Mechanism based screening discovery of t

International Journal for Parasitology 29 (1999) 105-I 12

Mechanism-based screening: discovery of the next
generation of anthelmintics depends upon more basic
research
Timothy
Animal

Health

G. Geary*, David P. Thompson,

Discovery

Research,

Pharmacia

& Upjohn.

301 Henrietta

Ronald D. Klein
Street,

Kalamazoo,

MI 49007,

USA

Received 24 March 1998; accepted 17 September 1998

Abstract
The therapeutic

arsenal for the control of helminth

infections

contains only a few chemical classes. The development

and spreadof resistancehaserodedthe utility of most currently available anthelmintics,at least for someindications,
and is a constant threat to further reducethe options for treatment. Discovery and development of novel anthelmintic
templatesis strategicallynecessaryto preservethe economicand health advantagesnow gainedthrough chemotherapy.
As the costsof developmentescalate,the questionof how bestto discovernew drugsbecomesparamount. Although
random screening in infected animals led to the discovery of all currently available anthelmintics, cost constraints and
a perceptionof diminishingreturns require newapproaches.Taking a cue from drug discoveryprogrammesfor human

illnesses,
we suggestthat mechanism-based
screeningwill provide the next generationof anthelminticmolecules.Critical
to success
in this venture will be the exploitation of the Caenorhabditis
elegnnsgenomethrough bioinformatics and
genetic technologies.

The greatest obstacle to success in this endeavour

is the paucity of information

available about the

molecular physiology of helminths, making the choice of a discovery target a risky proposition. 0 1998Australian
Society for Parasitology.Publishedby ElsevierScienceLtd. All rights reserved.
Keywords;

Nematode; Anthelminlic;

Mechanism-based screening; Drug discovery; Caenorhabditis

1. State of the art in drug discovery
The history of drug discovery can be arbitrarily
separated into four overlapping eras, separated by
paradigm shifts in technology. The first medicines
were botanicals, identified presumably by trial and
error; from them came (directly or indirectly) many
important drugs, including aspirin, digitalis, morphine and quinine. The concept that chemical compounds could reproduce the beneficial effects of

*Corresponding author: Tel: I-616-833-0916; Fax: 1-616-8331149; e-mail: tggeary@jam.pnu.com.

e/egans

medicinal plants led to the first paradigm shift in
drug discovery: that it should be possible to find
therapeutically useful agents among collections of
synthetic compounds, in addition to compounds
derived from traditional botanical medicines. Perhaps the first recognisable example of the principle
of screening was Paul Ehrlich’s search for trypanosomicidal compounds (see[l]), which initiated
the second era of drug discovery. This assay measured the survival of parasites in infected rodents
dosed with compounds available from a dye-manufacturing concern. Although the paradigm of testing random collections of compounds in whole
organisms, whole tissues,or whole cells repeatedly

0020-7519/99/S - see front matter ia 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved.
PILSOO20-7519(98)00170-2

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proved useful in many different therapeutic areas,
it eventually fell from favour and was replaced largely by a new paradigm: drug design. Within the
past two decades, however, another paradigm shift
has occurred. Screening of random compounds has
enjoyed a remarkable
renaissance in the pharmaceutical industry. Three developments have been
key to this most recent revolution: adoption of the
strategy of mechanism-based
screening; advances
in technology that permit testing of tens of thousands of samples per day; and the ability to provide
millions of test compounds through combinatorial
chemistry. These advances have led to the construction of the fourth discovery paradigm, one that
rewards programmes
which can advance a validated target suitable for automated, miniaturised
ultra-high-throughput
screening.
As industrial
investment in drug discovery becomes increasingly
devoted to projects that fit this new paradigm,

opportunities for projects that cannot meet the new
requirements will diminish. How can we ensure that
anthelmintic
discovery is not left behind? This
review will address the key components of the new
paradigm and the barriers that limit its application
to the discovery of parasiticides.
1 .I. The fading paradigm
of anthelmintic
whole-organism-based
screening

screening:

