BACKGROUND
eukaryotic signal transduction is dependent upon various secondary messenger signaling molecules in particular the cellular phospholipids called phosatidylinositols .
these phospholipids activate a spectrum of intracellular pathways that regulate multiple core functions including cellular metabolism, cell cycle and survival, protein synthesis, cell polarity and motility, and vesicle trafficking.
phosphorylations around the inositol ring generates various phosphoinositides  which allow for functional specificity in cell signaling and cellular membrane remodeling  <cit> .
known pis include ptdins-3-phosphate , ptdins-4-phosphate , ptdins-5-phosphate , ptdins- <dig> -bisphosphate , ptdins- <dig> -bisphosphate , ptdins- <dig> -bisphosphate , and ptdins- <dig> ,5-trisphosphate .
cellular syntheses of these various pis are regulated by certain phosphatases and distinct families of lipid-specific kinases called phosphoinositide lipid kinases or piks .
piks can be broadly categorized into three major protein types: ptins 3-kinases , ptdins 4-kinases , and ptdins-p  kinases .
pi3ks has eight known isoforms in mammals which are differentiated into three to four classes by amino acid sequence homology, regulator domains and activation cascades  <cit> .
mammals have three class ia pi3ks sharing homologous catalytic polypeptide subunits p110α , p110β  and p110δ .
class ia pi3ks are heterodimeric protein complexes consisting of the catalytic and regulatory subunits.
class ia kinases are multi-domain proteins having, in order from the n-terminus, a binding domain for p <dig> regulatory proteins, a ras binding domain  as well as c <dig>  helical  and catalytic domains.
the sole class ib pi3k, pik3cg, has a distinctive p <dig> regulatory domain as well as homologous, rbd, c <dig>  helical and p110γ catalytic domains.
class ii pi3ks lack either p <dig> or p <dig> regulatory domains yet have a p <dig> catalytic domain and the other domains.
in humans, there are three class ii isoforms, pik3c2α , pik3c2β  and pik3c2γ .
class iii pi3k is represented by a single isoform, pik3c <dig> , and is the least complex pi3k kinases having only c <dig>  helical and catalytic domains.
there are two types of pi4ks each comprised of two known paralogous isoforms in vertebrates.
these are known as type ii, pi4kiiα  and pi4kiiβ , and type iii pi4kiiiα  and pi4kiiiβ   <cit> .
pi4kiiis share sequence homology with pi3k and together comprise the largest family of phosphoinositide lipid kinases.
pi4kii isoforms do not share sequence homology with either pi4kiii or pi3k kinases.
the third pik family is the phosphatidylinositol-4-phosphate 5-kinases  or pip kinases which lack primary sequence or structural homology to either pi3ks or any type of pi4ks  <cit> .
in mammals, there are three types of pip kinases or pipks.
type i pip5k occurs as three homologs known as pip5k1-α , pip5k1-β  and pip5k1-γ .
type ii phosphatidylinositol-4-phosphate 5-kinase also has three isoforms called pip4k2-α , pip4k2-β  and pip4k2-γ .
both type i and ii pipks are homologs to yeast mss4p.
a third pipk is pikfyve has been recently designated as pip5k <dig> and includes yeast fab1p.
pi3k-akt-mtor signaling is a central regulatory axis for many key cellular functions including cell cycle, protein synthesis and glucose metabolism.
thus it is not surprising that many diverse disease etiologies have been associated with dysfunctional piks including cancer, diabetes and heart disease  <cit> .
phosphatidylinositol 3-kinase α peptide  is highly mutated in colon, brain and gastric cancers where apparent gain-of-function mutations confer increased activity for this lipid kinase  <cit> .
several small molecule inhibitors of pik3ca are in anti-cancer clinical trials  <cit> .
the paralog pik3c-γ or pik3cg is broadly implicated in many diseases due to its role as a downstream signaling component of chemokine receptors that modulate inflammatory pathways  <cit> .
for example, pik3cg has been suggested as a heart disease target because of its roles in atherosclerosis related inflammation  <cit>  and pathogenesis of cardiac hypertrophy and heart failure  <cit> .
recent studies also suggest that pi4kiiis are essential host factors utilized by the hepatitis c virus  to remodel the intracellular matrix for virus replication and release  <cit> .
ptdins and piks are evolutionary well conserved.
in fact, inositol precursors are found in bacteria and archaea although phosphorylated derivatives are strictly eukaryotic  <cit> .
homologs for all three piks groups  occur across major taxonomic clusters of eukaryotes although the number of isoforms is highly variable with greater complexity among metazoans, especially the vertebrates  <cit> .
clearly, the emergence and diversification of piks second messenger signaling is a hallmark, and potential facilitator, of eukaryotic evolution.
however, questions remain about the antiquity of the various pik subfamilies and the timing of the radiation and divergence of these lipid kinases relative to the emergence of major taxonomic groups.
this knowledge is potentially relevant to biomedical interests in piks such as in the interpretation and design of model organism studies, characterization of mutations and polymorphic variants and evaluation of potential polypharmacology  <cit> .
in addition, kinase inhibitors have been suggested as novel therapeutics against devastating eukaryotic parasitic diseases such as malaria, caused by the apicomplexa protists, plasmodium falciparum and p. vivax  <cit> , as well as leishmaniasis and trypanosomiasis that result from infections of leishmania sp., and trypanosoma sp., respectively, both members of the kinetoplastids  <cit> .
here, we provide a comprehensive genomic survey and phylogenetic analyses of the major types of piks.
we show the significance of these results in understanding the evolution of second messenger signaling as well as exploiting piks as drug targets for various therapeutic areas.
