Medical Mycology
November 2009, 47, 671–680
Review Article
Nutrient sensing G protein-coupled receptors: interesting
targets for antifungals?
PATRICK VAN DIJCK
VIB Department of Molecular Microbiology, K.U. Leuven, and Laboratory of Molecular Cell Biology, Institute of Botany and
Microbiology, Katholieke Universiteit Leuven, Leuven-Heverlee, Flanders, Belgium
G protein-coupled receptors (GPCRs) are important targets for various drugs that are on
the market. An important area in which we urgently need novel drug targets is the field of
antifungals. Recently a new class of nutrient sensing G protein-coupled receptors have
been identified in fungi. The founding member of this novel class of GPCRs is Gpr1, the
sucrose/glucose sensing receptor of Saccharomyces cerevisiae. This receptor activates
the cAMP-PKA pathway that is important for the yeast-to-pseudohypha transition and
for stress tolerance. The capacity to change morphology is an important virulence factor for pathogenic fungi and it has been shown in these fungi that morphogenesis also
depends on the cAMP-PKA pathway. Therefore, functional Gpr1 homologous receptors in these fungi may be interesting targets for antifungals. Despite the fact that these
receptors are not essential for growth, modification of their activity may affect fitness
of the fungus allowing the human or plant defense systems to win the battle. In addition, synthetic avirulent interactions, such as the one observed between the C. albicans
Gpr1 and the trehalose-6-phosphate phosphatase enzyme, may be a tool to come up
with a good combination therapy approach. It will be necessary to identify antagonists
or inverse agonists for these receptors, which will require good screening systems and
large compound libraries.
Keywords GPCR, Saccharomyces cerevisiae, filamentous fungi, nutrient sensing,
antifungal drug
Introduction
The superfamily of G-protein coupled receptors (GPCRs)
is one of the largest and most studied families of proteins,
largely because many of these seven-transmembrane
spanning domain containing proteins are important targets for drug development. GPCRs transduce a large variety of signals from the environment to the cellular
machinery controlling metabolism, growth and development. These signals include hormones, growth factors,
Received 23 July 2008; Received in final revised form 1 December 2008;
Accepted 26 December 2008
Correspondence: Patrick Van Dijck, VIB Department of Molecular
Microbiology, Katholieke Universiteit Leuven, Laboratory of Molecular
Cell Biology, Kasteelpark Arenberg 31, B-3001 Heverlee, Belgium.
Tel: ⫹32 16321512; fax: ⫹32 16321979; E-mail: patrick.vandijck@bio.
kuleuven.be
© 2009 ISHAM
neurotransmitters, light, odorants, taste compounds, etc.
[1]. Many of these ligands function as agonists which
means that the receptor only activates the downstream
signaling pathway when the ligand is bound to the ligandbinding site. Drugs targeting GPCRs can be divided in
two major classes: inverse agonists and antagonists.
Inverse agonists bind to the same binding site as the agonists but induce constitutive negative signaling whereas
an antagonist blocks the agonist or inverse agonist mediated response to prevent proper signaling. Since an antagonist can bind sites other than those occupied by the
agonist, an inverse agonist may be a better choice in
situations where the receptors undergo spontaneous activation without ligand stimulation.
