recent studies on the molecular basis of various diseases have revealed that the deregulation of protein phosphorylation reactions by kinases is clearly associated with many disease states , especially cancer ( noble et al 2004 ) . thus modulating aberrant kinase activities in cancer cells may form an effective cancer therapy , with fewer side effects than traditional cytotoxic drugs . a number of kinases have been studied as potential targets for cancer therapy ( collins and workman 2006 ) . kinases regulating the cell cycle , including cdk ( cyclin - dependent kinase ) , aurora kinases , and chk1 kinase , have also been the focus of many studies on cancer - treating agents ( fischer et al 2003 ; collins and garrett , 2005 ; warner et al 2006 ; de carcer et al 2007 ) in part because these cell cycle - regulating kinases are overproduced in cancer cells . cdc7 is a conserved serine - threonine kinase , which was originally identified in budding yeast and shown to be required for the initiation of dna replication ( hartwell 1971 ; patterson et al 1986 ; sclafani 2000 ) . subsequent studies showed that cdc7 is required not only for initiation but also for stable maintenance of replication forks ( snaith et al 2000 ; takeda et al 2001 ; fung et al 2002 ; matsumoto et al 2005 ; sommariva et al 2005 ) . this brief article summarizes the latest knowledge on the functions of cdc7 kinase , and current efforts on the design of a drug based on this interesting molecule . initial characterization of a budding yeast cdc7 temperature - sensitive mutant indicated that cdc7 is required to initiate dna synthesis . cdc7 is a serine - threonine kinase , which belongs to a unique group in the kinase family ( figure 1 ) . cdc7 forms a complex with dbf4 ( johnston and thomas 1982 ; kitada et al 1992 ; jackson et al 1993 ) , an activation subunit . both cdc7 and dbf4 are temperature - sensitive mutants of budding yeast and arrest with 1c dna content at a non - permissive temperature , suggesting a defect in the initiation of dna replication . in 1995 , the first functional homologue of cdc7 was identified in fission yeast ( masai et al 1995 ) . after this discovery , conserved presence of both cdc7 and dbf4 subunits was demonstrated across the species ( masai et al 1999 ; masai and arai 2002 ) . knockout of cdc7 genes in mice leads to early embryo death ; the mutant embryos die between e3.5 and 6.5 . conditional knockout of cdc7 genes in mouse embryonic stem cells resulted in the arrest of dna synthesis , accumulation of nuclear dna damage , and eventual p53-dependent cell death ( kim et al 2002 ) . these results suggest that cdc7 kinase has critical roles in dna replication , which may be conserved across species . the conserved targets for cdc7 kinase are mcm subunits , and phosphorylation of the n - terminal non - conserved tails of mcm2 , 4 , and 6 proteins has been shown to facilitate the association of cdc45 and other replisome factors with pre - replicative complex ( pre - rc ) ( masai et al 2000 , 2006 ; sheu and stillman 2006 ) . this step is crucial for the generation of active and efficient replication fork structures ( figure 2 ) . the replication fork is under continuous attack both internally and externally , clearly indicated by the fact that a recombinational repair system is essential for the viability of vertebrate cells ( sonoda et al 1998 ) . proper processing of stalled replication forks and the resumption of dna replication are essential for completion of the entire genome duplication within the given s phase . a defect in checkpoint regulation poses serious threats to the stable maintenance of the genome . indeed mutations in checkpoint regulators have been demonstrated to be responsible for various tumors or diseases ( michelson and weinert 2000 ; dandrea and grompe 2003;narek and lukas 2003 ) . the former is involved in activating checkpoint kinases , while the latter is involved in executing the checkpoint effects ( niida and nakanishi 2006 ) . accumulating evidence indicates the critical roles of cdc7 kinase in both phases of dna replication checkpoint responses . chk1 is activated in response to replication fork arrest , and cdc7 appears to be required for this activation ( kim et al 2008 ) . claspin is a mediator of checkpoint responses and is hyper - phosphorylated in response to stalled replication forks . cdc7 is required for this step ( kim et al 2008 ) . in fission yeast , the activation of cds1 , a checkpoint effector kinase , and hyperphosphorylation of mrc1 ( a fission yeast counterpart of claspin ) in