RESULTS
phylogeny of pi3k and pi4kiii kinases
in this study, we focused on the phylogenetic analyses of the phosphoinositide lipid kinases from representative species across seven major taxonomic groups: the mammalia, vertebrata , chordata/deutrostomia, protosomatia , pseudocoelomata , viridiplantae , fungi as well as various protists with an emphasis on human eukaryotic parasitic groups apicomplexa and kinetoplastids.
we retrieved amino acid sequences from genbank nonredundant  and species-specific databases via blastp  <cit>  searches using human pik proteins as the initial queries .
as necessary, sequences from other species were used as queries to obtain further homologs.
while all homologs were initially collected, representative species for each taxonomic major group were selected for phylogenetic analyses on the basis of sequence completeness which was not consistent across all genomes sequences.
for example, mammalian kinases could be consistently represented by human , rat  and mouse  sequences while chordata/deutrostomia were represented by an aggregate of one or more homologs from the genome sequences available for species of deutrostomia , tunicata  and/or cephalochordata .
for each of the three major piks families, sequences were aligned, edited to the core conserved amino acids and phylogenetic analyses were performed.
pi3k and pi4k type iii  kinases form the largest homologous group which collectively comprise of  <dig> distinct isoforms in vertebrates.
phylogenetic analysis shows a distinct clustering of the different subfamilies with high bootstrap and bayesian posterior probability for most clades .
pi4ka and pi4kb  are significantly diverged from each other as well as pi3k subfamily proteins.
both pi4ka and pi4kb are represented throughout the major taxonomic groups as single genes .
the eight isoforms of the pi3k family have a specific hierarchy with pik3c <dig>  the most divergent relative to pik3c <dig>  and pik3c  proteins.
pik3c <dig> occurs in all major taxonomic groups and is the sole pi3k class protein encoded in the genomes of plants, fungi and apicomplexan .
however, kinetoplastids species have a second pi3k which appears intermediate in homology to class i and ii kinases of metazoans.
pik classes ia, ib and ii cluster together with high bootstrap/probability values and, within each class, the nodes clustering the various isoforms also have strong bootstrap and bayesian posterior probability support.
class ii kinases are specific to metazoans.
of the three isoforms, pik3c2g  is the most divergent and appears common to all species.
nematodes and arthropods are not monophyletic, which is likely an artifact of tree reconstruction resulting from too few phylogenetically informative sites to resolve the evolutionary relationships among these invertebrate species.
pik3c2a  and pik3c2b  are both vertebrate specific and form a strongly supported clade.
deutrostomia/chordata species have a single pik3c <dig> ortholog which, although not entirely well resolved in the overall pi3kc <dig> cluster, appears to be ancestral to both pik3c2a and pik3c2b suggesting the latter two proteins evolved from a gene duplication event in early cold-blooded vertebrates.
similarly, class i pi3ks are mainly found in metazoans with a single isoform in invertebrates.
phylogenetic analysis shows two distinct subclusters of duplicated vertebrate sequences, one of pik3cb and pik3cd while the other is comprised of pik3ca and pik3cg.
each subcluster has a single ancestral ortholog gene which is found in all three species representing the deutrostomia/chordata.
therefore, at least two further gene duplications occurred after the emergence of true vertebrates, one lead to pik3cb and pik3cd while the other resulted in pik3ca and pik3cg.
phylogeny of class ii pi4k kinases
type ii pi4k kinases  do not share amino acid sequence homology with either pi4kiii or pi3k kinases .
mammalians and other vertebrates have two isoforms, pi4k2a and pi4k2b  which appear to have evolved from an early vertebrate gene duplication as similarly seen for pi3k subfamilies .
blastp database searches revealed single copy pi4k homologs in all other species except plants having multiple isoforms while kinetoplastids and other protists appear to be missing this protein.
fungal pi4kii are basal to metazoan isoforms while plant and apicomplexa pi4kii appear more closely related .
both phylogenetic relationships are consistent with current views on the fungal origin of metazoans  <cit>  and the role of secondary acquisition of a plastid-like endosymbiont by a single-cell eukaryote during the evolution of the apicomplexa  <cit> .
phylogeny of pip <dig> kinases
the pip5k family consists of three distantly related groups of proteins, pip5k <dig> , pip4k <dig>  and pip5k <dig>  or pikfyve.
in contrast to pi3k/pi4kiii and pi4kii kinases, the relative evolutionary relationships between these major subfamilies are poorly resolved.
although these proteins share recognizable sequence similarity, in comparison to pi3k/pi4kiii and pi4kii, fewer amino acids could be confidently aligned thus reducing the phylogenetic signal.
furthermore, most eukaryotes have multiple proteins with fyve finger motifs that are not active kinases but rather kinase substrates  <cit> .
therefore, we removed from our analysis any proteins with low kinase domain homology .
nonetheless, all metazoan pip5ks were resolved into one of the three subfamilies .
all invertebrate species as well as deutrostomia/chordata had a single copy of pip5k <dig>  pip4k <dig> and pip5k <dig>  for pip5k <dig>  the three known human isoforms had orthologs in cold-blooded vertebrates, again suggesting early vertebrate specific gene duplication events.
moreover, pip5k1a  and pip5k1b  appear to be more similar to each other than either to pip5k1c  thus might be the result of a more recent divergence event.
similarly, all three vertebrate pip4k <dig> proteins were monophyletic with pip4k2c  and pip4k2a  as sister groups relative to pip4k2b .
pip5k <dig> is a single copy isoform in all metazoans as well as fungi where the yeast prototype is known as fab1p.
invertebrates are depicted as paraphyletic and outside of the metazoan cluster in figure  <dig>  however, both the bootstrap neighbor-joining consensus and bayesian trees supported the monophyly of metazoan pip5k <dig>  fungi also have a second pip <dig> kinase, the yeast ortholog mss4p, which is more similar to pip5k <dig>
plants have multiple, duplicated enzymes which are most similar to pip5k <dig> and pip4k <dig> kinases although phylogenetic analyses could not confidently cluster plant kinases within either subtype.
no pip5k <dig> homologs were found in plants.
kinetoplastid species have lineage specific duplications of pip5k1-like kinases as well as a single pip5k <dig> kinase.
in contrast, apicomplexa have a single isoform that is most similar to pip5k <dig> kinases.
the major nodes connecting plant, yeast, metazoan, kinetoplastid and apicomplexa isoforms were not well supported by bootstrap or probability values, therefore no inference can be made about the relative order of evolutionary emergence of the major pipk types.
discussion
evolution of second messenger signaling
as central components of second messenger signaling, the evolutionary relationships of piks are indicative of the emergence and divergence of this critical signaling pathway in eukaryotes.
integration of multiple pik phylogenies across major eukaryotic taxonomic groups permits an overview of the evolutionary stages of second messenger signaling from unicellular protists to mammals .
based on their universal conservation across eukaryotes, there is a core group of four piks which are type iii pik4a and pik4b, and at least one homolog from pi3k  and pip5k families.
