Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling

Abstract Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo‐crosslinking motif and a peptide stapling reagent. Using double‐click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo‐crosslinking amino acid. When applied to a p53‐derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull‐down assays, and can be used to investigate the target selectivity of stapled peptides.

Photoaffinity labelling is au seful methodf or studying how proteins interactw ith ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We presentt he design and synthesis of a novel multifunctional benzophenonel inker that serves as both ap hoto-crosslinking motif and ap eptides tapling reagent. Using double-click stapling, we attached the benzophenonet o the peptidev ia the staple linker,r ather than by modifying the peptides equence with ap hoto-crosslinking amino acid. When appliedt oap 53-derived peptide, the resultingp hotoreactive stapled peptidew as able to preferentially crosslink with MDM2 in the presence of competingp rotein. This multifunctional linker also features an extra alkyne handle ford ownstream applicationss uch as pull-down assays, and can be used to investigate the target selectivityo fs tapled peptides.
Protein-protein interactions (PPIs) are inextricably involved in ahost of cellular functions, with aberrantactivity linked to avariety of humand iseases. [1] Tool compounds that can probe specific PPIs are vital for unravelling the functions of individual proteins within acomplex protein network, and can potentially identify and characterise new targets for drug development. [2] In particular, compounds that can be used for photoaffinity labelling are powerful tools for studying the interactions of proteins with ligands or other biomolecules. [3] Photoaffinity labelling involves the use of ap hotoactivatable functionality that, on exposure to UV light, can form ac ovalent linkaget ob iomolecules within close proximity. [4] This irreversible process enables subsequent analysisoft he interaction. [5] As part of our work on stapled peptides to inhibit PPIs, we were interested in developing photoaffinity tools to character-ise our peptides and their target PPIs. In peptides tapling, ap romising strategy for designing a-helix mimetic inhibitors, two amino acid side-chains are joined to form am acrocycle. [6] Non-proteogenic amino acids are commonly used for macrocylisation;h owever,t echniques involving all native residues are also available. [7] The resulting peptide is stabilised in ah elical conformation, which can lead to improved bindinga ffinity and pharmacokinetic properties, relative to those of the linear analogues. [8] Walensky and co-workersu sed photoaffinity probes to covalently trap proteins in the BH3/BCL2 complexb yi ncorporatingu nnatural benzophenone-bearing amino acids into hydrocarbon-stapled BH3 peptides. [9] We have previously reported ad ouble-click stapling technique where the peptidea nd linker are two separatec omponents.T his allowed facile modification of the linker to tailor the reactivity of the overall stapled peptide. [8b, 10] In this study,w es ought to incorporate ap hotoactive benzophenone moietyi nto the staplingl inker itself. By installing the photoactive group in the linker the peptide sequence is not modified, thus possibly minimising disruption of the overall binding affinity.W er easoned thatc ombining double-click stapling groups with photoaffinity labelling within one linker could providearapid meanso fa ssessing the target selectivity of stapled peptides as well as the identification of potential off-target interactions. In addition to the double-click stapling motif andp hotoactivatable group, we also designed an extra protected alkyne handle for downstream applications such as pull-downa ssays (Scheme 1). As ap roofo fc oncept, we applied our photolabelling linker to ap 53 peptidef or binding MDM2, aP PI that has received significant attention in anticancer therapeutics. Overexpression of the E3 ubiquitin ligase MDM2i ns ome cancer cell lines leads to loss of function of p53, ac rucial tumour suppressor protein. [11] This in turn can result in uncontrolled abnormal cell growth and subsequent cancer progression.
The novel benzophenone linker 1 was synthesised in four steps (Scheme 2), commencing with aS onogashira coupling of (commercially available) 3,5-dibromobenzaldehyde (2)w ith trimethylsilylacetylene to give the bis-TMS-protected intermediate 3 in good yield. Deprotectiono ft he acetylene groups under basic conditions resulted in the dialkynei ntermediate 4. Nucleophilic addition with pre-prepared (4-((trimethylsilyl)ethylnyl)phenyl)lithium reagent afforded the secondary alcohol 6. Oxidation of the alcohol with Dess-Martin periodinane (DMP) gave the final dialkynyl benzophenone linker 1 in an overall yield of 12 %for the four-step synthesis.
