Monobodies as tool biologics for accelerating target validation and druggable site discovery

Akkapeddi, Padma; Teng, Kai Wen; Koide, Shohei

doi:10.1039/d1md00188d

Cited by 13 publications

(11 citation statements)

References 166 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Currently, the cell membrane and endosomal entrapment of proteins limit the ability to deliver antibodies intracellularly . Intracellular antibodies have been used as PPI modulators in a few cases, such as the signaling pathway of RAS proteins, and we now show their utility for selectively blocking one PPI of a hub protein that binds to many proteins at the same site. − Development of efficient and robust methods for delivering recombinant antibodies to intracellular PPI targets will further extend the applicability of the technology for biomedical applications, ideally by perturbing the formation of malfunctional protein complexes that are associated with diseases and disorders.…”

Section: Discussionmentioning

confidence: 99%

Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein–Protein Interactions

et al. 2022

View full text Add to dashboard Cite

Protein−protein interactions (PPIs) form complex networks to drive cellular signaling and cellular functions. Precise modulation of a target PPI helps explain the role of the PPI in cellular events and possesses therapeutic potential. For example, valosin-containing protein (VCP/p97) is a hub protein that interacts with more than 30 adaptor proteins involved in various cellular functions. However, the role of each p97 PPI during the relevant cellular event is underexplored. The development of small-molecule PPI modulators remains challenging due to a lack of grooves and pockets in the relatively large PPI interface and the fact that a common binding groove in p97 binds to multiple adaptors. Here, we report an antibody fragment-based modulator for the PPI between p97 and its adaptor protein NSFL1C (p47). We engineered these antibody modulators by phage display against the p97-interacting domain of p47 and minimizing binding to other p97 adaptors. The selected antibody fragment modulators specifically disrupt the intracellular p97/p47 interaction. The potential of this antibody platform to develop PPI inhibitors in therapeutic applications was demonstrated through the inhibition of Golgi reassembly, which requires the p97/p47 interaction. This study presents a unique approach to modulate specific intracellular PPIs using engineered antibody fragments, demonstrating a method to dissect the function of a PPI within a convoluted PPI network.

show abstract

Section: Discussionmentioning

confidence: 99%

Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein–Protein Interactions

et al. 2022

View full text Add to dashboard Cite

show abstract

“…Together, these studies demonstrate that synthetic proteins can be developed to target small-molecule binding sites and vice versa. We envision the utility of Mbs and other synthetic binding proteins as tool biologics for identifying and validating a potential binding site for small molecules and also for establishing drug-screening assays ( 25 ).…”

Section: Discussionmentioning

confidence: 99%

“…Such reagents can be used as functional probes to identify sites on a target for inhibition. For example, synthetic proteins, such as monobodies, have been successfully developed against a diverse array of targets, including RAS, and tend to bind to underappreciated functional sites ( 21 – 28 ). Whereas inhibitors like sotorasib, adagrasib, and MRTX1133 bind near the Switch region of RAS, the monobody (Mb) NS1 marked the first inhibitor to bind to RAS outside of the Switch region ( 27 ).…”

mentioning

confidence: 99%

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket

Wallon

Khan

Teng

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

show abstract

“…Here, we create an adaptable and genetically encoded switch by applying the AFF mechanism to the 10 th domain of FN3, termed monobody, a small protein that has been evolved by in vitro selection methods to specifically bind ∼30 protein targets 14,15 including epidermal growth factor receptor 16 , maltose-binding protein (MBP) 17 , mixed lineage kinase domain-like protein 18,19 , and severe acute respiratory syndrome coronavirus 2 spike protein 20,21 . Like the antibody light chains they resemble, monobodies achieve molecular recognition by presenting different residues at surface positions (1-3 CDR-like loops as well as several βstrands) while maintaining a constant amino acid sequence at other positions 14 . Monobodies have been developed for use in degradation systems 22–25 , light-controlled applications 26,27 , cancer therapeutics 28–30 , and as inhibitors 14,31–33 and biosensors 34,35 .…”

mentioning

confidence: 99%

“…Like the antibody light chains they resemble, monobodies achieve molecular recognition by presenting different residues at surface positions (1-3 CDR-like loops as well as several βstrands) while maintaining a constant amino acid sequence at other positions 14 . Monobodies have been developed for use in degradation systems 22–25 , light-controlled applications 26,27 , cancer therapeutics 28–30 , and as inhibitors 14,31–33 and biosensors 34,35 .…”

mentioning

confidence: 99%

An adaptable, monobody-based biosensor scaffold with FRET output

Presti

Loh

2022

Preprint

View full text Add to dashboard Cite

Protein-based fluorescent biosensors are powerful tools for analyte recognition in vitro and in cells. Numerous proteinaceous binding scaffolds have been developed that recognize ligands with affinity and specificity comparable to those of conventional antibodies, but are smaller, readily overexpressed, and more amenable to engineering. Like antibodies, these binding domains are useful as recognition modules in protein switches and biosensors, but they are not capable of reporting on the binding event by themselves. Here, we engineer a small binding scaffold—a consensus-designed fibronectin 3 monobody—such that it undergoes a conformational change upon ligand binding. This change is detected by Förster resonance energy transfer using chemical dyes or cyan and yellow fluorescent proteins as donor/acceptor groups. By grafting substrate recognition residues from different monobodies onto this scaffold, we create fluorescent biosensors for c-Abl Src homology 2 (SH2) domain, WD40-repeat protein 5 (WDR5), small ubiquitin-like modifier-1 (SUMO), and h-Ras. The biosensors bind their cognate ligands reversibly, with affinities consistent with those of the parent monobodies, and with half times of seconds to minutes. This design serves as generalizable platform for creating a genetically-encoded, ratiometric biosensors by swapping binding residues from known monobodies, with minimal modification.

show abstract

Monobodies as tool biologics for accelerating target validation and druggable site discovery

Abstract: Despite increased investment and technological advancement, new drug approvals have not proportionally increased. Low drug approval rates, particularly for new targets, are linked to insufficient target validation at early stages....

Cited by 13 publications

References 166 publications

Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein–Protein Interactions

Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein–Protein Interactions

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket

An adaptable, monobody-based biosensor scaffold with FRET output

Contact Info

Product

Resources

About