Following the identification of 4a as a functional compound, derivatives in which the (${}_D$)-naphthylalanine moiety was replaced with other aromatic (${}_D$)-amino acids (tryptophan, phenylalanine and tyrosine) were envisaged. As a result, a set of four compounds (Fig. 1B) were synthesized (see Supplementary material) based on 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide, with a view to examining their structure-activity relationship.

Prior to testing of the biological potential of the synthesised compounds in cells, the structure and purity of the compounds were confirmed. Purification was achieved via column chromatography using Merck 200-300 mesh silica gel, and for thin layer chromatography (TLC) Merck 60 mesh size pre-coated aluminium plates were used. Visualization of TLC bands was achieved using a UV lamp at 254 nm. NMR spectra were obtained using a Jeol JNM ECP400 spectrometer (Welwyn Garden City, UK). ES-MS data were collected on HCT ultra ETD II mass spectrometer (KRSS Europe BV, Veenendaal, Netherlands) and melting points were recorded using a Fisher-Johns apparatus in open capillary tubes. CHN combustion elemental microanalyses were performed using a Carlo-Erba EA1108 CHN Analyzer (Fisons, Loughborough, UK).

MDA-MB-231 breast cancer cell lines (ATCC, Teddington, UK) were cultured in DMEM containing 10% (v/v) FCS. Human dermal blood microvascular endothelial cells (HDBEC; PromoCell, Heidelberg, Germany), devoid of endogenous TF were cultured in MV media containing 5% (v/v) foetal calf serum (FCS) and growth supplements (PromoCell). Cells (5 $\times$ 10${}^4$) were seeded out into 48-well plates and treated with the set of inhibitor compounds (100 $\mu$M) or the DMSO vehicle for 24 h. Cell numbers were determined by staining with crystal violet as previously described [37, 38] and calculated from a standard curve. In addition, cellular apoptosis was quantified using the TiterTACS™ Colorimetric Apoptosis Detection Kit (AMS Biotechnology, Abingdon, UK) according to the manufacturer’s instructions [26, 39].

We previously showed that active Pin1 was capable of binding a synthetic phosphorylated substrate peptide corresponding to the last 18 amino acids of the cytoplasmic domain of TF [4]. A biotinylated form of the phosphorylated peptide (biotin-RKAGVGQSWKENpSPLNVS) was synthesised (Biomatik, Ontario, Canada) and used here as a means of detecting Pin1 binding activity in vitro. An additional scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) was also included alongside as control. The measurements were carried out as outlined previously [4]. Briefly, the peptides (5 $\mu$M final concentration) were diluted to 100 $\mu$L with PBS and distributed (50 $\mu$L per well) in a NeutrAvidin-coated 96-well plate (Thermo Scientific, Warrington, UK) and incubated for 2 h at room temperature to allow binding. The wells were washed four times, each time with 300 $\mu$L of PBST. Sets of wells were supplemented with a range of synthetic inhibitor compounds (0–100 $\mu$M) or DMSO vehicle. HRP-conjugated recombinant Pin1 protein was then diluted 1:500 (v/v) in PBST, added to the wells (100 $\mu$L) and incubated for 1 h at room temperature. The wells were then washed a further four times and developed with TMB One Solution (100 $\mu$L; Promega Corporation, Southampton, UK). Once the colour was developed the reactions were stopped by the addition of 2M sulphuric acid (50 $\mu$L) and absorptions measured at 450 nm using a plate-reader. The concentrations of Pin1 were determined from a standard curve made using HRP-conjugated recombinant Pin1 protein.

The isomerase activity of Pin1 was also examined using a previously described procedure [4, 40]. Pin1 was incubated with penta-peptides, Succ-ENSPL-pNitroanilide and Succ-ENpSPL-pNitroanilide and alterations in spectroscopic absorption of the solution analysed. Briefly, samples (100 $\mu$L) of the substrate peptides (0.5 $\mu$M) were in turn placed in a microcuvette. Recombinant HRP-conjugated Pin1 (10 nM final concentration) was then pre-incubated (5 min) with the inhibitors and then added to the cuvettes. The change in absorption at 315 nm over time was then immediately monitored.