According to the GPCR data base web site
(www.gpcr.org/7tm/) the superfamily of GPCRs can be
subdivided into six major classes: Class A (rhodopsin
DOI: 10.3109/13693780802713349
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Van Dijck
like), Class B (secretin like), Class C (metabotropic
glutamate/pheromone), Class D (fungal pheromone),
Class E (cAMP receptors) and Class F (the Frizzled/
Smoothened family). Many GPCRs have been identified
based on similarity searches using bioinformatics tools
or by PCR-based approaches with the consequence that
no information about the ligand was known at the time
of cloning. A whole new area of research was developed
in order to deorphanize these receptors. This was called
reverse pharmacology in which high throughput methods
were developed to identify putative ligands. Initially, this
was done using radioligand binding assays with membrane fractions of eukaryotic cells transfected with
orphan GPCRs. As more knowledge about the mechanism of action of these GPCRs became clear, novel types
of assays were developed in which the level of downstream effectors of the GPCR-activated pathways were
monitored. A classical example is the increase in calcium
levels in the cell upon activation of a GPCR. These calcium levels can then be converted to a light signal, which
is measured by a luminometer. This and similar assays
have been developed into high-throughput screening systems allowing tens of thousands of compounds to be
tested [2]. Yeast has also been used extensively as a
cheap and simple high-throughput system to identify
ligands of human GPCRs. This is mainly due to the high
homology that is present between the yeast pheromone
signaling pathway and the pathways activated by mammalian GPCRs. When expressed in yeast, mammalian
GPCRs have been shown to couple functionally to the
endogenous yeast Gα protein Gpa1 or to co-expressed
mammalian Gα subunits (or hybrid mammalian/yeast
Gα proteins). Activation through the pathway is monitored by the presence of either pheromone-regulated
promoter-reporter (e.g.,luciferase) constructs or by pheromone-regulated promoter-auxotrophic marker constructs allowing selection on minimal medium [3–7]. The
ease of working with S. cerevisiae makes this approach
very suitable for high throughput screening of new drugs
as well as allowing the investigation of the still unknown
ligands of orphan receptors. Apart from S. cerevisiae,
alternative systems such as S. pombe have been explored
as not all mammalian GPCRs could be functionally
expressed in S. cerevisiae [8].
Since the 1990s, bioinformatics methods have been
used for predicting the ligands of orphan GPCR. An early
example was the prediction that neuromedin U was the
ligand of the orphan receptor FM3 [9]. Since then, many
other orphan receptors have been deorphanized using
this approach. The progress on GPCR de-orphanization
can be followed on the IUPHAR homepage (http://www.
iuphar-db.org/). However, from the about 850 predicted
GPCRs in the human genome, there are still about 100
receptors of which nothing is known about the ligand.
This may open the possibility that some GPCRs may
not have an endogenous ligand but play other roles. An
example is the GPR50 receptor. By using BRET (bioluminescence resonance energy transfer), which is a
method to show interactions between two proteins, it was
shown that GPR50 heterodimerizes with two GPCR
melatonine receptors MT1 and MT2 [10]. Dimerization
of GPR50 with MT1 prevents melatonine binding at
MT1 and prevents melatonine-induced signal transduction by blocking the recruitment of the G protein
to the MT1 receptor. This shows that some orphan
GPCRs may regulate the function of non-orphan GPCRs
through heterodimerization or other mechanisms
[11,12].
Approximately 30% of the currently used drugs have
a GPCR as target, which makes them the most
important family of proteins in disease treatment.
Pathogenic fungi, such as Candida albicans, Aspergillus
fumigatus, Magnapothe grisea and Cryptococcus
neoformans also contain a number of GPCR encoding
genes in their genomes that play a wide array of roles in
morphogenesis, mating, and virulence. However, these
receptors have not been considered as the interesting
targets for antifungals as they are not essential for
growth. In general fungi contain two types of GPCRs,
pheromone receptors (class D, see above) and nutrient
sensing receptors [13]. Upon binding of pheromones, the
pheromone receptors undergo conformational changes
and activate the signalling cascades leading to growth
arrest and mating, as in the case of the class D receptors
Ste2 and Ste3 of S. cerevisiae. Their functioning
has been elucidated in great detail [14]. Sensing of
pheromones activates a MAP-kinase pathway that results
in the activation of transcription factors that will
stop cell proliferation and that induce mating. Homologues of these mating factor receptors have been
found in many fungi, including basidiomycetes,
although in this latter group only Ste3 homologues are
present.
