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PDK1 P70S6K AKT RSK2 0.003 0.007 0.074 0.130 0.163 0.182 0.186 0.195 0.215 0.227 0.279 0.354 0.453 0.476 0.684 0 1 2 3 4 5 6 7 8 9 10 11 12 EC 50 (uM) SNS-510 (PDK1 inhibitor) MK-2206 (Akt inhibitor) GDC-0941 (PI3K inhibitor) pRSK2 S227 pPDK1 S241 β-actin [nM] SNS-510 300 100 30 10 GSK-2334470 300 100 30 10 MK-2206 300 100 30 10 pP70 S6K T229 a B a C ABSTRACT ● Background: Phosphatidyl-inositol (PI) dependent kinase 1, PDK1, is a master kinase that activates members of the AKT, PKC, RSK and SGK families ● PDK1 can interact with its substrates through PI-dependent (PH-mediated) or PI-independent (PIF-mediated) mechanisms. PIF refers to PDK1 Interacting Fragment also known as Hydrophobic Motif (HF) (Fig 1). ● Results: Here we report characterization of two potent PDK1 kinase inhibitors, SNS-229 and SNS-510 (Fig 2), that block both PH-mediated (AKT) and PIF-mediated (RSK) substrate phosphorylation and have broad anti- tumor activity in hematologic cancers ● SNS-229/-510 bind the inactive conformation of PDK1 as determined by X-ray crystallography (Fig 3). The compounds bind deep in the adaptive pocket, distorting the N-terminal domain and perturbing the PIF-pocket, thereby inhibiting PIF-mediated substrate binding (Fig 4) ● SNS-510 was evaluated in several hematologic cancer cell lines and showed strong anti-proliferative activity with EC 50 s ranging from 3 nM to 900 nM (Fig 5) and produced substantial apoptosis after 24 hours (Fig 6) ● Anti-proliferative activity correlated with pathway modulation as assessed by inhibition of phosphorylation of PDK1, RSK2, P70S6K, and AKT (Fig 7) ● In mice, SNS-229 and SNS-510 showed good oral bioavailability (%F>40%) with a Tmax of 4 hours and prolonged exposure (Fig 8) ● Pathway modulation was evaluated in vivo in a MV4-11 xenograft mouse model. Potent, dose-dependent pathway modulation was observed at 4 and 8 hours after a single oral dose of SNS-229 and SNS-510 (1 to 25 mg/kg; Fig 8) ● SNS-510 was compared to the PDK1 inhibitor GSK-2334470, showing comparable biochemical potency, but SNS-510 was 10 to 30 fold more potent at inhibiting PDK1, RSK2, P70S6K, and AKT phosphorylation in cells and 3 to 50 fold more potent in viability assays. In vivo, GSK-2334470 had negligible activity compared to SNS- 229/-510 even at a much higher dose ● In 21-day MV4-11 xenografts, SNS-229/-510 showed dose-related efficacy with >95% tumor growth inhibition and partial regression (>50% tumor shrinkage) in 70% and 100% of animals at the highest dose (Fig 9) PDK1 inhibitors SNS-229 and SNS-510 cause pathway modulation, apoptosis and tumor regression in hematologic cancer models in addition to solid tumors Stig K. Hansen 1 , Johan Enquist 1 , Jeff Iwig 1 , Minke E. Binnerts 2 , Gene Jamieson 2 , Judy A. Fox 2 , Adam R. Craig 2 ; 1 Carmot Therapeutics, Inc,. San Francisco, CA. 2 Sunesis Pharmaceuticals, Inc., South San Francisco, CA FIG 9: TUMOR GROWTH INHIBTION AND PARTIAL REGRESSION IN MV4-11 XENOGRAFTS FIG 2: SNS-229/-510 