pi4kii while present in metazoans, plants, fungi and plasmodium sp., is apparently absent from kinetoplastids and further searches failed to find significant homologs in the genomes of other protists such as trichomonas vaginalis, giardia lamblia and entamoeba histolytica.
plasmodium sp.
belongs to the apicomplexa which are believed to have evolved from the secondary endosymbiosis of a plastid-bearing protist by a primordial eukaryotic cell  <cit> .
significant evidence supporting this hypothesis are that all apicomplexa have a non-photosynthetic plastid organelle called the apicoplast and many genes with plant-like origins are integrated into their primary genome  <cit> .
therefore the occurrence of pi4kii in plasmodium sp.
might also be due to the secondary acquisition from a plastid-bearing eukaryotic lineage.
another possible core pik candidate is pip5k <dig> or pikfyve which is found throughout metazoans, fungi and some kinetoplastids.
however, we could not find in plants any true pip5k <dig> homologs  that could be included confidently included our phylogenetic analyses  <cit> .
among the eight known pi3k family proteins, pik3c <dig> is the most likely candidate ancestral kinase of this subfamily.
two pieces of evidence in our analysis support this hypothesis.
first, single pik3c <dig> orthologs are found in all eukaryotic groups  <cit> .
second, phylogenetic analysis firmly establishes the early divergence of pik3c <dig> from class i and ii pi3ks.
this assessment is congruent with the early pathway catalytic function of pik3c <dig> in the generation of ptdins-3-phosphate  from a ptdins precursor.
in addition, pik3c <dig> has the simplest protein structure among all pi3k kinases insofar as it lacks the ras binding domains of class ia, ib and ii pi3ks as well as the p85/p <dig> regulatory domains of class ia pi3ks.
although only the core pik and catalytic domains were used for the construction of the pi3/4k phylogenetic tree, the branching order of the three pi3k classes corresponds well with the general trend towards increasing kinase complexity in terms of the number of regulatory domains and the capability to phosphorylate more complex ptdins derivatives.
with the exception of some kinetoplastid sequences, class i and ii pi3ks are metazoan specific proteins.
our phylogenomic analysis suggests an evolutionary trend where increasing organism complexity is associated with two major waves of pik diversification .
first, in early metazoans gene duplications lead to the emergence of class i and ii piks as well as pip5k <dig> and pip4k <dig> families.
this is evident from the wider repertoire of pik isoforms in invertebrates relative to plants, fungi and protists.
the second gene expansion occurred in early, true vertebrates.
for most piks, a series of gene duplications occurred in cold-blooded vertebrates after their divergence from early urochordates but before the emergence of mammals.
representative species of the deutrostomia , tunicata  and cephalochordata  mostly have the same pik complement as arthropods and other invertebrates.
possible exceptions are the class ia and class ib piks where a single corresponding ortholog for each subclass can be found in the deutrostomia/chordata species.
thus the initial diversification of class i pi3ks likely occurred in the early deutrostome lineage followed by two additional gene duplications in vertebrates resulting in contemporary complement of four class i piks throughout cold-blooded vertebrates and mammals.
many different genes spanning wide functional categories have undergone major gene duplications in vertebrates and mammals.
however, given the crucial role of second messenger signaling, it is tempting to speculate that increasing multi-cellular complexity was accompanied and, perhaps facilitated, by the evolution of more diverse and specialized piks signaling cascades.
evolutionary context for cancer mutations
the gene for phosphoinositide-3-kinase α peptide  is highly mutated in colon, brain and gastric cancers where apparent gain-of-function mutations confer increased activity for this lipid kinase  <cit> .
our phylogenetic analysis shows that pik3ca is most closely related to other class ia and ib kinases, pi3kc-ß , pi3kc-δ , and pi3kc-γ  .
thus further knowledge about tumor-related mutations in pik3ca might be gained by comparing those variants to orthologs in other species as well as paralogs of related kinases.
alignment of a consensus sequence of non-synonymous cancer mutations reported in the cosmic database  <cit>  with normal human pik3ca as well as orthologs from mammals and human paralogues for pik3cb, pik3cd and pik3cg are shown in figure  <dig>  several mutations occur in regions of pik3ca which are conserved throughout mammalian isoforms.
at least seven mutations, while non-conserved among pik3ca orthologs, are conservative changes matching residues in one or more of the three other corresponding human pik3c paralogs.
according to the cosmic database, one of the most frequent variants observed in cancer is h1047r in the terminal end of the kinase domain which has been shown to be an activating or gain-of-function mutation.
the variants h1047l and more rarely, h0147y, have also been recovered from clinical tumor samples.
gymnopoulos et al.
<cit>  measured the oncogenic potential of the  <dig> most common pik3ca mutations found in tumors by introducing retroviral expression vectors with each of the variants into avian cells and measuring their individual efficiencies for tumorigenic transformation.
their functional assays confirmed that the mutation h1047r strongly conferred oncogenic potency while moderate and weak potency was induced by the variants h1047l and h1047y, respectively.
interestingly, pik3ca h1047r mutation aligns with an arginine found in normal human pik3cg while h1047l corresponds to leucine in wild-type pik3cb and pik3cd .
h1047y is rarely found in tumors, appears to convey much weaker oncogenic potency than the other two mutations and is not conserved in any class i pi3k paralog.
the correspondence of certain cancer mutations in pik3ca to those found in normal paralogs suggests that selection pressures in the tumor might limit the range of acceptable amino acid changes.
one might speculate that these constrained amino acid changes in a mutated pik3ca protein could lead to convergence in function, protein interactions or regulation with those of pik3cb, pik3cg or pik3cd.
gymnopoulos and co-workers mapped h1047r/l mutations onto a structural model of pik3ca which suggested that these changes are located near the hinge region of the activation loop and could serve to increase catalytic activity  <cit> .
given the importance of mutated kinases in tumor cell viability and their increased exploitation as cancer drug targets, better insights into delineating between passenger and driver mutations might be gained through broader sequence comparisons across different species as well as related protein family members  <cit> .
pik inhibitor polypharmacology
knight et al.
reported inhibition data for compounds screened across the entire pik3c family as well as several more distantly related pi3k-like kinases such as atm , atr , prkdc  and mtor   <cit> .
