach for systematically identifying and screening the full complement of
kinases for drug discovery purposes11–13. As receptors
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and kinases represent the ‘most druggable’ targets in
the genome, representing 5% and 2.8% of the genome,
respectively14, screens focused on these segments of the
genome are most likely to yield new drugs14. Both of
these more focused approaches suffer from the risks
related to optimizing and validating novel chemical
probes for therapeutic uses.
We would like to highlight an approach more
likely to be successful in reaching the ultimate goal of
providing new drugs, one in which already available
medications — most of which are off-patent — are
simultaneously screened using various in vitro and
in vivo model systems. This approach utilizes existing medications that are subsequently used as probes
for preclinical molecular target- or phenotype-based
drug discovery efforts. This differs from the standard
‘repositioning’ approaches that have recently been
reviewed15. Repositioning typically occurs when an
interesting side effect of an approved medication is
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Table 1 | Selected public-sector small-molecule screening resources
Name of screening
resource
Website of resource
Users
Comments
NIMH Psychoactive Drug
Screening Program
http://pdsp.cwru.edu
A/G/NP
Free and confidential
screening to qualified users
ChemBank
http://chembank.broad.harvard.edu
A/G/NP
Large database of chemical
compounds available
NIH Chemical Genomics
Center
http://www.genome.gov/12512295
G
Advertises screening capacity
of up to
1 × 106 compounds per day
Scripps Research
Institute HTS Facility
None yet
NP/A
To be installed summer 2005
Rockefeller University
HTS Core Facility
http://www.rockefeller.edu/
highthroughput/highthroughput.php
A/NP
One of the oldest NP HTS
facilities
Kansas University HTS
Lab
http://www.hts.ku.edu
A
Funded by NIH COBRE
mechanism
Johns Hopkins University
HTS Core
http://www.molecularinteraction.org/
HIT%20center.htm
A
A, academic; C, commercial; COBRE, Centers of Biomedical Research Excellence; G, government; HTS, high-throughput screening;
NIH, National Institutes of Health; NIMH, National Institute of Mental Health; NP, non-profit.
RECEPTOROMICS AND
RECEPTOROME
Receptorome refers, ultimately,
to the entire complement of
‘receptors in the genome’,
although it is more commonly
applied to that portion of the
genome encoding G-proteincoupled receptors.
Receptoromics represents tools
and methods of analysis to
study the receptorome.
KINOMICS AND KINOME
The kinome refers to the entire
complement of protein kinases
in a particular genome.
Kinomics represents tools and
methods of analysis to study
the kinome.
identified during clinical trials, which then provides
the impetus for a new therapeutic indication. The best
known example is that of sildenafil (Viagra; Pfizer),
which was initially developed as an anti-angina
medication but had the side effect of producing
prolonged penile erections in human volunteers15.
Repositioning can also arise from technology platforms established to identify repositioning opportunities, such as CombinatoRx’s cHTS system (see
REF. 16). However, under either of these conditions a
compound is first identified for a known target, then
similar compounds are screened against that target.
Our proposed approach, by contrast, blindly screens
existing compounds against a multitude of targets,
and therefore identifies either possible therapeutic
benefits or side effects in a non-biased fashion.
Both of these approaches, repositioning and screening of approved medications, benefit from the information obtained from the original clinical trials. When
screening current medications for novel applications,
most of the identified ‘hits’ will already have been used
in humans and will have detailed information related
to dosing, in vivo pharmacokinetics and toxicity, and
so clinical trials can be performed sooner and more
economically using the more limited resources of the
public sector. According to the 2005 Pharmaceutical
Industry Profile17carried out by the Pharmaceutical
Researcher Manufacturers of America (PhRMA),
member companies spent US$38.8 billion in research
and development in 2004. One-third of these funds
were spent on preclinical/prehuman research and
development. In the ideal case using this paradigm, a
novel target for an off-patent medication will be discovered and its therapeutic indication validated using
public-sector resources. Because the medications are
off-patent, the cost savings for consumers are potentially enormous because of savings related to medication cost. Furthermore, the majority of research and
development costs for de novo therapeutics are due to
NATURE REVIEWS | DRUG DISCOVERY
the large dependence on medicinal chemistry, pharmacokinetic and toxicology expertise. In the public
or academic setting, the infrastructure for this type of
large-scale therapeutic development is absent using
the approach we are describing. The benefit of the
approach we describe of screening current medications
for novel therapies bypasses the dependence on such
an infrastructure. Additionally, because of the inherent financial opportunities associated with trials for
orphan diseases (which allows for 7 years of exclusivity
even with off-patent medications) abundant financial
opportunities will exist for smaller companies that use
this approach.