ARE POTENT INHIBITORS OF PDK1 FIG 5: SNS-229/-510 INHIBIT HEMATOLOGIC CANCER CELL S RESISTANT TO AKT INHIBITION FIG 4: SNS-229/-510 PERTURB THE PIF-POCKET AND PIF BINDING FIG 8: STRONG PATHWAY MODULATION IN MV4-11 XENOGRAFT CONCLUSIONS FIG 7: GROWTH INHIBITION BY SNS-510/-229 CORRELATES WITH PATHWAY MODULATION FIG 1: PDK1 INTERACTS WITH SUBSTRATES THROUGH PH OR PIF MEDIATED MECHANISMS The PH domains of PDK1 and AKT bind PIP3 and co-localize PDK1 and AKT at the cell membrane The PDK Interacting Fragment (PIF) or Hydrophobic Motif (HF) binds to the PDK1 PIF pocket to stabilize PDK1 substrate interaction PH PIP 3 P-S241 PI3K PIP 3 RTK PH PH PH PIP 3 PIP 3 PIF-pocket P-T308 AKT P-S241 PH PIP 3 PTEN PI PDK1 PH RSK2 P-S227 PIF PH PH P-T308 PIF PDK1 P-S241 P-S241 mTOR AKT P-S473 PDK1 PIF- pocket PIF mediated substrate recognition: RSK, AKT-C-term, SGK, SP706K PH-domain mediated substrate recognition (PI dependent) GSK-2334470 2.4 nM 1.9 nM BX795 5.4 nM 6.6 nM SNS-510 3.9 nM 3.1 nM SNS-229 2.9 nM 1.9 nM 5 nM AKT, 0.225 nM (de-phos)/0.07 nM (phos) PDK1 full length, 100 nM FITC-crosstide, 2 hr kinase rxn, 30 min assay development compound SNS-229 SNS-510 IC 50 (nM) BX795 GSK-2334470 IC 50 (nM) P-S241-PDK1 3.9 2.9 5.4 2.4 3.1 1.9 6.6 1.9 PDK1 ● The selectivity of SNS-229 and SNS-510 was evaluated in a panel of 270 different human kinases at 10 mM (1,000-fold IC 50 ) and showed: ● SNS-229: >90% inhibition for 18 kinases including PDK1 ● SNS-510: >90% inhibition for 20 kinases including PDK1 ● Main off-targets: Abl, Lck, LIMK, Met, TrkA, TrkB (>95% inhibition at 10 mM) ● SNS-229/-510 inhibit both phos- and un-phos PDK1 and affect less than 8% of kinases at 1,000-fold IC 50 for PDK1 ● In the active (DFG-in) conformation of PDK1, the aB and aC helices form a binding pocket (indicated by a black circle in panel A) for the PDK Interacting Fragment (PIF) ● The conformation of the PIF pocket is preserved in the GSK and BX-320 structures (yellow and purple, A) ● SNS PDK1 inhibitors push out the aB and aC helices and thereby perturb the PIF binding pocket (teal, A) ● In a fluorescent PIF-binding assay, BX-795 (a close analog of BX-320) does not compete with PIF binding (B-C), consistent with the crystal structure of BX-320 ● SNS-991/-229/-510 inhibit PIF binding and block PIF- dependent substrate binding to PDK1 The binding mode of the SNS-series is exemplified by the crystal structure of PDK1 bound to SNS-991 (A), a close analog of SNS-229/-510. Conformations unique to SNS-991 are shown in teal. Key features of the structure include: ● SNS-991 binds to the inactive (DFG-out) conformation of PDK1 (A) ● SNS-991 occupies the binding pocket for Phe of the DFG-loop and thus prevents formation of the active (DFG-in) conformation as seen in the overlay with the ATP-bound structure (orange, B) ● Binding of SNS-991 changes the conformation of the aB and aC helices relative to the ATP-bound structure (B) E) SNS-991 DFG-loop A) aB aC SNS-991 Phe of DFG-loop B) aB aC ATP C) GSK aB aC D) aB aC BX-320 ● SNS-229/-510 inhibit