SUPPLEMENTARY INFORMATION In format provided by Giordano et al. (JULY 2008)

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Osborne Byrd
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1 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) up plement ary i nfor mation 2 elected inhibitors of cell cycle in preclinical screening Inhib itor (co m pan y)* Main ta rge ts ther ta rge ts tud y da ta Cycl in-de pend ent kina ses (C D Ks) CDKi-277 (RE. 33) (Amgen) CDK6 GK3β In vitro kinase assay: IC 50 = 4 nm ( CYCE), 5 nm ( p25), 8 nm (CDK1 CYCB), 51 nm (CDK6 CYCD) 41 nm (GK3β) Low activity (IC 50 > 5 µm) against numerous serine threonine tyrosine Mediated G1 G2 phase arrest induced apoptosis in a panel of tumour cell lines, independently of the RB p53 status of the cells (mean cellular IC 50 = µm) uppressed the tumour growth in human tumour xenografts models in mice R-3306 (RE. 45) CDK1 CDK4 In vitro kinase assay: K i = 35 nm (CDK1 CYCB), 110 nm (CDK1 CYCA), 340 nm ( CYCE) > 2 μm (CDK4 CYCD) >15-fold in vitro selectivity for CDK1 against a panel of eight non-cdk human Reversibly arrested cells at the G2 M phase border at 9 μm concentration, was well tolerated up to 24 h induced apoptosis after 72 h; produced a larger apoptotic fraction in cancer cell lines (30 40%) than in non-transformed cells ( 10%) Purvalanol A (RE 28,29,175) Cl CDK4 In vitro kinase assay: IC 50 = μm ( CYCA), 0.1 μm ( CYC), 0.24 μm ( p25), 0.4 μm (CDK1 CYCB) 8.2 μm (CDK4 CYCD) Inhibited progression through G1 phase G2 phase Induced apoptosis downregulation of anti-apoptotic proteins by inhibition of JAK2 TAT3 RA polymerase II 24 h exposure (10 μm concentration) induced apoptosis or necrosis in exponentially proliferating cancer cells, without affecting quiescent (nontransformed) cells ATURE REVIEW DRUG DICVERY

2 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) Reinforced inhibition of proliferation by combined treatment with the cytotoxic agent doxorubicin U6140 (RE. 29) CDK4, In vitro kinase assay: IC 50 = 0.42 μm ( CYCA), 3.9 μm ( CYC), 5.5 μm (CDK4 CYCD), 6.6 μm (CDK1 CYCB) 15 μm ( p25) Induced a concentration-dependent G2 M phase arrest apoptosis in cancer cells, with down-regulation of the anti-apoptotic protein survivin Enhanced the cytotoxicity pro-apoptotic activity of paclitaxel (~fourfold increase) in cervical carcinomas ovarian cancer cell lines, with inhibition of survivin expression or phosphorylation as the potential mechanism -CR8 (RE. 31) CDK3, CK1, DYRK1A, GK3α β, ERK2 Displayed a twofold to fourfold higher kinase inhibitory activity against than R-roscovitine In vitro kinase assay: IC 50 = 60 nm ( CYCE), 80 nm ( CYCA), 110 nm ( CYCT), 120 nm ( p25) 150 nm (CDK1 CYCB). In vitro selectivity toward non-cdk protein : IC 50 = 0.61 μm (CK1), 0.9 μm (DYRK1A), 2.1 μm (ERK2) 30 μm (GK3) fold more potent than R-roscovitine at inducing apoptosis in a panel of 25 human cancer cells -&-1, also known as GP0210 (RE 30,32) (Greenpharma), CK1, GK3α β, ERK2, PDXK Displayed a 3 5-fold higher kinase inhibitory activity against than R-roscovitine, but was approximately equipotent against In vitro kinase assay: IC 50 = 40 nm ( CYCA), 43 μm ( CYCT), 70 nm ( p25), 73 nm (CDK1 CYCB) 500 nm ( CYC) howed a threefold to fivefold higher inhibitory activity against CK1 GK3α β than R-roscovitine, but weaker activity against ERK2 PDXK 14-fold more potent than R-roscovitine at inhibiting cell proliferation in a panel of 60 tumour cell lines, but only modestly more potent at inducing apoptosis AZ703 (RE 176, 177) (AstraZeneca) CDK1 CDK4, In vitro kinase assay: IC 50 = 29 nm (CDK1 CYCB), 34 nm ( CYCE), 521 nm ( CYCT), 2.1 μm ( CYC) >10 μm (CDK4 CYCD). Induced apoptosis in several tumour cell lines in vitro caused a reversible ATURE REVIEW DRUG DICVERY