In the following sections, we provide several case
histories that validate this approach and suggest that
this approach will allow the public sector to achieve
the largest return on its investment.
Case histories
Antibiotics as neuroprotective agents. The first antibiotic was identified by Alexander Fleming in 1929
when a culture plate of Staphylococcus bacteria became
contaminated by a mold. Penicillin was merely the
first in a series of antibiotics classified as β-lactam
antibiotics, due to their functional requirement of
an intact β-lactam ring structure. This class of antibiotics, which includes aminocillins, cephalosporins,
carbapenems and monobactams, acts via inhibition
of bacterial cell-wall synthesis. The widespread use of
this class of antibiotics has grown exponentially since
its discovery more than 70 years ago. According to the
Centers for Disease Control and Prevention’s National
Center for Health Statistics, 126 million antimicrobial
prescriptions were written in ambulatory care settings
in 2000 alone18.
More recently, β-lactams were found to also have
a role in regulating glutamate levels, via interaction
with the glutamate transporter GLT1 (also known as
EEAT2)19. GLT1 is responsible for the inactivation
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and prevention of neurotoxicity via reuptake of the
primary excitatory neurotransmitter glutamate20.
This astroglial protein has been implicated in multiple neurological disorders, including amyotrophic
lateral sclerosis (ALS), stroke, brain tumours and
epilepsy21.
The discovery of the neuroprotective effects of
β-lactam antibiotics represents an exemplary case
study on drug screening methodology. Rothstein
and colleagues19 systematically screened 1,040 FDAapproved compounds for interaction with GLT1.
The screens were performed in multiple model
b Identification of JC virus co-receptor
a Normal viral life-cycle
JC virus coreceptor identified
as 5-HT2A
Clathrincoated pit
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT2A
5-HT2C
5-HT3
5-HT5
5-HT6
5-HT7
D1
D2
D3
D4
D5
α2A
α2B
α2C
α1A
α1B
M1-muscarinic
M2-muscarinic
M3-muscarinic
M4-muscarinic
M5-muscarinic
γ-A
γ-B
BZP
NMDA
PCP
SERT
NET
DAT
H1
H2
H3
H4
EP-3
EP-4
CB1
Ca2+ channel
NAR a2/b2
NAR a2/b4
NAR a3/b2
NAR a3/b4
NAR a/b2
NAR a7
β1-AR
β2-AR
I1-imidazoline
mGlu1
mGlu2
mGlu4
Atypicality
5-HT2A
Weight gain 5-HT2C
Endosome
Endocytosis of
JC virus with
5-HT2A receptors
Antipsychotic D2
efficacy
Adrenergic
Side
effects
Degradation
Muscarinic
JC viral Transcription
DNA
Translation
Viral replication
Lysosome
Infection
Weight gain
c Infection blocked by 5-HT2A antagonist
H1
Pretreated with 5-HT2A
receptor antagonist
before JC virus infection
Clathrincoated pit
Internalized
vesicles
Internalized
receptors
are sorted to
endosome
1 nM
10 nM
100 nM
1,000 nM
10,000 nM
Ki
Antagonist treatment
causes endocytosis
of cell surface
5-HT2A receptors
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Figure 2 | Identification of the 5-hydroxytryptamine 2A receptor as the JC virus co-receptor via a combination of
phenotypic and receptorome-based screening reveals a novel strategy for treating progressive multifocal
leukoencephalopathy. a | The presumed normal life cycle of JC virus in which it interacts with cell-surface glycoproteins51 and
with an unknown G-protein-coupled receptor (GPCR) that can be blocked by antipsychotic drugs28. Following internalization via
the endosome pathway, virions are released and a new cycle of infection begins leading, ultimately, to progressive multifocal
leukoencephalopathy (PML). b | A receptorome profile of typical and atypical antipsychotic drugs shows a large number of
potential molecular targets with which antipsychotic drugs interact, which identified the 5-hydroxytryptamine 2A (5-HT2A)
receptor as the likely JC virus co-receptor. c | A likely mechanism by which 5-HT2A receptor antagonists block JC virus infection
via antagonist-induced intracellular sequestration to remove potential co-receptors from the cell surface. If other GPCRs are
identified as viral co-receptors, this mechanism could lead to a novel viral treatment strategy. Panel b reprinted, with permission,
from REF. 10 © Macmillan Magazines Limited.
