SU5416 is a Ligand of the AHR
To confirm that this molecule is a direct ligand of the AHR and not working through some other agonist, we performed competitive binding assays of the AHR using a radioligand. Photoaffinity experiments incubating 125IBr2N3DpD with the hepatic cytosolic fraction from C57BL/6J mice (AHRb isoform) were conducted as described in the Methods [21]. Increasing concentrations of SU5416, TCDD, BNF, and 1,2-Benzanthracene (a ligand of low receptor affinity) were added. As shown in figure 2C, SU5416 competitively displaced the radiolabel with efficacy similar to TCDD.
Figure 2. Induction of DRE-mediated transcription by SU5416 is AHR dependent. A. The AHR-mutant C35 cell line was transfected with the AHRb, lacZ gene and a 36DRE-Luc construct. Controls were transfected with the empty pSPORT vector plus the reporter constructs. After 24 h, the cells were treated with 3 mM SU5416 or 0.3% (v/v) DMSO, then incubated for 18 more h. Induction of AHR activity was determined by normalizing the luciferase activity to b-galactosidase activity. White bars: Empty vector. Grey bars: AHR. Error bars: SD; (n = 3). B. Induction of DRE-mediated transcription by SU5416 is ARNT dependent. The ARNT-deficient C4 cell line was transfected with the human ARNT or the pSPORT parent vector. These cells were also cotransfected, treated and assayed as in A. White bars: Empty vector. Grey Bars: ARNT. Error bars: SD; (n = 3). C. SU5416 is a ligand of the AHR. The hepatic cytosolic fraction from C57BL/6J mice was incubated with 1 nM of the radioligand 125BR2N3DpD, in the presence of increasing concentrations of competitor, SU5416, TCDD, BNF or 1,2-Benzanthracene. Ordinate: Specifically bound radioligand in the presence of competitor divided by specifically bound radioligand in the absence of competitor. Abscissa: The concentration of competing ligand, represented as log of molar concentration. Each data point represents the average of two determinations. Competitive binding to the C57BL/6J
cytosol produced the IC50 values of SU5416 = 2.1 nM, TCDD = 1.5 nM, BNF = 2.8 nM, and 1,2-Benzanthracene = 13.7 nM.

In utero Exposure to SU5416 Stimulates Closure of DV
We have previously shown that genetically altered mice that express only 10% of the AHR display a patent ductus venosus (DV) in the liver in nearly all cases [22]. We additionally identified that in utero activation of the receptor in the hypomorphs with TCDD successfully closed the DV [5]. To test the role of SU5416 as an in vivo ligand and its potential effect on embryology and vascular development, we performed timed matings of female AHRfxneo/+ mice to male AHRfxneo/fxneo mice. The pregnant dams were treated at embryonic day E18.5 with a single dose of SU5416 at 110 mg/kg, or an equivalent volume of the vehicle, corn oil. At 4 weeks of age, the pups were sacrificed, and DV status was examined by hepatic perfusion with trypan blue. As seen in Table 1, only 1 of 25 AHRfxneo/fxneo pups treated with corn oil possessed a closed DV. In the experimental group, 13 of 22 animals of this phenotype exposed to SU5416 had a closed DV.activity indicating loss of binding to the DRE, which is clearly in contrast to the long duration DRE-binding seen with TCDD. Of note, when we did titrate SU5416 doses as high as 10 mM, we did observe as much as 20% of TCDD response (1 nM) as far out as 96 hours (data not shown). This SU5416 data is similar to the known plasma half-life of 30 minutes, although VEGF-receptor inhibitor effects have been shown to last as much as 72 hours in culture [23]. We further analyzed whether the AHR antagonist CH223191 could inhibit the ability of SU5416 to activate the DRE in 101L-hepatoma cells. It has previously been shown that this antagonist inhibits TCDD but not some of the other ligands of the AHR including some polycyclic aromatic hydrocarbons. We first performed a titration of the AHR antagonist in culture with either 1 nM TCDD or 100 nM SU5416. As can be seen in figure S2B, the effects of TCDD are inhibited whereas minimal inhibition is shown for SU5416. In figure S2C, we show a titration of SU5416 with only a small amount of inhibition of activity by the antagonist (10 mM).

