Schirmer et al. 10.1073/pnas.0600445103. |
Supporting Table 4
Supporting Table 5
Supporting Figure 3
Supporting Figure 4
Supporting Table 6
Supporting Figure 5
Fig. 3.
Typical analysis of hypothemycin inactivation kinetics. (A) Progress curves of inactivation of 2.8 nM kinase insert domain-containing kinase (KDR) at various concentrations of inhibitor using 1 mM ATP and 1 mg/ml Poly(E4Y1). Data were fit to the appropriate equation as described in Materials and Methods to obtain kobsd. (B) Replot of kobsd vs. [I] determined a Kapp of 0.11 ± 0.02 mM and kinact of 0.14 ± 0.01 min–1.Fig. 4.
Inhibition of resorcylic acid lactone (RAL) target kinases in cell-based assays. Cell lines HT29 (A and E), MV4-11 (B), EOL-1 (C), P815 (D), A549 (F), and HeLa (G) were treated with either vehicle or hypothemycin in vehicle at indicated nanomolar concentrations for 1 h. (E and F) For induction of p38 (E) and c-Jun N-terminal kinase (JNK) (F) phosphorylation, cells were subsequently treated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) for 30 min. (G) For induction of IkB kinase b (IKKb) phosphorylation, cells were subsequently treated with 10 ng/ml IL-1 for 15 min. Phosphorylated kinase levels and total kinase levels were detected by Western blot analysis; levels of total kinases did not change. IC50 values were estimated by densitometry, and the doses needed to cause 50% growth inhibition (GI50) for hypothemycin with each of the cell lines are shown for comparison. Anti-p44/42 (extracellular signal-regulated kinase, ERK1/2), anti-PO4-p44/42 (ERK1/2), anti-PO4-MEK1/2 (MEK, mitogen-activated protein kinase kinase), anti-PO4-FLT3, anti-PO4-p38, anti-PO4-JNK (JNK, c-Jun N-terminal kinase), and anti-PO4-IKKb antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-MEK1, anti-FLT3, anti-PDGFRa (PDGFR, platelet-derived growth factor receptor), anti-PO4-PDGFRa, and anti-PO4-c-KIT antibodies were purchased from Santa Cruz Biotechnology. Anti-c-Kit antibody was from Calbiochem.Fig. 5.
Rapid and persistent effects of hypothemycin on PO4-ERK (ERK, extracellular signal-regulated kinase) levels in BRAF V600E mutant cell lines. (A) BRAF V600E mutant cell line COLO829 was treated with 1 mM hypothemycin for 2, 5, 10, 15, 30, and 60 min, followed by lysis, SDS/PAGE, and analysis of PO4-ERK levels by Western blot. (B and C) BRAF V600E mutant cell line HT29 (B) and KRAS mutant cell line A549 (C) were treated with 1 mM hypothemycin (H), 1 mM U0126 (U), or a DMSO control (D) for 1 h. Cells were then washed extensively with fresh media and incubated for a further 3-24 h, followed by lysis in radioimmunoprecipitation assay (RIPA) buffer, SDS/PAGE, and analysis of PO4-ERK levels by Western blot. RIPA buffer contained 50 mM Tris·HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 1% (wt/vol) Nonidet P-40, 0.1% (wt/vol) SDS, 12 mM sodium deoxycholate, 0.9 mM Na3VO4, and Roche Complete protease inhibitor mixture.Table 4. Screen of a 124 kinase panel for inhibition by hypothemycin