Cell surface TF-fVIIa activity was measured by modification of previously described procedures [4]. MDA-MB-231 cells (5 $\times$ 10${}^4$) were incubated with the compound inhibitors (100 $\mu$M) diluted in the reaction buffer (100 $\mu$L; HEPES-buffered saline (HBS) pH 7.4, containing 1% (w/v) bovine serum albumin (BSA) and 5 mM CaCl${}_2$) for 60 min. The cells were washed and incubated with fVIIa (20 nM; Enzyme Research Labs, Swansea, UK) in the reaction buffer for an additional 10 min and then supplemented with fX (100 nM), together with fXa substrate (0.2 mM; Hyphen) diluted in the same buffer (100 $\mu$L). The samples were incubated for 60 min to develop the colour. Aliquots (150 $\mu$L) were then transferred to a 96-well plate containing 2% (v/v) acetic acid (50 $\mu$L) and the absorptions measured immediately at 405 nm. The amount of fXa generated was determined using a standard curve prepared using fXa (Enzyme Research Labs, Swansea, UK). To confirm the cell surface TF activity, cells were pre-incubated with the inhibitory anti-TF antibody HTF1 (40 $\mu$g/mL; eBioscience/Thermo Scientific, Warrington, UK) prior to the addition of fVIIa.

MDA-MB-231 cells (5 $\times$ 10${}^4$) were seeded out into 48-well plates and incubated with the inhibitor compounds as above (100 $\mu$M) for 18 h. The cells were then washed with PBS and fixed with 3% (v/v) formaldehyde. The cells were incubated with an HRP-conjugated sheep anti-human TF antibody (100 $\mu$L, Enzyme Research Labs., Swansea, UK) diluted 1:1000 (v/v) in PBS for 1 h. The cells were then washed four times with PBS and developed using TMB One Solution substrate Solution (100 $\mu$L). Once the colour was developed the reactions were stopped by the addition of 2M sulphuric acid (50 $\mu$L) and absorptions measured at 450 nm using a plate-reader. Microvesicle-associated TF antigen was measured using the Quantikine TF-ELISA kit (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions.

Cells were incubated overnight with the compound inhibitors as above. The cells were then lysed in Laemmli’s buffer containing a protease inhibitor cocktail (Sigma Chemical Company, Poole, UK) and equal amounts were separated by 12% (w/v) SDS-PAGE. The protein bands were transferred onto nitrocellulose membranes and blocked with TBST (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20). The membranes were then probed with a polyclonal rabbit anti-human p53 antibody (Cell Signalling Technologies/New England Biolabs, Hitchin, UK) or a mouse monoclonal anti-human Bax antibody (202; Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1:3000 (v/v) in TBST. The membranes were then washed with TBST and probed with a goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1000 (v/v) and incubated for 90 min. The bands were then visualised using the Western Blue stabilised alkaline phosphatase-substrate (Promega Corporation Ltd, Southampton, UK) and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:2000 (v/v).

Total RNA was isolated using the TRI-reagent system (Sigma Chemical Company, Poole, UK) from 2 $\times$ 10${}^5$ cells and 100 ng of total RNA was used for each reaction. The relative amounts of TF or bax mRNA were determined using QuantiTect primer sets to detect either TF or bax, in conjunction with $\beta$-actin (Qiagen, Manchester, UK). The reaction was carried out at an annealing temperature of 60 ${}^{\circ }$C for 1 min using the GoTaq® 1-Step RT-qPCR System (Promega Corporation Ltd, Southampton, UK) on an iCycler thermal cycler (Bio-Rad, Hemel Hempstead, UK) for 40 cycles. Following amplification, the amounts of mRNA were determined using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }CT}$ method and ratios were calculated [41].

