1 Design and Development of Stapled Transmembrane Peptides That

Design and development of stapled transmembrane peptides that disrupt the activity of G-protein coupled receptor oligomers Joaquín Botta1,2, Lucka Bibic2, Patrick Killoran3, Peter J. McCormick*1 and Lesley A. Howell*4 From the 1Centre for Endocrinology, William Harvey Research Institute, Bart’s and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ; 2School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ; 3School of Pharmacy and Biomolecular Sciences, James Parsons Building, Byrom Street, Liverpool, L3 3AF; 4School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS Running Title: Stapled TM peptides modulate GPCR oligomers * To whom correspondence should be addressed: Lesley A. Howell: School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS; [email protected]; Tel. (+44) 207 8826625 or Peter J. McCormick: Centre for Endocrinology, William Harvey Research Institute, Bart’s and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ; [email protected]. Keywords: G protein‐coupled receptor (GPCR), cannabinoid receptor type 1 (CB1), dimerization, cell‐ penetrating peptide (CPP), peptide chemical synthesis, serotonin receptor type 2A (5HT2A), NanoLuc binary technology (NanoBiT), hydrocarbon stapling, transmembrane peptide, bimolecular fluorescence complementation assessments, we developed a NanoLuc binary ABSTRACT technology (NanoBiT)-based reporter assay to Membrane proteins can associate into screen a small library of aryl-carbon–stapled larger complexes. Examples include receptor transmembrane mimicking peptides produced by tyrosine complexes, ion channels, transporters solid-phase peptide synthesis. We found that and G-protein coupled receptors (GPCRs). For these stapling peptides have increased α-helicity the latter, there is abundant evidence indicating and improved proteolytic resistance without any that GPCRs, assemble into complexes, through loss of disrupting activity in vitro, suggesting both homo or heterodimerization. However, the that this approach may also have utility in vivo. tools for studying and disrupting these In summary, our results provide proof of complexes, GPCR or otherwise, are limited. concept for using NanoBiT to study membrane Here we have developed stabilized interference protein complexes and for stabilizing disrupting peptides for this purpose. We have previously peptides to target such membrane complexes reported that tetrahydrocannabinol-mediated through hydrocarbon-mediated stapling. We cognitive impairment arises from homo- or propose that these peptides could be developed hetero-oligomerization between the GPCRs to target previously un-druggable GPCR cannabinoid receptor type 1 (CB1R) and 5- heteromers. hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1–5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP The discovery that G-protein coupled measurements, bimolecular fluorescence receptors (GPCRs) could oligomerize, termed complementation assays, and CD-based helicity homo or heterodimerization, sparked an intense 1 Stapled TM peptides modulate GPCR oligomers debate that has moved on from not if they can overcome these limitations; the bioactive but to why, how, how frequently and how do we conformation of the peptide is maintained, and target them for therapeutic purposes. Several X- careful positioning and choice of staple can ray resolved GPCRs crystal structures have result in a high affinity binder with improved revealed common dimeric interfaces stabilizing cellular uptake and stability. Stapled peptides oligomeric arrangements within the Rhodopsin- therefore represent an attractive approach to like family receptors. Dimers having an interface developing more drug-like peptides. For a involving the transmembrane (TM) domains recent review on stapled peptides see Ali et al TM1, TM2 and H8 appear to be a commonly (15). We have previously shown that the conserved organization, including the structures undesired effect of cognitive impairment in the of the rhodopsin, opsin, metarhodopsin II, μ and presence of trans-Δ9-tetrahydrocannabinol κ opioid, and the β1 adrenergic receptors (1–6). (THC) is driven by homo/hetero-oligomerization An additional interface involving the TM4 and between CB1 and 5-HT2A receptors (16). To TM5 domains was also shown in the squid disrupt this interaction, we developed a rhodopsin and the β1 adrenergic receptors (2, 7). NanoBiT-based assay for the screening of a Furthermore, the crystal structures of the small library of aryl-hydrocarbon stapled CB1R CXCR4 and μ-opioid receptors revealed a TM5 mimicking peptides to target CB1-5-HT2A substantial buried