Kinase | % activity | Kinase | % activity | Kinase | % activity |
Abl(h) | 98 | Fms(h) | 91(90) | PKBg(h) | 102 |
Abl(T315I)(h) | 90 | Fyn(h) | 87 | PKCa(h) | 96 |
ALK(h) | 101 | GSK3a(h) | 90(88) | PKCßI(h) | 99 |
Arg(h) | 98 | GSK3ß(h) | 90(40) | PKCßII(h) | 97 |
ASK1(h) | 100 | Hck(h) | 78(91) | PKCg(h) | 98 |
Aurora-A(h) | 87(91) | IGF-1R(h) | 107 | PKCd(h) | 96 |
Axl(h) | 110 | IKKa(h) | 101 | PKCe(h) | 106 |
Bmx(h) | 99 | IKKß(h) | 81(94) | PKCh(h) | 97 |
BRK(h) | 89 | IR(h) | 100 | PKCi(h) | 108 |
BTK(h) | 104 | IRAK4(h) | 84 | PKCμ(h) | 34(6) |
CaMKIV(h) | 102 | JNK1a1(h) | 97 | PKCq(h) | 104 |
CDK1/cyclinB(h) | 89 | JNK2a2(h) | 93 | PKCz(h) | 94 |
CDK2/cyclinA(h) | 634 (106) | JNK3(h) | 76 | PKD2(h) | 31(-2) |
CDK2/cyclinE(h) | 105 | KDR | nt(15) | Plk3(h) | 101 |
CDK3/cyclinE(h) | 95 | Lck(h) | 98 | PRAK(h) | 20(1) |
CDK5/p35(h) | 103 | Lyn(h) | 82(83) | PRK2(h) | 108 |
CDK6/cyclinD3(h) | 95 | MAPK1(h) | 79(10) | Pyk2(h) | 84 |
CDK7/cyclinH/MAT1(h) | 101 | MAPK2(h) | 79(5) | Ret(h) | 104 |
CHK1(h) | 94 | MAPKAP-K2(h) | 98 | RIPK2(h) | 101 |
CHK2(h) | 97 | MAPKAP-K3(h) | 98 | ROCK-I(h) | 121 |
CK1d(h) | 105 | MEK1(h) | 54(8) | ROCK-II(h) | 104 |
CK2(h) | 99 | Met(h) | 96 | Ron(h) | 91 |
cKit(h) | 93(21) | MINK(h) | 88 | Ros(h) | 97 |
cKit(D816V)(h) | 57(0) | MKK6(h) | 43(8) | Rse(h) | 90 |
c-RAF(h) | 103 | MKK7ß(h) | 85(41) | Rsk1(h)* | 100 |
CSK(h) | 104 | MSK1(h) | 87 | Rsk2(h)* | 91 |
cSRC(h) | 75(49) | MSK2(h) | 101 | Rsk3(h)* | 94 |
DDR2(h) | 92 | MST2(h) | 94 | SAPK2a(h) | 107 |
EGFR(h) | 106(94) | NEK2(h) | 99 | SAPK2b(h) | 104 |
EphA2(h) | 87 | NEK6(h) | 104 | SAPK3(h) | 104 |
EphB2(h) | 118 | NEK7(h) | 98 | SAPK4(h) | 101 |
EphB4(h) | 82 | p70S6K(h) | 94 | SGK(h) | 99 |
ErbB4(h) | 124 | PAK2(h) | 104 | Syk(h) | 105 |
Fer(h) | 102 | PAK4(h) | 92 | TAK1(h) | 12(5) |
Fes(h) | 92 | PAR-1Ba(h) | 93 | Tie2(h) | 93 |
FGFR1(h) | 99 | PDGFRa(h) | 77(20) | TrkA(h) | 22(1) |
FGFR3(h) | 102 | PDGFRß(h) | 73(40) | TrkB(h) | 58(18) |
FGFR4(h) | 87 | PDK1(h) | 93 | Yes(h) | 99 |
Fgr(h) | 100 | Pim-1(h) | 100 | ZAP-70(h) | 111 |
Flt1(h) | 2(7) | PKA(h) | 108 | ZIPK(h) | 96 |
Flt3(h) | 6(3) | PKBa(h) | 153 |
|
|
Flt3 D835Y h | 4 2 | PKBß h | 91 |
|
|
The screen was performed by Upstate Biotechnology as described by their protocol using a 40-min assay. Underlined kinases contain the resorcylic acid lactone (RAL) target Cys residue. Percent activity is of control at 0.2 (2.0) µM hypothemycin.
*RSK assays measured activity of N-terminal kinase domains, which do not have the RAL-targeted Cys on the C-terminal kinase domains of these enzymes.
