Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

Martin, Heather L.; Bedford, Robert; Heseltine, Sophie J; Tang, Anna A; Haza, Katarzyna Z.; Rao, Ajinkya; McPherson, Michael J.; Tomlinson, Darren C.

doi:10.1016/j.nbt.2018.02.008

Cited by 22 publications

(13 citation statements)

References 79 publications

(151 reference statements)

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“…In this case, we have used an AlphaScreen in vitro assay but other direct interaction assay such as FRET could be envisaged. We have used an intracellular antibody fragment in this case but similar approaches could be used for other macromolecules such as affimers 16 , monobodies 17 or DARPins 18 . An advantage of antibodies or antibody fragments is that dematuration does not require structural information as the primary sequence identifies the CDRs for mutagenesis and glycine/alanine scanning can be undertaken with just the knowledge of the primary antibody sequence.…”

Section: Discussionmentioning

confidence: 99%

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody

Tanaka

Thomas

Montfort

et al. 2021

Sci Rep

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Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

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Section: Discussionmentioning

confidence: 99%

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody

Tanaka

Thomas

Montfort

et al. 2021

Sci Rep

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“…cyclin E and c-Myc) [37]. In these and additional studies [5,6,38], the ubiquitin scaffold was demonstrated to be a versatile tool to inhibit Ub/enzyme complexes and test the biological functions of particular DUBs and Ubligases.…”

Section: Dub Inhibition Leads To Degradation Of Oncogenes and Stabilimentioning

confidence: 99%

“…The past twenty years of research have generated a diverse set of PPI inhibitors, including proteins [5][6][7] and small molecules [8][9][10][11][12] that act through orthosteric or allosteric mechanisms. Current questions can now focus on the effect of these PPI modulators on the broader networks.…”

Section: Introductionmentioning

confidence: 99%

Modulating protein–protein interaction networks in protein homeostasis

Zhong

Lee

Sijbesma

et al. 2019

Current Opinion in Chemical Biology

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Protein-protein interactions (PPIs) occur in complex networks. These networks are highly dependent on cellular context and can be extensively altered in disease states such as cancer and viral infection. In recent years, there has been significant progress in developing inhibitors that target individual PPIs either orthosterically (at the interface) or allosterically. These molecules can now be used as tools to dissect PPI networks. Here, we review recent examples that highlight the use of small molecules and engineered proteins to probe PPIs within the complex networks that regulate protein homeostasis. Researchers have discovered multiple mechanisms to modulate PPIs involved in host/viral interactions, deubiquitinases, the ATPase p97/VCP, and HSP70 chaperones. However, few studies have evaluated the effect of such modulators on the target's network or have compared the biological implications of different modulation strategies. Such studies will have an important impact on next generation therapeutics.

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“…An alternative stabilization strategy is to graft peptides onto a robust protein scaffold. These scaffolded peptides can readily facilitate detection and modulation of aberrant interactions, crucial for understanding underlying biological processes and also have great therapeutic potential ( Binz and Plückthun, 2005 ; Weidle et al , 2013 ; Reverdatto et al , 2015 ; Tiede et al , 2017 ; Martin et al , 2018 ). Protein scaffolds under active development have expanded beyond conventional antibody fragments to include other non-antibody alternatives such as affimers, lipocalins, DARPins, kunitz domains, knottins and fibronectins.…”

Section: Introductionmentioning

confidence: 99%

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function

Sobota

et al. 2018

Protein Engineering, Design and Selection

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Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

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Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

Cited by 22 publications

References 79 publications

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody

Modulating protein–protein interaction networks in protein homeostasis

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function

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