Article Figures & Data
Figures
- Figure 1.
Interactions of compound 1 with the human AKT1 PH domain. A, schematic representation of the interaction of compound 1 (NSC 348900, 4-[1-(4-chlorobenzoyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-ylazo]-N-pyrimidine-2-yl-benzenesulfonamide, C21H16ClN7O4S) with amino acid residues of the AKT1 PH domain (Arg86, Asn53, Arg23, and Ile19). B, the docking pose of compound 1 with the AKT1 PH domain. The AKT1 PH domain (red); residues Arg23, Arg25, and Arg86 are colored according to atom type. Compound 1 is colored according to atom type (capped stick). Hydrogen bonding interactions (dotted lines). The sulfamido group interacts with Arg86 through a hydrogen bonding interaction whereas similar hydrogen bonds are involved in the interaction of the diazopyrazolyl group with Arg23. These two arginines are intimately involved in the interaction with the phosphate head groups of the substrate phosphoinositol-1,3,4,5-tetrakisphosphate. C and D, the binding mode of compound 1 in the binding pocket of the PH domain of PDK1 with the amino acids involved in the binding pocket (D). Note that compound 1 exhibits the reverse binding pose in the PDK PH domain similar to compound 2 in the PH domain of AKT1. Supplementary Fig. 1A and B show the binding mode of compound 1 in the binding pocket of the PH domain of IRS1.
- Figure 2.
Synthesis of compounds 1 to 6.
- Figure 3.
Binding of the compounds (1–2) to the PH domain of AKT1 and IRS1. The proteins were immobilized on a CM5 Sensorchip as described in Materials and Methods. The drugs were injected over the surface at the indicated concentrations and binding to the proteins was measured by surface plasmon resonance. A, the binding curves of compound 1 (left) and compound 2 (right) to the PH domain of AKT. B, the binding curves of compound 1 (left) and compound 2 (right) binding to the PH domain of IRS1. Overlay plot of typical sensorgrams obtained with increasing concentrations of compound 1 or 2 (arrows; A).
- Figure 4.
ELISA competitive binding assay. Competition binding curves for compounds 1 (A) and 2 (B), respectively, to the (□) AKT PH domain and (•) IRS1 PH domain. Points, mean of three determinations; bars, SD (**, P < 0.05). Compound 1 binds the PH domains of AKT1 and IRS1 in a similar manner. Compound 2 binds only the PH domain of AKT1.
- Figure 5.
Inhibition of AKT in cancer cells. A, HT-29 colon cancer cells were treated with compounds 1 to 6, at 20 μmol/L for 2 h and stimulated with 50 ng/nL of epidermal growth factor for 30 min. Akt activity was measured by Western blotting using anti–phospho-Ser437 AKT antibody. All other downstream targets of AKT were also detected by Western blotting using specific anti–phospho-antibodies. β-Actin was used as a loading control. Note that compounds 2 and 6 inhibit AKT phosphorylation and downstream GSK3 phosphorylation. B, apoptosis measured as described in Materials and Methods. Note that both compounds 2 and 6 at 20 μmol/L induce significant apoptosis significantly as compared with controls. Columns, mean of three determinations; bars, SD (**, P < 0.05 and ***, P < 0.001). Compound 1 (C) or its analogue compound 2 (D) were also tested at the concentrations shown for 2 h, and in HT-29 cells, stimulated with 50 ng/nL of epidermal growth factor for 30 min. Akt activity was measured by Western blotting using anti–phospho-Ser437 AKT antibody, PDK activity by anti–phospho-Ser241 PDK antibody as well as downstream target PKC using pan–phospho-PKC antibodies. β-Actin was used as a loading control.
- Figure 6.
In vivo activity of compound 1. A, pharmacokinetics of compound 1 in mice. Female scid mice were administered compound 1 at a dose of 250 mg/kg either (•) i.p. or (○) p.o. by oral gavage and plasma concentrations measured. Points, means of three mice; bars, SE. B, antitumor activity in female scid mice with HT-29 colon cancer xenografts treated p.o. daily for 5 days (arrows) with (•) vehicle alone or (□) compound 1 at 250 mg/kg daily. Points, means of 10 mice; bars, SE. C. Effect on tumor phospho-AKT in female scid mice with HT-29 colon cancer xenografts treated p.o. with (open columns) vehicle alone or (filled columns) compound 1 at 250 mg/kg. Tumors were removed at various times for Western blotting. Columns, mean of four mice; bars, SE (*, P < 0.05; **, P < 0.01).