Nutrient sensing GPCRs detect the presence or
absence of nutrients thereby inducing signaling pathways that control metabolism, morphogenesis and virulence [13,15–17]. Interestingly, these fungal nutrient G
protein-coupled receptors do not belong to any of the
above-mentioned classes and therefore seem to be fungal specific, an important characteristic for an antifungal drug [18–22]. Although nutrient sensing GPCRs are
not essential for growth, which makes them less attractive as fungicidal drug targets, inappropriate signaling
through these receptors can cause lower virulence
or aberrant mating, thereby lowering the fitness of the
fungus. In this review we will focus on this group of
© 2009 ISHAM, Medical Mycology, 47, 671–680
GPCRs as antifungal targets?
nutrient sensing G protein-coupled receptors and their
potential as a novel class of antifungal targets.
Fungal Gpr1 homologues can be divided into
three classes
The fungal nutrient-sensing GPCRs apparently define a
novel evolutionary distant subfamily of GPCRs [23]. We
have performed extensive BLAST and TBLASTN [24]
searches in various public databases and in the A. niger private database, using the S. cerevisiae Gpr1 and the S. pombe
Git3 and Stm1+ sequences as queries. ClustalX [25] was
used to make multiple alignments and TreeView (http://
taxonomy.zoology.gla.ac.uk/rod/rod.html) was used to make
a phylogenetic tree (Fig. 1). The phylogenetic tree clearly
indicates the presence of three subfamilies of Gpr1-related
proteins. The first class (red) contains GPCRs with close
homology to the yeast Gpr1 receptor. A second class (blue)
contains GPCRs closely related to the GprD receptor of
A. nidulans. Genes homologous to this receptor are also present in other Aspergillus species, including A. fumigatus and
A. niger. A 3rd class (green) of putative nutrient sensing GPCRs
shows homology to the S. pombe Stm1+ receptor, although
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it is not very clear whether these receptors interact with a G
protein. Apart from fungi, homologues of this third class are
also present in Arabidopsis thaliana, C. elegans, D. melanogaster and humans. Furthermore, most organisms seem
to have at least two members of this class. In this phylogenetic analysis we did not include the G protein-coupled
cAMP receptors, originally described in Dictyostelium
discoideum or the pheromone receptors.
ClassI: ScGpr1, a glucose/sucrose sensing
receptor in S. cerevisiae
Gpr1 was identified in two-hybrid screens with the
Gα protein Gpa2 as bait [18,26] and in a screen for
mutants deficient in glucose-induced loss of heat
resistance, a property controlled by the cAMP-PKA
pathway [18]. The activity of Gpa2 is controlled by the
RGS protein Rgs2 [27]. Hence, a GPCR system composed of Gpr1, Gpa2 and Rgs2 has been proposed to
act as a glucose sensing system for control of the cAMP
pathway [13,28]. Recently we have obtained evidence
that Gpr1 is a sensor for sucrose and glucose and that
mannose acts as an antagonist [29]. This detection of
Fig. 1 Phylogenetic tree showing three subclasses of the new class of fungal nutrient sensing G protein-coupled receptors. The S. cerevisiae Gpr1, the
S. pombe Git3 and Stm1+ and the A. nidulans GprD protein sequences served as queries for the different BLASTP and TBLASTN searches. BLAST
hits of e-10 or lower were aligned using ClustalX and Treeview was used to produce the phylogenetic tree. The full name of the different species is
mentioned in the text. Putative intron sequences have been removed from the genomic sequences. The sequences are obtained from the Genome projects
going on at the Stanford Genome Technology Center, funded by the NIAID/NIH under cooperative agreement AI47087, the Institute for Genomic
Research (http://www.tigr.org), funded by the NIAID/NIH under cooperative agreement UO1 AI48594, the genome project of Ashbya gossypii [74], the
genome project of 5 different Saccharomyces spp. [75], the Syngenta proprietary database (G. zeae), the DSM proprietary database (A. niger; courtesy
to Dr Gert S. P. Groot) and the Broad Institute (http://www.broad.mit.edu).