response to fork arrest , depend on hsk1 , the fission yeast homologue of cdc7 kinase ( takeda et al 2001 ; shimmoto et al unpublished data ) . claspin / mrc1 is efficiently phosphorylated by cdc7/hsk1 in vitro , suggesting that possibly cdc7 directly regulates the checkpoint activation ( kim et al 2008 ; kakusho et al unpublished data ) . the requirement of cdc7 for continuous activation of the rad53 checkpoint kinase in response to hu has also been reported in budding yeast ( ogi et al 2008 ) . on the other hand , the role of cdc7 in the effector phase was suggested by the fact that cdc7 is inactivated in xenopus egg extracts in response to etopside , a dna - damaging agent introducing single - stranded nicks ( costanzo et al 2003 ) . cdc7 was also shown to be down - regulated by etopside in human cells ( dierov et al 2004 ) . evidence showing that the fork block inhibits cdc7 kinase was also reported in budding yeast ( weinreich and stillman 1999 ) . these results strongly suggest the dual roles of cdc7 kinase in the mediator and effector phases of checkpoint responses . however researchers report that cdc7 activity is unaltered by replication fork blocks in human cells ( tenca et al 2007 ) . furthermore , we can not rule out the possibility that defects in the dna replication fork caused by cdc7 depletion contributes to the apparent defect in checkpoint activation . whatever the mechanisms are , loss of cdc7 functions in actively proliferating cells causes replication fork arrest which results in dna damage , and simultaneously leads to defective checkpoint activation . unique features of cdc7 include the presence of two or three amino acid segments which interrupt the kinase domains at conserved locations ( figure 3 ) . although the functional significance of these kinase insert sequences has not been defined , association of nuclear localization and nuclear export activities with the second and third inserts , respectively , was reported for human cdc7 ( kim et al 2007 ) . cdc7 associates with chromatin during the s through g2 phase , and dissociates from chromatin during the m phase ( masai et al 2006 ) when it is phosphorylated , presumably by cdc2 kinase . kinase activity may not be regulated by cdk - mediated phosphorylation , since escherichia coli - generated cdc7 kinase is highly active and the activity is not affected by cdk ( kakusho et al unpublished data ) . cdc7 kinase is generally active only when it associates with an activation subunit ( johstone et al 1999 ; masai et al 1999 , 2000 ; masai and arai 2002 ) . the dbf4-like activation subunits share three conserved motifs , namely dbf4-motif - n , -m and -c ( masai and arai 2000 ) . the dbf4-motif - n , a brct - like motif , is not essential for kinase activation , nor for cell viability in yeasts , although mutations in motif - n result in sensitivity to replication fork blockages induced by hu and other agents in yeasts ( ogino et al 2001 ; varrin et al 2005 ; gabrielse et al 2006 ) . the dbf4-motif - m ( proline - rich novel motif ) or motif - c ( c2h2-type zinc - finger motif ) is capable of binding to cdc7 on its own , but the maximum activation , and viability in fission yeast cells through meiosis , require the presence of both motifs connected by a non - conserved linker segment ( ogino et al 2001 ) . on the other hand , survival through mitosis in fission yeast can be maintained only by the motif - m , albeit with reduced growth rate , whereas motif - c is required for resistance to the alkylating reagent , methyl methanesulfonate ( fung et al 2002 ) . in mammalian cells , in addition to motif - m and motif - c , motif - n is also required for viability ( yamashita et al 2005 ) . although the precise substrate recognition code is not known for cdc7 kinase , it is clear that cdc7 is an acidophylic kinase , preferring the presence of acidic residues near the phosphorylation site . it was reported recently that positively charged polymers such as polyamines strongly stimulate cdc7 kinase activity , whereas the negatively charged polymers such as nucleic acids severely inhibit cdc7 kinase activity ( kakusho et al 2008 ) . these characters of cdc7 are also shared by casein kinase ( hathaway and traugh 1984 ) , reinforcing the structural similarity between cdc7 and casein kinase . sirna - mediated downregulation of cdc7 expression in various cancer cells induced retardation of s phase progression , followed by accumulation of nuclear damage , presumably induced by a stalled replication fork which was not properly stabilized and collapsed ( montagnoli et al 2004 ; yoshizaw - sugata et al 2005 ; i m and lee 2008 ; kim et al 2008 ) . cells then undergo chromosome fragmentation and die , which occurs in both p53-positive and -minus cells , indicating that cell death is p53-independent ( montagnoli et al 2004 ) . in contrast , normal fibroblast cells do not die in significant quantities under the same conditions . s phase cells slow down dna synthesis and some dna damage appears in nuclei , but is eventually repaired . cells in other cell cycles may be arrested in the g1 phase in a p53-dependent manner ( montagnoli et al 2004 ; ishii et al unpublished data ) . the precise mechanisms of cancer - specific cell death are not clear . depletion of cdc7 would cause defects in firing of the replication origins as well as acute destabilization of replication forks which would eventually collapse . furthermore , checkpoint defects may trigger uncoupling of the m phase with the s phase . therefore we suggest that the combined effect of replication fork arrest and deficient checkpoint responses might augment cell death by inducing aberrant cell cycle progression . whereas normal cells could cope with these situations through backup checkpoint mechanisms , cancer cells , in which checkpoint mechanisms are very often partially abrogated , researchers reported recently that atr - induced activation of p38 map kinase causes caspase activation in cdc7-depleted cancer cells ( i m and lee 2008 ) . a ligand atp binds to the atp - binding pocket through hydrogen bonds with the hinge region connecting the n- and c - terminal lobes of the kinase catalytic domain ( figure 4 ) . most kinase inhibitors reported so far also bind to this hinge region with additional interactions with two hydrophobic regions ( sites b and e in figure 4 ) adjacent to the atp - binding pocket . the hydrophobic region i ( e in figure 4 ) is commonly referred to as the selectivity pocket , because the size of a single residue in the interior of this region , termed the gatekeeper , has been shown to be a critical determinant of inhibitor sensitivity ( cherry and williams 2004 ; vieth et al 2004 ) . based on our in - house human kinome database , approximately 90 kinases possess a small threonine gatekeeper , and 190 kinases have a large methionine residue . according to a homology model of cdc7 we built in - house , the large gatekeeper residue ( met134 ) appears to make the entrance to the hydrophobic region i very narrow , and the back pocket of this site is occupied by residues val120 and ile132 ( figure 5 ) . the central portion of the adenine - binding region is formed by a hydrophobic sandwich with two residues , met118 and ile64 , suggesting that a flat hydrophobic core may be preferred as an inhibitor scaffold . in the same region , two ionic residues , lys90 and asp196 , could provide opportunities for the design of potent inhibitors with appropriate substituent targeting these residues . only a handful of cdc7 kinase inhibitors have been reported so far ( figures 6 and 7 ) . a series of pyrrolopyridinone compounds with potent activity against cdc7 has been reported by nerviano medical sciences ( montagnoli et al 2008a ; vanotti et al 2008 ) . pha-767491 ( # 1 ) was identified from high throughput screening ( hts ) and characterized as an atp - competitive inhibitor of cdc7 ( ic50 = 10 nm ) . this compound recapitulates the most relevant molecular phenotypes that are expected from cdc7 inhibition ( montagnoli et al 2008a ) . it inhibited cdc7-directed phosphorylation of a substrate protein ( mcm2 ) as well as dna synthesis , and induced cell death in multiple cancer cell lines , but did not affect the viability of normal cells ( nhdf ; normal human dermal fibroblasts ) . treatment of nhdf with pha-767491 indicates that cdc7 inhibition may prevent the activation of replication origins but does not trigger a sustained dna damage response . subsequent optimization of this scaffold led to 3-aminopyrimidine analog ( compound # 2 ; ic50 = 7 nm ) . the structure activity relationship ( sar ) study suggested that the aminopyrimidine group probably interacts with the hinge . although the compound # 2 showed antiproliferative activity against the a-2780 ovarian cancer cell line , it exhibited poor membrane permeability in the pampa assay which would cause low oral absorption . further optimization led to ( s)-2-(2-aminopyrimidiny-4-yl)-7-(2-fluoroethyl)-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridinone ( compound # 3 ; menichincheri et al 2007 ) . compound # 3 , which had an ic50 value of 2 nm