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. 2017 Jun 9:8:15772.
doi: 10.1038/ncomms15772.

A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action

Elena Campaner  1   2 , Alessandra Rustighi  1 , Alessandro Zannini  1   2 , Alberto Cristiani  1 , Silvano Piazza  1 , Yari Ciani  1 , Ori Kalid  3 , Gali Golan  3 , Erkan Baloglu  3 , Sharon Shacham  3 , Barbara Valsasina  4 , Ulisse Cucchi  4 , Agnese Chiara Pippione  5 , Marco Lucio Lolli  5 , Barbara Giabbai  6 , Paola Storici  6 , Paolo Carloni  7 , Giulia Rossetti  7   8   9 , Federica Benvenuti  10 , Ezia Bello  11 , Maurizio D'Incalci  11 , Elisa Cappuzzello  12 , Antonio Rosato  12   13 , Giannino Del Sal  1   2
Affiliations

A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action

Elena Campaner et al. Nat Commun. .

Abstract

The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.

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Conflict of interest statement

Erkan Baloglu and Sharon Shacham are employees and shareholders of Karyopharm Therapeutics. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1. KPT-6566 specifically inactivates PIN1 PPIase activity in vitro.
(a) Scheme representing the screening conducted with the drug like collection, indicating the stepwise approach that ends up with nine potential covalent inhibitors of PIN1. (b) Bar plot indicating PPIase activity K of PIN1 (black), PIN1 S67E (white) and PIN1 with 30 μM of the indicated compounds. Positive hit is marked in bold. (c) Chemical structure of KPT-6566 (1): the compound contains a polycyclic aromatic hydrocarbon bound to a sulfanyl-acetic acid group (-S-CH2-COOH) and to a tert-butylphenyl group through a sulfonamide moiety (N-SO2). (d) Mass spectrum deconvolution of DMSO (left) and KPT-6566 treated (right) PIN1. A MW increase of 90 Da can be appreciated in the compound treated sample. (e) 3D image showing the docking of KPT-6566 in the catalytic pocket of PIN1. KPT-6566 is shown in green licorice representation. The surface of PIN1 is shown as solid representation and coloured by heteroatom. The binding cavity is magnified in the right insert. (f) Plot of the observed rate constants for inhibition (kobs) against inhibitor concentration of KPT-6566 from which estimations of kinetic parameters for covalent inhibition of PIN1 in vitro were made. The corresponding kinact and Ki values are reported. (g) Bar plot indicating PPIase activity of GST-FKBP4 incubated with DMSO, 30 μM FK506 or 30 μM KPT-6566. (h) Same as in (g) for GST-PPIA incubated with DMSO, 30 μM Cyclosporin A or 30 μM KPT-6566. Data shown in b,g,h, are the means±s.d. of n=3 independent experiments, *P<0.05, **P<0.01; two-tailed Student's t-test.
Figure 2
Figure 2. KPT-6566 impacts on cell proliferation and viability in a PIN1-dependent manner.
(a) Growth curves of WT (left) or Pin1 KO (right) MEFs treated with the indicated concentrations of KPT-6566 or DMSO. (b) Immunoblotting of the indicated cell cycle-related proteins in WT or Pin1 KO MEFs treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h. (c) Growth curves in empty-vector transduced Pin1 KO MEFs (Vector, left) or Pin1 KO MEFs reconstituted with HA-PIN1 (PIN1, right) treated with KPT-6566 or DMSO. (d) Immunoblotting of the indicated proteins in Pin1 KO MEFs or Pin1 KO MEFs reconstituted with HA-PIN1 treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h. (e) Left, immunoblotting of the indicated proteins in cell lysates from MCF10A and MCF10AT1 cells. Right, cell viability (WST) assay of MCF10A and MCF10AT1 treated with the indicated concentrations of KPT-6566 for 48 h. (f) Immunoblotting of PIN1 in normal breast epithelial cells (MCF10A, HMEC) and in the indicated cancer cell lines. (g) Cell viability (ATPlite) assay of the same cell lines as in (f) treated with the indicated concentrations of KPT-6566 for 48 h. b,d–f Actin levels are reported as loading control; size markers are indicated. Data shown in a,c,e,g are the means±s.d. of n=3 independent experiments, **P<0.01, n.s. not significant; two-tailed Student's t-test.
Figure 3
Figure 3. KPT-6566 interferes with PIN1 oncogenic functions.
(a) Immunoblotting of PIN1 client proteins expressed in MDA-MB-231 breast cancer cells treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h. (b) Quantitative RT-PCR analysis of mut-p53 and NOTCH1 target genes (BUB1, DEPDC1 and HEY2, BIRC5, respectively) in MDA-MB-231 cells treated with indicated concentrations of KPT-6566 or DMSO (−) for 48 h. (c) Left, representative pictures of MDA-MB-231 colonies in the indicated experimental conditions. Right, histogram showing colony formation efficiency of MDA-MB-231 cells in the indicated experimental conditions. Cells were transfected with control siRNA (siRNA Ctrl) or with PIN1 siRNA#1. After 24 h cells were trypsinized, plated for colony forming assay and treated with 5 μM PiB, 1 μM KPT-6566 or DMSO every two days. Colonies ≥50 pixels were counted 10 days after seeding using ImageJ software. (d) Histogram showing invasive ability and proliferation (cell number) of MDA-MB-231 breast cancer cells plated in Matrigel-coated Boyden chambers in the