surface area of 850 Å2 and receptor heteromers. NanoBiT is a luciferase 1,492 Å2, respectively, comprised of the TM5 based complementation assay designed to and TM6 domains (3, 8). Selective disruption of interrogate protein-protein interactions in live dimers using synthetic peptides harbouring the cells (17). Using this approach we found that same amino acid sequence as the interacting TM stapling peptides led to increased α-helicity and domains, has helped to validate and understand improved proteolytic resistance without any loss the functional consequences of receptor of function, suggesting this approach may oligomerization, including the 2 adrenergic, improve these molecules chances in vivo. CXCR4, oxytocin and apelin receptors homo- oligomers (9–12). Synthetic peptides have also Results provided unique tools to map the interfaces and Establishing the NanoBiT system for understand the biological relevance of Class A heteromer screening purposes GPCRs heteromerization (13, 14). An additional The CB1 and 5-HT2A receptor heteromer advantage of TM peptides is that, unlike knock- has been recently characterized both in vivo and out studies or deletions, the peptides preserve in heterologous expression systems using a the functional single protomers and allow one to broad range of biochemical approaches (16). In discriminate between those effects driven by the the case of these heteromers, the cognitive interacting receptors and those derived from the impairment induced by THC is abrogated after individual protomers. An example of the former treatment with CB1R TM5 peptides, while is the in vivo disruption of the cross-class maintaining its antinociceptive properties (16). angiotensin receptor subtype 1a (AT1aR) and Therefore, for therapeutic purposes there needs secretin receptor heteromers with a TM1 AT1aR to be prevention of heteromer formation but mimetic peptide, reducing hyperosmolality- preservation of the individual protomer’s induced drinking behaviour (13). function. Thus, we first sought to develop a In cases where heteromer disruption NanoBiT-based assay for the screening of a might serve a therapeutic purpose, it would be small library of hydrocarbon stapled CB1R TM5 advantageous to translate TM peptides not only mimicking peptides. To assess whether as tools, but into drug-like entities. Peptides in NanoBiT BiLC may be a suitable system to general are considered poor drug like molecules, study GPCR oligomerization, we generated although this view is changing. Efficacy is often CB1R and 5-HT2AR constructs with the small compromised in vivo due to a loss of secondary and large BiT pairs (SmBiT and LgBiT, structure; cellular uptake is poor and finally respectively) attached to the C-terminus end of peptides are highly susceptible to proteolysis. both receptors. A total of four fusion proteins Stapling of the peptide backbone can help to were generated, with Sm/LgBiT fused after the 2 Stapled TM peptides modulate GPCR oligomers Gly/Ser rich flexible linker and under the control (CMV) promoter. Interestingly, all 5-HT2AR of the herpes simplex virus thymidine kinase constructs in an HSV-TK context failed to elicit gene promoter (HSV-TK) (Figure S1A). Next, intracellular calcium release (canonical we performed conformational screenings to signalling pathway downstream to the Gq/11- assess the optimal configuration for all receptor coupled 5-HT2AR) after agonist stimulation (18). pairs. Accordingly, when analysing CB1R-5- However, both 5-HT2AR-LgBiT and 5-HT2AR- HT2AR heteromers, HEK293 cells were SmBiT displayed similar efficacies and transiently transfected with all possible potencies in comparison to the wild type (WT) combinations of 5-HT2AR Lg/SmBiT and CB1R receptor when expressed under the control of the Lg/SmBiT at two different DNA ratios (Figure CMV promoter (Figure S2A). Similarly, we S1B). Surprisingly, none of the analysed assessed CB1R-driven adenylate cyclase (AC) configurations yielded a positive interaction. inhibition with analogous results. Both CMV- Similarly, when addressing the formation of regulated CB1R constructs inhibited the CB1R and 5-HT2AR homodimers, none of the Forskolin (FK)-induced cAMP release with examined orientations nor DNA ratios exhibit equivalent potencies and efficacies to the WT significant differences in comparison with the CB1R receptor. However, under the HVS-TK individual receptors when expressed by promoter, CB1R-LgBiT failed to signal through themselves (Figures S1C and D). Importantly, to heterotrimeric Gi/o proteins and CB1R SmBiT rule out whether these negative results might exhibited

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