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systems, each closely approximating in vivo conditions, to assure proper functional relevance. Initial
drug screens were carried out in organotypic spinal
cord slices, which mimic cellular metabolism and
cell–cell interactions present in vivo. More than 20
compounds were found to increase GLT1 protein
expression by more than twofold, including penicillin and cephalosporin. After discovering that this
class of compounds increased protein expression, a
smaller subgroup was then screened in astrocytes
and non-neuronal tissues expressing the GLT1 promoter. These studies allowed determination of the
specificity and time-dependent effects of β-lactams
on GLT1. To further elucidate the clinical potential
of this effect, in vivo assays were performed in normal rats, as well as in mouse models of neurological
disease states. This series of experiments provides
both molecular and in vivo evidence to support the
conclusion that β-lactams could have potential as
neuroprotective agents, as well as their antibiotic
effects.
In addition, this study provides pharmacological evidence to support this conclusion. Although
β-lactams such as ceftriaxone increased GLT1 protein
expression, non-β-lactam antibiotics such as fluconazole had no effect, indicating a specific effect of this
sub-class of compounds19.
This group subsequently investigated the direct
neuroprotective effects of ceftriaxone, such as cell
survival under ischaemic conditions and extended
lifespan in transgenic mouse models of neurodegenerative diseases. In doing so, these studies indicate the
potential use of antibiotics in slowing the progression
of, or preventing, neurodegenerative diseases such as
ALS19. These findings and the proposed clinical trials
have also generated controversy22 because ceftriaxone was previously reported, in at least two Italian
clinical trials, to be ineffective in curing ALS23,24.
These earlier trials can be criticized because of inadequate statistical power, trial design (for example,
not double-blinded) and trial length22. A Phase II/III
‘bridging study’ is now in preparation to begin in
2005 to test this hypothesis more adequately (see The
Robert Packard Center for ALS Research at Johns
Hopkins in Further information). At the very least,
these studies support GLT1 as a potential molecular
target for commercial drug discovery efforts for ALS
and related disorders.
Atypical antipsychotics, PML and Tysabri. Here the
recent discovery of the potential role of serotonergic
antagonists in blocking the cellular entry, and therefore
spread, of the human polyomavirus JC virus (JCV), in
HIV-infected patients and individuals who have been
administered natalizumab (Tysabri; Elan Biogen Idec)
will be discussed. In addition, the possibility that other
viruses use a similar cellular apparatus to invade host
cells will be discussed.
Progressive multifocal leukoencephalopathy
(PML), a usually fatal disorder characterized by
demyelination, typically occurs in immunosuppressed
NATURE REVIEWS | DRUG DISCOVERY
patients, such as those with AIDS25. Recently, multiple
cases of PML have been reported in patients treated
with natalizumab for multiple sclerosis26 and Crohn’s
disease and in individuals who have been treated
with tumour-necrosis factor-α (TNFα) antagonists27
for rheumatoid arthritis. JCV has been shown to
infect oligodendrocytes and astrocytes, cells within
the central nervous system (CNS), which results in
the initiation of PML (FIG. 2). Recently, Elphick and
colleagues28 found that this infection of glial cells
could be blocked by antipsychotic drugs (which are
normally used to treat schizophrenia). Importantly,
selective serotonin (5-hydroxytryptamine: 5-HT)
5-HT2A receptor inhibitors, such as M100907, along
with antipsychotic drugs that also are potent 5-HT2A
receptor antagonists — including clozapine and
chlorpromazine (see FIG. 2 and PDSP Ki Database
in Further information) — were able to block infection of glial cells by JCV. In addition, infection
could be blocked by 5-HT2A receptor-neutralizing
antibodies, suggesting that the 5-HT 2A receptor
is probably a co-receptor for JCV (FIG. 2). Finally,
Elphick et al.28 demonstrated that JCV was co-internalized with 5-HT2A receptors in cells that overexpressed receptors tagged with green fluorescent
protein. It is likely that 5-HT2A receptor antagonists
block JCV infection by inducing internalization of
5-HT2A receptors and loss of cell-surface receptors
(FIG. 2)29, rather than by directly blocking viral receptor binding, but further studies will be needed to test
this hypothesis.