© 2009 ISHAM, Medical Mycology, 47, 671–680
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glucose and sucrose is important for the rapid activation
of the PKA pathway resulting in induction of growth,
thereby providing an advantage for yeast cells that have
such a receptor. Since a G-protein is normally heterotrimeric, many laboratories have tried to identify the Gβ
and Gγ proteins that act together with Gpa2. In the yeast
genome there is only one Gβ protein Ste4 functioning
in the pheromone-sensing pathway. Based on twohybrid and GST in vivo pull down assays it was shown
that Gpa2 interacts with Gpb1/Krh2 and Gpb2/Krh1,
two proteins that have been proposed to act as Gβ mimicking subunits based on structural resemblance with
classical Gβ proteins [30–32]. In addition, genetic and
biochemical evidence has clearly indicated that Krh1
and Krh2 proteins not only interacted with Gpa2 but
also with the catalytic subunits of PKA [32–34] and
with the Ira1 and Ira2 proteins, which are the stimulators of the Ras GTPase activity [35]. Recently Asc1 has
been proposed as a Gβ-like protein for Gpa2. The Asc1
protein is the yeast ortholog of RACK1, the receptor of
activated protein kinase C1 [36]. Taken together it
seems that the Gpr1-induced signal transduction pathway is a highly complex one with a central role for the
Krh1 and Krh2 proteins as putative scaffold proteins of
the cAMP-PKA pathway, similar to Ste5 in the MAPK
pathway. A critical overview of the putative functions
of the Krh proteins as well as other non-conventional
Gβ proteins in a number of fungi was recently published [37].
Gpr1-dependent activation of the cAMP-PKA pathway
plays an important role in the induction of genes involved
in pseudohyphal and invasive growth [38–40]. For instance
diploid gpr1Δ or gpa2Δ strains cannot undergo the yeastto-pseudohyphae transition under certain environmental
conditions [38,41]. However, other environmental conditions, such as growth in the presence of maltose as the sole
carbon source can also stimulate Gpr1-independent hyphal
growth [42].
Another protein clustered within the class of Gpr1like receptors is the S. pombe Git3 receptor. Git3 was
identified in a screen for mutants affected in glucose
repression of fbp1 transcription and gluconate uptake
[43], which also suggest a function as glucose receptor.
In addition, all three subunits of the heterotrimeric
guanine-nucleotide binding protein were also identified
in this screen. Therefore, contrary to the situation in S.
cerevisiae, S. pombe has classical Gβ and Gγ subunits
in this pathway [44]. Another difference between the
two species is the fact that the Ras proteins are strong
stimulators of the adenylate cyclase in S. cerevisiae but
not in S. pombe [45]. More recently, Hsp90 has been
identified as a novel regulator of adenylate cyclase
activity in S. pombe. Specific mutations have been
identified that differentiate between its general chaperone function and its role in mediating the activity of the
cAMP pathway [46]. Whether the specific role of Hsp90
in this pathway is specific to S. pombe or whether it
exerts the same function in other fungi, including S.
cerevisiae remains to be investigated.
To investigate the importance of the class I Gpr1
receptor for virulence in pathogenic yeast, we as well as
others have generated C. albicans gpr1 and gpa2
knockout strains. We have tested these strains in a mouse
model for systemic infection as well as on reconstituted
tissues. Because other components of the cAMP-PKA
pathway, such as Cdc35 and Ras1, are essential for
virulence [47,48], our hypothesis was that GPR1 deletion
strains would be severely affected in virulence. However,
this was not the case. A C. albicans GPR1 deletion strain
is only marginally affected in virulence, despite a
strong effect on morphogenesis on solid media, which is
an important virulence factor [19,49]. The reason for the
low effect on virulence is probably that other pathways
involved in morphogenesis may be activated by external
signals and they may overcome the absence of Gpr1 [50].