Tables
- Table 1.
Structures and calculated properties of lead compound 1 (NSC 348900) and analogues (2–6)
Compounds AKT1 FlexX score AKT1 G score AKT1 cKD(μmol/L) PDK1 FlexX score PDK1 G score PDK1 cKD(μmol/L) IRS1 FlexX score IRS1 G score IRS1 cKD(μmol/L) 
NS NS 4.0 NS NS NS NS NS NS 1 −31.0 −96.9 1.2 −17.4 −109.0 1.74 −16.0 −128.0 1.99 2 −29.6 −31.9 2.4 −17.0 −40.0 2.60 −17.1 −96.2 2.40 3 −28.2 −99.5 1.2 −17.1 −103.4 1.70 −14.8 −79.7 10.70 4 −29.1 −71.9 3.0 −17.5 −88.6 2.20 −17.9 −145.5 1.80 5 −33.0 −120.6 1.3 −20.1 −137.1 2.40 −14.6 −90.1 10.70 6 −24.3 −132.0 0.85 −21.0 −109.1 1.45 −14.5 −140.6 0.52 Abbreviation: NS, not shown.
- Table 2.
Sequence alignments of the PH domain of AKT
Alignment of human AKT PH domain isoforms AA numbers 14 20 23 25 27 52 55 AKT1 PH MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVA 58 AKT2 PH MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVA 58 AKT3 PH MSDVTIVKEGWVQKRGEYIKNWRPRYFLLKTDGSFIGYKEKPQDVDLP-YPLNNFSVA 57 AA numbers 71 74 76 AKT1 PH QCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGL 110 AKT2 PH QCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGL 110 AKT3 PH KCQLMKTERPKPNTFIIRCLQWTTVIERTFHVDTPEEREEWTEAIQAVADRL 109 Alignment of human (h) and mouse (m) AKT PH domains AA numbers 14 20 23 25 27 52 55 hAKT PH MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQC mAKT PH MNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQRESPLNNFSVAQC AA numbers 71 74 76 hAKT PH QLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGL 110 mAKT PH QLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQTVADGL 110 The PH domain binding residues are shown underlined in the sequence alignment above obtained using CLUSTAL W (1.82) multiple sequence alignment.
- Table 3.
Selectivity for other PH domains
Compounds AKT1 PH mKD (μmol/L) IRS1 PH mKD (μmol/L) PDK1 PH mKD (μmol/L) PtdIns(3,4,5)P3 3.08 ± 0.49 ND ND DPIEL 5.04 ± 0.48 31.56 ± 8.49 NB 1 0.37 ± 0.04 0.39 ± 0.01 31.28 ± 9.54 2 3.66 ± 0.03 NB 0.17 ± 0.10 3 1.37 ± 0.25 NB 3.57 ± 0.96 4 0.51 ± 0.06 0.14 ± 0.02 NB 5 1.35 ± 0.02 1.74 ± 0.41 0.42 ± 0.17 6 1.62 ± 0.02 NB 0.98 ± 0.48 NOTE: Binding affinities were measured by surface plasmon resonance spectroscopy as described in Materials and Methods and are referred to as mKD (μmol/L) for measured.
Abbreviations: NB, no measurable binding; ND, not determined.
- Table 4.
Biological properties of compound 1 and analogues
Compound AKT inhibition (IC50 μmol/L) Cytotoxicity (IC50 μmol/L) Log P Metabolic half-life (min) Solubility (μmol/L) Permeability (nm/s) NIH3T3 HT-29 Caco2 MDCK 1 4 13 24 2.1 62 17.9 90 91 2 11 20 14 1.9 62 28.3 83 34 3 >20 >20 25 3.2 91 28.6 95 39 4 ND >20 NI 1.2 >480 12.9 23 8 5 5 >20 NI 1.5 138 13.1 185 200 6 3 5 ND 1.9 ND <0.1 14 5 Abbreviations: NI, not inhibitory for IC50 >100 μmol/L; ND, not determined.
NOTE: For each of the analogues, phospho-Ser473 AKT inhibition was measured in either mouse NIH3T3 or human HT-29 colon cancer cells. Cytotoxicity was measured in HT-29 cells. Metabolic stability was measured by incubating with HT-29 cells at the maximum concentration in DMEM at 37°C. The apparent permeability (nm/s) in Caco-2 and MDCK cells was obtained using the QikProp software (Schrodinger Inc.). Values ranged between <25 for poor and >500 for great permeability.
Volume 7, Issue 9