Table 5. Source, substrates, and ATP kinetic parameters of kinases tested
Kinase | Source | Substrate [concentration] | Km of ATP, M | k cat, sec–1 | k cat/Km,M–1·sec–1 |
MEK1 | Upstate | H2O [—] | 5.80E-06 | 0.28 | 4.83E+04 |
MEK2 | Upstate | H2O [—] | 2.30E-05 | 0.19 | 8.26E+04 |
ERK1 | Upstate | Erktide* [500 μM] | 1.56E-04 | 4.50 | 2.88E+04 |
ERK2 | Kosan | Erktide* [500 μM] | 1.06E-04 | 2.80 | 2.64E+04 |
PDGFR a | Upstate | Poly(E4:Y1) [5 mg/ml] | 4.74E-04 | 0.07 | 1.48E+02 |
PDGFR b | Upstate | Poly(E4:Y1) [5 mg/ml] | 4.82E-04 | 0.31 | 6.43E+02 |
FLT3 | Invitrogen | Poly(E4:Y1) [10 mg/ml] | 2.47E-04 | 0.63 | 2.55E+03 |
FLT1 (VEGFR1) | Invitrogen | Poly(E4:Y1) [5 mg/ml] | 9.00E-05 | 0.50 | 5.56E+03 |
KDR (VEGFR2) | Invitrogen | Poly(E4:Y1) [2 mg/ml] | 8.10E-05 | 4.80 | 5.93E+04 |
PKD1 | Upstate | MK pep. subs.† [100 μM] | 9.00E-05 | 0.78 | 8.67E+03 |
MAPKAP5 | Invitrogen | PRAK pep. subs.‡ [200 μM] | 1.90E-04 | 0.36 | 1.89E+03 |
TRKA | Invitrogen | Poly(E4:Y1) [2 mg/ml] | 1.00E-03 | 0.10 | 9.50E+01 |
TRKB | Invitrogen | Poly(E4:Y1) [2 mg/ml] | 2.25E-04 | 1.87 | 8.31E+03 |
SRC | Invitrogen | Poly(E4:Y1) [5 mg/ml] | 8.80E-05 | 0.32 | 3.64E+03 |
GSK3 a | Invitrogen | GSK3 peptide§ [200 μM] | 5.00E-05 | 0.60 | 1.20E+04 |
GSK3 b | Invitrogen | GSK3 peptide§ [200 μM] | 3.50E-05 | 0.82 | 2.34E+04 |
Kinases were analyzed using progress curve analysis from a continuous fluorometric assay. Km and kcat values are averages of two determinations; standard errors were 5-40% for Km and 2-20% for kcat. The significant ATPase activity associated with MEK1 and MEK2 was used for assays. —, not applicable; Upstate, Upstate Biotechnology; MEK, mitogen-activated protein kinase (MAPK) kinase; ERK, extracellular signal-regulated kinase; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor; GSK, glycogen synthase kinase.
*ATGPLSPGPFGRR (1)
†
KKLNRTLSVA (2)‡
KKLRRTLSVA (2)§
RRRAAEELDSRAGpSPQL (3)1. Prowse, C. N., Hagopian, J. C., Cobb, M. H., Ahn, N. G. & Lew, J. (2000) Biochemistry 39, 6258–6266.
2. Stokoe, D, Caudwell, B., Cohen, P. T. W. & Cohen, P. (1993). Biochem. J. 296, 843–849.
3. Leclerc, S., Garnier, M., Hoessel, R., Marko, D., Bibb, J. A., Snyder, G. L., Greengard, P., Biernat, J., Wu, Y. Z., Mandelkow, E. M., Eisenbrand, G. & Meijer, L. J. (2001) Biol. Chem. 276, 251–260.
Table 6. Sensitivity of BRAF mutant cancer cell lines to hypothemycin
Cell Line | Origin | BRAF status | IC50, μM | ||
Hypo | BAY43-9006 | SU11248 | |||
A549* | NSCLC | wt (KRAS mutant) | 6.0 | 5.5 | nt |
SKOV3* | Ovarian | wt (HER2 overexpression) | 7.0 | 5.5 | 6.7 |
HT29* | Colon | V600E | 0.10 | 4.7 | 4.2 |
DU4475 | Breast | V600E | 0.018 | 3.6 | 4.0 |
WM-266-4 | Melanoma | V600D | 0.066 | 5.4 | 8.2 |
COLO829 | Melanoma | V600E | 0.049 | 6.0 | 7.1 |
A375 | Melanoma | V600E | 0.048 | 4.3 | 5.4 |
UACC-903 | Melanoma | V600E | 0.078 | 5.3 | 5.2 |
LS411N | Colon | V600E | 0.32 | 4.7 | 4.2 |
nt, not tested.
*In NCI60 panel.