In addition, there is also evidence that the function of the
C. albicans Gpr1 receptor diverged from the function of
the homologous receptor in S. cerevisiae. Deletion of the
S. cerevisiae GPR1 receptor results in absence of a cAMP
signal upon addition of glucose. In C. albicans, deletion
of GPR1 still results in a normal cAMP signal upon addition of glucose [19]. This indicates that the C. albicans
cAMP pathway can be activated independent of Gpr1.
We have shown, however, that overexpression of downstream components of the cAMP-PKA pathway as well
as the addition of cAMP can suppress the morphogenesis
phenotype observed in the GPR1 deletion strain [19,51].
So it seems that CaGpr1 may activate more than one
pathway. The same observation can be made for CaGpa2.
Recently it has been shown that apart from
its function in the cAMP pathway, C. albicans Gpa2
also plays an important role in the mating pathway in
C. albicans [52].
The Gpr1 homologue of N. crassa is GPR-4. Similar
to S. cerevisiae, deletion of this receptor results in the
absence of a glucose-induced cAMP increase [21]. No
functional information is available for the Gpr1 homologues of Ashbya gossypii, M. grisea and Gibberella
zeae. As mentioned before, no sequence-based homologous receptors seem to be present in basidiomycetes.
Recently, however, a functional homolog of the C. albicans Gpr1 receptorhas been described in Cryptococcus
neoformans and named Gpr4 [20]. This receptor has no
apparent sequence homology to either the yeast or the
C. albicans Gpr1 protein. However, the gene structure,
including a long 3rd intracellular loop is quite similar.
© 2009 ISHAM, Medical Mycology, 47, 671–680
GPCRs as antifungal targets?
The C. neoformans Gpr4 receptor is also situated
upstream of the cAMP pathway and is required for the
methionine-induced cAMP signal, indicating that methionine may be the ligand for Gpr4. Also in C. albicans
there are indications that methionine may be the ligand
for CaGpr1 as addition of methionine results in a rapid
internalization of the Gpr1 receptor and methionineinduced morphogenesis requires Gpr1 [19]. Despite the
fact that the C. neoformans Gpr4 receptor is situated
upstream of the cAMP pathway, this receptor is not
required for virulence. This is in contrast with its coupled Gα protein Gpa1, of which a deletion strain is
strongly affected in virulence [20,53].
ClassII: A. nidulans GprD, a putative nutrient
receptor in A. nidulans, required for normal
germination and growth
A. nidulans has nine putative GPCR encoding genes,
classified in four classes [54]. Two are similar to the
pheromone-sensing GPCRs, three are similar to the
yeast Gpr1 receptor, two are similar to the Stm1+ receptor (see further) and two are homologous to the D. discoideum cAMP receptor [55]. The three proteins with
homology to Gpr1 have been named GprC, GprD and
GprE. In our phylogenetic tree these three sequences,
together with homologous sequences from A. fumigatus
and A. niger, form a second class of GPCRs which are
clearly evolutionary distant from Gpr1. Disruption of
GprC or GprE did not result in any phenotype indicating
possible redundancy. Disruption of the homologous
GprD, however, resulted in a dramatic restriction of
hyphal growth and a strong increase in the number of
fruiting bodies [54]. The authors have obtained further
evidence that signaling through GprD is important for
inhibition of sexual development. The ligand of these
receptors is not yet known. However, the glucose-induced
cAMP increase in A. nidulans requires the G-protein
GanB, one of the three G proteins present in the A. nidulans genome [56]. In the future, it will be interesting
to investigate whether the A. fumigatus Gpr1 homologous receptors are required for virulence.
Class III: SpStm1+, a low nitrogen sensing
receptor in S. pombe
The third class of putative GPCRs contains Stm1+ homologues, which belong to the PQ-loop family (pfam04193.6).
The members of this family also have seven transmembrane domains and two of the loops between these domains
contain a conserved sequence, called the PQ motif [57].
A multiple alignment of the two PQ motifs of a number of
© 2009 ISHAM, Medical Mycology, 47, 671–680
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Stm1+ homologues is shown in Fig. 2. The glutamine residue seems especially important as it is conserved in all
members of this class of receptors (Fig. 2) [57].