against cdc7 with greater than 60-fold selectivity over other kinases , showed excellent inhibition of cell proliferation in various tumor lines with submicromolar ic50 values . in addition , compound # 3 was found to be effective in an a2780 cell line mouse xenograft model , when administered twice daily at an oral dose of 60 mg / kg for 10 days . in the dmba - induced mammary carcinoma rat model , compound # 3 was also active at a dose of 20 mg / kg . the nerviano group also examined other possible modifications to the pyrrolopyridinone scaffold and disclosed novel pyrrole compounds , such as compound # 4 ( vanotti et al 2007 ) . a clinical candidate , nms-354 , which is derived from the same chemical class as compound # 4 , was reported as an orally available cdc7 inhibitor ( ic50 = 3 nm ; montagnoli et al 2008b ) . nms-354 is potent in blocking proliferation and inducing apoptosis in a broad panel of 120 cancer cell lines with ic50 values in the submicromolar range . in vivo , nms-354 has a broad range of activities when administered singly ( 20 mg / kg ) in different xenograft tumor models ( ovarian , colon , mammary , leukemia ) and also in a rat mammary carcinogenic - induced tumor model ( dmba ) with tumor growth inhibition of more than 80% without toxic effects . researchers at hoffmann - la roche explored a series of tricyclic cdc7 inhibitors and identified potent and selective compounds ( zhao et al 2005 ) . a pyridothienopyrimidine was identified from hts and subsequent structural optimization resulted in the discovery of compound # 5 which is a potent cdc7 inhibitor with ki of 2 nm ( figure 7 ) . recently , novartis reported a series of 4-indazolylpyrimidin-2(1h)-one as potent cdc7 inhibitors ( shafer et al 2007 , 2008 ) . compound # 6 was identified as a potent inhibitor of cdc7 with an ic50 = 5 nm ( figure 7 ) . they predict the binding mode of this series of compounds on the basis of the co - crystal structure of the parent compound , phenol pyrimidin-2(1h)-one , with pim-1 kinase . the structure shows that the phenol and pyrimidinone are coplanar , and this flat shape is expected to fit well with the cdc7 atp - binding site as described in the previous section . although the details of the interactions may be different between pim-1 and cdc7 , the structure suggested that the nh group in the indazole ring is likely to bind to the hinge region of cdc7 . unfortunately , this series of compounds exhibits low membrane permeability as well as metabolic liability , and , in fact , showed weak cellular activity . various cdc7 kinase inhibitors have also been disclosed in the patent literatures recently ( figure 7 ) . thienopyrazoles have been claimed as cdc7 inhibitors by pharmacia ( now pfizer ) ( tonani et al 2004 ) . in this application , compound # 7 was described as possessing cdc7 kinase - inhibiting activity with ic50 < 1000 nm . the 2-pyrimidyl-5-amidothiophenes were identified as an akt inhibitor by chiron ( now novartis vaccines and diagnostics ; lin et al 2005 ) . the patent application for this compound series claims that these analogs also have inhibitory activities against cdc7 as exemplified by compound # 8 . however , no activity data were presented . a series of imidazolones was recently disclosed in a patent application by sanofi - aventis ( leroy et al 2008 ) . they claim that the members of this series , as represented in compound # 9 , have inhibitory activities against cdc7 . although cdc7 is essential for the viability of eukaryotic cells , its inhibition or depletion leads to cell death specifically in cancer cells . this is caused by acute genomic instability induced by the combined effects of replication fork arrest , its destabilization and aberrant m phase due to checkpoint defect . cancer cells are generally associated with defects in checkpoint response pathways , and thus exhibit greater sensitivity to s phase perturbation than normal cells . potent compounds capable of inhibiting cdc7 kinase with nm ic50 were recently reported , and were shown to be effective in reducing tumor growth in mouse xenograft models . only a few patent applications have been disclosed so far for cdc7 kinase inhibitors , and it is not yet possible to determine which compounds will prove most promising , because detailed evaluation of the compounds at the cellular level or in animal models has not been conducted for most of these compounds . nevertheless , because of the unique nature of its structure and action , inhibitors of cdc7 kinase would be promising candidates for novel classes of cancer drugs .