3 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) cell cycle arrest in normal cells JJ (RE 34, 35, 178) (Johnson & Johnson) 2 2 RGB (RE 179, 180) (GPC Biotech) aurora A aurora B CDK3,, CDK4, CDK6, GK3, VEGR2 GR2 CDKrelated (CCRK, PCTK1 PCTK3 PTK1) In vitro kinase assay: IC 50 = 3 nm ( CYCE), 4 nm ( CYCA), 9 nm (CDK1 CYCB), 11 nm (aurora A), 15 nm (aurora B), 175 nm (CDK6 CYCD1) 253 nm (CDK4 CYCD1). Active against GK3 (IC 50 = 254 nm), VEGR2 (IC 50 = 154 nm) GR2 (IC 50 = 226 nm); exhibited lower (sub-μm) inhibition of VEGR3, TIE2 GR1 receptor tyrosine, was poorly active or inactive against several other tyrosine serine threonine Induced G1 phase delay G2 M phase arrest in various human cancer cells, was tenfold less active in normal cells; induced AC impairment in nocodazole-synchronised cells In vivo efficacy in an A375 human melanoma xenograft model in mice at intermittent dosing regimens rally bioavailable 16-fold resistance observed in a human epithelial cervical carcinoma cell line exposed to increasing doses; cellular sensitivity restored by administration of the ABCG2 transporter inhibitor fumitremorgin C Induced cell cycle arrest in G1 G2 phases apoptosis at low nm concentrations in tumour cell lines in vitro Antitumour efficacy in vivo using human ovarian cancer prostate cancer xenograft models Activity against quiescent tumour cells but not normal cells, suggesting solid tumour activity ,4-diaminocyclohexylsubstituted derivative 18 (unensis) CDK1 In vitro kinase assay: IC 50 = 4 nm ( CYCT), 38 nm ( CYCA), 62 nm ( CYC) 480 nm (CDK1 CYCB). howed cellular activity against at sub-μm concentrations (IC 50 = ATURE REVIEW DRUG DICVERY

4 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) -032 nipecotic amide derivative 19 (unensis) D A da mage chec kp oint kin ases KU (RE 56, 57, 181) 2 howed a comparable CDK selectivity, but over fivefold higher permeability in μm) MDCK cells increased bioavailability in mice compared with its parent compound, -032 CDK4 In vitro kinase assay IC 50 = 4 nm ( CYCT), 20 nm ( CYCA), 110 nm ( CYC), 120 nm (CDK1 CYCB) 250 nm (CDK4 CYCD) howed comparable activity against to its parent compound, -032, but improved cellular permeability in vitro. table in mouse liver microsomes Antitumour activity pharmacokinetics bioavailability studies in vivo are anticipated ATM A howed >100-fold higher potency against ATM than other PI3K family members no significant activity against several unrelated Reversibly inhibited cellular ATM function in response to IR-induced DA damage at a concentration of 10 μm ensitized human melanoma cells to TRAIL-mediated apoptosis CP (RE. 57) 2 CBP501 (RE. 182) ATM A Inhibited ATM, but not PI3K, PI3K-like proteins or BCR ABL kinase in cells; its potential activity against RC kinase other possible off-target proteins are yet to be characterized Reversibly inhibited cellular ATM function in response to IR-induced DA damage at a concentration of 6 μm In clonogenic survival assays, transient exposure was sufficient to sensitize tumour cells to IR CK1 CK2 er216- specific, such as MAPK 2 ynthetic peptide with 12 D-type amino acid residues In vitro kinase assay: IC 50 = 3.4 μm (CK1) 6.5 μm (CK2) howed cancer cell-selective enhancement of the cytotoxicity of DAdamaging drugs ATURE REVIEW DRUG DICVERY

