15 research outputs found
Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries
We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 105 per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cell
Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries
We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 105 per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cell
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Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites
Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies
The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19)
Hypothesis A novel strategy to design binding molecules harnessing the modular nature of repeat proteins
Abstract Repeat proteins, such as ankyrin or leucine-rich repeat proteins, are ubiquitous binding molecules, which occur, unlike antibodies, intra- and extracellularly. Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target-binding surfaces. Based on this modularity, we developed a novel strategy to generate combinatorial libraries of polypeptides with highly diversi¢ed binding speci¢cities. This strategy includes the consensus design of self-compatible repeats displaying variable surface residues and their random assembly into repeat domains. We envision that such repeat protein libraries will be highly valuable sources for novel binding molecules especially suitable for intracellular applications
Designing Repeat Proteins: Modular Leucine-rich Repeat Protein Libraries Based on the Mammalian Ribonuclease Inhibitor Family
r as characterized by gel-filtration, and a-helical CD spectra, confirming the success of our design approach. q 2003 Elsevier Ltd. All rights reserved. Keywords: combinatorial library; consensus sequence; leucine-rich repeat; protein design; ribonuclease inhibitor *Corresponding author Introduction The search for protein frameworks with binding properties similar to antibodies was initiated once technologies for synthetic library generation and selection systems became available. In the past decade, many alternative framework libraries have been generated. A number of desirable properties for novel designed binding molecules can be defined: Besides high thermodynamic stability and high-level soluble expression in bacteria, the stability of designed binding molecules should be independent of disulfide bonds in order to work in intracellular and extracellular environments. In addition, designed binding molecules should possess a sufficiently large surface suitable for target bin
Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins
scaffold *Corresponding author Introduction Repeat proteins mediate numerous key protein -- protein interactions in nature. 1,2 Their repetitive architecture permits the adaptation of their size and thus their variable and modular binding surface to a target protein, leading to high-affinity interactions. We developed a strategy that exploits this modular architecture for the generation of combinatorial libraries of repeat proteins with novel binding specificities (Figure 1). Our strategy consists of designing a self-compatible repeat module for a given repeat type. In such a repeat module, residues important for maintaining the repeat structure (i.e. framework residues) are defined, while potential target interaction residues are randomized. Such self-compatible repeat modules can then be joined repetitively to yield a stack of repeats. To form repeat domains, the continuous hydrophobic core of this stack is sealed by N and C-terminal capping repeats (Figure 1). We hypothesiz