nature chemical biology ARTICLES A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity ra Vulsasinal, Alessia Montagnoli!, Barbs lor Cruci!, Maria Menichincheri', Sonia Rainoldi!, ‘Vanessa Marchesi!, Marcello Tibolla!, Pierluigi Tenca*, Deborah Brotherton, Clara Albanese}, ‘Veronica Patton!, Rachele Alzani', Antonella Ciavolella’, Francesco Sola’, Antonia Molinari, Daniele Volpi, Nilla Avanai', Francesco Fiorentini', Marina Cattoni!, Sandra Healy', Dario Ballinari', Enrico Pesenti', Antondlla Isa ¢ that promotes DNA replication by ac phosphorylation sites, Unlike does not impede replication fork progress and it does not ti I and cell-hased assays, and we testes that the compound blocks DNA synthesis and affects the phosphoryl rent DNA synths inhibilors, PHA-767491 prevents the activation of replication origins but ‘hi!, Jurgen Moll!, Aaron Bensimon’, Ermes Vanoiti) & Corrado Santocanale!* 5 of replication. Here, we characterized the potent ts anUiumar activity in rodents. We found ive DNA helicase at Cae7-lependent 1 of the rep ger a sustained DNA damage response, Treatment with PHAL 767491 results in apoptotic cell death in multiple cancer cell types anc tumor growth inhibition in preclinical cancer models, To our knowledge, PHA-767491 is the first clongation af anticas DNA synthesis iviites Goo aruiple oubins of sepication on cheoroosoenes. Ce? is am esteolial Rinse eeqbite Loe ie: ativa Ui it ghosghorvlales one ae rae sun Le eaiscomnenone zainsianesproléis 2? {MCMs), sid coc the rephiclve LOA eins, and stich ane hownd ta origin DNA in a “pee seplcarive compa fined ding the previnns C1 phase of the scl este, Phasphorlaiew is thought ages the MEM helio, us leding .o the kcal unscnctng of double sanaded DNA at bovis of plication, slo Ube lua of DA poles wd axcustory Lacan t pariipae iv Uhe secretive spin 0 ew DIA assur chain ongation . Deregula! entry inka § phase af the call cycle fs a halina-k of exer ls. and drags tha tutget DNA vepliston Sergtion ate wep used in chsmatorap— “ar exoriple, gematabing 2, ative meteboltes of 3 Fugue! fF TU, 34 au! bsdioasune CHU, These compeurals ll ale de polpinesiration aap of TINA replication, by enbslancing the poo aNNTP. endion y beng inamporatal by TN polymnatise nethe saison sands and acting a: chain tenvinators, Orker anicareer ébnge afer elongation inden ither “yp targing ence shat ‘acts cepisron fork progrestin, such os epoisnmneise ehh torso by xeadaga phic. alock o fouk sunpiesson, sucks DNA Merci ageao?. A blockade ol eeplcaion fake olen esl tsakage othe TINA moles and activation of an ATREATMe ays Wie Sai 5 sy a vey Nelo saa loaleinag 68 NATURE CHIFAUCAL BIOLOGY VOLT 2 RYSATEG JLT 2026 wT, 2081 Rares, ta alecule that directly affects the mechanisms controlling initiation as opposed to hibition could he a new strategy for the development cepandent 8 phase cbeckpoiat anheray tan senses tbe damage gd ‘meciates cellu responses to dup ueaumen?4, Howeres La oliival beulit ot these ceugs i Finite by sir tiv’ webich eoap be rela! Ur the recanise of ae.ion ‘ior example, gatrirtetinal end Fons marrow toxiciyt ar to eamepousdsspecinie effets, such a8 renroroxicin:. tomar eels cen also Recame resistant 0 “hese agents i anaeipl vgs, thus suppartng she need forthe develapirent compounals Lat cae Lae soe process theouga a eferenk ect ‘The yene ennsiding Ca? an fist isola it beg yous? The urna homolog wae identied simost bso Gecades Ize an ts volvement in DSA repliesron and cell pre'Feraion has ea Come rated! 1" “cplinedcpemors kinases, Fe? acing Fequites the binding of either one af two reguezary subunits, DBF sod Dll fake scone as DUAB)HE, Periodic, eetumulatios v! Cade, DO and TC TIDE durin § pase is eet wo bethe maior Dechranises that rogulaus Cae? setiity dung the ell ye! The MEM DSU lion is the best chamestired Ce? thar! sin yg hw-nan CA°7 can phowphorgloe several MCR snomits swith some e-ence for cin’ praein, avi in celle Men? phosptoesktion a S140 and SeS3 is completely depends, v2 Cale? avtsite!