Previous medications developed to treat PML have
largely failed due to poor bioavailability within the
CNS. However, because 5-HT2A receptor antagonists
are often used to treat psychological disorders, they
could provide a novel direction of drug development
to treat HIV-infected patients. Unfortunately, drugs
such as clozapine and chlorpromazine have serious
and occasionally fatal side effects and are not likely
to be well tolerated by immunocompromised individuals. To identify compounds with high affinity
for 5-HT 2A receptors that can be easily tolerated,
we searched the National Institute of Mental Health
(NIMH)-Psychoactive Drug Screening Program’s Ki
Database (see Further information; FIG. 3) for compounds with sub-nanomolar affinity for 5-HT 2A
receptors. Identified compounds included many
atypical antipsychotic drugs, as expected, along with
several antihistamines, including cyproheptadine
(K i = 0.46 nM) and mirtazepine (K i = 2.0 nM),
which we have identified as potent 5-HT2A receptor
antagonists (FIG. 3). Cyproheptadine is a well-tolerated, generic medication frequently used to treat
HIV-wasting syndrome30, which preliminary studies have indicated suppresses JCV infection in vitro
(W. Atwood, personal communication). Cyproheptadine or mirtazepine would, therefore, qualify as
a candidate medication for prophylactic treatment of
PML, which occurs as a consequence of HIV infection
as well as treatment with various biological response
modifiers including natalizumab and TNFα.
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KiDB
PubChem
N
NIMH-PDSP
86
RFU (x 1,000)
76
66
56
46
36
26
16
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (sec)
Clinical trials
Pretreated with 5-HT2A
receptor antagonist
before JC virus infection
Clathrincoated pit
Internalized
vesicles
Internalized
receptors
are sorted to
endosome
=
=
Antagonist treatment
causes endocytosis
of cell surface
5-HT2A receptors
Endosome
Lysosome
Degradation
ASN
Figure 3 | Identification of a potentially prophylactic treatment for progressive multifocal leukoencephalopathy.
Because antipsychotic drugs have serious and potentially life-threatening side-effects, they are not suitable for prophylactic
treatment of progressive multifocal leukoencephalopathy (PML). A search of a public database of drug–receptor affinity
values (see PDSP Ki Database Search Page in Further information) facilitated the identification of cyproheptadine (Periactin)
— an off-patent sub-nanomolar affinity 5-hydroxytryptamine 2A (5-HT2A) receptor ligand (structure via link to PubChem). The
middle panel shows that 1 nM cyproheptadine is able to abolish 5-HT2A receptor signalling, and the bottom panel shows the
presumed mechanism by which cyproheptadine might block JC virus infection (courtesy of National Institute of Mental
Health-Psychoactive Drug Screening Program (NIMH-PDSP)). Clinical trials are currently being planned to test this
hypothesis. Because PML is now also linked to treatment with various immunomodulators, including natalizumab (Tysabri;
Elan/Biogen Idec), prophylactic treatment with 5-HT2A receptor antagonists such as cyproheptadine could be considered.
RFU, relative fluorescent units.
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Table 2 | Commercial and non-commercial sources of available medication
Name
Library characteristics
Website
Prestwick Chemical
Co.: Prestwick
Chemical Library
1,120 off-patent with >85% marketed
pharmaceuticals
http://www.prestwickchemical.com
Microsource: The
Spectrum Collection
2,000 biologically active and structurally
diverse compounds from libraries of known
drugs, experimental bioactives and pure
natural products
http://www.msdiscovery.com/spect.html
Sequoia
A large collection of available medications
— many currently ‘on-patent’ and includes
compounds not normally available elsewhere
http://www.seqchem.com
NIH Brain Bioactive
Compound Collection
(BIBCC)
A planned large collection of available
medications, known CNS active compounds
as well as compounds with likely CNS
activity. Collection will be available via the
NIH Roadmap Initiative.
http://nihroadmap.nih.gov
CNS, central nervous system; NIH, National Institutes of Health.
COUNTERSCREENING
The approach of screening
candidate medications against
an array of molecular targets to
identify ‘off-target’ actions.
When side-effects are lethal: ‘Fen/phen’ and the
human valvulopathy receptor. Recently, the inability
of regulatory agencies to rapidly identify the serious and potentially life-threatening side effects of
approved drugs has received a great deal of attention.
