NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay

Peier, Andrea; Ge, Lan; Boyer, Nicole; Frost, John R.; Duggal, Ruchia; Biswas, Kaustav; Edmondson, Scott D.; Hermes, Jeffrey D.; Yan, Lin; Zimprich, Chad; Sadruddin, Ahmad; Kaan, Hung Yi Kristal; Chandramohan, Arun; Brown, Christopher J.; Thean, Dawn; Lee, Xue Er; Yuen, Tsz Ying; Ferrer-Gago, Fernando J.; Johannes, Charles W.; Lane, David P.; Sherborne, Brad; Corona, Cesear; Robers, Matthew B.; Sawyer, Tomi K.; Partridge, Anthony W.

doi:10.1021/acschembio.0c00804

Cited by 54 publications

(63 citation statements)

References 70 publications

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“…Accordingly, we sought to identify a peptide that maintained KRAS binding affinity but with a redox insensitive cross-link. Using a template with an N-terminal 6-azido-L-lysine residue, to enable cellular permeability measurements with the NanoClick assay [16], we explored a series of disulfide bridge replacements (Table 1). To assess the peptide binding affinities in high-throughput format, we used a TR-FRET assay employing GDP-loaded KRAS G12D and a FAM-labeled KRpep-2d family member tracer peptide (see Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Lim¹,

Boyer

Boo³

et al. 2021

Preprint

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RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C- terminal arginine residues with D-arginines and introduction of an α- methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP- 3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non- binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS.

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Section: Resultsmentioning

confidence: 99%

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Lim¹,

Boyer

Boo³

et al. 2021

Preprint

Self Cite

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“…Adding 6-azido-L-lysine (AzK) (8) or substituting Ser with AzK (9), at a position close to the N-terminus, did not affect their binding affinity as compared to 7. The freedom to incorporate AzK allowed us to introduce a bioorthogonal reactive handle for NanoClick 40 a cellular assay for measuring peptide permeability (vide infra). Cyclization often enhances the affinity of linear peptides, owing to reduced conformational freedom and a smaller entropic penalty paid upon target binding.…”

Section: Structure-activity Relationship Of Peptidementioning

confidence: 99%

“…Accordingly, we employed NanoClick, a target-agnostic assay developed previously, to measure the cellular uptake of molecules via different mechanisms, including but not limited to endocytosis, membrane translocation, and passive permeability. 40 This assay quantifies an azide-containing test compound, e.g., peptide, such that it will pre-compete with a permeable azide-containing dye molecule which covalently labels an intracellular protein sensor in HeLa cells. Accordingly, test compounds with higher permeability in NanoClick will exhibit smaller EC50 value in competing off the permeable dye molecule.…”

Section: Cellular Permeability Of Macrocyclic Peptidesmentioning

confidence: 99%

Discovery and Structure-Based Design of Macrocyclic Peptides Targeting STUB1

Ng¹,

Brueckner

Bahmanjah

et al. 2021

Preprint

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STIP1 homology and U-Box containing protein 1 (STUB1) plays a key role in maintaining cell health during stress and aging. Recent evidence suggested STUB1 also helps regulate immunity with the potential of clearing malignant cells. Indeed, we and others have shown that STUB1 is a pivotal negative regulator of interferon gamma sensing – a process critical to the immunosurveillance of tumors and pathogens. Thus far, investigation of STUB1’s role relies mostly on genetic approaches as pharmacological inhibitors of this protein are lacking. Identification of a STUB1 tool compound is important as it would allow therapeutically relevant target validation in a broader sense. Accordingly, we leveraged phage display and computational modeling to identify and refine STUB1 binders. Screening of >10E9 macrocyclic peptides resulted in several conserved motifs as well as structurally diverse leads. Co-crystal structure of the peptide hit and STUB1 has enabled us to employ structure-based in silico design for further optimization. Of the modifications employed, replacing the hydrophilic solvent-exposed region of the macrocyclic peptides with a hydrophobic scaffold improved cellular permeability, while the binding conformation was maintained. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to certain extent while improving permeability, suggesting a path forward. The current lead, although not optimal for cellular study, provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.

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“…Existing methods to quantify uptake into cells include fluorescence, immunoblot, and mass spectrometry, but it is still challenging to distinguish between endosomal and cytosolic localization. Several assays can differentiate between endosomal and cytosolic localization using indirect quantification via a readout generated by a delivered cargo, including the chloroalkane penetration assay (CAPA), 28 GFP complementation assays, 29 and more recently the NanoClick 30 assay and SLEEQ 31 assay. Direct quantification of the cytosolic concentration of a fluorescentlylabeled protein construct is possible using fluorescence correlation spectroscopy.…”

Section: Introductionmentioning

confidence: 99%

Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

Schissel

Farquhar

Malmberg

et al. 2021

Preprint

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Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise non-penetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMO) resulted in higher muscle concentration, increased exon-skipping and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical L-peptides. We hypothesized that mirror-image D-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities, while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency which may be useful for determining which peptide sequence to pursue in further pre-clinical studies.

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NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay

Cited by 54 publications

References 70 publications

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Discovery and Structure-Based Design of Macrocyclic Peptides Targeting STUB1

Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

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