their study did not include a phylogenetic or comparative sequence analyses but rather used separate principal component analysis  plots to compare the statistical space of target similarity versus compound-target inhibition values.
more recently, the same group published in apsel et al.
the results of several dual inhibitors of both tyrosine and pi3k and pik4a kinases  <cit> .
compounds were assayed for inhibition against all class ia and ib pi3ks, mtor, prkdc and pik4a as well as several tyrosine kinases although not all screening panels had a complete kinase set.
studying and predicting the target spectrum and specificity of any drug is important for determining potential polypharmacology  as well as evaluating possible drug toxicities .
in these respects, we were interested in melding phylogenetic analyses of lipid kinases with compound activity data to as a potential approach to complement chemogenomics studies.
we aligned the core kinase domains of pi3ks, pi4kiiis, atm, atr, mtor and prkdc  and reconstructed a phylogenetic tree .
selected compound inhibition data from both studies were mapped to the terminal branches of the respective kinase in the phylogenetic tree .
pi3kcd is the most common, potently inhibited kinase in both studies and several compounds shown high specificity such as the compound pik <dig> from knight and co-workers  <cit> .
other compounds, such as pik <dig> and pi <dig> from knight et al.
<cit>  or pp <dig> and pp <dig> of apsel et al.
<cit> , with high potencies across multiple pi3ks also inhibit one or more distantly related kinases such as atm, atr, prkdc, or mtor.
interestingly, several compounds show higher potency against the kinase pair pik3ca and pik3cg versus pik3cd and pik3cb.
pik3ca, pik3cg and pik3cb are considered as class ia while pik3cg is classified as class ib.
however, the compound inhibition profiles generally correlate with the closer evolutionary relationships between pik3ca and pik3cg kinases as shown in our trees .
the published compound data has some limitations insofar that not all possible lipid kinase homologs were assayed.
for example, apsel et al.
<cit>  do not report any compound assay data for pik3c <dig> or pik3c <dig> kinases.
however, there is an overall trend for compounds with broad class i pi3k spectrum to inhibit other piks as well as more distantly related kinases.
conversely, compounds with high potency to a subset of class i pi3ks tend to be more specific.
selective polypharmacology of piks might be an important goal for certain disease indications.
foukas et al.
suggest that class ia pi3ks have functional redundancy in cancer cells, so inhibition of multiple isoforms might be desirable  <cit> .
conversely, selective inhibition of a particular pi4kiii isoform might be necessary for developing a well-tolerated anti-hepatitis c viral therapy  <cit> .
further molecular modeling and testing with additional compound chemotypes might help illuminate the particular binding interactions that are involved in compound specificities.
piks as anti-parasite drug targets
some of the world's most important infectious diseases are caused by eukaryotic single-cell parasites.
for example, approximately  <dig> million cases of malaria and  <dig>  attributed deaths were reported globally in  <dig>  <cit> .
the causative agents of malaria are the apicomplexa parasites of which plasmodmium falciparum followed by p. vivax are the two most important infectious agents.
globally, millions are afflicted by the diseases leishmaniasis  and trypanosomiasis  which result from infections of two genera of kinetoplastids, leishmania sp., and trypanosoma sp., respectively  <cit> .
new drugs against these parasites are urgently required because of toxicities of existing therapies, the lack of pediatric formulations and the emergence of drug-resistant strains.
the need for novel drugs is particularly acute for malaria where there have been increasing reports of p. falciparum strains with reduced clinical response to the latest class of artemisinin-containing drug combinations  <cit> .
several previous studies have suggested that kinases are novel target opportunities for these pathogens because of their essential roles in metabolism, signaling and pathogenesis  <cit> .
in several ways, our study extends that rationale.
the lower complement of piks in unicellular eukaryotes relative to metazoans suggests poor pathway redundancy and enhanced probability of essential function to the parasite.
a recent screen of a large pharmaceutical compound collection revealed thousands of potential leads including compounds known to inhibit human pi3ks  <cit> .
the anti-plasmodial activity of these compounds might be through direct inhibition of plasmodium pi3k, although blocking the human host target cannot be discounted because the screens were performed on p. falciparum cultured in blood erythrocytes.
a comparison of human and plasmodium kinases of the pi3k and pi4kiii types show overall conservation of key catalytic amino acids which suggests that screening human pik <dig> compound collections might find further inhibitors of malarial kinases .
as shown in our phylogenetic trees, parasite kinases are divergent from human host homologs suggesting potential scope for developing compounds with specificity to bind leishmania, trypanosoma or plasmodium piks.
however, kinetoplastids and apicomplexa are evolutionary distinct groups.
the apicomplexa, including plasmodium sp., have been proposed to evolve from an ancestor that acquired a secondary plastid through endosymbiosis with a photosynthetic alga  <cit> .
in contrast, comparative genomic analyses of trypanosoma cruzi, t. brucei and leishmania major could not find significant evidence supporting similar plastid invasion into those genomes  <cit> .
our findings of type ii pik <dig> kinases being present in plants and apicomplexa yet absent in kinetoplastids, additional pi3k genes in kinetoplastids and the overall non-clustering of these groups in our phylogenetic trees supports the varied evolutionary history of these protists.
as such, differential development of kinase inhibitors for the treatment of malaria, leishmaniasis and trypanosomiasis might need to be considered.
malarial genes of putative plastid origins have been suggested to be potential drug targets because of their divergence from human homologs  <cit> .
in our analysis, the type iii pik4cb and type ii pik4b proteins of plasmodium sp.
cluster with plant orthologs with significant posterior probabilities and high bootstrap values suggesting a plant or cyanobacteria origin for those kinases.
understanding the evolutionary relationships of trypanosoma, leishmania and plasmodium piks relative to each other and their human host will further facilitate both drug target selection and the design of counter-screen assays.
CONCLUSIONS
phylogenomic analyses of piks show the progressive complexity of second messenger signaling from unicellular eukaryotes through metazoans and, finally, vertebrates.
furthermore, an evolutionary perspective is a potentially useful framework for characterizing piks as drug targets in cancer and parasitic diseases as well as providing insights into the polypharmacology of this important group of kinases.