The first fungal gene product identified as a member of
the Stm1+ gene family was S. cerevisiae Ers1. It was isolated as a multicopy suppressor of an ER Retention Defective (erd1) mutant [58]. In our analysis we have identified
two other yeast 7 TM proteins that contain the PQ motif
in an analogous position. These proteins, Ybr147w and
Yol092w, have not yet been characterized in great detail.
Genome-wide assays show that Ybr147 may be located in
the mitochondria and its deletion results in a strain that is
resistant to the antifungal drug fluconazole [59,60]. This
latter phenotype makes this protein not an interesting target
for antifungals.
The S. pombe Stm1+ gene was isolated as a multi-copy
suppressor of a synthetic lethal mutant carrying mutations
in Ras1 and an unknown gene. Two-hybrid analysis indicated an interaction between Stm1+ and the Gα-protein
Gpa2, the same protein to which the Git3 receptor binds.
This interaction has not yet been confirmed by in vivo
GST pull down assays. Stm1+ was suggested to control
vegetative growth by sensing the availability of nitrogen
sources [61].
Contrary to the class I and class II proteins, Stm1+
sequence homologues are present in basidiomycetes such
U. maydis, a plant pathogen [62] and in humans (Fig. 1).
Mutations in the human homologous gene cystinosin
(CTNS) have been associated with the renal Fanconi syndrome, a lysosomal storage disorder caused by a defect in
cystine transport across the lysosomal membrane [63].
There is also evidence that this receptor may function as a
H(+)-driven transporter that can export cystine from the
lysosomes [64]. To date, none of these class III receptors
have been investigated in view of their potential use as
antifungal targets. As they share quite some sequence
homology, it will be important to identify drugs that
are specific for fungal class III proteins with no affinity
for the human homologues.
Screening assays using G protein-coupled
receptors for the development of antifungal
drugs
In the introduction I have described a number of
approaches to identify the ligands of GPCRs. So far,
most of these assays turned out to be unsuccessful in
the hunt for ligands of fungal nutrient sensing GPCRs.
The major problem is that per definition, these nutrients
will not only be sensed by the GPCRs but they will also
be transported by a number of specific and general transporters and metabolized. In our hunt for the characterization
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Van Dijck
Fig. 2 Multiple alignment of the two PQ-loop regions in the different class III receptors presented in Fig. 1. The numbers indicate the position in the
protein.
of the ligand of Gpr1 we have tried radioligand binding
assays or BIAcore interaction analysis. To avoid binding
of the ligand (e.g., glucose) to transport proteins, one
would need to use a strain deleted for all glucose transporters with and without additional deletion of Gpr1 but even
in such a strain a classical binding assay as is done for
mammalian GPCRs is not possible because of the high
Km. Bioinformatics approaches have also not yet been used
as none of these nutrient sensing receptors has been crystallized. No publications appeared where fungal nutrient sensing
receptors have been expressed in Xenopus laevis oocytes in
order to identify the ligand.
Whereas the MAPK pathway has been used extensively, the use of the Gpr1 controlled nutrient-signaling
pathway as an alternative system for the characterization of heterologous expressed GPCRs has not been
described in the literature. Previously we have tried to
functionally complement the yeast gpr1? strain with the
S. pombe Git3 receptor, but this receptor was unable
to complement the Gpr1 dependent glucose-induced
cAMP increase or other downstream targets of the pathway [65]. Additional introduction of the S. pombe
Gpa1 protein did not result in complementation. More
recently, we observed a similar lack of complementation when we expressed the C. albicans Gpr1 receptor
in S. cerevisiae (Serneels et al., unpublished). This indicates that the elements of the nutrient sensing pathway
in S. cerevisiae likely require highly specific protein
interactions, making this pathway less amenable for
identification of the ligands for heterologously expressed
GPCRs.
