Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

Cheloha, Ross W.; Fischer, Fabian A.; Woodham, Andrew W.; Daley, Eileen; Suminski, Naomi; Gardella, ThomasJ.; Ploegh, Hidde L.

doi:10.26434/chemrxiv.9619823.v1

Cited by 3 publications

(4 citation statements)

References 44 publications

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“…Protein-peptide C-to-C conjugates have previously been generated in multiple steps, relying on transpeptidase-mediated attachment of a click handle and a subsequent click reaction for peptide attachment. [13] Here we do so in a single step, obviating the need for specialized click handle building blocks and intermediate purification steps.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic C‐to‐C Protein Ligation

et al. 2022

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Transpeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions.

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Section: Resultsmentioning

confidence: 99%

Enzymatic C‐to‐C Protein Ligation

et al. 2022

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“…Previous studies involving PTHR1 ligands have demonstrated enhanced potency upon conjugation with Nbs that bind to the receptor. 8 Whether this trend extended to other receptors was unknown prior to these studies. Lastly, we investigated the influence of the Nb epitope's location on the observed signaling properties of Nb-ligand conjugates.…”

Section: Discussionmentioning

confidence: 98%

“…2,4 The linkage of peptide agonists of GPCRs with Nbs can yield conjugates with improved signaling potency and selectivity compared to free peptides. 8 For example, the conjugation of weakly active fragments of parathyroid hormone (PTH) to nanobodies that bound the type-1 PTH-receptor (PTHR1), or epitope tags incorporated therein, provided conjugates with significantly improved biological potencies (up to 7800-fold). 8,9 These conjugates also showed improved specificity for a single receptor subtype relative to the index ligand from which they were derived.…”

Section: Introductionmentioning

confidence: 99%

Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1

Emidio,

Cheloha

2023

Preprint

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Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands. The neurokinin-1 receptor (NK1R), a GPCR targeted for the treatment of pain, is activated by peptide agonists such as Substance P (SP) and neurokinin A (NKA), which induce signaling through multiple pathways (Gs, Gqand β-arrestin). In this study, we investigated whether conjugating NK1R ligands with Nbs that bind to a separate location on the receptor would provide chimeric compounds with distinctive signaling properties. We employed sortase A-mediated ligation to generate several conjugates consisting of Nbs linked to NK1R ligands. Many of these conjugates exhibited divergent and unexpected signaling properties and transcriptional outputs. For example, some Nb-NKA conjugates showed enhanced receptor binding capacity, high potency partial agonism, prolonged cAMP production, and an increase in transcriptional output associated with Gssignaling; whereas other conjugates were virtually inactive. Nanobody conjugation caused only minor alterations in ligand-induced upstream Gqsignaling with unexpected enhancements in transcriptional (downstream) responses. Our findings underscore the potential of nanobody conjugation for providing compounds with advantageous properties such as biased agonism, prolonged duration of action, and enhanced transcriptional responses. These compounds hold promise not only for facilitating fundamental research on GPCR signal transduction mechanisms but also for the development of more potent and enduring therapeutics.

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“…Besides direct inhibition, nanobodies have been used to investigate antigen function by targeted proteasomal degradation via recruitment of the cellular ubiquitination machinery [12–16] or by controlling the antigen‘s subcellular localisation [3,4,7,8,17–20] . The binding specificity of nanobodies and ease of intracellular expression has been exploited to improve ligand affinity to receptors for observation of rare binding events [21] . Additionally, nanobodies have been used to deliver photo‐switchable ligands to receptors for optical control of receptor activation [22] .…”

Section: Introductionmentioning

confidence: 99%

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering**

et al. 2022

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Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

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Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

Abstract: Here we describe a new method for improving signalling potency and selectivity for suboptimal GPCR ligands through conjugation with receptor-targeting nanobodies<br>

Cited by 3 publications

References 44 publications

Enzymatic C‐to‐C Protein Ligation

Enzymatic C‐to‐C Protein Ligation

Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering**

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