Ehrlich’s screenfor trypanosomicides established
a paradigm for discovery that employed the target
organism/tissue as the tool. This philosophy was
extended to assays that measured survival, in the
presence of test compounds, of tumour cells,
bacteria, fungi and parasites (or a surrogate such

as Caenorhabditis
elegans), either in culture or in
animal models of disease. In addition, animal
models were developed for compounds that were
useful in cardiovascular diseases,mental illnesses
and metabolic diseasessuch as diabetes, among
many others. These screens were relatively low
volume (by current standards) and labour-intensive. Typically, only analogues of known drugs or
endogenous substanceswere tested. Notable exceptions were screens for new chemotherapeutic
agents, which evolved to accommodate large numbers of compounds or fermentations selectedat random. It is sobering but useful to realise that

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essentially all of the available classesof chemotherapeutic agents for treatment of cancer, prokaryotic and eukaryotic infections are derived from
screensof random-compound collections in wholeorganism/whole-cell bioassays.Despite the unquestioned successof this discovery strategy, it became
associatedwith a rather low intellectual status, and

fell from favour throughout most of the industry.
In addition to the perception that broad screening
was not really scientific, two other factors contributed to its demise: the senseof a diminishing
rate of return in new compound discovery, and the
appearance of another paradigm shift in discovery,
the more cerebral approach to drug identification
termed rational drug design [2].
1.2. Drug design in anthelmintic

discovery

In a nutshell, drug design promised that one
could deduce the structure of an active compound,
given a sufficiently accurate understanding of the
three-dimensional structure of the target site and
enough computational power. While this promise
has yet to be fulfilled, the investment in computational chemistry has had practical consequences. Very frequently, analysis of ligandreceptor interactions with X-ray crystallographic
techniques has facilitated synthesis of more active
analogues [3, 41. An alternative strategy based on
the theme of computational chemistry involves

what may be considered electronic screening. Data
on the three-dimensional charge-shape structures
of millions of chemical compounds are available
in databases. One can screen these databases for
compounds that are most closely complementary
to the shape-charge structure of the target site on
the protein of interest. The compounds predicted
to have the best fit can be synthesised or acquired
and tested for activity in a low-throughput screen.
This sophisticated electronic high-throughput
screen successfully identified several inhibitors of
a schistosome protease [5]. Despite considerable
investment in drug design technology, discovery
of a new template is not a likely event. Though
unquestionably useful in refining leads (though
threatened in this regard by combinatorial chemistry; seebelow), its attractiveness as a strategy for
new template discovery was diminished by the

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length of time typically needed from investment to
payoff. Like the situation for parasite vaccines, the
technology seemed obviously destined for great success, which led some proponents to prematurely
predict a scientific revolution based upon it. The
surprising demonstration
that broad, blind screening in a mechanism-based
assay could generate
novel templates for an unexploited drug target provided an alternative to drug design for new template
discovery and set off the fourth paradigm of drug
discovery.
1.3. Mechanism-based

high-throughput

screens

Research beginning in the mid- 1970s proved that
morphine and related opiates were non-peptide
ligands for receptors for the endogenous neuropeptides termed enkephalins and endorphins
[6].
Morphine was, of course, isolated from a traditional botanical medicine, the poppy. While it was
evident that receptors for neuropeptides and peptide hormones were excellent targets for drug discovery, the problem of how to identify the next
morphine
remained
unsolved.
Neither
lowthroughput animal screens for novel analgesics nor
drug design technologies had succeeded in uncovering novel, non-peptide ligands for peptide receptors. An alternative strategy had to emerge from
basic research.
Receptor binding assays were first proposed in
1971 [7] and the concept was brought to fruition a
few years later as high specific activity radiolabelled
ligands became available. These assays were used in
the pharmaceutical industry mostly to discriminate
intrinsic activity among limited numbers of analogues of known compounds [8,9]. A key advance
was the conceptual extension of receptor binding
assays to random screening. Mechanism-based
screening searches for compounds that selectively
act on a defined receptor, instead of looking for
compounds that affect tissues or organisms through
unknown mechanisms. This concept was brought to
fruition by the demonstration that high-throughput
blind screening of microbial fermentations (a natural products collection), coupled with traditional
medicinal chemistry, identified novel, highly potent,
non-peptide ligands for mammalian cholecystokinin receptors [ 10, 111. The discovery of the fungal

