vFLIP vs cFLIP Cleft 1

1. vFLIP vs cFLIPCleft 1 Edith Chan WIBR

2. cFLIP – sequence and structure • One of our objectives in this project is to find binders for the p22-cFLIP IKKg interaction. • Sequence identity between the vFLIP and cFLIP is low, about 30%. However, p22-cFLIP has been shown to be a strong activator for NF-kB. • cFLIP homology model was built using Modeler and this 3D structure has confirmed that the two binding clefts can be clearly identified and the mutation of residues can be accommodated easily within the structure. Green – conserved residues Cyan – binding sites Magenta – insertion/deletion of MC159

3. Cleft1 Cleft2 vFLIP – Clef1 review • SURFNET locates two clefts on the protein surface - Cleft1 and Cleft2. • In Cleft1, mainly F238, D242, and K246 from IKKg interact with vFLIP, with F238 reaching the deepest pocket. • In Cleft2, Q236 and E240 are pointing into the pocket. Binding site relatively more shadow. • Cleft1 is relatively deeper than Cleft2, making it a better target for molecular design.

4. vFLIP vs cFLIP - Cleft1 sequence • Below shown the residues that are involved in interaction with IKKg. • Two residues are conserved – A57 and H83. • Mutation study on vFLIP • A57L has impaired the forming of the complex and the activation of IKKg. • P54G shows a reduction in affinity • Y90F retains affinity • Mutation study on IKKg • D242R mutant has rendered IKKg incapable of forming a complex. 53 57 79 83 87 90 Mutation GL F vFLIP: FP--A---------------------FL--H---TMSY cFLIP: VG--A---------------------AV--H---NPHL 52 56 78 82 86 89 Mutation done on vFLIP A-L impaired effect P-G reduction in affinity Y-F retain affinity

5. Y90(v) L89(c) L80(v) V79(c) S89(v) H88(c) F238 Yellow cFLIp Red vFLIP Size of Cleft1 • The proposed Cleft1 binding site of cFLIP is larger than vFLIP, especially in two areas. • The area around where F238 binds is much deeper in cFLIP • L80(v) to V79(c): Leu sidechain is larger than Val. • Similarly, a slightly larger pocket around where D242/K246 binds, although somewhat near the surface of the binding site • S89(v) to H88(c) and Y90(v) to L89(c) • Both of these may imply cFLIP can accommodate bigger groups.

6. vFLIP cFLIP P54 G53 Y90 L89 P87 N86 H82 M88 T87 H83 Pharmacophore – HA • In both proteins, HA interactions (e.g. CO2-, C=O) are localized around where Asp242 binds due to His82/Y90 or His82/Asn86. • Since His, Tyr, and Asn are also capable of HD and HA, HD pharmacophore is also detected at that region, although the contribution is much smaller at cFLIP. Red sphere - HA and negative charge, Blue sphere - HD and positive charge

7. vFLIP cFLIP P54 G53 Y90 L89 P87 N86 H82 M88 T87 H83 Pharmacophore - HD • In vFLIP, HD interactions (N-H, sp3 N) can be found in two regions: • lys246 binds (C=O from Met88) • Near pro54 (C=O) - small probe • In cFLIP, HD interaction is near Gly53 (from C=O) – small probe • The loss of a HD contribution around where lys246 binds is due to a change from M88(vFLIP) to P87(cFLIP). The backbond C=O is no longer available from Pro87. Red sphere - HA and negative charge, Blue sphere - HD and positive charge

8. vFLIP cFLIP P54 F53 G53 V52 L89 Y90 A56 A57 P87 N86 H82 V79 A78 M88 T87 H83 L80 F79 Pharmacophore - Hydrophobic • sp3 C (hydrophobic/lipophlic interaction) and sp2 C (aromatic interaction) are all favourable in both proteins. • However, aromatic is slightly favourable near where Phe binds at vFLIP (due to aromatic residues), while hydrophobic is slightly favourable in the cFLIP binding site. Green sphere – sp3 type C, Purple sphere – sp2 type C aromatic

9. Cleft 1 - Summary

10. Compounds to buy • Aryl sulfonamides • Isoindolinones – have not found suitable compounds to buy yet (ChemDiv) • Nutlins – wait for more compounds to be ordered at the same time

11. Sulfonamides • ChemBridge • 1mg or 5mg - $38 • 10 compounds selected.