Peter Blurton

Activation of mesolimbic dopamine function by phencyclidine is enhanced by 5-HT2C/2B receptor antagonists: neurochemical and behavioural studies

Administration of the non-competitive NMDA receptor antagonists phencyclidine (PCP) (0.6-5 mg/kg s.c.) and MK-801 (0.1-0.8 mg/kg s.c. ) dose-dependently increased locomotor activity in the rat. Pre-treatment of rats with SB 221284 (0.1-1 mg/kg, i.p.) a 5-HT(2C/2B) receptor antagonist or SB 242084 (1 mg/kg, i.p.) a selective 5-HT(2C) receptor antagonist, doses shown to block mCPP induced hypolocomotion, significantly enhanced the hyperactivity induced by PCP or MK-801. Neither compound altered locomotor activity when administered alone. Furthermore, systemic administration of PCP (5 mg/kg s.c.) increased nucleus accumbens dopamine efflux in the rat to a maximum of approximately 220% of basal, 40-60 min after administration. Pre-treatment with the 5-HT(2C/2B) receptor antagonist SB 221284 (1 mg/kg, i.p.) and the 5-HT(2C) receptor antagonist SB 242084 (1 mg/kg i.p.) failed to affect nucleus accumbens dopamine efflux per se but significantly enhanced the magnitude and duration of the increase induced by PCP. However, the time course of the neurochemical and behavioural effects were qualitatively and quantitatively different, suggesting the potential involvement of other neurotransmitter pathways. Nevertheless, the present results provide behavioural and neurochemical evidence which demonstrate that, in the absence of effects per se, blockade of 5-HT(2C) receptors enhanced the activation of mesolimbic dopamine neuronal function by the non-competitive NMDA receptor antagonists PCP and MK-801.

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.

Abstract LB-037: Development and preclinical assessment of a first-in-class small molecule inhibitor of the major cell death regulator protein FLIP

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 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 cytotoxic chemotherapeutics. 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). Lead compounds have been shown to block recruitment of FLIP to the TRAIL-R2 death-inducing signalling complex (DISC), confirming their on-target activity. Moreover, 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 standard-of-care chemotherapeutics and radiotherapy. To further confirm the mechanism of action, FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors. 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 xenograft models Conclusions The novel, first-in-class inhibitors of FLIP developed in this study have the potential for broad application in a range of cancers, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a grant from the Wellcome Trust9s Seeding Drug Discovery Initiative (reference: 099470). Reference 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 Boffey, Trevor R. Perrior, Patrick G. Johnston, David Haigh, Timothy Harrison, Daniel B. Longley. Development and preclinical assessment of a first-in-class small molecule inhibitor of the major cell death regulator protein FLIP. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-037. doi:10.1158/1538-7445.AM2015-LB-037