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107

metabolite asperlicin and its more potent derivatives changed the image of screening in a profound
way. Random high-throughput
screening meant
that chemical insight or design was no longer
needed at the start of a discovery project; no preconceptions about potential templates were necessary or appropriate.
Asperlicin
proved that this
strategy could lead to the discovery of entirely
unanticipated templates. Important developments
in technology, including computer storage and
analysis of data, miniaturisation
and automation,
drastically reduced labour costs and, at least in
theory, sped up the essential process of new lead
discovery [12]. The considerable expense of hardware and the need to maintain a cadre of trained
technicians to run the sophisticated equipment led
to the centralisation
of screening operations in
many companies. How useful this technology will
prove for anthelmintic discovery is a moot point,
but it has become the standard for antibacterial
discovery. a process that is conceptually identical
[131.
The sophistication and capacity of modern highthroughput screening operations is remarkable [14].
New technologies that promise ultra-high-throughput and reductions in the amount of test compound
needed for screening are being introduced at an
increasing rate [15-l 71. For instance, microplate
formats were once standardised
for a 96-well
design; 384-well plates are now in use (the same size
as the 96-well plates) and 1536-well plates are in
development. Assays that can accommodate nanolitre volumes and assay rates of 1000 000 per week
are envisioned. While the benefits in efficiency of
operation are obvious, the technology utilised for
such screens will impose further limits on the types
of targets that can be addressed.
Additional improvements in ultra-high-throughput screening are predicted to arise from developments in combinatorial chemistry [IS-201. In combinatorial chemistry, myriad compounds are synthesised simultaneously as defined mixtures, rather
than one at a time as in traditional medicinal chemistry. Mixtures or single compounds derived from
this exercise can then be tested, with the potential
for generating and testing over a million compounds within a short time. Currently,
diversity
within any given combinatorial library is limited by

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the use of one or a few templates as scaffolds for
modification; it is not clear that available libraries
will prove broadly
useful for new template
discovery. However, efforts to increase the diversity
of libraries are underway [21] and may very well
serve to make available many millions of novel
compounds
for screening. This situation only
underscores the need to make sure that anthelmintic
screening is compatible with the rapidly changing
state-of-the-art.
Since a large number of analogues
can be made combinatorially
based upon a lead
derived from screening, the sometimes slow process
of single analogue synthesis and testing can be eliminated. The ability to look at thousands of random
analogues based upon a template of interest quickly
and efficiently also lessens that value of sophisticated drug design techniques to guide analogue
synthesis; it is much cheaper and quicker to test a
few thousand analogues than to attempt to make
better
compounds
through
rational
design.
Although diminishing the role of the intellect in
the discovery process may seem counterintuitive,
it
must be remembered that the real work begins when
a good lead compound is available. How that lead
is obtained is almost irrelevant from the industrial
perspective.

may be prohibitively expensive to routinely collect
the amount of parasites needed to supply tissue for
non-recombinant
assays. It is also sadly true that,
although one can suggest many attractive targets
for anthelmintic discovery, few have been validated
(see below). One example of a cheap and simple
alternative system that does work is the use of
recombinant micro-organisms
as screening tools [ 1,
221. Expression of genes encoding parasite enzymes
in bacteria or yeast that lack the homologous
enzyme provides an efficient tool for high-throughput screening [I]. Remarkably, heterologous receptor expression and function can be linked to a
viability phenotype in yeast, providing a powerful
means of screening for agonists or antagonists of
hormones, neurotransmitters
or neuropeptides [22].
Assays that use microbial viability as an end-point
have a long and honoured tradition in drug discovery and provide an approach that can be used
by almost any industrial research group to screen
outside a core, high-tech facility.
None the less, the long-term preservation
of
anthelmintic screening will require participation in
the current discovery paradigm. How can we keep
up?