 eukaryotic signal transduction is dependent upon various secondary messenger signaling molecules in particular the cellular phospholipids called phosatidylinositols. these phospholipids activate a spectrum of intracellular pathways that regulate multiple core functions including cellular metabolism, cell cycle and survival, protein synthesis, cell polarity and motility, and vesicle trafficking. phosphorylations around the inositol ring generates various phosphoinositides which allow for functional specificity in cell signaling and cellular membrane remodeling. known pis include ptdins-3-phosphate, ptdins-4-phosphate, ptdins-5-phosphate, ptdins- -bisphosphate, ptdins- -bisphosphate, ptdins- -bisphosphate, and ptdins-,5-trisphosphate. cellular syntheses of these various pis are regulated by certain phosphatases and distinct families of lipid-specific kinases called phosphoinositide lipid kinases or piks. piks can be broadly categorized into three major protein types: ptins 3-kinases, ptdins 4-kinases, and ptdins-p kinases. pi3ks has eight known isoforms in mammals which are differentiated into three to four classes by amino acid sequence homology, regulator domains and activation cascades. mammals have three class ia pi3ks sharing homologous catalytic polypeptide subunits p110α, p110β and p110δ. class ia pi3ks are heterodimeric protein complexes consisting of the catalytic and regulatory subunits. class ia kinases are multi-domain proteins having, in order from the n-terminus, a binding domain for p regulatory proteins, a ras binding domain as well as c helical and catalytic domains. the sole class ib pi3k, pik3cg, has a distinctive p regulatory domain as well as homologous, rbd, c helical and p110γ catalytic domains. class ii pi3ks lack either p or p regulatory domains yet have a p catalytic domain and the other domains. in humans, there are three class ii isoforms, pik3c2α, pik3c2β and pik3c2γ. class iii pi3k is represented by a single isoform, pik3c, and is the least complex pi3k kinases having only c helical and catalytic domains. there are two types of pi4ks each comprised of two known paralogous isoforms in vertebrates. these are known as type ii, pi4kiiα and pi4kiiβ, and type iii pi4kiiiα and pi4kiiiβ. pi4kiiis share sequence homology with pi3k and together comprise the largest family of phosphoinositide lipid kinases. pi4kii isoforms do not share sequence homology with either pi4kiii or pi3k kinases. the third pik family is the phosphatidylinositol-4-phosphate 5-kinases or pip kinases which lack primary sequence or structural homology to either pi3ks or any type of pi4ks. in mammals, there are three types of pip kinases or pipks. type i pip5k occurs as three homologs known as pip5k1-α, pip5k1-β and pip5k1-γ. type ii phosphatidylinositol-4-phosphate 5-kinase also has three isoforms called pip4k2-α, pip4k2-β and pip4k2-γ. both type i and ii pipks are homologs to yeast mss4p. a third pipk is pikfyve has been recently designated as pip5k and includes yeast fab1p. pi3k-akt-mtor signaling is a central regulatory axis for many key cellular functions including cell cycle, protein synthesis and glucose metabolism. thus it is not surprising that many diverse disease etiologies have been associated with dysfunctional piks including cancer, diabetes and heart disease. phosphatidylinositol 3-kinase α peptide is highly mutated in colon, brain and gastric cancers where apparent gain-of-function mutations confer increased activity for this lipid kinase. several small molecule inhibitors of pik3ca are in anti-cancer clinical trials. the paralog pik3c-γ or pik3cg is broadly implicated in many diseases due to its role as a downstream signaling component of chemokine receptors that modulate inflammatory pathways. for example, pik3cg has been suggested as a heart disease target because of its roles in atherosclerosis related inflammation and pathogenesis of cardiac hypertrophy and heart failure. recent studies also suggest that pi4kiiis are essential host factors utilized by the hepatitis c virus to remodel the intracellular matrix for virus replication and release. ptdins and piks are evolutionary well conserved. in fact, inositol precursors are found in bacteria and archaea although phosphorylated derivatives are strictly eukaryotic. homologs for all three piks groups occur across major taxonomic clusters of eukaryotes although the number of isoforms is highly variable with greater complexity among metazoans, especially the vertebrates. clearly, the emergence and diversification of piks second messenger signaling is a hallmark, and potential facilitator, of eukaryotic evolution. however, questions remain about the antiquity of the various pik subfamilies and the timing of the radiation and divergence of these lipid kinases relative to the emergence of major taxonomic groups. this knowledge is potentially relevant to biomedical interests in piks such as in the interpretation and design of model organism studies, characterization of mutations and polymorphic variants and evaluation of potential polypharmacology. in addition, kinase inhibitors have been suggested as novel therapeutics against devastating eukaryotic parasitic diseases such as malaria, caused by the apicomplexa protists, plasmodium falciparum and p. vivax, as well as leishmaniasis and trypanosomiasis that result from infections of leishmania sp., and trypanosoma sp., respectively, both members of the kinetoplastids. here, we provide a comprehensive genomic survey and phylogenetic analyses of the major types of piks. we show the significance of these results in understanding the evolution of second messenger signaling as well as exploiting piks as drug targets for various therapeutic areas.  phylogeny of pi3k and pi4kiii kinases in this study, we focused on the phylogenetic analyses of the phosphoinositide lipid kinases from representative species across seven major taxonomic groups: the mammalia, vertebrata, chordata/deutrostomia, protosomatia, pseudocoelomata, viridiplantae, fungi as well as various protists with an emphasis on human eukaryotic parasitic groups apicomplexa and kinetoplastids. we retrieved amino acid sequences from genbank nonredundant and species-specific databases via blastp searches using human pik proteins as the initial queries. as necessary, sequences from other species were used as queries to obtain further homologs. while all homologs were initially collected, representative species for each taxonomic major group were selected for phylogenetic analyses on the basis of sequence completeness which was not consistent across all genomes sequences. for example, mammalian kinases could be consistently represented by human, rat and mouse sequences while chordata/deutrostomia were represented by an aggregate of one or more homologs from the genome sequences available for species of deutrostomia, tunicata and/or cephalochordata. for each of the three major piks families, sequences were aligned, edited to the core conserved amino acids and phylogenetic analyses were performed. pi3k and pi4k type iii kinases form the largest homologous group which collectively comprise of distinct isoforms in vertebrates. phylogenetic analysis shows a distinct clustering of the different subfamilies with high bootstrap and bayesian posterior probability for most clades. pi4ka and pi4kb are significantly diverged from each other as well as pi3k subfamily proteins. both pi4ka and pi4kb are represented throughout the major taxonomic groups as single genes. the eight isoforms of the pi3k family have a