Recently, some screening assays to identify modulators of fungal nutrient G protein-coupled receptors have
been described. One system is based on the uncontrolled
cell division cycle of S. pombe cells by overexpression
of the S. pombe Stm1 receptor, described above [66].
This results in a strong growth defect, a phenotype that
can be used for high throughput screening for inhibitors
of Stm1. As mentioned previously, this Stm1 receptor
senses the nutritional state thereby driving the cell to
enter meiosis upon nutritionally deficient conditions. It
was observed that overexpression of Stm1+, in an ade6
mutant, results in the conversion from diploid to haploid
cells and this was associated with pink or red colonies
respectively on low adenine containing medium. Chung
and coworkers have now utilized the growth defect and
the color-based system to develop a high throughput
screening system to identify modulators of Stm1. Out of
413 compounds they identified four potent modulators
including Biochanin A, which possess strong inhibitory
activity against uncontrolled cell division and which was
included as a kind of control [66]. As it was previously
shown that Stm1 requires Gpa2 in order to exert its effect
on cell growth, this screening could be used to test heterologous G protein-coupled receptors on condition that
they could interact with the S. pombe Gpa2 [61]. Alternatively, similar to the screenings using the yeast pheromone
receptor system, it should be possible to generate hybrid
G proteins in S. pombe in order to couple the heterologous
GPCR with the downstream signalling pathway.
In my laboratory we are currently screening for antagonists or inverse agonists of the C. albicans Gpr1 receptor
which may tell us more about the agonist. As mentioned
above, deletion of this receptor has only a slight effect on
virulence in a mouse model for systemic infection. This
would indicate that an antagonist or inverse agonist of this
CaGpr1 receptor would not be very effective as an antifungal drug. However, there are conditions under which the
presence of a functional Gpr1 receptor is required. Recently
we have shown that Gpr1 has a strong synthetic effect with
Tps2, the C. albicans trehalose-6-phosphate (T6P) phosphatase enzyme [67].
© 2009 ISHAM, Medical Mycology, 47, 671–680
GPCRs as antifungal targets?
Previously we have shown that deletion of C. albicans
TPS2 has a strong effect on virulence in a mouse model
for systemic infection. The hypothesis is that C. albicans
cells will encounter stress by the host defence mechanisms when they become pathogenic. Under stress, the
C. albicans tps2 mutant accumulates high levels of trehalose-6-phosphate, which deregulates central metabolism
and becomes toxic for the cells [68]. In an effort to make
the phenotype even stronger, we generated a double deletion strain in which both GPR1 and TPS2 are deleted.
Trehalose biosynthesis is situated downstream of and is
negatively regulated by the cAMP-PKA pathway. Inhibiting the activity of the pathway, e.g., by inactivating Gpr1,
will result in higher trehalose levels or in the case of a
tps2 mutant in even higher T6P levels under stress conditions. This is indeed what we have observed. In addition,
the double deletion strain is avirulent in a mouse model
for systemic infection [67]. A gpr1 tps2 double mutant
has a very severe growth defect at high temperatures, such
as 43°C. When one copy of GPR1 is present, then the
strain grows much better at this temperature. Therefore,
the GPR1/gpr1 tps2/tps2 strain represents an easy assay
for high throughput screening for inhibitors of Gpr1 as
these would result in absence of growth at 43°C. In a
second screen we then have to identify drugs that inhibit
the Tps2 enzyme. This could be achieved in two ways.
On the one hand we can use the gpr1/gpr1 TPS2/tps2
strain, which grows quite well and select compounds that
inhibit growth. On the other hand, we can purify the Tps2
enzyme and perform a high-throughput screening assay
for T6P phosphatase inhibitors, by measuring the level of
free phosphate released from T6P. Potent inhibitors should
block the release of free phosphate. Recently we have
screened 10000 compounds from ChemBridge (DiverseSet) and identified eight compounds that inhibited
growth of a GPR1/gpr1 TPS2/tps2 mutant strain by more
than 95%. These compounds will now be investigated
further.