MDA-MB-231 cells (10${}^3$) were seeded out into 35-mm glass-bottom with 10 mm $\mu$-well dishes and incubated for 4 h at 37 ${}^{\circ }$C. The medium was aspirated and replaced with 100 $\mu$L of medium supplemented with test reagents (100 $\mu$M). Sets of cells were treated with of TNF-$\alpha$ (10 ng/mL) or DMSO and were used as positive and negative controls, respectively. The media were removed after 18 h and the cells were washed twice with PBS (200 $\mu$L) and fixed using 4% (v/v) paraformaldehyde. After three further washes with PBS, the cells were permeabilised with 0.2% (v/v) Triton X-100 diluted in PBS and incubated at room temperature for a further 10 min. The samples were then blocked for 1 h with PBS containing 3% (w/v) bovine serum albumin (BSA). The cells were then washed a further three times and probed with a rabbit polyclonal anti-human p53 antibody diluted 1:250 (v/v) in PBS/BSA buffer (100 $\mu$L) and incubated overnight, at 4 ${}^{\circ }$C. After a further three washes with PBS, the samples were incubated for 1 h with a Northern Lights-637 donkey anti-rabbit antibody (R&D Systems, Abingdon, UK) diluted 1:100 (v/v) in PBS/BSA buffer (100 $\mu$L), in the dark. The cells were then washed another two times with PBS and stained with DAPI (2 $\mu$g/mL). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a $\times$40 magnification (Carl Zeiss Ltd, Welwyn Garden City, UK). The localisation of p53 within the nuclei were determined using ImageJ program (version 1.48v, LOCI, University of Wisconsin, Madison, WI, USA), in 10 fields of view from each assay and Mander’s coefficient determined [42, 43].

The structures of the four compounds were constructed using the Alchemy program (Tripos Associates Inc., St Louis, USA) and then saved in Brookhaven format (PDB). The crystal structures of Pin1 (1PIN) was obtained from Brookhaven format (PDB). The location and efficiency of binding of the compounds to Pin1 was estimated using Autodock 4v2.6 [44]. The Autodock graphical interface AutoDockTools 1.5.6 was used, the polar hydrogens were retained and partial charges added to the proteins using the Gasteiger charges. The search space was limited to an area of 20 $\times$ 20 $\times$ 20 Å, centred around the hydroxyl group of Ser18 in the enzymatic site of Pin1. For each enzyme, 25 $\times$ ligand orientations (poses) were examined and ranked according to the scoring-function and inhibition coefficient calculated.

All data represent the calculated mean values from the number of experiments stated in each figure legend $\pm$ the calculated standard error of the mean. Statistical analysis was carried out using the Statistical Package for the Social Sciences v21 (SPSS Inc. Chicago, IL, USA). Significance was determined using one-way ANOVA (analysis of variance) and Tukey’s honesty significance test or where appropriate, by paired t-test.

To determine the inhibitory potential of the synthesised compounds, the ability of the compounds to prevent the substrate binding and isomerase activities of Pin1 was examined. The enzyme used was recombinant HRP-conjugated Pin1 and the target substrate peptide was biotin-RKAGVGQSWKENpSPLNVS (from the cytoplasmic domain of TF) which was described and confirmed previously as a suitable target for Pin1 binding [4]. Inclusion of ${}_D$-tryptophan (substance 4b) and ${}_D$-tyrosine (substance 4d) head-groups inhibited the Pin1 binding to the substrate peptide with latter being the more efficient inhibitor (Fig. 2A). In contrast, inclusion of ${}_D$-phenylalanine (substance 4c) head-group had no significant outcome on Pin1 binding while the addition of 3-(2-naphthyl)-${}_D$-alanine (substance 4a) marginally enhanced Pin1 binding. In addition to the binding assay, the isomerase activity of Pin1 towards a pentapeptide (Succ-ENpSPL-pNitroanilide) was measured spectroscopically in the presence of the synthesised inhibitor substances (Fig. 2B). Analyses of these samples showed a clear decrease in Pin1 isomerase activity with the ${}_D$-tyrosine derivative (substance 4d) but not phenylalanine (substance 4c) derivative. In contrast, an increase in Pin1 isomerase activity was detected on inclusion of 3-(2-naphthyl)-${}_D$-alanine (substance 4a) and ${}_D$-tryptophan (substance 4b) head-groups (Fig. 2B).

Fig. 2.