indicated experimental conditions for 20 h. (e) Histogram showing percentage of secondary mammosphere formation efficiency (%M2FE) of MDA-MB-231 cells in the indicated experimental conditions. (f) Immunoblotting of the indicated proteins expressed in MDA-MB-231 breast cancer cells treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h. (g) %M2FE of MCF10AT1 cells with stable control- (shRNA Ctrl) or PIN1 silencing (shRNA PIN1), treated as indicated. (h) %M2FE of MCF10A cells transduced with empty- (Vec) or HA-PIN1-expressing vectors (PIN1), treated as indicated. (a,f) Actin levels are reported as loading control; size markers are indicated. Data shown in b–e,g,h are the means±s.d. of n=3 independent experiments, *P<0.05, **P<0.01, n.s. not significant; two-tailed Student's t-test.
Figure 4
Figure 4. Interaction of KPT-6566 with PIN1 promotes its structural change and degradation.
Immunoblotting of the indicated proteins in cell lysates from (a) MDA-MB-231 cells treated with the indicated compounds (+) or DMSO (−) for 48 h; (b) PC3, PANC1 and H1299 cells treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h; (c) PIN1 KO MDA-MB-231 cells transduced with empty (−) or PIN1 vectors (+) treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h; (d) PIN1 KO MDA-MB-231 cells reconstituted with HA-PIN1, treated with increasing amounts of KPT-6566 or DMSO; (e) PIN1 KO MDA-MB-231 cells reconstituted with HA-PIN1, treated with 5 μM KPT-6566 (+) or DMSO (−) followed by cycloheximide (CHX) chase for the indicated hours; (f) PIN1 KO MDA-MB-231 cells reconstituted with HA-PIN1, treated with 5 μM KPT-6566 (+), 10 μM MG132 (+) or DMSO (−) for 16 h. a–f Actin levels are reported as loading control; size markers are indicated.
Figure 5
Figure 5. Global transcriptional effects of KPT-6566.
(a) Venn diagram of the Illumina microarray analysis with common and differentially up- and down-regulated genes of MDA-MB-231 cells treated with siRNA PIN1#1 vs. KPT-6566, using a cutoff of 0.75 logFC and P-value ≤0.05. (b) Venn diagram of the Gene Ontology (GO) analysis with common and differential enrichment for functional terms of MDA-MB-231 cells treated with siRNA PIN1 vs. KPT-6566. (c) Histogram indicating comparison of IPA analysis of genes regulated by PIN1 siRNA (white) or KPT-6566 (red) in function of their activation Z-scores. (d) Quantitative RT-PCR analysis of NRF2 pathway members in MDA-MB-231 cells treated with the indicated compounds for 48 h. Data are indicated as means±s.d. of n=3 independent experiments, *P<0.05, **P<0.01; two-tailed Student's t-test.
Figure 6
Figure 6. KPT-6566 induces DNA damage in a PIN1-dependent manner.
Immunoblotting of the indicated proteins from (a) MDA-MB-231 cells treated with increasing amounts of KPT-6566 or DMSO for 48 h; (b) MCF10A, MDA-MB-231 and MDA-MB-468 cells treated with 5 μM KPT-6566 (+), 10 μM Bleomycin (+) or DMSO (−) for 48 h; (c) MDA-MB-231 cells transfected with control- (siRNA Ctrl) or two different PIN1 siRNAs (siRNA PIN1#1 and PIN1#2) and treated as indicated for 24 h. (d) Histogram representing the percentage of γH2AX positive nuclei of MDA-MB-231 cells treated as in c. (e) Immunoblotting of the indicated proteins from MDA-MB-231 cells treated with the indicated compounds (+) or DMSO (–) for 48 h. a–c,e Actin levels are reported as loading control; size markers are indicated. Data shown in (d) are the means±s.d. of n=3 independent experiments, *P<0.05, **P<0.01; two-tailed Student's t-test.
Figure 7
Figure 7. KPT-6566 induces cell death of cancer cells and reduces metastasis growth in vivo.
(a) Histogram representing respectively the percentage of early apoptotic and late apoptotic/dead MDA-MB-231 cells in the indicated experimental conditions. Results are indicated as means±s.d. of n=3 independent experiments, **P<0.01; two-tailed Student's t-test. (b) Left, immunoblotting of the indicated proteins of MDA-MB-231 cell lysates untreated (−) or treated (+) with the indicated compounds. Right, immunoblotting of HMGB1 from supernatant of the same cells as in the left panel; actin levels are reported as loading control; size markers are indicated. (c) Left, hematoxylin and eosin staining of representative sections of entire pulmonary lobes from mice inoculated with MDA-MB-231 cells and treated with vehicle (n=8) or KPT-6566 (n=7); arrows indicate representative metastases. Scale bar, 4 mm. Right, computer-aided assessment of percentage of lung tissue area occupied by metastases in the indicated conditions. Data are reported in histograms as means±s.e.m.; *P<0.05; two-tailed Mann–Whitney test. (d) Proposed model of KPT-6566 mechanism of action. After entering the cell, KPT-6566 binds to PIN1 and elicits a cytostatic effect associated to covalent inhibition and degradation of PIN1 with consequent decrease of oncogenic circuitries. In addition, KPT-6566 might induce intracellular ROS production (upper part). After reaction with PIN1, KPT-6566 has a simultaneous cytotoxic effect releasing KPT-6566-B, which generates further ROS, induces DNA damage and cancer cell death (lower part).
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