1.4. Mechanism-based

2. Caenorhabditis elegans: the future of
anthelmintic discovery?

anthelmintic

screens

The enormous investment being made in the new
technology is not without drawbacks.
Alternative
methods of discovery will be discouraged in order
to recoup the costs of establishing, staffing and
maintaining core screening facilities. Projects that
benefit from a large knowledge base can propose
candidate drug targets with confidence that a “hit”
will be valuable. Fields that suffer from an inadequate knowledge base cannot readily offer validated targets suitable for the technology and will
be left out of the discovery arena unless simple
alternative techniques can be identified, developed
and implemented. Since adaptation of a putative
target for screening technology can be an expensive
proposition, cost can also prevent screening against
non-validated targets. With regard to anthelmintic
screening, it is not obvious that invertebrate targets
will be amenable to expression in systems developed
for mammalian genes and proteins. In addition, it

The free-living nematode C. elegans can easily
be propagated in culture, unlike parasitic species.
Essentially all commercially available anthelmintics
have detectable effects on C. elegans at reasonable
concentrations, which prompted its development as
a model for parasitic nematodes for drug screening
[23]. Visual inspection of wells in 96-well microtitre
plates for drug-induced changes in behaviour, survival or reproduction. though labour-intensive, permitted the screening of up to a few thousand
samples per week. Compatible with the discovery
paradigm available in the early 1980s the C. elegans
screen became an essential part of most industrial
anthelmintic programmes.
Samples that affected
the worm were typically tested in an animal model
[24]; disappointingly
few demonstrated
sufficient
activity to pass on to further testing in target
animals. It is surprising and sobering to note that,

T.G. Geury

et al. ! Intermtional

Joumal,fbr

of the many initial positives detected in C. elegans
random screens, none (to our knowledge)
representing a new anthelmintic template is even in
advanced development. The high labour investment
required, the failure of the screen to identify valuable new leads, and the shift in discovery paradigm
to high-throughput,
mechanism-based
screens,
markedly reduced the use of this whole-organism
screen.
Although it may seem paradoxical, the importance of C. elegans will only increase with the paradigm shift. The entire genome of this organism,
some lOOmillion bases, should be completely
sequenced by the end of 1998. The data promise
to be of enormous significance for many areas of
research, including anthelmintic discovery.
2.1. Genome scanning
targets

to iden@

potential

drug

Analysis of the nucleotide sequence of the C.
elegans genome includes diagnosis of predicted
open reading frames, preliminary characterisation
of the predicted protein (hydropathy
analysis, etc.)
and an attempt to identify the most closely related
proteins
by comparison
to other sequences
deposited in public databases (see [25]). Indeed,
such “bioinformatics”
approaches are currently of
intense interest [26]. The database can be scanned
with amino-acid motifs conserved among potential
drug targets (ion channels, receptors, enzymes) to
identify
nematode
homologues.
Alternatively,
annotation provided by the homology analysis can
be used to identify members of such groups. This
kind of query, or instance, reveals that there are
already > 200 putative members of the G proteincoupled receptor family in C. elegarzs (DE Lowery,
personal communication).
Several steps can be
taken to prioritise candidate drug targets identified
in such an exercise, including:
1. Are homologous sequences present in important
parasitic species?
2. Is the candidate gene expressed in adult nematodes?
3. Is it expressed in a target tissue?
4. Can the candidate gene be functionally expressed in a system suitable for screen development?

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109

It must be emphasisedthat, as yet, little has been
reported about the physiological function of C. elegans proteins, and the small size of this animal
makes most physiological experiments problematic.
However, the reverse genetic approach available for
C. elegansprovides an alternative way to assessthe
importance and function of candidate drug targets.

