Nanobody Loop Mimetics Enhance Son of Sevenless 1-Catalyzed Nucleotide Exchange on RAS.

RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active guanosine triphosphate (GTP)-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14 (Nb14), which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of Nb14 with an EC50 value of 29 µM. Altogether, this study demonstrated that peptides able to modulate a protein-protein interaction can be obtained by structural mimicry of a Nb paratope.

Nature-Inspired Dual Purpose Strategy toward Cell-Adhesive PCL Networks: C(-linker-)RGD Incorporation via Thiol-ene Crosslinking.

In an attempt to mimic nature's ability to adhere cells, PCL is often coated with nature-derived polymers or its surface is functionalized with a cell-binding motif. However, said surface modifications are limited to the material's surface, include multiple steps, and are mediated by harsh conditions. Here, we introduce a single-step strategy toward cell-adhesive polymer networks where thiol-ene chemistry serves a dual purpose. First, alkene-functionalized PCL is crosslinked by means of a multifunctional thiol. Second, by means of a cysteine coupling site, the cell-binding motif C(-linker-)RGD is covalently bound throughout the PCL networks during crosslinking. Moreover, the influence of various linkers (type and length), between the cysteine coupling site and the cell-binding motif RGD, is investigated and the functionalization is assessed by means of static contact angle measurements and X-ray photoelectron spectroscopy. Finally, successful introduction of cell adhesiveness is illustrated for the networks by seeding fibroblasts onto the functionalized PCL networks.

Propargylamine Amino Acids as Constrained Nε-Substituted Lysine Mimetics.

Herein, alkylated propargylamines are reported as constrained lysine mimetics and constructed in a single step using a copper(I)-catalyzed A3-coupling reaction. Using multiple secondary amines, the reaction allowed the generation of a structurally diverse set of N-Fmoc protected amino acid derivatives. In addition, the A3-reaction was applied on solid phase via the assembly of short model tripeptides. Moreover, the internal alkyne moiety allowed further functionalization toward novel 1,4,5-trisubstituted 1,2,3-triazole-based amino acids.

Downsizing antibodies: Towards complementarity-determining region (CDR)-based peptide mimetics.

Monoclonal antibodies emerged as an important therapeutic drug class with remarkable specificity and binding affinity. Nonetheless, these heterotetrameric immunoglobulin proteins come with high manufacturing and therapeutic costs which can take extraordinary proportions, besides other limitations such as their limited in cellulo access imposed by their molecular size (ca. 150 kDa). These drawbacks stimulated the development of downsized functional antibody fragments (ca. 15-50 kDa), together with smaller synthetic peptides (ca. 1-3 kDa) derived from the antibodies' crucial complementarity-determining regions (CDR). Despite the general lack of success in the literal translation of CDR loops in peptide mimetics, rational structure-based and computational approaches have shown their potential for obtaining functional CDR-based peptide mimetics. In this review, we describe the efforts made in the development of antibody and nanobody paratope-derived peptide mimetics with particular focus on the used design strategies, in addition to highlighting the challenges associated with their development.

Structure-Based Design of Melanocortin 4 Receptor Ligands Based on the SHU-9119-hMC4R Cocrystal Structure†.

The melanocortin receptors (MC1R-MC5R) belong to class A G-protein-coupled receptors (GPCRs) and are known to have receptor-specific roles in normal and diseased states. Selectivity for MC4R is of particular interest due to its involvement in various metabolic disorders, including obesity, feeding regulation, and sexual dysfunctions. To further improve the potency and selectivity of MC4R (ant)agonist peptide ligands, we designed and synthesized a series of cyclic peptides based on the recent crystal structure of MC4R in complex with the well-characterized antagonist SHU-9119 (Ac-Nle4-c[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2). These analogues were pharmacologically characterized in vitro, giving key insights into exploiting binding site subpockets to deliver more selective ligands. More specifically, the side chains of the Nle4, DNal(2')7, and Trp9 residues in SHU-9119, as well as the amide linkage between the Asp5 and Lys10 side chains, were found to represent structural features engaging a hMC4R/hMC3R selectivity switch.

The 1,3-diyne linker as a rigid "i,i+7" staple for α-helix stabilization: Stereochemistry at work.

Short alphahelical peptide sequences were stabilized through Glaser-Hay couplings of propargylated l- and/or d-serine residues at positions i and i+7. NMR analysis confirmed a full stabilization of the helical structure when a d-Ser (i), l-Ser (i+7) combination was applied. In case two l-Ser residues were involved in the cyclization, the helical conformation is disrupted outside the peptide's macrocycle.

Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.