specific hierarchy with pik3c the most divergent relative to pik3c and pik3c proteins. pik3c occurs in all major taxonomic groups and is the sole pi3k class protein encoded in the genomes of plants, fungi and apicomplexan. however, kinetoplastids species have a second pi3k which appears intermediate in homology to class i and ii kinases of metazoans. pik classes ia, ib and ii cluster together with high bootstrap/probability values and, within each class, the nodes clustering the various isoforms also have strong bootstrap and bayesian posterior probability support. class ii kinases are specific to metazoans. of the three isoforms, pik3c2g is the most divergent and appears common to all species. nematodes and arthropods are not monophyletic, which is likely an artifact of tree reconstruction resulting from too few phylogenetically informative sites to resolve the evolutionary relationships among these invertebrate species. pik3c2a and pik3c2b are both vertebrate specific and form a strongly supported clade. deutrostomia/chordata species have a single pik3c ortholog which, although not entirely well resolved in the overall pi3kc cluster, appears to be ancestral to both pik3c2a and pik3c2b suggesting the latter two proteins evolved from a gene duplication event in early cold-blooded vertebrates. similarly, class i pi3ks are mainly found in metazoans with a single isoform in invertebrates. phylogenetic analysis shows two distinct subclusters of duplicated vertebrate sequences, one of pik3cb and pik3cd while the other is comprised of pik3ca and pik3cg. each subcluster has a single ancestral ortholog gene which is found in all three species representing the deutrostomia/chordata. therefore, at least two further gene duplications occurred after the emergence of true vertebrates, one lead to pik3cb and pik3cd while the other resulted in pik3ca and pik3cg. phylogeny of class ii pi4k kinases type ii pi4k kinases do not share amino acid sequence homology with either pi4kiii or pi3k kinases. mammalians and other vertebrates have two isoforms, pi4k2a and pi4k2b which appear to have evolved from an early vertebrate gene duplication as similarly seen for pi3k subfamilies. blastp database searches revealed single copy pi4k homologs in all other species except plants having multiple isoforms while kinetoplastids and other protists appear to be missing this protein. fungal pi4kii are basal to metazoan isoforms while plant and apicomplexa pi4kii appear more closely related. both phylogenetic relationships are consistent with current views on the fungal origin of metazoans and the role of secondary acquisition of a plastid-like endosymbiont by a single-cell eukaryote during the evolution of the apicomplexa. phylogeny of pip kinases the pip5k family consists of three distantly related groups of proteins, pip5k, pip4k and pip5k or pikfyve. in contrast to pi3k/pi4kiii and pi4kii kinases, the relative evolutionary relationships between these major subfamilies are poorly resolved. although these proteins share recognizable sequence similarity, in comparison to pi3k/pi4kiii and pi4kii, fewer amino acids could be confidently aligned thus reducing the phylogenetic signal. furthermore, most eukaryotes have multiple proteins with fyve finger motifs that are not active kinases but rather kinase substrates. therefore, we removed from our analysis any proteins with low kinase domain homology. nonetheless, all metazoan pip5ks were resolved into one of the three subfamilies. all invertebrate species as well as deutrostomia/chordata had a single copy of pip5k pip4k and pip5k for pip5k the three known human isoforms had orthologs in cold-blooded vertebrates, again suggesting early vertebrate specific gene duplication events. moreover, pip5k1a and pip5k1b appear to be more similar to each other than either to pip5k1c thus might be the result of a more recent divergence event. similarly, all three vertebrate pip4k proteins were monophyletic with pip4k2c and pip4k2a as sister groups relative to pip4k2b. pip5k is a single copy isoform in all metazoans as well as fungi where the yeast prototype is known as fab1p. invertebrates are depicted as paraphyletic and outside of the metazoan cluster in figure however, both the bootstrap neighbor-joining consensus and bayesian trees supported the monophyly of metazoan pip5k fungi also have a second pip kinase, the yeast ortholog mss4p, which is more similar to pip5k plants have multiple, duplicated enzymes which are most similar to pip5k and pip4k kinases although phylogenetic analyses could not confidently cluster plant kinases within either subtype. no pip5k homologs were found in plants. kinetoplastid species have lineage specific duplications of pip5k1-like kinases as well as a single pip5k kinase. in contrast, apicomplexa have a single isoform that is most similar to pip5k kinases. the major nodes connecting plant, yeast, metazoan, kinetoplastid and apicomplexa isoforms were not well supported by bootstrap or probability values, therefore no inference can be made about the relative order of evolutionary emergence of the major pipk types. discussion evolution of second messenger signaling as central components of second messenger signaling, the evolutionary relationships of piks are indicative of the emergence and divergence of this critical signaling pathway in eukaryotes. integration of multiple pik phylogenies across major eukaryotic taxonomic groups permits an overview of the evolutionary stages of second messenger signaling from unicellular protists to mammals. based on their universal conservation across eukaryotes, there is a core group of four piks which are type iii pik4a and pik4b, and at least one homolog from pi3k and pip5k families. pi4kii while present in metazoans, plants, fungi and plasmodium sp., is apparently absent from kinetoplastids and further searches failed to find significant homologs in the genomes of other protists such as trichomonas vaginalis, giardia lamblia and entamoeba histolytica. plasmodium sp. belongs to the apicomplexa which are believed to have evolved from the secondary endosymbiosis of a plastid-bearing protist by a primordial eukaryotic cell. significant evidence supporting this hypothesis are that all apicomplexa have a non-photosynthetic plastid organelle called the apicoplast and many genes with plant-like origins are integrated into their primary genome. therefore the occurrence of pi4kii in plasmodium sp. might also be due to the secondary acquisition from a plastid-bearing eukaryotic lineage. another possible core pik candidate is pip5k or pikfyve which is found throughout metazoans, fungi and some kinetoplastids. however, we could not find in plants any true pip5k homologs that could be included confidently included our phylogenetic analyses. among the eight known pi3k family proteins, pik3c is the most likely candidate ancestral kinase of this subfamily. two pieces of evidence in our analysis support this hypothesis. first, single pik3c orthologs are found in all eukaryotic groups. second, phylogenetic analysis firmly establishes the early divergence of pik3c from class i and ii pi3ks. this assessment is congruent with the early pathway catalytic function of pik3c in the generation of ptdins-3-phosphate from a ptdins precursor. in addition, pik3c has the simplest protein structure among all pi3k kinases insofar as it lacks the ras binding domains of class ia, ib and ii pi3ks as well as the p85/p regulatory domains of class ia pi3ks. although only the core pik and catalytic domains were used for