In order to synthesise the photoactive stapled peptide, we followed our previously reported, optimised copper-catalysed double-click method with ap eptides equence (A0)d erived from an alpha-helix in the N-terminal transactivation domain of p53. [8b, 10, 12] This approach involved the installation of azidecontaining non-naturala minoa cids at defined positions (i, i+ +7) within the peptides equence. These azide functionalities were then reactedw itht he bis-alkyne-benzophenone linking unit 1 by coppercatalysis. Formation of the TMS-protected stapled peptide product was seen initially ( Figure S2 in the Supporting Information). Subsequently,f ull TMS deprotection was observeda fter six hours under the click-reaction conditions to yield the desired photoaffinity probe A1 in one step (Scheme 3, Ta ble 1).
We envisioned that incorporation of ab iotin moiety would facilitate future pull-down experiments, so at est click reaction of A1 with commerciallya vailable biotin-PEG3-azidew as carried out (Scheme 3). The click reaction generatedt he expected tris-triazole product C cleanly,asm onitored by HPLC and LCMS (see Sections 2and 5i nt he SupportingI nformation).
Isothermal calorimetry experiments werec arriedo ut to test whether our synthesised photoaffinity probe A1 achieved bindinga ffinity comparable to that of similar stapled analogues (e.g.,t he non-TAMRA-labelled A0 sequence stapled with 1.3-diethynylbenzene; K d = 6.7 AE 2.8 nm). [8b, 10] The binding affinity of A1 for MDM2 was 18 AE 6nm,t hus suggesting that the staple modificationd id not significantly impact target binding. We also synthesised an F3A negative control B1, which was significantly less potent as ar esult of mutating one of the key binding residues to alanine.   The crosslinking ability of A1 was investigated by incubation with recombinantM DM2. Upon UV irradiationa t3 65 nm, successfulc rosslinking to MDM2 was observed by in-gel fluorescence of the TAMRA label after SDS-PAGE (Figure 1). A1 crosslinking was time-dependent over the course of an hour,whereas B1 (F3A controlp eptide) showed no crosslinking to the target (SectionS7).
In order to determine if the observed crosslinking was specific for MDM2, A1 was incubated with mixtures of MDM2 and bovine serum albumin( BSA). Clear bands reflected preferential labellingo fM DM2 by A1 (not B1), and the addition of BSA did not significantly impact the ability of A1 to crosslink to MDM2 (Figure 2).
In summary,wedesigned and synthesised anovel multifunctional linker to serve as both ap eptides tapling reagent and ap hotoaffinity probe with pulldown capability.T he benzophenone linker successfully underwent copper-catalysed doubleclick stapling to generate A1.S ubsequentr eaction of the terminal alkyne on the linker with ab iotinylated azide demonstrated the potential to carry out pull-down assaysw ith the probe.T he binding affinity of the stapled probe was comparable to those of previously studied p53 stapled peptides. Finally, the probe effectively crosslinked with MDM2 after UV irradia-tion, andt he crosslinking was specific for MDM2 over competing BSA. This methodology is currently limited to labelling purified protein and known PPIs. The next step is MDM2 labelling and pulldown in cell lysate or live cells. We envisage that this linker strategy could be applied to study other PPIs and their correspondingpeptidic inhibitors.
Photoaffinity labelling of recombinant MDM2: Am ixture of benzophenone stapled peptide A1 or B1 (0.1 nmol) and recombinant MDM2 (17 nmol) in Tris buffer (100 mL) was incubated for 15 min at RT,a nd then irradiated at 365 nm in aL ongwave Ultraviolet Crosslinker (model CL-1000 L; UVP,U pland, CA) for the indicated time. Irradiated samples were analysed by SDS-PAGE on 4-20 %t ricine gels (Expedeon, San Diego, CA) and visualised by in-gel fluorescence imaging in aT yphoon FLA 9500 (555 nm;G EH ealthcare) and InstantBlue protein staining (Expedeon). Binding specificity experiments with BSA (Fraction V, pH 7.0;G EH ealthcare) were performed as above with the indicated concentrations of BSA in PBS buffer.