2.2. Reverse genetics ill C. elegans: connecting
phenotype to genotype
The powerful genetic system available in C.
elegans,amplified by the nearly complete nucleotide
sequence of the genome [27], can be exploited to
identify the effects of inactivating a suspecteddrug
target through analysis of mutations in the gene
that encodes it. If the resulting phenotype suggests
that the potential target must be functional for survival of an adult parasite in a host, one can initiate a
screenwith someconfidence that active compounds
will be valuable. Several methods are available to
identify specifically mutated C. elegans genes;
unfortunately, it is not yet possible to specifically
knock out C. elegansgenes. However, useful techniques have been developed to isolate desired
mutants from populations of randomly mutated
worms. The original method relied on mutations
randomly induced by transposon insertion [28,29];
transposons are short sequences of DNA that
“jump” or translocate somewhat randomly into the
genome and typically inactivate genes into which
they have been inserted. Pools of worms that have
an active transposon can be screenedby PCR with
primers specific for the gene of interest and for the
transposon (in two pairs). Pools that contain worms
with a transposon inserted into the target gene will
produce an amplified DNA fragment, and individuals carrying the desired mutation isolated by
repeated rounds of pool dilution and PCR. Mutants
isolated this way are typically homozygous, though
this must be verified before phenotypic characterisation [29].
An alternative method involves exposure of
worm populations to mutagens that induce
deletions [30, 311, such as trimethylpsoralen and
U.V. light or ethyl methanesulfonate (EMS). Again,
pools of mutated worms are screened by PCR. In
this strategy. primers specific for the genomic locus

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of interest are used, and PCR conditions
are
adjusted so that the wild-type gene is very poorly
amplified. However, if a pool contains an individual
with a signficant deletion in the target gene, an
amplified fragment will be readily available and the
individual can be isolated by standard sib-selection
protocols [29, 301.
A very recent finding provides a simpler and faster method to analyse the consequences of gene
knock-out.
Inexplicably, dsRNA prepared from a
target gene and injected into C. elegans has been
found to specifically suppress expression of that
gene [32]. Progeny of worms injected with dsRNA
demonstrate a knock-out phenotype, with eventual
reversion to a wild-type phenotype in succeeding
generations. Although the mechanism behind this
“genetic interference” has not been elucidated, the
apparent robustness of the technique suggests that
it will prove remarkably
useful in evaluating, at
least in this free-living organism, the value of candidate anthelmintic targets.
2.3. Pharmacophenotype

screening

Since a great deal of experience has been gained
in the pharmaceutical industry in the use of wholeorganism C. elegans screens, it is worthwhile
to note
that the exquisite understanding of its genome may
resurrect such screens for extremely specific discovery purposes. By carefully defining the phenotype of worms with mutations in target genes, one
can construct a screen in wild-type animals to discover compounds
that exactly reproduce
the
mutant phenotype [33]. A considerable advantage
of this strategy is that any active compound will
be, almost by definition, specific for the target of
interest. The strategy avoids the problem of high
rate of false positives that haunted previous C. eleguns screens, which is also an important advantage.
2.4. Lethal mutations define drug targets
Analysis of mutagenised populations of C. eleguns has identified a number of mutations which are
lethal. Almost by definition, the protein encoded by
the mutated gene in these animals is a drug target,
since interference with its function is associated
with lethality. This is particularly true for loss-of-