The influence of the synthesised inhibitors on Pin1 binding and activity. (A) A biotinylated form of the phosphorylated TF peptide (biotin-RKAGVGQSWKENpSPLNVS) was used to capture Pin1 in vitro along with a scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) as control. The peptides (5 $\mu$M final concentration) were diluted to 100 $\mu$L with PBS and distributed (50 $\mu$L per well) in a NeutrAvidin-coated 96-well and incubated for 2 h at room temperature to allow binding. The wells were washed four times, each time with 300 $\mu$L of PBST. Sets of wells were supplemented with a range of synthetic inhibitors (0–100 $\mu$M) or DMSO vehicle. HRP-conjugated recombinant Pin1 protein was diluted 1:500 (v/v) in PBST, added to the wells (100 $\mu$L) and incubated for 1 h at room temperature. The wells were then washed a further four times and developed with TMB One Solution (100 $\mu$L). Once the colour was developed the reactions were stopped by the addition of 2M sulphuric acid (50 $\mu$L) and absorptions measured at 450 nm using a plate reader. The concentrations of Pin1 were determined from a standard curve made using HRP-conjugated recombinant Pin1 protein (n = 3, * = p 0.05). (B) Pin1 was incubated with penta-peptides, Succ-Glu-Asn-Ser-Pro-Leu-pNitroanilide and Succ-Glu-Asn-phosphoSer-Pro-Leu-pNitroanilide and alterations in spectroscopic absorption of the solution analysed. Briefly, samples (100 $\mu$L) of the substrate peptides (0.5 $\mu$M) were in turn placed in a microcuvette. Recombinant HRP-conjugated Pin1 (10 nM final concentration) was then pre-incubated (5 min) with the inhibitors and then added to the cuvettes. The change in absorption at 315 nm over time was then immediately monitored (n = 3, * = p 0.05).

To assess the direct influence of the inhibitors on TF activity, MDA-MB-231 cells were pre-incubated for 60 min with the synthesised substances and TF activity measured using a chromogenic fXa-generation assay. Inclusion of ${}_D$-tryptophan (substance 4b) and ${}_D$-tyrosine (substance 4d) head-groups in the compounds inhibited the thrombin generation by 38% and 31% respectively (Fig. 3A). In contrast, reductions in TF activity, on inclusion of 3-(2-naphthyl)-${}_D$-alanine (substance 4a) and ${}_D$-phenylalanine (substance 4c) derivatives were not significant. Therefore, only the alterations observed on with ${}_D$-tryptophan (substance 4b) and ${}_D$-tyrosine (substance 4d) derivatives were assumed to be specific.

Fig. 3.

The influence of the synthesised inhibitors on TF activity, antigen and mRNA levels. (A) MDA-MB-231 cells (5 $\times$ 10${}^4$) were incubated with the inhibitors (100 $\mu$M) diluted in the reaction buffer (100 $\mu$L; HEPES-buffered saline (HBS) pH 7.4, containing 1% (w/v) bovine serum albumin (BSA) and 5 mM CaCl${}_2$) for 60 min. The cells were washed and incubated with fVIIa (20 nM) in the reaction buffer for an additional 10 min and then supplemented with fX (100 nM), together with fXa substrate (0.2 mM) diluted in the same buffer (100 $\mu$L). The samples were incubated for 60 min to develop the colour. Aliquots (150 $\mu$L) were then transferred to a 96-well plate containing 2% (v/v) acetic acid (50 $\mu$L) and the absorptions measured immediately at 405 nm. The amount of fXa generated was determined using a standard curve prepared using fXa (n = 5, * = p 0.05). (B) MDA-MB-231 cells (5 $\times$ 10${}^4$) were seeded out into 48-well plates and incubated with the inhibitors as above (100 $\mu$M) for 18 h. The cells were then washed with PBS and fixed with 3% (v/v) formaldehyde. The cells were then incubated with an HRP-conjugated sheep anti-human TF antibody (100 $\mu$L) diluted 1:1000 (v/v) in PBS for 1 h. The cells were then washed four times with PBS and developed using TMB One Solution substrate Solution (100 $\mu$L). Once the colour was developed the reactions were stopped by the addition of 2M sulphuric acid (50 $\mu$L) and absorptions measured at 450 nm using a plate reader (n = 5, * = p 0.05). (C) The TF antigen associated with the microvesicles was measured using the Quantikine TF-ELISA kit according to the manufacturers’ instructions (n = 3, * = p 0.05). (D) Total RNA was isolated using the TRI-reagent system from 2 $\times$ 10${}^5$ cells and 100 ng of total RNA was used for each reaction. The relative amounts of TF mRNA were determined using QuantiTect primer sets to detect TF in conjunction with $\beta$-actin. The reaction was carried out at an annealing temperature of 60 ${}^{\circ }$C for 1 min using the GoTaq® 1-Step RT-qPCR System on an iCycler thermal cycler for 40 cycles (n = 3, * = p 0.05).