SU5416-induced Upregulation of CYP1A1 is Similar in Murine AHRb and AHRd Splenocytes
As the above in vitro experiments were performed in cell lines, we next utilized AHRb (C57BL/6J) and AHRd congenic mice (on a C57BL/6J background). Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of TCDD and SU5416. These data are presented in figure 4, where the graphs show normalized data from 0 to 100% response. Normalized data was chosen to allow comparison of CYP1A1 upregulation to its maximum in AHRb versus AHRd mice. After 4 hours of culture, TCDD induced CYP1A1 more rapidly and to a higher degree in wild-type than AHRd splenocytes, with an EC50 of 0.461 nM in wild-type and 1.894 nM in AHRd animals. Figure 4B shows that SU5416 induced CYP1a1 similarly in AHRb and AHRd mice, with an EC50 of 0.682 nM in wild-type and 0.730 nM in AHRd mice. Figures S3A and B show the total fold change seen by qPCR analysis of splenocytes after exposure to TCDD and SU5416, to allow an assessment of the potency of AHR activation of these two ligands with CYP1A1 induction as the readout. As can be seen in the figure, TCDD elicits more CYP1A1 in AHRb compared to AHRd mice, whereas SU5416 leads to the same or more CYP1A1 in AHRd mice. By this readout, TCDD and SU5416 have similar potency in AHRd cells, and TCDD is a stronger ligand in AHRb cells.

SU5416 Upregulates CYP1A1 and CYP1B1
The above data clearly shows that SU5416 is a ligand of the AHR. We now focused our attention on the strong response of SU5416 to the AHRd polymorphism in the screening assay, and compared the activity of this ligand in the high and low affinity polymorphisms. We utilized the wild type rat hepatoma cell line, 5L, which harbors the high affinity AHR isoform, and our newly created AHRd-15 cell line. As seen in figure 3A, we first performed a titration with TCDD and measured EROD activity. As expected, the activity of TCDD was shifted by 1.5 orders of magnitude to the left for the AHRb isoform. In contrast, when SU5416 was tested in vitro, the two curves virtually overlapped (figure 3B), showing equal potency for cytochrome P450 induction using the two cell lines. We also tested BNF, which as expected, showed a strong response with the 5L cell line and no response with the AHRd-15 cell line (figure 3C). As these experiments were done in cell lines, and in addition the AHRd-15 line combines a rat cell line with a transfected murine AHR, we further tested the ability of SU5416 to activate the AHR in vivo. Six-week old C57BL/6J mice (AHRb) and DBA/2J (AHRd) were orally administered 30, 80, or 120 mg of SU5416 per kg of body weight.

Phosphorylations were also attempted with di-tert-butyl or dicyanoethyl phosphoramidites to produce di-tert-butyl or dicyanoethyl instead of dibenzyl phosphate. Neither of these phosphates was stable on silica gel, and b-elimination products were obtained after chromatography. TFA deprotection of crude di-tert-butyl phosphate, and NH4OH deprotection of crude dicyanoethyl phosphate both gave b-elimination products as well. Thus, the dibenzylphosphate was chosen to carry through to the final products 1 and rac-2. Hydrogenation of the crude dibenzyl phosphate (1S,3R,4R)13 went very slowly, giving a complex crude mixture. Thus, (1S,3R,4R)-13 was purified by reverse-phase semi-preparative high performance liquid chromatography (HPLC). With pure dibenzyl phosphate, hydrogenation at atmospheric pressure worked very well, and gave a very clean final product 1, similar to our experience with a-ketoamides [14].X-ray crystallography
During the synthesis of the inhibitors, Michael addition of tristhiomethyl methide to an a,b-unsaturated ketone 8 produced three stereoisomers of 9, which could not be readily separated (Figure 3). Two diastereomers of a subsequent synthetic intermediate, (1S,3R,4R)-11 and rac-11, were separated by chromatography. Each diastereomer was crystallized, and the relative stereochemistry was determined.