®. Prelesinary suulie indicate et Cele? is tes coverespresed in tuo: well fins and in wumiar agocnens! and nei So, Fo co, a Deki Pe Ugo as wo ver ol Selsey2008 Nature Publishing Group httpu/mww.nature.comvnaturechomicalbiology g ARTICLES a e cate oe iin the init alicstion Hel Figure 1 PHA-767, reaction DRS stu {ay HE PE7E41 mn anu arr sera, 8) Cal ste xe nouted ml Ey 76/41 uF ay ne rnd ied lire Beare ha. on vals ware abelee fr 30 maxi Bra, ane. DN thesis wae analyzed by FACS, Pasennags of Bil-pes m ese. sale Ce) a inst PI 2275/49] eae cll, Calls mas cubated sic € 41 P=2-767491 or 3 0 t hsngr2a CH. fer the ica ime. Pra eats ee sae td an Ad 92 tor mths ol Ge? Ue 60 aid 38°83 Ne) or marks ot apoptes Ib--2RX, ache caspase-S and 22RPI and fr mmatkes of DHA ca nage c-ackpol“ sponte [Sora Shel and plmube OE. 9) Go se villosa eae SH 2 ale wh Lue Pom TET] ive vese-oe or atueree 0 8938 intcare fe? fant alten 2c ete analiza: by es Dt wih tne sca iba es Le ol ve cescl >” al relicaicy nte~necales ut fs ne SFA rclcile ty the CNA corr ng ohn ef The sewage diane hetseen ~2 sed seen cL wy cu nes cost be Ue fo ile bers) and 21 gry bs) eck ry Eas: 9° Eh (lack bars ater “armen! it inciate are tof ETE TAN. (gh 16 pape 96 Ivana seer tele 920) ar 2 ih ay (MM expresiua ligsdy eorelies 6° aggresive pease sl a ‘a ralignances®™ ppowerial marker of efnical eoune iv a vara ‘We have rhanwer hat sal: inerfering WNA-anedined Cee? deple- Vs ro enfor apoptosis in a pS ndependlant rnannat, while simply arsing ell egcle progecasion ie ngemal sels", Ditfgeertil cling, of ta nar eersus ng-mal cls appears ta be w Ustieive featuce uf Ute inlibition origi wtivaioe, as it ssas abo sea by blocking tbe activa 0 Cas an Cad treb. 21,271, ‘which are imtlved im che londing of MEMS el origins, tucthertcone, (GA depletion, im enotras to rep “cation Karki ceade, dd nal aus a sustainad actvation oF the Sep'wnge chockpnirr kinases. hose sF ene sces betwoen ¢he eallclar respences to Tae inbibiton of CAF ‘od the inbibifioa of DNA sepLcation elangaton by conventional shercolherapectisn promple us bo searea (ar saall anal alibi los he CHE? Kinase, an 4 explore Car potential fn preclinical cnodl's at a new css oF anticancer drug RESULTS Discovery of a smallmolectle Cac? kinase inhibitor The sereetny ol cur eompouné Ebvary tapproxircriely $20,000 scorapounls! wilh a biveaemicsl Cie? Kinane assay revealed hit aprening seven! chamotypes Among these. PILATATEST [L) hig. Tay seal ese forthe proparazioa of MIK2 inhibtar, seat rsonpniend af a potene initte oF Cac? Kerase, sani af ae sompourd yieldad a haFmvion! inhibitory cenccnt ation 11C5 wae of 1D aM in the pence of 15 uM ATE, which i approximately Cove its caleulated Kyy valueS Substrate and ATP Uuetios “ni ‘alee hat PITA 76°43 comptes withthe aloe tr nding (Supple mentary Fig. Tonlirs. thus behaving asa pen: ATV competitor We hem prafled this commoned against a pans] of 3% huntan seiner ti hicat yor st eh oe be Ee Eien tend wee ain celta ER BORER rote sh neva aud tyrosine Kies i stuulardize. cocuiiousy hiss while allows us direcy etmpare ioehetrical Ty, sued hroting Ural these Ischemia values do rot else flo the slecrivity of the componmé once ina cellar enrvest"™ PIAS PATA ws “Gund to inhibit CAT, Cie and GSKB, although its pte ny teas 20.44 leer against these fo, Savas ese: Zena: te Dee lage of Ces 1 DOE SO 1 meets the sonsiily oF elle bo eal ele ses and other cellular stress by irereasing apaptosie" Indeed, ca proliferation 3233 inhibited in 9 past 61 human cell lines challenged seh low mictomoir concectiatioes of