growth of multiple cancer cell lines at concentrations ranging from 3 to 500 nM (14 out of 15 cell lines shown in panel A (MTS proliferation assay) ● SNS-229/-510 are active in cell lines that are resistant to PI3K inhibition (GDC-0941) or AKT inhibition (MK-2206) FIG 6: SNS-229/-510 INDUCE APOPTOSIS IN AKT INHIBITOR RESISTANT CELL LINES pRSK2 S227 GSK SNS-510 MK-2206 [nM] B) E) 9% pPDK1-Ser241, 4 hrs G1 G2 G3 G4 G5 G6 0 25 50 75 100 125 G1 Vehicle G2 SNS-229 1mg/kg G3 SNS-229 5mg/kg G4 SNS-229 25mg/kg G5 SNS-510 1mg/kg G6 SNS-510 5mg/kg G7 SNS-510 25mg/kg G8 GDC0941 50mg/kg G9 GSK2234470 50mg/kg treatment group % (treated/untreated) pRSK2-Ser227, 8 hrs G1 G2 G3 G4 G5 G6 0 25 50 75 100 125 G1 Vehicle G2 SNS-229 1mg/kg G3 SNS-229 5mg/kg G4 SNS-229 25mg/kg G5 SNS-510 1mg/kg G6 SNS-510 5mg/kg G7 SNS-510 25mg/kg G8 GDC0941 50mg/kg G9 GSK2234470 50mg/kg treatment group % (treated/untreated) 0.5 4 17 pPDK1-Ser241, 8 hrs G1 G2 G3 G4 G5 G6 G7 G8 G9 0 25 50 75 100 125 G1 Vehicle G2 SNS-229 1mg/kg G3 SNS-229 5mg/kg G4 SNS-229 25mg/kg G5 SNS-510 1mg/kg G6 SNS-510 5mg/kg G7 SNS-510 25mg/kg G8 GDC0941 50mg/kg G9 GSK2234470 50mg/kg treatment group % (treated/untreated) 0.5 3 10 pAkt-Thr308, 8 hrs G1 G2 G3 G4 G5 G6 G7 G8 G9 0 25 50 75 100 125 150 G1 Vehicle G2 SNS-229 1mg/kg G3 SNS-229 5mg/kg G4 SNS-229 25mg/kg G5 SNS-510 1mg/kg G6 SNS-510 5mg/kg G7 SNS-510 25mg/kg G8 GDC0941 50mg/kg G9 GSK2234470 50mg/kg treatment group % (treated/untreated) 0.5 3 10 A) B) D) C) 0.6 2.5 20 0.5 2 11 1.4 0.8 0.4 0.4 0.5 3 10 0.6 2.5 20 0.4 0.4 0.6 2.5 20 0.4 0.4 • SNS-229/-510 show superior pathway modulation in vivo and at lower doses than GSK-2334470 and GDC0941 A) MV4-11 median tumor volume day 1-22 B) Tumor volume distribution day 22 ● SNS-229/-510 induce a dose dependent reduction in tumor growth. Median tumor growth for all groups was delayed compared with that for controls (A), with the two highest dose groups achieving statistical significance on day 22 (B) ● SNS-229 treatment resulted in 69%, 80% and 96% tumor growth inhibition (TGI) at 5, 11, and 25 mg/kg, with 77%, 92% and 97% TGI observed for SNS-510 at 5, 11, and 25 mg/kg ● Maximum mean weight loss on day 22 was 4% ● Partial tumor regression was observed in 70% of animals treated with SNS-229 at 25 mg/kg, 33% of animals treated with SNS-510 at 11 mg/kg and 90% of animals treated with SNS-510 at 25 mg/kg ● Comparison with PD study (Fig. 8) suggests that >50% P-PDK1 and >90% P-RSK and P-AKT inhibition may be required for PRs SNS-229/-510 were dosed orally QD for 21 days at the doses of 5, 11, and 25 mg/kgStatistical Significance for Kruskal Wallis Dunn's test: ns = non- significant, * = 0.01 < P < 0.05, ** = 0.001 < P < 0.01, *** = P < 0.001 when compared to Group 1. TGI > 60% indicates potential therapeutic activity. PR: tumor volume less than 50% of its day 1 volume for three consecutive measurements PH P-S241 PIFtide + inhibitor + PH P-S241 PIFtide + inhibitor PH P-S241 PIFtide Measure bound/un-bound PIF-tide in FP-assay B) PIF-tide binding assay: A) B) EC 50 