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function mutations. The convergence of the physical and genetic maps with the genome sequence
allows one to quickly identify cosmid clones that
overlap the site of a lethal mutation [34]. Transformation of the lethal mutant with clones that
encode regions of the cosmid can complement the
mutation; the wild-type gene will rescue the lethal
phenotype. The sequence of the rescuing gene may,
by homology analysis, define its function. Further
experiments can determine if homologues of this
gene are present in parasitic species and if it is
expressed in the currently acccepted target (adult)
stage for anthelmintic chemotherapy and thus set
the stage for screen development.
2.5. Future opportunities from genome projects
Comparisons
among target pathogens will be
made simpler, at least on a computational level, as
information
about other genomes becomes more
available. Unlike the case for C. elegans, in which
the entire genome will be sequenced, most parasitic
organisms
will be characterised
only by compilations of sequences of genes that are expressed,
as Expressed Sequence Tags (or ESTs) [35, 361. It
is possible to search the C. elegans database with
sequences derived from parasite EST projects. If
homologous
genes are found in C. elegans, any
phenotypes associated with mutations will be readily apparent. Cloning of the gene encoding the parasite homologue and its expression in the mutant
will show if the genes share functions [37] helping to
validate the target insofar as the C. elegans mutant
phenotype may be inferred to be inducible by drug
exposure in the parasite. In this regard, C. elegans
can provide a shortcut to target identification and
validation. Finally, recent molecular analyses have
led to the proposal that organisms which molt, such
as the arthropods
and nematodes, form an evolutionary clade and may be more closely related
than previously suspected [38]. In this regard, the
unanticipated arthropod-insect
spectrum of action
exhibited by the macrocyclic lactones may make
more sense. The genome of a “model” arthropod,
Drosophila melanogaster, is also of intense interest
in the global scientific community. It may soon be
possible to compare the C. elegans and D. melanogaster databases to identify common targets,

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perhaps speeding the purposeful discovery of the
next template with an avermectin-like
spectrum.
3. Barriers
discovery

to mechanism-based

anthelmintic

3.1. Ignorance is not bliss
It is astonishing to realise that the first metazoan
to have its genome completely sequenced will be a
nematode. This prodigious accomplishment
bodes
well for the future of anthelmintic discovery, but
there is a long way to go before we understand how
the database can be mined for useful anthelmintic
targets. We know too little about how nematodes
work to be confident that a C. elegans gene with
some homology to a gene known from a mammal
(for instance) would be a good site for chemotherapeutic intervention. A marriage between the
physiology of parasitic nematodes and the genome
of C. elegans must be arranged; how that is to be
done remains to be seen. Among the parasites, only
the physiology of Ascaris suum and Ascaris lumbricoides has been investigated to any significant
extent, and we still cannot say how closely those
species
resemble
the trichostrongylids
(for
instance). Many areas of nematode physiology
remain baffling (at least to us; [39]), and how insight
can be gained into such areas simply by reading
nucleotide sequence is not clear. It is likely that,
as attention shifts from compiling the sequence to
interpreting it, answers will be found as the functions of C. elegans genes are determined by knockout technology. Choosing likely drug targets for
such studies should be a priority for industrial
research groups. In addition, the availability of this
database should encourage governments and other
monetary sources that investments in nematode
physiology are timely, appropriate and necessary
to derive full benefit from the C. elegans genome
project. The need for increased spending of publicsector funds on basic aspects of parasitic nematode
biology has never been clearer or stronger.
3.2. Target validation
Proposing targets for anthelmintic discovery is
not a problem; every biochemist who studies these

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111

creatures has at least one [40]. The problem is in
deciding how to prioritise candidates. If one accepts
that the only truly validated target is a target for a
known efficacious drug, then there are only a few
such targets available to us. The techniques for
analysing C. elegans genes described above will be
useful for assigning function to them, but the question remains how well these data predict function
of homologous parasite genes. One can determine
if candidate genes are present in important species
of parasites with a variety of molecular tools, but
establishing a conserved physiology between C. eleguns and a parasite, or among parasite species, will
remain a formidable task. In the end, it is likely that
we will know if a target is valid only when a screen
uncovers a compound that potently affects it; if the
compound is efficacious in vivo, a target is born. If
not, another target must be selected for screening.
Only organisations
that can tolerate risk on this
level will remain in the field. We believe that the
potential rewards which will come from a new
anthelmintic template justify the endeavour.

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