the construction of the pi3/4k phylogenetic tree, the branching order of the three pi3k classes corresponds well with the general trend towards increasing kinase complexity in terms of the number of regulatory domains and the capability to phosphorylate more complex ptdins derivatives. with the exception of some kinetoplastid sequences, class i and ii pi3ks are metazoan specific proteins. our phylogenomic analysis suggests an evolutionary trend where increasing organism complexity is associated with two major waves of pik diversification. first, in early metazoans gene duplications lead to the emergence of class i and ii piks as well as pip5k and pip4k families. this is evident from the wider repertoire of pik isoforms in invertebrates relative to plants, fungi and protists. the second gene expansion occurred in early, true vertebrates. for most piks, a series of gene duplications occurred in cold-blooded vertebrates after their divergence from early urochordates but before the emergence of mammals. representative species of the deutrostomia, tunicata and cephalochordata mostly have the same pik complement as arthropods and other invertebrates. possible exceptions are the class ia and class ib piks where a single corresponding ortholog for each subclass can be found in the deutrostomia/chordata species. thus the initial diversification of class i pi3ks likely occurred in the early deutrostome lineage followed by two additional gene duplications in vertebrates resulting in contemporary complement of four class i piks throughout cold-blooded vertebrates and mammals. many different genes spanning wide functional categories have undergone major gene duplications in vertebrates and mammals. however, given the crucial role of second messenger signaling, it is tempting to speculate that increasing multi-cellular complexity was accompanied and, perhaps facilitated, by the evolution of more diverse and specialized piks signaling cascades. evolutionary context for cancer mutations the gene for phosphoinositide-3-kinase α peptide is highly mutated in colon, brain and gastric cancers where apparent gain-of-function mutations confer increased activity for this lipid kinase. our phylogenetic analysis shows that pik3ca is most closely related to other class ia and ib kinases, pi3kc-ß, pi3kc-δ, and pi3kc-γ. thus further knowledge about tumor-related mutations in pik3ca might be gained by comparing those variants to orthologs in other species as well as paralogs of related kinases. alignment of a consensus sequence of non-synonymous cancer mutations reported in the cosmic database with normal human pik3ca as well as orthologs from mammals and human paralogues for pik3cb, pik3cd and pik3cg are shown in figure several mutations occur in regions of pik3ca which are conserved throughout mammalian isoforms. at least seven mutations, while non-conserved among pik3ca orthologs, are conservative changes matching residues in one or more of the three other corresponding human pik3c paralogs. according to the cosmic database, one of the most frequent variants observed in cancer is h1047r in the terminal end of the kinase domain which has been shown to be an activating or gain-of-function mutation. the variants h1047l and more rarely, h0147y, have also been recovered from clinical tumor samples. gymnopoulos et al. measured the oncogenic potential of the most common pik3ca mutations found in tumors by introducing retroviral expression vectors with each of the variants into avian cells and measuring their individual efficiencies for tumorigenic transformation. their functional assays confirmed that the mutation h1047r strongly conferred oncogenic potency while moderate and weak potency was induced by the variants h1047l and h1047y, respectively. interestingly, pik3ca h1047r mutation aligns with an arginine found in normal human pik3cg while h1047l corresponds to leucine in wild-type pik3cb and pik3cd. h1047y is rarely found in tumors, appears to convey much weaker oncogenic potency than the other two mutations and is not conserved in any class i pi3k paralog. the correspondence of certain cancer mutations in pik3ca to those found in normal paralogs suggests that selection pressures in the tumor might limit the range of acceptable amino acid changes. one might speculate that these constrained amino acid changes in a mutated pik3ca protein could lead to convergence in function, protein interactions or regulation with those of pik3cb, pik3cg or pik3cd. gymnopoulos and co-workers mapped h1047r/l mutations onto a structural model of pik3ca which suggested that these changes are located near the hinge region of the activation loop and could serve to increase catalytic activity. given the importance of mutated kinases in tumor cell viability and their increased exploitation as cancer drug targets, better insights into delineating between passenger and driver mutations might be gained through broader sequence comparisons across different species as well as related protein family members. pik inhibitor polypharmacology knight et al. reported inhibition data for compounds screened across the entire pik3c family as well as several more distantly related pi3k-like kinases such as atm, atr, prkdc and mtor. their study did not include a phylogenetic or comparative sequence analyses but rather used separate principal component analysis plots to compare the statistical space of target similarity versus compound-target inhibition values. more recently, the same group published in apsel et al. the results of several dual inhibitors of both tyrosine and pi3k and pik4a kinases. compounds were assayed for inhibition against all class ia and ib pi3ks, mtor, prkdc and pik4a as well as several tyrosine kinases although not all screening panels had a complete kinase set. studying and predicting the target spectrum and specificity of any drug is important for determining potential polypharmacology as well as evaluating possible drug toxicities. in these respects, we were interested in melding phylogenetic analyses of lipid kinases with compound activity data to as a potential approach to complement chemogenomics studies. we aligned the core kinase domains of pi3ks, pi4kiiis, atm, atr, mtor and prkdc and reconstructed a phylogenetic tree. selected compound inhibition data from both studies were mapped to the terminal branches of the respective kinase in the phylogenetic tree. pi3kcd is the most common, potently inhibited kinase in both studies and several compounds shown high specificity such as the compound pik from knight and co-workers. other compounds, such as pik and pi from knight et al. or pp and pp of apsel et al., with high potencies across multiple pi3ks also inhibit one or more distantly related kinases such as atm, atr, prkdc, or mtor. interestingly, several compounds show higher potency against the kinase pair pik3ca and pik3cg versus pik3cd and pik3cb. pik3ca, pik3cg and pik3cb are considered as class ia while pik3cg is classified as class ib. however, the compound inhibition profiles generally correlate with the closer evolutionary relationships between pik3ca and pik3cg kinases as shown in our trees. the published compound data has some limitations insofar that not all possible lipid kinase homologs were assayed. for example, apsel et al. do not report any compound assay data for pik3c or pik3c kinases. however, there is an overall trend for compounds with broad class i pi3k spectrum to inhibit other piks as well as more distantly related kinases. conversely, compounds with high potency