Incubation of MDA-MB-231 cells with compound 4d, containing ${}_D$-tyrosine head-group, resulted in low but significant reduction in cell-surface TF antigen (Fig. 3B) and TF release within microvesicles (Fig. 3C). Moreover, treatment of cells with either substances containing 3-(2-naphthyl)-${}_D$-alanine (substance 4a) or ${}_D$-tryptophan (substance 4b) head groups resulted in substantial increases in the release of TF within cell-derived microvesicles (Fig. 3C). Incubation of MDA-MB-231 cells for 6 h with any of the four synthesised compounds, did not result in significant changes in TF mRNA expression (Fig. 3D).

Incubation of MDA-MB-231 cells with substances containing the 3-(2-naphthyl)-${}_D$-alanine (substance 4a), ${}_D$-tryptophan (substance 4b) and ${}_D$-tyrosine (substance 4d) head-groups resulted in approximately 22%, 48% and 55% reduction in cell numbers respectively (Fig. 4A) and were associated with significant increases in DNA-fragmentation as measured by the end-labelling TUNEL assay (Fig. 4B). In contrast, the inclusion of ${}_D$-phenylalanine (substance 4c) was ineffective. Importantly, incubation of the HDBEC primary cells which are devoid of TF, did not have any detectable influence on cell numbers of the rate of cell apoptosis (Fig. 4C).

Fig. 4.

Examination of the pro-apoptotic potential of the synthesised inhibitors. (A) MDA-MB-231 cells (5 $\times$ 10${}^4$) were seeded out into 48-well plates and treated with the set of inhibitors or the DMSO vehicle. Cell numbers were determined by staining with crystal violet and calculated from a standard curve (n = 5, * = p 0.05). (B) Cellular apoptosis was quantified using the TiterTACS™ Colorimetric Apoptosis Detection Kit according to the manufacturer’s instructions (n = 3, * = p 0.05). (C) HDBEC (5 $\times$ 10${}^4$) were seeded out into 48-well plates and treated with the set of inhibitors or the DMSO vehicle. Cell numbers were determined by staining with crystal violet and calculated from a standard curve. (n = 3). (D) Cells (5 $\times$ 10${}^4$) were treated as above and lysed in Laemmeli’s buffer containing a protease inhibitor cocktail. Equal amounts were separated by 12% (w/v) SDS-PAGE and the protein bands were transferred onto nitrocellulose membranes and blocked with TBST. The membranes were then probed with a mouse monoclonal anti-human Bax antibody (202), diluted 1:3000 (v/v) in TBST. The membranes were washed with TBST and probed with a goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and incubated for 90 min. The bands were then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded (Images are representative of 3 separate experiments). (E) All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v) (n = 3, * = p 0.05). (F) Total RNA was isolated using the TRI-reagent system from 2 $\times$ 10${}^5$ cells and 100 ng of total RNA was used for each reaction. The relative amounts of bax mRNA was determined using QuantiTect primer sets to detect bax in conjunction with $\beta$-actin. The reaction was carried out at an annealing temperature of 60 ${}^{\circ }$C for 1 min using the GoTaq® 1-Step RT-qPCR System on an iCycler thermal cycler for 40 cycles (n = 3, * = p 0.05).

To further confirm the mechanism of apoptosis in MDA-MB-231 cells, following treatment with the inhibitors the expression of Bax protein and bax mRNA were measured. ${}_D$-tyrosine derivative (substance 4d) and to a lesser extent ${}_D$-tryptophan derivative (substance 4b) resulted in increased expression of both Bax protein (Fig. 4D,E) and bax mRNA (Fig. 4F) while the 3-(2-naphthyl)-${}_D$-alanine (substance 4a) and ${}_D$-phenylalanine (substance 4c) derivatives were ineffective.