So far, the ligand of Gpr1 in C. albicans is not yet
known. There is evidence that methionine or a downstream metabolite may be the ligand but whether this is
sensed intra-cellular or extra-cellular remains to be investigated. In our search for ligands we have tested many
compounds and interestingly, we observed a strong synergistic effect of histatin 5 in the growth of the gpr1
mutant. In humans, histatins are produced by the submandibular and parotid glands and secreted in saliva [69].
They form part of the innate immune system and play an
important role in maintaining oral health. Histatins are
small, cationic, histidine-rich peptides of 3–4 kDa with
potent antimicrobial activity [70]. The most important
members of the histatin family are histatin 3 and histatin
5 (32 and 24 amino acids, respectively). Histatin 5, which
© 2009 ISHAM, Medical Mycology, 47, 671–680
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has the strongest antimicrobial activity, has received most
of the attention. It has potent activity against the pathogenic fungi C. albicans, C. neoformans and A. fumigatus
[71]. The mechanism of action of histatin 5 in C. albicans
is not completely clear. The most recent data seem to
indicate that it would initiate an osmotic stress response
by activation of the Hog1 pathway [72]. We have now
shown that cell defense to histatin 5 is dependent on Gpr1
(unpublished data). A homozygous gpr1 mutant is very
sensitive to histatin 5. At concentrations above 8 μM
histatin 5, this strain does not grow anymore, whereas a
heterozygous or wild type strain still grows [73]. This
would indicate that a combination of histatin 5 and an
antagonist of Gpr1 would be an interesting antifungal
cocktail. It would be interesting to see whether a gpr1
mutant strain is more affected in an oropharyngeal candidiasis model system compared to the systemic infection
model system. We are currently screening the heterozygous gpr1 mutant for compounds that inhibit the growth
when added in the presence of histatin 5.
Once the ligand is known, it may be important to identify the exact binding sites. An interesting approach to use
is the substituted scanning accessibility method (SCAM),
which requires the generation of point mutations in the
putative binding sites and the availability of a screening
system to follow receptor activation. This method was
used in order to identify the binding sites of sucrose in
the Gpr1 receptor [29].
Conclusions
Compared to higher eukaryotes, fungi seem to have a
very limited number of genes encoding real or putative
G protein-coupled receptors. Apart from the well-known
pheromone receptors a new class of fungal GPCRs that
specifically seem to sense nutrients has recently been
discovered. The first characterized member was the glucose/sucrose sensing GPCR Gpr1 in S. cerevisiae. It is
now clear that Gpr1 is the founding member of a novel,
seventh class of GPCRs. Functional homologues seem
to be present in most fungi with the C. neoformans Gpr4
receptor as a clear example. This receptor functions
similarly to the C. albicans Gpr1 receptor, despite the
absence of sequence homology. As this class of GPCRs
is fungal specific and GPCRs form the largest groups of
therapeutic targets, two important criteria for a good
drug target are fulfilled (specificity and possibility to
screen for drugs). However, those receptors investigated
so far are not essential and might therefore be considered
uninteresting targets. Despite this, it seems that they
may still be good targets in combination with other targets. As combination therapy may become the golden
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Van Dijck
standard to treat fungal diseases, it is still valid to identify antagonists or inverse agonists of nutrient G proteincoupled receptors. In addition, such molecules may
advance fundamental research on the function/structure
analysis of these receptors.
Acknowledgements
I wish to thank the various consortia that sequenced
the fungal genomes and that made these sequences
available to the scientific community. Original research
was supported by grants from the Fund for Scientific
Research Flanders (G.0242.04), and by the Marie
Curie Research Training network CanTrain (MRTN-CT2004-512841).
Declaration of interest: The author reports no conflicts of
interest. The author alone is responsible for the content and
writing of the paper.
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This paper was first published online on iFirst on 31 January 2009.
© 2009 ISHAM, Medical Mycology, 47, 671–680