Kilian Colas

Peptide lasso-grafting strategies enable the transfer of established pharmacophores into compact protein scaffolds but often require optimization to preserve target binding and biological activity. Here, we report Random in-vitro Protein single-Loop Engineering (RiPLE), an mRNA-display strategy for the direct discovery of engineered ubiquitin binders, termed U-bodies, from a library containing a fully randomized surface loop of variable length. Using receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a model target, we generated U-bodies through RiPLE and compared them with lasso-grafted U-bodies derived from a previously identified ROR1-binding macrocyclic peptide. Both approaches yielded U-bodies bearing single-digit nanomolar affinity to ROR1; however, RiPLE converged on de novo binding sequences that were distinct from the parental peptide. Notably, most selected U-bodies exhibited strong selectivity for ROR1 over the closely related receptor ROR2, whereas the parental peptide bound both receptors with high affinity. Representative ROR1-binding U-bodies also inhibited wound closure in ROR1-expressing MDA-MB-231 cells without detectable cytotoxicity. Applying RiPLE to the heterotrimeric G-protein subunit (GαS) similarly yielded high-affinity binders and revealed sequence convergence patterns distinct from lasso-grafting. Together, these results establish RiPLE as a powerful and complementary alternative to peptide-guided lasso-grafting, enabling de novo evolution of functional binding surfaces directly within their final protein scaffold.

We report the de novo discovery of a cyclic peptide inhibitor of ROR1/2 from the RaPID (random nonstandard peptides integrated discovery) system. Multiple independent affinity selection campaigns converged on a highly conserved family of sequences exhibiting single-digit nanomolar binding affinities to ROR1. One peptide from this family containing three nonproteinogenic residues, including a key cyclic β-amino acid, displayed pronounced activity in cellular assays. Strikingly, this peptide induced potent growth inhibition across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines, whereas other family members had no measurable cellular effects. Mechanistic analysis revealed that the active peptide uniquely competes with Wnt5a, the endogenous ligand of ROR1, for receptor binding. To our knowledge, this report provides the first evidence implicating ROR1/2 as a viable therapeutic target in DIPG and establishes macrocyclic peptides as a promising modality for its pharmacological inhibition.

We report the de novo discovery of a cyclic peptide inhibitor of ROR1 from the RaPID (Random nonstandard Peptides Integrated Discovery) system. Multiple independent affinity selection campaigns converged on a highly conserved family of sequences exhibiting single digit nanomolar binding affinities to ROR1. One peptide from this family containing three non-proteinogenic residues, including a key cyclic β-amino acid, displayed pronounced activity in cellular assays. Strikingly, this peptide induced potent growth inhibition across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines, whereas other family members had no measurable cellular effects. Mechanistic analysis revealed that the active peptide uniquely competes with Wnt5a, the endogenous ligand of ROR1, for receptor binding. To our knowledge, this report provides the first evidence implicating ROR1 as a visible therapeutic target in DIPG and establishes macrocyclic peptides as a promising modality for its pharmacological inh

Technological advances and breakthrough developments in the pharmaceutical field are knocking at the door of the "undruggable" fortress with increasing insistence. Notably, the 21st century has seen the emergence of macrocyclic compounds, among which cyclic peptides are of particular interest. This new class of potential drug candidates occupies the vast chemical space between classic small-molecule drugs and larger protein-based therapeutics, such as antibodies. As research advances toward clinical targets that have long been considered inaccessible, macrocyclic peptides are well-suited to tackle these challenges in a post-rule of 5 pharmaceutical landscape. Facilitating their discovery is an arsenal of high-throughput screening methods that exploit massive randomized libraries of genetically encoded compounds. These techniques benefit from the incorporation of non-natural moieties, such as non- proteinogenic amino acids or stabilizing hydrocarbon staples. Exploiting these features for the strategic architectural design of macrocyclic peptides has the potential to tackle challenging targets such as protein-protein interactions, which have long resisted research efforts. This Review summarizes the basic principles and recent developments of the main high-throughput techniques for the discovery of macrocyclic peptides and focuses on their specific deployment for targeting undruggable space. A particular focus is placed on the development of new design guidelines and principles for the cyclization and structural stabilization of cyclic peptides and the resulting success stories achieved against well-known inaccessible drug targets.

Protein engineering techniques have vastly expanded their domain of impact, notably following the success of antibodies. Likewise, smaller peptide therapeutics have carved an increasingly significant niche for themselves in the pharmaceutical landscape. The concept of grafting such peptides onto larger protein scaffolds, thus harvesting the advantages of both, has given rise to a variety of protein engineering strategies that are reviewed herein. We also describe our own “Lasso‐Grafting” approach, which combines traditional grafting concepts with mRNA display to streamline the production of multiple grafted drug candidates for virtually any target.

Invited for the cover of this issue is the group of Abraham Mendoza at Stockholm University. The image depicts a Grignard reagent “turbo‐charged” with a magnesium anilide additive. Read the full text of the article at 10.1002/chem.202104053.

The synthesis of ketones through addition of organometallic reagents to aliphatic carboxylic acids is a straightforward strategy that is limited to organolithium reagents. More desirable Grignard reagents can be activated and controlled with a bulky aniline‐derived turbo‐Hauser base. This operationally simple procedure allows the straightforward preparation of a variety of aliphatic and perfluoroalkyl ketones alike from functionalized alkyl, aryl and heteroaryl Grignard reagents.

We present an extensive photophysical study of a series of fluorescent indolylbenzothiadiazole derivatives and their ability to specifically image lipid droplets in astrocytes and glioblastoma cells. All compounds in the series displayed positive solvatochromism together with large Stokes shifts, and π-extended derivatives exhibited elevated brightness. It was shown that the fluorescence properties were highly tunable by varying the electronic character or size of the N-substituent on the indole motif. Three compounds proved capable as probes for detecting small quantities of lipid deposits in healthy and cancerous brain cells. In addition, all twelve compounds in the series were predicted to cross the blood–brain barrier, which raises the prospect for future in vivo studies for exploring the role of lipid droplets in the central nervous system.