Catherine Higgins

Mechanistic and anti-proliferative studies of two novel, biologically active bis-benzimidazoles

We have previously synthesised a number of novel head-to-head bis-benzimidazole derivatives that are structurally related to the fluorochrome, Hoechst 33258, and which possess strong affinity for A:T sites in the minor groove of duplex DNA. Initial studies revealed these compounds to exhibit potent antiproliferative activity against a range of ovarian cell lines and to inhibit transcription in an in vitro setting. In this study, we have examined their cellular behaviour in detail and have shown that two of these compounds (ABA13 and ABA833) potently inhibit the proliferation of a range of human tumour cell lines, and show some specificity towards breast carcinoma cell lines. In most of the cell lines investigated, ABA833 was the more potent of the two compounds. Flow cytometric analysis revealed that ABA13 and ABA833 (50-500 nM) induced an S phase block and increased the pre-G1 population in MCF-7 and MDA 468 human breast cancer cells. An increase in the pre-G1 population of RKO colon carcinoma cells was seen only at 500 nM with ABA833, reflecting the reduced sensitivity of this cell line to the bis-benzimidazoles in comparison to the breast cancer cell lines. Mechanistic studies revealed that neither ABA13 or ABA833 act as topoisomerase I (topo I) or topoisomerase II (topo II) poisons in plasmid or kinetoplast DNA (kDNA) relaxation assays, but both compounds do inhibit the catalytic activity of these enzymes. Drug uptake studies showed that reduced sensitivity of MCF-7adr and RKO cells compared with MCF-7 to both ABA13 and ABA833 correlated with a markedly reduced intracellular drug accumulation.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages DEDs in procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD’s α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

Synthesis and in Vitro and in Vivo Evaluation of a Series of Dihydroisocoumarin Derivatives Conjugated with Fatty Acids, Alcohols, and Amines as Potential Anticancer Agents

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to the dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1,3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo in a number of human tumor xenograft models.

Growth Inhibitory Activity of Extracted Material and Isolated Compounds from the Fruits of Kigelia pinnata

A study of the components of the fruits of Kigelia pinnata was undertaken to identify compounds with potential growth inhibitory activity against human melanoma cells, since extracts from the fruits of this plant have been described in traditional medicine to have application in the treatment of skin cancer and other skin ailments. A bioactivity-guided fractionation process yielded a number of crude fractions, which demonstrated cytotoxicity in vitro against human melanoma cells. Compounds isolated and identified included the isocoumarins, demethylkigelin (1) and kigelin (2), fatty acids, oleic (3) and heneicosanoic acids (4), the furonaphthoquinone, 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione (5), and ferulic acid (6). A number of structurally related synthetic compounds were also tested using the MTT assay. The most potent series of these compounds, the furonaphthoquinones, also demonstrated a cytotoxic effect in two human breast cancer cell lines tested.

FLIP: a targetable mediator of resistance to radiation in non-small cell lung cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non–small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor FLIP is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were downregulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the antitumor activity of IR in vivo. Therefore, FLIP downregulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR. Mol Cancer Ther; 15(10); 2432–41. ©2016 AACR.

P3 SAR exploration of biphenyl carbamate based Legumain inhibitors

This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification of a sub-nanomolar inhibitor of Legumain.

Development of a potent and selective cell penetrant Legumain inhibitor

This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.

Abstract LB-305: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP

Background Evasion of cell death is a major cause of resistance to anti-cancer therapies, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We have found that there are important differences between FLIP and procaspase-8 in terms of their preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD interaction; cell-free screening assays; cell-based activity assays; biophysical binding assays; on-target mechanism-of-action studies, in vivo studies. Results Molecular modelling of the FLIP-FADD interaction identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimization of these hits identified a series of compounds that are able to disrupt FLIP’s interaction with the DISC and display nanomolar activity in NSCLC and CRC cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutics such as e.g. cisplatin (NSCLC) or 5FU (CRC). FLIP overexpression and CRISPR-mediated procaspase-8 deletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. In addition, using peripheral blood mononuclear cells (PBMCs), we demonstrate that FLIP inhibitors have selectivity against cancer cells. Finally, we identified lead molecules with ADME profiles suitable for in vivo evaluation and using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models. Conclusions The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of cancer, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust. 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD9s DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Jennifer Fox, Greti E. Fiedler, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2017-LB-305