Finally, since Pin1 can influence cell apoptosis through altering the activity and stability of p53 protein, the influence of synthesised inhibitor substances on the expression and nuclear localisation of p53 was examined. Incubation of MDA-MB-231 cells with ${}_D$-tyrosine (substance 4d) containing compound resulted in increased nuclear localisation of p53 compared to the control sample, but was not significant with compounds containing the 3-(2-naphthyl)-${}_D$-alanine (substance 4a), ${}_D$-tryptophan (substance 4b) and ${}_D$-phenylalanine (substance 4c) head-groups (Fig. 5A,B). Moreover, incubation of the cells with any of the four compounds did not alter the amount of p53 proteins as measured by western blot (Fig. 5C).

Fig. 5.

The influence of the synthesised inhibitors on nuclear localisation of p53. Cells were seeded out into 35-mm glass-bottom with 10 mm $\mu$-well dishes were seeded 10${}^3$ MDA-MB-231 cells and incubated for 4 h at 37 ${}^{\circ }$C in an incubator. The medium was aspirated and replaced with 100 $\mu$L of medium supplemented with test agents (100 $\mu$M). Sets of cells were treated with of TNF-$\alpha$ (10 ng/mL) or used untreated and used as positive and negative controls, respectively. The media were removed after 18 h and the cells were washed twice with PBS (200 $\mu$L) and fixed using 4% (v/v) paraformaldehyde. After three washes with PBS, the cells were permeabilised with 0.2% (v/v) Triton X-100 diluted in PBS and incubated at room temperature for a further 10 min. The samples were then blocked for 1 h with PBS containing 3% (w/v) bovine serum albumin (BSA). The cells were then washed a further three times and probed with a rabbit polyclonal anti-human p53 antibody diluted 1:250 (v/v) in PBS/BSA buffer (100 $\mu$L) and incubated overnight, at 4 ${}^{\circ }$C. After a further three washes with PBS, the samples were incubated with a Northern Lights-637 donkey anti-rabbit antibody diluted 1:100 (v/v) PBS/BSA buffer (100 $\mu$L), for 1 h in the dark. The cells were washed another two times with PBS and stained with DAPI (2 $\mu$g/mL). (A) Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a $\times$40 magnification (Images are representative of 10 field of view from 3 separate experiments). (B) The localisation of p53 within the nuclei were determined using ImageJ, in 10 fields of view from each assay and Mander’s coefficient determined (n = 3, * = p 0.05). (C) Cells (5 $\times$ 10${}^4$) were treated as above and lysed in Laemmeli’s buffer containing a protease inhibitor cocktail. Equal amounts were separated by 12% (w/v) SDS-PAGE and the protein bands were transferred onto nitrocellulose membranes and blocked with TBST. The membranes were then probed with a polyclonal rabbit anti-human p53 antibody. The membranes were washed with TBST and probed with a goat anti-rabbit or goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and incubated for 90 min. The bands were then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. (Images are representative of 3 separate experiments).

The interactions of the four compounds with Pin1 enzyme were examined using the crystal structure of Pin1 (1PIN). The location (Fig. 6) and efficiency of binding of the four compounds to Pin1 was examined using Autodock 4v2.6 software. Estimation of the binding efficiencies indicated a comparable binding energy for all four compounds (Table 1). However, calculated binding constants indicated a higher affinity for the substances 4a and 4b (5.03 $\mu$M and 10.97 $\mu$M respectively) but lower for substances 4c and 4d (28.18 $\mu$M and 47.44 $\mu$M respectively). A visual inspection of the complexes with Pin1 illustrated similar conformations in three of the docked molecules but indicated interaction with additional residues in the molecule containing the ${}_D$-tyrosine (substance 4d) head-group.

Fig. 6.

The influence of the synthesised inhibitors on nuclear localisation of p53. The structure of the four molecules were constructed using the Alchemy program and saved in Brookhaven format (PDB). The crystal structures of Pin1 (1PIN) was obtained from Brookhaven format (PDB). The location and efficiency of binding of the molecules to Pin1 was estimated using the Autodock 4v2.6, the polar hydrogens were retained and partial charges added to the proteins using the Gasteiger charges. The search space was limited to an area of 20 $\times$ 20 $\times$ 20 Å, centred around the hydroxyl group of Ser18 in the enzymatic site of Pin1.