5 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) MARK3 C (RE. 65) 2 2 CK2 BRK, GR, IGR LCK In vitro kinase assay: IC 50 ~0.2 nm (CK2); no effects on CK1 elective for CK2 against a panel of 20 other (IC 50 values were over 6.5- fold higher against other ) Br VRX (RE. 64) Au ro ra kina ses esperadin 70 (Boehringer Ingelheim) CK2 Aurora A In vitro kinase assay: IC 50 = 120 nm (CK2) > 10 μm (CK1) uppressed the DA damage-induced intracellular activation of CK2 at 10 μm o effect on cytotoxicity of doxorubicin, cisplatin or taxol at 10 μm Aurora B Aurora A In vitro kinase assay: IC 50 = 250 nm (aurora B) > 2.8 μm (various CDKs) Produced a cellular phenotype consistent with aurora B inhibition TC-28 (RE. 183) Aurora A A igh specificity in vitro for aurora A (IC 50 = 2.7 μm) compared with aurora B (IC 50 > 100 μm) Inactive (IC 50 > 100 μm) against various tyrosine, including EGR, IGR, VEGR2, VEGR3, GR TIE2) Delayed G2 M phase progression, compatible with aurora A inhibition, in human tumour cells VE-465 (RE. 184) Aurora A aurora C Aurora B In vitro kinase assay: K i = 1.0 nm (aurora A), 26.0 nm (aurora B) 8.7 nm (aurora C) Growth-inhibitory effects on several human leukaemia cell lines expressing wildtype BCR-ABL or its T315I mutant form ATURE REVIEW DRUG DICVERY

6 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) ZM (RE. 70) (AstraZeneca) Aurora B Aurora A aurora C In vitro kinase assay: IC 50 ~50 nm (aurora B), 250 nm (aurora C) 1 μm (aurora A). electivity over a range of other, including CDK1 PLK1 (IC 50 > 10 μm) Inhibited histone 3 phosphorylation at 100 nm in cells Polo -lik e k ina se s (PLKs) Cyclapolin 1 (RE. 70) (Cyclacel) 2 2 DAP-81 (RE. 70) (Rockefeller University) 2 PLK1 A In vitro kinase assay: IC 50 = 20 nm (PLK1); showed little activity against 20 other Cellular effects consistent with PLK1 inhibition were observed only at concentrations > 10 μm in Drosophila cells PLK1 A In vitro kinase assay: IC 50 = 0.9 μm (PLK1) Induced a cellular phenotype consistent with RAi-mediated PLK1 ablation, including strong impairment of spindle bipolarity Poloxin (RE. 108) PLK1 PLK2 PLK3 Inhibited the PLK1 polo box domain with an apparent IC 50 of 4.8 μm (fluorescence polarization assay) Blocked PLK2 polo box domain with an IC 50 of 18.7 μm PLK3 polo box domain with an IC 50 of 53.9 μm ATURE REVIEW DRUG DICVERY

7 UPPLEMETARY IRMATI In format provided by Giordano et al. (JULY 2008) TAL (RE. 92) PLK1 PLK2 PLK3 Prevented PLK1 from binding to its intracellular anchoring sites substrates, inducing mitotic arrest apoptosis in tumour cells A lead compound for development of thymoquinone derivatives as specific PLK inhibitors for anticancer therapy A useful tool for studying the function of the polo box domain of PLKs in mammalian cells In vitro kinase assay: IC 50 = 19 nm (PLK1) Also inhibited PLK2 PLK3 with IC 50 < 100 nm, but showed little or no activity against 93 other serine threonine tyrosine Induced mitotic arrest cytokinesis failure with an IC 50 of μm in human mouse tumour cell lines *Where no company is listed, the compound was developed by one or more academic groups; see references for details. ABCG2, ATP-binding cassette sub-family G member 2; ATM, ataxia telangiectasia mutated; BRK, breast tumour kinase (also known as PTK6); CCRK, cell cycle-related kinase (also known as p42); CK, checkpoint kinase; CYC, cyclin; DYRK1A, dual specificity tyrosine-phosphorylation-regulated kinase 1A; EGR, epidermal growth factor receptor; ERK2, extracellular signalregulated kinase 2 (also known as MAPK1); GR, fibroblast growth factor receptor; G0, quiescent; G1, first gap; G2, second gap; GR, GR, hepatocyte growth factor receptor (also known as MET); IGR, insulin-like growth factor receptor; IR, ionizing radiation; M, mitosis; GK, glycogen synthase kinase; IC 50, compound concentration that causes 50% inhibition of kinase activity; JAK2, janus kinase 2; K i, inhibition constant; LCK, lymphocyte-specific protein tyrosine kinase; MAPK2, mitogen-activated protein kinase 2; MARK3, MAP microtubule affinity-regulating kinase 3 (also known as CTAK1); A, not available; PCTK, PCTAIRE protein kinase ; PDXK, pyridoxal kinase; PTK1, PTAIRE protein kinase 1; PI3K, phosphoinositide 3-kinase; RB, retinoblastoma protein; AC, spindle assembly checkpoint; TAT3, signal transducer activator of transcription 3; TIE2, tunica interna endothelial cell kinase (also known as TEK); TRAIL, T-related apoptosis-inducing lig; VEGR, vascular endothelial growth factor receptor. ATURE REVIEW DRUG DICVERY

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