FLIPP-FADD: PPI inhibitors for the treatment of solid tumours
FLIP is a non-redundant inhibitor of caspase 8 activation which mediates apoptosis. Over-expression of FLIP is a tumour cell survival mechanism, and hence FLIP is of interest for the treatment of a number of cancers including lung cancer.
Challenge
When this project was started, the FLIP-FADD protein-protein interaction (PPI) was widely thought to be 'undruggable', partly because the structural details of this binding were unknown, but also due to a further problem: it was believed that the procaspase 8 and FLIP binding modes were analogous, and so it would be impossible to find a compound that would inhibit FLIP binding, but be permissive to the procaspase 8–FADD interaction that is required for apoptosis to proceed. When Prof. Dan Longley’s group at Queen’s University Belfast used extensive protein mutagenesis data and molecular modelling to understand these two binding events, they realised that there were significant differences in their binding modes (Figure 2), and a therapeutic opportunity was recognised.1 The aim was therefore to use rational design based on this understanding in order to find selective inhibitors of the FLIP-FADD PPI.
Figure 1: FLIP inhibits caspase 8 mediated apoptosisFigure 2: FLIP binds to the DED of FADD using its DED2, whereas procaspase 8 binds to FADD using its DED1
Solution
Longley’s group identified a druggable cleft on the surface of the DED2 domain of FLIP by molecular dynamics studies on the protein, and their modelling suggested that the binding of an α-helix of FADD into this cleft was a key element of the PPI. This hypothesis was supported by mutagenesis studies and offered an opportunity to use a rational design approach to find selective binders to FLIP that would inhibit the PPI with FADD, but would not bind to procaspase 8. Domainex’s CADD scientists used the proposed binding cleft to design pharmacophoric search protocols with their proprietary virtual screening platform, LeadBuilder. This was used to select a compound screening set of around 1000 compounds, covering multiple chemotypes, which were evaluated in a medium-throughput screen. A handful of structurally unrelated compounds were identified which showed binding to FLIP at 10uM.Three of the hits were selected for further investigation, and optimisation of their potency and pharmacokinetic properties led to a viable lead and back-up series.
Figure 3: Optimisation of FLIP inhibitors
The Longley group showed that the compounds had excellent cell activity (Figure 5), good in vivo PK (Figure 4) and in vivo activity in murine xenograft studies (e.g. Figure 6). Their selectivity with respect to procaspase 8 binding was confirmed in mechanistic cellular assays, and broader specificity was established in a Eurofins Safety 44 panel.
Figure 4: Representative example inhibitor (FLIPi_2) shows excellent drug-like properties
FLIPi_2
CLint (Hep, mouse)*
55
PPB (% bound, mouse)
69
Caco-2, Papp A-B (efflux)§
7.6 (1.4)
MW
<350
Log D (measured)
1.0
Log P (calculated)
2.6
*μL/min/106cells; § x 10-6 cm-1
Figure 5: Activity of FLIP inhibitor (FLIPi_1) in HCT116 colorectal cancer model. HCT116 colorectal cancer cells were treated with FLIP inhibitor at the concentrations indicated above, in the absence or presence of 5 ng/mL TRAIL 6h post-treatment (i) caspase-8 and (ii) caspase-3/7 activity was assessed. (iii) Caspase-3 activity was assessed in HCT116 cells cultured in the absence or presence of 5 µM 5FU for 48h, after which time, FLIPi_1 was added for 6h at the concentrations indicated above.Figure 6: A549 NSCLC xenograft study of FLIP inhibitor (FLIPi_2) dosed P.O. @75 mg/kg q.d. as a single agent in Balb/c nude mice. Balb/c nude mice were inoculated with 2x106 cells in each flank. After 8 days, mice were treated P.O. with 75mg/kg FLIPi_2 or vehicle. Treatment was repeated every day, and tumour volume was monitored using digital calipers. Significant differences (p<0.05) between vehicle control and FLIPi_2 treated groups were observed from day 15 onwards, and T/C ratios of ~0.5 observed from day 19 onwards.
This programme is currently at the pre-clinical candidate nomination stage and several companies are carrying out due diligence with regards to taking it on for further development.
Conclusions
Starting with an innovative hypothesis developed by our academic collaborators in Belfast, virtual screening using LeadBuilder, and hit optimisation by the Domainex medicinal chemistry team led to the world’s firstinhibitors of the FLIP-FADD interaction. These compounds have been designed and shown to have high specificity for FLIP, and to have a good PK profile. Testing in murine xenograft experiments has provided proof of concept for this anti-tumour mechanism, and further development of the chemistry has provided a range of pre-clinical candidates.
This programme was funded in part by the Wellcome Trust Seeding Drug Discovery scheme.
Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC Assembly. J. Majkut, M. Sgobba, C. Holohan, N. Crawford, A.E. Logan, E. Kerr, C.A. Higgins, K.L. Redmond, J.S. Riley1, I. Stasik, D.A. Fennell, S. Van Schaeybroeck, S. Haider, P.G. Johnston, D. Haigh and D.B. Longley. Nat Commun., 2014, 5, 3350.
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