to a subset of class i pi3ks tend to be more specific. selective polypharmacology of piks might be an important goal for certain disease indications. foukas et al. suggest that class ia pi3ks have functional redundancy in cancer cells, so inhibition of multiple isoforms might be desirable. conversely, selective inhibition of a particular pi4kiii isoform might be necessary for developing a well-tolerated anti-hepatitis c viral therapy. further molecular modeling and testing with additional compound chemotypes might help illuminate the particular binding interactions that are involved in compound specificities. piks as anti-parasite drug targets some of the world's most important infectious diseases are caused by eukaryotic single-cell parasites. for example, approximately million cases of malaria and attributed deaths were reported globally in. the causative agents of malaria are the apicomplexa parasites of which plasmodmium falciparum followed by p. vivax are the two most important infectious agents. globally, millions are afflicted by the diseases leishmaniasis and trypanosomiasis which result from infections of two genera of kinetoplastids, leishmania sp., and trypanosoma sp., respectively. new drugs against these parasites are urgently required because of toxicities of existing therapies, the lack of pediatric formulations and the emergence of drug-resistant strains. the need for novel drugs is particularly acute for malaria where there have been increasing reports of p. falciparum strains with reduced clinical response to the latest class of artemisinin-containing drug combinations. several previous studies have suggested that kinases are novel target opportunities for these pathogens because of their essential roles in metabolism, signaling and pathogenesis. in several ways, our study extends that rationale. the lower complement of piks in unicellular eukaryotes relative to metazoans suggests poor pathway redundancy and enhanced probability of essential function to the parasite. a recent screen of a large pharmaceutical compound collection revealed thousands of potential leads including compounds known to inhibit human pi3ks. the anti-plasmodial activity of these compounds might be through direct inhibition of plasmodium pi3k, although blocking the human host target cannot be discounted because the screens were performed on p. falciparum cultured in blood erythrocytes. a comparison of human and plasmodium kinases of the pi3k and pi4kiii types show overall conservation of key catalytic amino acids which suggests that screening human pik compound collections might find further inhibitors of malarial kinases. as shown in our phylogenetic trees, parasite kinases are divergent from human host homologs suggesting potential scope for developing compounds with specificity to bind leishmania, trypanosoma or plasmodium piks. however, kinetoplastids and apicomplexa are evolutionary distinct groups. the apicomplexa, including plasmodium sp., have been proposed to evolve from an ancestor that acquired a secondary plastid through endosymbiosis with a photosynthetic alga. in contrast, comparative genomic analyses of trypanosoma cruzi, t. brucei and leishmania major could not find significant evidence supporting similar plastid invasion into those genomes. our findings of type ii pik kinases being present in plants and apicomplexa yet absent in kinetoplastids, additional pi3k genes in kinetoplastids and the overall non-clustering of these groups in our phylogenetic trees supports the varied evolutionary history of these protists. as such, differential development of kinase inhibitors for the treatment of malaria, leishmaniasis and trypanosomiasis might need to be considered. malarial genes of putative plastid origins have been suggested to be potential drug targets because of their divergence from human homologs. in our analysis, the type iii pik4cb and type ii pik4b proteins of plasmodium sp. cluster with plant orthologs with significant posterior probabilities and high bootstrap values suggesting a plant or cyanobacteria origin for those kinases. understanding the evolutionary relationships of trypanosoma, leishmania and plasmodium piks relative to each other and their human host will further facilitate both drug target selection and the design of counter-screen assays.  phylogenomic analyses of piks show the progressive complexity of second messenger signaling from unicellular eukaryotes through metazoans and, finally, vertebrates. furthermore, an evolutionary perspective is a potentially useful framework for characterizing piks as drug targets in cancer and parasitic diseases as well as providing insights into the polypharmacology of this important group of kinases. 
mammals have pik isoforms spread across three major families: the ptins 3-kinases, ptdins 4-kinases, and ptdins-p kinases. we found conservation of activating mutations of pik3ca in paralogous isoforms suggesting specific functional constraints on these residues. piks are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets. by mapping published compound inhibition data onto a phylogeny of pi3ks, type ii pi4ks and distantly related, mtor, atm, atr and prkdc kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. our analyses reveal four core eukaryotic piks which are type iii pik4a and pik4b, and at least one homolog each from pi3k and pip5k families. phosphoinositide lipid kinases generate specific phosphorylated variants of phosatidylinositols that are critical for second messenger signaling and cellular membrane remodeling. here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three pik families across major eukaryotic groups and suggest potential ramifications for drug discovery. we postulate two waves of pik diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of deutrostomia\chordata but prior to the appearance of mammals.
 phosphoinositide lipid kinases generate specific phosphorylated variants of phosatidylinositols that are critical for second messenger signaling and cellular membrane remodeling. mammals have pik isoforms spread across three major families: the ptins 3-kinases, ptdins 4-kinases, and ptdins-p kinases. other eukaryotes have fewer yet varying pik complements. piks are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three pik families across major eukaryotic groups and suggest potential ramifications for drug discovery.  our analyses reveal four core eukaryotic piks which are type iii pik4a and pik4b, and at least one homolog each from pi3k and pip5k families. we also applied evolutionary analyses to pik disease ontology and drug discovery. mutated pik3ca are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. we found conservation of activating mutations of pik3ca in paralogous isoforms suggesting specific functional constraints on these residues. by mapping published compound inhibition data onto a phylogeny of pi3ks, type ii pi4ks and distantly related, mtor, atm, atr and prkdc kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. finally, we extended the rationale for drugs targeting piks of malarial plasmodium falciparum, and the parasites, leishmania sp. and trypanosoma sp. by identifying those piks highly divergent from human homologs.  our phylogenomic analysis of piks provides new insights into the evolution of second messenger signaling. we postulate two waves of pik diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of deutrostomia\chordata but prior to the appearance of mammals. reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets. 