s in MTS proliferation assay ● SNS-510 is active in AKT inhibitor (MK-2206) resistant cell lines (MV4-11 above and panel G) pointing to RSK and or P70S6K as important for cell growth ● In AKT inhibitor (MK-2206) sensitive cell lines (H), SNS-510 show more robust growth inhibition probably by inhibition of RSK and P70S6K that are largely unaffected by MK-2206 ● SNS-510 is more potent in all assays and cell lines compared to GSK-2334470 ● Together, these data show that SNS-510 is more potent than GSK-2334470, is active in AKT inhibitor resistant cell lines and outperforms MK-2206 in AKT dependent cell lines B-F): MV4-11 Cells treated for 24 hrs with compound. Western blot analysis and quantification by LICOR A-D): MV4-11 tumor bearing mice received a single dose of compound and tumors were harvested at 4 or 8 hrs. Tumors were homogenized and analyzed by Western blot and LC-MS/MS for compound concentration, shown above each bar graph (mM) ● SNS-229/-510 show dose and time dependent inhibition of P-PDK1 (A-B) and strong suppression of RSK2 and AKT phosphorylation with up to 80-90% inhibition 8 hrs post dose (C-D) ● GSK-2334470 and GDC0941dosed at 50 mg/kg show moderate PDK1 and RSK2 inhibition and no AKT inhibition (B-D) ● The structures of PDK1 with GSK-2334470 (yellow, C) and BX-320 (purple, D) closely resemble the ATP-bound structure and do not show perturbation of aB and aC helices ● Top view of the N-terminal kinase domain with overlay of the three inhibitors (E): SNS-991 occupies the ATP pocket and reaches into the core of PDK1, whereas BX-320 and GSK- 2334470 occupy the ATP pocket and bind along the surface of the protein ● SNS-229/-510 induces apoptosis in MV4-11 cells (annexin positive), whereas treatment with GSK-2334470 does not produce significant apoptosis beyond vehicle (A) ● SNS-229/-510 also produce robust apoptosis in C1498 cells, but no effect of GSK-2334470 was observed even after 72 hrs (B) ● Thus, SNS-229/-510 inhibit growth and induce apoptosis in Akt inhibitor resistant cells (Fig. 5B) A) A-B): Apoptosis assay: 24 hrs compound treatment. Annexin V labeling analyzed by FACS B) ● Inhibition of PDK1 auto- and substrate-phosphorylation (A) was examined in several cell lines ● SNS-510 inhibits PDK1, RSK2 and pP70S6K phosphorylation in MV4-11 cells (B, D-F) with 10-fold better potency than GSK-2334470 ● No pAKT T308 signal (not shown) and no significant pathway modulation is observed with MK2206, indicating that Akt is not activated in MV4-11 cells ● Inhibition of PDK1 auto-phosphorylation by SNS-510 increases over time (4 to 24 hrs) while RSK2 inhibition is rapid (C) ● GSK-2334470 mediated inhibition of PDK1 auto-phosphorylation is only partial and reaches a plateau after 8 hours (C) pPDK1 S241 GSK SNS-510 MK-2206 [nM] C) G-H): 72 hrs MTS data. Western blot analysis (24 hrs) and quantification by LICOR. Color coding was performed within cell lines and separately for MTS and Western data. H) • PK analysis in CD/1 mice (E) shows that SNS-229/-510 have good pharmacokinetic