Figure 4. X-ray crystal structures of intermediates (1S,3R,4R)-11 and rac-11 are shown above as displacement ellipsoid drawings (50%). The positional disorder of the benzyl group in rac11 is shown as lighter lines. Hydrogen atoms are omitted for clarity. Structural depiction of the stereochemistries of (1S,3R,4R)-11 and rac11 are shown below each crystal structure. ?after geometry optimization was 3.16 A; with the trans-pyrrolidine torsion angle fixed during geometry optimization, the distance was ?3.67 A (Figure 6).Discussion Stereochemical results of inhibitor synthesisFigure 3. Cyclohexyl ketone inhibitor 1 was synthesized by the method shown. Thermodynamic control in the Michael addition resulted in the anti-Ser-trans-cyclohexyl stereoisomer of 9 as the major product (Figure 4). The chiral center adjacent to the Ser carbonyl was easily epimerized due to the electron-withdrawing effects of both the a-amide and a-ketone, resulting in an enantiomeric mixture of a second diastereomer, rac-9. Because the unnatural D-Thr-the original Ser configuration intact (Figure 4). The minor isomer, rac-11, proved to be a racemic mixture. The absolute configurations were assigned as (1R,3R,4R)-11 and (1S,3S,4S)-11, in which the stereocenter of the Ser analogue was partially epimerized to the syn-Ser-trans-cyclohexyl configuration (Figure 4).Pin1 PPIase Enzyme Assays
The a-chymotrypsin protease-coupled assay was used to evaluate inhibition of Pin1 by compounds 1 and rac-2 with the same substrate concentration as described previously [10,14]. The IC50 values of the two diastereomers were determined to be 260630 mM for 1, and 6168 mM for rac-2. Preincubation with Pin1 for 15 minutes did not result in improved inhibition.

Molecular modeling
Each of the three cyclohexyl ketone inhibitors was docked flexibly, with geometry minimization, into the Pin1 active site. The resulting docked stereoisomers, (1S,3R,4R)-1, (1R,3R,4R)-2, and (1S,3S,4S)-2, are shown in Figure 5. The total energies, Cys113?S–C = O ketone distances, and angles are reported in Table 1. Figure 5. Models of cyclohexyl ketone inhibitors were docked with dynamic minimization. (A) (1S,3R,4R)-1 in orange, (B) (1R,3R,4R)-2 in blue, (C) (1S,3S,4S)-2 in green, and (D) superposition of all atoms of 1 and rac-2. Models were based on PDB 2Q5A [32], and minimized using Sybyl 8.1.1 [42]. Images were prepared using MacPyMol [44]. Figure 6. Pin1 is proposed to stretch the prolyl ring by binding phosphate and C-terminal residues tightly, creating a transpyrrolidine conformation of the substrate and forcing pyramidalization of the prolyl nitrogen in the twisted-amide mechanism. Distance measurements are from calculated structures of AcroH in the ground state and the trans-pyrrolidine transition state.
containing inhibitors were more potent than the L-Thr in work by Zhang et al [32], both diastereomers 1 and rac-2 were tested for Pin1 inhibition. Inhibitor 1, corresponding to the native L-Ser-LPro stereochemistry of Pin1 substrates, had an IC50 value of 260 mM, while rac-2, an enantiomeric mixture of D-Ser-L-Pro and L-Ser-D-Pro analogues, had an IC50 value of 61 mM. Preincubation did not result in improved inhibition, suggesting that they are not slow-binding inhibitors. We obtained a crystal structure of the similarly substituted, reduced amide inhibitor 4 bound in the Pin1 active site, suggesting that the ketones also bind in the active site [27].

conformation, and the trans-pyrrolidine AcroH conformation ?was 0.51 A (Figure 6). This effect of stretching the ring conformation may provide insight into the mechanism of Pin1. In either of the proposed mechanisms: (1) nucleophilic-addition [26], or (2) twisted-amide [25], the nitrogen of the prolyl ring must become pyramidalized and deconjugated from the carbonyl in the transition state [22,24,25]. If binding of substrate to the catalytic site forces the Pro ring into a trans-pyrrolidine conformation, the nitrogen lone pair and the carbonyl p-bond would no longer be conjugated (Figure 6).