Abstract C4: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP for treatment of NSCLC

Background Evasion of cell death is a major cause of resistance to cancer therapy, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We recently reported that there are important differences between FLIP and procaspase-8 in terms of both their binding affinities and preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. We now report our subsequent work leading to the development and pre-clinical characterisation of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD complex; virtual small molecule library screening; cell-free screening assays; cell-based activity assays; biophysical binding assays; in vivo anti-tumor studies. Results Molecular modelling of the FLIP-FADD complex identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimisation of these hits afforded lead and back-up series with nanomolar activity in cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutic cisplatin. FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. Lead molecules have been identified with ADME profiles suitable for in vivo evaluation. Using these compounds, single-agent anti-tumor effects have been demonstrated in NSCLC xenograft models. Conclusions The novel first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of NSCLC, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust (reference: 099470). 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD9s DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Adnan Malik, Zsusannah Nemeth, Peter Blurton, Ray J. Boffey, Trevor R. Perrior, David Haigh, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP for treatment of NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C4.

Abstract C5: FLIP protein-protein interaction inhibitors enhance sensitivity of colorectal cancer cells to chemotherapy and TRAIL

Background Colorectal Cancer (CRC) is the second most common cause of cancer death, with 40% of patients with this disease obtaining no benefit from current chemotherapy. Novel therapeutic strategies are needed to improve CRC patient response rates and survival. FLIP is an inhibitor of the extrinsic apoptotic pathway that binds to FADD at death-inducing signalling complexes (DISCs), such as those formed by the TNF-α-related apoptosis inducing ligand (TRAIL) receptors TRAIL-R1 and TRAIL-R2, thereby blocking homodimerization and activation of procaspase-8 and inhibiting apoptosis induction. We previously reported that FLIP blocks apoptosis induced by TRAIL and standard-of-care chemotherapeutics (5-Fluorouracil, oxaliplatin and SN38) in CRC models. Moreover, FLIP is frequently overexpressed in CRC and its overexpression correlates with poor prognosis. Subsequently, we have developed novel small molecule inhibitors that target FLIP9s critical protein-protein interactions, preventing its interaction with FADD and therefore promoting activation of caspase-8 and apoptosis induction. Methods A DISC recruitment assay was used to assess levels of FLIP at the TRAIL-R2 DISC. Caspase activity, cell viability and apoptosis induction were assessed in CRC models treated with FLIP inhibitors alone and in combination with TRAIL or standard-of-care chemotherapeutics. Mechanism-of-action was assessed using caspase-8-targeted siRNA and FLIP overexpressing models. Results Using CRC cell line models, it was demonstrated that FLIP recruitment to the DISC is inhibited by FLIP inhibitors from the lead series. As a result, TRAIL-induced caspase-8 and caspase-3/7 activity were enhanced and increased levels of apoptosis cells were observed. Decreased cell viability was observed that was proportionate to the levels of apoptosis induced. Cell death triggered by FLIP inhibitors was shown to be caspase-8-dependent consistent with the expected mechanism-of-action. Importantly, inhibitors of FLIP also enhanced apoptosis induction in response to 5-Fluorouracil, oxaliplatin and SN38. Conclusion We have developed inhibitors of FLIP that decrease its recruitment to the TRAIL-R2 DISC and increase TRAIL-induced caspase activation and apoptosis. Moreover, these inhibitors synergise with 5-Fluorouracil, oxaliplatin and SN38, suggesting that this novel class of agents has therapeutic potential in CRC when used in conjunction with standard-of-care chemotherapeutic agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust (reference: 099470). Citation Format: Jennifer P. Fox, Joanna Majkut, Catherine Higgins, Zsuzsanna Nemeth, Adnan Malik, Christopher J. Scott, Peter Blurton, Ray J. Boffey, Trevor R. Perrior, Timothy Harrison, Daniel B. Longley. FLIP protein-protein interaction inhibitors enhance sensitivity of colorectal cancer cells to chemotherapy and TRAIL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C5.