PHA 787491, ssid a average IC velue of 3.17 Nia lee thses KOM “al ae, Tre & smaller purel of 1 aliferemt call pes, using sub-G1 TSA, ‘sintent aso parurseter i apples, we Round Ural 24 Wf expotire Us fither Sor 1 IM PLASFA341 wes sufficient to cms cell each, albeit ta dite-ent extn (Table | and Supplementary Fig. 2 online) The same cel's generally survived expacie to § FU ar gemcitabine ‘a doses well abuse Lae avenge ICs) ateisured ia Lie pole tion assay {Table 1. Aunoiny Lae entep.ions, B96? eubesnia cel wore Jak sameithe Mh PUIALTST491 in the IRA replication andl povlfiration aoays, ard SCI NHDDE erlls sere meestant to. PRATER nduced pepsi In foo panel, gemeltebine caused apoptosis. onky in HT IS and Juskot ceb lings, ssheseas 5 FU only caused opoplosis in HCT 116 cells Table 1 Traportanly PUTA-707491 is capable of proven ing duplication, and hoik pSipeuite and pSneyative wumor calls of free argins, as ellos call Hos insite to linc ly rsa DIA rep eation falvbirs, sich as gemeitab’ae and 3-FL. Babe 1) nctesses of the Ise values io cell bived sss, compiced ila Ure bivehenical assays ace mostly enplained by Ure bight a Jinity ha Cae les for ATP iq of 0:7 pM andl lee higher levels of intr coll AIP {7ef, 271, Using Ike Chengitusot? equation far ALP caumpottars 9 elcubsts the Mreortical shift in potency Pa is dus tx semply snersasing the AIP concemtatior, an Cap oF 4.4 pM at T mM ATP ig expecta —a value thac it compatible ith chess experimentally observed (Table 1 148) 8 reaoured in a allel etal which sa also lyerved ond sam ki Ure corpo (1.68 ese remrane pureaaility say SPAMPAT™. as wall da other aepete tandem. alons, may alas somiuts to the higher Wag vals neal NATURE CHIFAUCAL BIOLOGY VOLT 2 RYSATEG JLT 2026 The suboptinal peecieailty 07 PHA-767491 decreases origin fring Ina ima onmvss experimen, Heta ell wore tated seit 3 pM PHA 74719", DNA semtbesis wns Sollwsed ty Auorescencoacregted call setting {EACS; amalyels, aud Mca phosphorshrion at the Ce? ‘epee sles SeraD au $e.33 vas anesoured ay 1 plisutseusouuaic pparumetar of Cle? inhibition, AL east 6 uo 9 1) of treatment were hecasury us observe a worked deersase in the number a hremusleos= sardine (Ih, Stpositve cells (Pig. Th, sich fs casters write the tims -equitsd to achices enmplcte Mem? dephospacey aian at Sor sd S058 (Fig, 1), Phesphorslarn at the adiacene CacF indepe> eu site Sed foe -€) sus ul alte Mp, Le}. The Wockade of DNA synthesis correlated lh entry Sols apoplenis aw 7 TIZAX, active ceapase-3 ond wis poly. ADe-riaose| pailymmerase (PARI Gelucable al @ band kept seanmulaing diver time (Hig, Te. previonsly shso-wed with Ciée7 deploy ENA Gr Vand ans "reatment with the TINA replication elgrgstioninhisigar HI, we led ta detect Chl phosphosslation al S345, wheteas only low eves of Chaz pliospborskstion al Tee were elected in PITA 76.491. Ueated lp (Fig. Te. The incusion ol a pan-eaypa inkilston, 7-VAD i proven] PIIA-T67491—induced vall Jeath [Supplementary Fig. 21 xspase-3 acvaticn and the indaccom of j-H2AX Utig. 143, hich fa rman ehat is alan slate to the pivscnar of DNA dans bveaks (DSB, This indicates bat PHA 787 DNA darnige, ace that y TRAX induction is a seomadicy elec, possiby related ur DNA Gagmen ation occurring ducing apaplesn, Convery, -112AX induction ay HU ia doug that dees nt pger sapuplosis in Dut Slueks repkeaor. fk amd may caine Ds a ars) nos imposes hy /-NADD (Hig. 1a Ty understsrd the consequsnces @ PHACTEMIST sraatne ar 63 DNA plication dsaamics, we used tbe DINA couubicg lechaique®, ‘which ively lable rassel TINA ie cells ut ten exlecting zn orsogencously streshing DNA fibers wr: plass sliées. Replication inermadiaies are siualied by immunefluurscance, allowing eee 5 streh can he caken asa 1 eloes aot eine cause surement ofthe average inteigin dst