Cell expansion/viability measurement
To evaluate the impact of unique medications, cell advancement was calculated using the alamarBlue staining process as encouraged by the company (Biosource). The take a look at incorporates a fluorescent oxidation-reduction indicator. Fluorescence depth is proportional to cellular metabolic reduction. Experiments had been completed by incubating 3000 cells/well in triplicate at Day in 96 effectively plates. AlamarBlue was calculated at Day five by incubating one/ ten volume of alamarBlue solution for 2 h at 37uC and read at 595 nm (FLUOstar Optima, BMG Labtech).

Cells had been taken care of according to the manufacturer’s suggestions (BD Pharmingen, BrdU (Bromodeoxyuridin) Move Package). FITC conjugated BrdU antibody was incubated for 20 min at area temperature. Cells have been washed and resuspended in a buffer containing seven-AAD. Cell cycle distribution was analyzed with a FACScalibur circulation-cytometer (BectonDickinson). Analysis was carried out employing the BD CellQuest move cytometry investigation software program.

Mice treatment by TMI-1
MMTV-ERRB2/neu mice were handled intraperitoneally with TMI-one (a hundred mg/kg/d) or with car or truck (.nine% NaCl, .5% methylcellulose, two% Tween 80) for a period of 30 days. Injections started out with the detection of the tumor. Tumor volume was calculated employing the formulation V = .52 (L6W2). Tumor volumes were blended when mice created multiple tumors. Mice had been weighted everyday. Following completion of the analysis, autopsy of mice was performed.

Measurement of apoptosis in mouse tumors Investigation of caspase activity
Caspase pursuits were being determined working with commercially available kits. For the measurement of caspase-three/seven action, 105 cells were incubated in triplicate in 96 plates. TMI-1 at indicated concentrations was incubated for 24 h at 37uC. Action was identified utilizing luminescence Caspase-Glo Assays (Promega) in accordance to the manufacturer’s instruction. For the measurement of caspase-eight and caspase-nine, 106 cells/mL were being incubated with TMI-one at indicated concentrations for 48 h at 37uC. One particular mL of Purple-IETD-FMK (caspase-eight test) or Pink-LEHD-FMK (caspase-nine exam) was added for one h at 37uC in accordance to the manufacturer’s tips (Biovision). Cells ended up washed then analyzed with the LSRII movement cytometer. DNA fragmentation in apoptotic cells was analyzed by the TUNEL assay (ApopTag detection kit, (Millipore)) as advised by the producer. Briefly, four mM sections of paraffinembedded set tissue were deparaffined with successive histolemon and ethanol washes, then handled with twenty mg/mL proteinase K for 15 min at RT. Endogenous peroxidase were quenched with 3% hydrogen peroxide. Digoxigenin-dNTPs have been enzymatically additional to the totally free 39OH DNA termini by terminal deoxynucleotidyl transferase (TdT) and discovered by the peroxidase antidigoxigenin antibody. Coloration was performed working with the diaminobenzidine combined substrate (Dako). Counterstaining was carried out with a answer of one% methyl eco-friendly for 5 min at RT. Soon after distilled h2o and N-butanol washes, specimens had been mounted in Pertex medium (CellPath). Observations have been carried out using the Leica DMD108 electronic microimaging product (Leica Microsystems GmbH).

Measurement of ROS output
Intracellular era of ROS was measured making use of dihydroethidium. Cells (106) were handled with TMI-one or CCCP at indicated concentrations for 48 h. Cells have been trypsinized then stained with 5 mM dihydroethidium for thirty min at 37uC. Ethidium fluorescence depth resulting from dihydroethidium oxidation was calculated working with the LSRII move cytometer.

Drug mix analyses
Mix in between TMI-one and docetaxel, doxorubicin and lapatinib was analyzed by combining drugs at consistent molar ratio. Focus assortment for every drug was identified from productive dose (ED) fifty values calculated working with nonlinear regression analysis. Cells were addressed with serial dilutions of drug by yourself or in mix at consistent molar ratio. Benefits were being analyzed utilizing the Chou and Talalay approach that integrates ED50 and the form of the dose-effect curve (Calcusyn software program, Biosoft) [11]. The Blend Index (CI) was calculated to appraise involving