In large measure, this failure comes from ignorance
of the molecular and cellular mechanisms responsible
for serious side effects and the subsequent inability to
reliably predict them. Large pharmaceutical companies do not ordinarily have the scientific resources nor
the motivation to uncover the cellular and molecular
mechanisms of drug toxicities of approved drugs.
Additionally, it does not seem that the US FDA and
similar regulatory agencies have the adequate scientific and monetary resources to perform the sort
of basic discovery-based research needed to reveal
the mechanisms responsible for serious side effects
of approved medications. The recent withdrawal of
rofecoxib (Vioxx; Merck) and many other cyclooxygenase-2 (COX2) inhibitors because of serious cardiovascular side effects is a pertinent example31. The
inability to identify the ‘toxic target’ responsible for
serious side effects of prescribed medications makes it
impossible to avoid these targets via COUNTERSCREENING
in subsequent drug discovery efforts. We will use the
case of the ‘Fen/Phen’ (fenfluramine/phentermine)
appetite suppressant combination as a pertinent
case in which the identification of the ‘toxic target’
using available medications as probes has allowed the
development of a new class of appetite suppressant
medications.
In 1997, Connolly and colleagues32 described valvular heart disease (VHD) as a common and serious
side effect of the widely prescribed appetite suppressant
combination of fenfluramine and phentermine. Later
reports indicated that VHD was specifically associated
with fenfluramine and its isomer dex-fenfluramine33
— findings that led to the voluntary withdrawal of fenfluramine and dex-fenfluramine and >US$20 billion
in potential liability against Wyeth Pharmaceuticals.
However, it was not until 3 years later that a mechanism
was identified.
NATURE REVIEWS | DRUG DISCOVERY
Using a systematic approach, we analysed the
receptorome profile of fenfluramine and its active
metabolite norfenfluramine34. Additionally, we examined several other off-patent medications known to
induce VHD, including methysergide and its active
metabolite methylergonovine34,35. Because fenfluramine was known to induce 5-HT release, we also
screened other drugs that elevate levels of 5-HT but
are not known to be associated with VHD, including
fluoxetine (Prozac; Eli Lilly) and its main metabolite norfluoxetine. This receptorome screen allowed
the rapid identification of the 5-HT2B receptor as
the likely culprit for the side effects of fenfluramine
and other drugs that induce cardiac valvulopathy. A
similar conclusion was reported by Fitzgerald and colleagues36 who independently showed an association
between 5-HT2B receptor agonist potency and valvulopathic propensity of several approved medications.
Subsequently, the 5-HT2B receptor was identified as
the likely molecular target responsible for fenfluramine-induced pulmonary hypertension — a fatal
side effect of fenfluramine administration37.
Since then, we used a commercially available
library (see TABLE 2 for sources of libraries containing
approved medications) to determine whether other
approved medications might be likely to induce
VHD of the fenfluramine-type by virtue of 5-HT2B
receptor agonism. Pergolide and dihydroergotamine
were identified by us as potential valvulopathogens35,
whereas cabergoline was identified as a 5-HT2B receptor agonist by others38. Subsequently these medications have been identified by the US FDA and others
as valvulopathogens and their use has been severely
restricted39–41.
The receptorome screen that identified the 5-HT2B
receptor as the target for the side effects of norfenfluramine also identified the 5-HT2C receptor as the likely
site responsible for the appetite-suppressing actions of
norfenfluramine (FIG. 4). Indeed, for many years it had
been appreciated by others that 5-HT2C receptor agonists are anorectic42,43 and that the anorectic actions
of fenfluramine in vivo are produced, in part, by
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that APD 356 suppresses appetite and induces weight
loss without detectable echocardiographic changes in
obese humans (see Arena Pharmaceuticals in Further
information).
From Alzheimer’s disease to cancer
With the commercial availability of libraries of
approved medications for in vitro and in vivo
EP-3
EP-1
Adenosine A2
Adenosine A1
α1B
α1A
β2
β1
α2C
α2B
α2A
AMPA
PCP
NMDA
H2
H1
Oxytocin
V3
V2
V1
BZP
GABAB
GABAA
DAT
NET
SERT
M5
M4
M3
M2
M1
Kappa
Delta
MU
D5
D4
D3
D2
D1
5-HT7
5-HT6
5-HT5
5-HT2C
5-HT2B
5-HT2A
5-HT1E
5-HT1D
5-HT1B
5-HT1A
5-HT2C receptor agonism44. These observations have
led to the proposition that 5-HT2C receptor-selective
agonists that are devoid at 5-HT2B receptor activity
will be safe and effective appetite suppressants 45.