properties, with t 1/2 of 4-5 hours, T max at 4-6 hours and oral bioavailability >90%. E) ● SNS-229/-510 are 5-10 fold more potent than the PDK1 inhibitor GSK2334470 (6 out of 7 cell lines, panel B) ● SNS-229/-510 show low nM potency in three cells lines that are resistant to AKT inhibitor MK-2206 A) pP70 S6K T229 GSK SNS-510 MK-2206 [nM] F) D) Cell line, conc. Assay SNS-510 GSK-2334470 MK-2206 U2932 MTS 34% 6% 18% DLBCL pPDK1 64% 64% 3% 300 nM pRSK 95% 88% 0% pP70S6K 90% 73% 36% pAKT 89% 57% 93% A20 MTS 83% 27% 74% BCL pPDK1 68% 44% 7% 300 nM pRSK 96% 67% 31% pP70S6K 67% 50% 32% pAKT 84% 24% 87% Cell line, conc. Assay SNS-510 GSK-2334470 MK-2206 RPMI-8226 MTS 54% 28% 5% MM pPDK1 78% 49% 30% 300 nM pRSK 91% 70% 3.5% pP70S6K 95% 69% 0% pAKT No signal No signal No signal C1498 MTS 93% 37% 33% AML pPDK1 71% 64% 0% 300 nM pRSK 94% 71% 0% pP70S6K 89% 49% 30% pAKT No signal No signal No signal G) Cell line/ mM cmpd SNS-229 SNS-510 MK-2206 GSK- 2334470 Molm-13 0.006 0.003 1.369 0.360 MV4-11 0.005 0.003 1.500 0.083 U-2932 1.603 0.559 5.076 3.422 RPMI- 8226 0.174 0.163 0.906 1.513 Jeko 0.286 0.279 0.980 2.250 A20 0.156 0.114 0.138 0.49 C1498 0.053 0.045 1.190 0.503 FIG 3: SNS -229/-510 BIND THE INACTIVE (DFG-OUT) CONFORMATION OF PDK1 ● Here we report novel PDK1 inhibitors with a PK/PD relationship that correlates with profound tumor growth inhibition in hematologic cancers. In particular, the compounds show ● Low single digit nM potency in kinase assay with good selectivity ● Binding to inactive PDK1 conformation and perturbation of PIF-substrate binding pocket ● Ability to drive PDK1 into un-phosphorylated, inactive state ● Growth inhibition in many cell lines at low-mid nM concentrations ● Induction of apoptosis at nM concentrations ● PDK1/RSK2/P70S6K/AKT pathway modulation correlating with growth inhibition and apoptosis ● Active in cells with no AKT activation and in cells that are resistant to AKT inhibitors ● Orally bioavailability with long half-life (potential for QD dosing) ● Strong pathway modulation in vivo after single oral dose ● Greater than 95% TGI and partial regression (>50% tumor shrinkage) when dosed PO QD with no significant weight loss or adverse clinical observations ● In summary, targeting the inactive conformation of PDK1 and inhibiting PIF-mediated substrate binding has broad potential for the treatment of cancer 0 10 20 30 40 50 60 70 80 90 100 % of vehicle SNS-510, 30 nM GSK2334470, 30 nM 0 20 40 60 80 100 120 DMSO BX-795 SNS-991 SNS-229 SNS-510 % PIFtide binding 20 nM PDK1, 5 nM FITC-PIFtide, 250 nM compound, 1 hr incubation Multiple of IC 50 : NA >40x >40x >40x >40x aB aC PIF binding pocket SNS-991 BX320 A) C) 1 10 100 1000 10000 0 4 8 12 16 20 24 Concentration (ng/mL) Time (hr) PO Average 0 100 200 300 0 10 20 30 40 vehicle doxorubicin SNS-229 SNS-510 GSK-2334470 [Drug], nM % annexinV positive 0 5 10 15 20 25 30 35 40 vehicle 30 nM Doxorubicin 1000 nM GSK-2334470 1000 nM SNS-229 % annexin V positive cells 24 hours 72 hours
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