Indeed, many pharmaceutical companies now have
active programmes for developing 5-HT2C receptor
agonists devoid of activity at 5-HT2B receptors, and
one, Arena Pharmaceuticals, has recently reported
(+/–) Fenfluramine HCl
Ki (nM)
0.1
1
10
100
1,000
10,000
5-HT2B
(+) Fenfluramine HCl
(–) Fenfluramine HCl
Methysergide maleate
Ergotamine tartrate
Phentermine
Fluoxetine
Norfluoxetine
(+/–) Norfenfluramine
(+) Norfenfluramine
(–) Norfenfluramine
Intraperitoneal accumulation (% of 5-HT max)
Side effects = 5-HT2B
Efficacy = 5-HT2C
NPY
120
Arcuate
nucleus
5-HT
Pergolide
DHE
100
Tuberomammilary
nucleus
80
Leptin
60
40
20
Raphe
nucleus
H3
0
Histamine
–20
–13
–12
–11
–10
–9
–8
–7
log [drug]
US FDA ‘black box’ warning
–6
–5
–4
H1
–3
5-HT2C
5-HT2C receptor
activation suppresses
appetite [AU: OK?]
APD 356
NPY
Paraventricular
hypothalamic
nucleus
Figure 4 | How to make a safe and effective anorectic agent. The top panel shows the receptorome profile that identified the
5-hydroxytryptamine 5-HT2B receptor as the molecular target responsible for the side effects of fenfluramine and the 5-HT2C
receptor as the molecular target likely to be responsible for the anorectic actions of fenfluramine. Subsequently, the medications
pergolide and dihydroergotamine (DHE) were identified in vitro and in humans as being valvulopathic. Importantly, Arena
Pharmaceuticals has developed a 5-HT2C receptor-selective agonist that proved effective at suppressing appetite in a Phase II
clinical trial without inducing heart-valve pathology as assessed by echocardiography. The bottom right figure represents multiple
pathways involved in appetite control in mammals. In addition to the 5-HT2C receptor pathways described, additional hormones
and neurochemicals have been described in the regulation of appetite control. Leptin interacts with leptin receptors within the
central nervous system resulting in stimulation of hypothalamic anorexigenic pathways. Increased levels of histamine have also
ben shown to suppress appetite via these pathways.
1012 | DECEMBER 2005
| VOLUME 4
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REVIEWS
screening campaigns, potential therapies have been
identified for several indications. For example,
non-steroidal anti-inflammatory drugs (NSAIDs;
for example, ibuprofen) and peroxisome proliferator-activated receptor-γ (PPARγ; thiazolidinediones)
inhibitors have emerged as potential therapeutic
agents for Alzheimer’s disease46,47 suitable for clinical
trials. A different approach by Kukar et al.48, using
a small library of 300 compounds (of which many
were approved medications), identified several new
compounds that mimic Alzheimer’s disease by elevating amyloid 42-β production. This chemical genetic
approach has identified potential pathways involved
in Alzheimer’s disease that could subsequently be
modulated by small molecules.
Likewise in cancer chemotherapeutics, the generic
medication fluphenazine — which has potent activities at various 5-HT and dopamine receptors — was
identified as a candidate medication for multiple
myeloma49 with clinical trials in progress. Immune
Control has proposed that the 5-HT1B receptor antagonist activity of fluphenazine (Ki~300 nM; see PDSP
Ki Database Search Page in Further information)
might be responsible for its actions. This approach
has begun to be widely adopted by the commercial
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Acknowledgements
The authors thank D. Sheffler for figures related to the JC virus life
cycle, W. Kroeze for editorial assistance and grants from the
National Institutes of Health and the National Institute of Mental
Health-Psychoactive Drug Screening Program for supporting the
work in the authors’ lab.
Competing interests statement
The authors declare competing financial interests: see Web version
for details.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
GLT1
OMIM:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
ALS | Crohn’s disease | epilepsy | stroke
FURTHER INFORMATION
Arena Pharmaceuticals:
http://www.arenapharm.com
PDSP Ki Database: http://kidb.case.edu
PDSP Ki Database Search Page:
http://kidb.case.edu/pdsp.php
The Robert Packard Center for ALS Research at Johns
Hopkins: http://www.alscenter.org/clinical_trials
Access to this interactive links box is free online.
www.nature.com/reviews/drugdisc