This review provides a detailed examination and comparison of the diverse non-covalent albumin-binding ligands developed until now to extend the half-life of different small biotherapeutics.

The current trend dealing with the production of per- and polyfluoroalkyl substances (PFASs) involves the shifting toward branched short-chain fluorinated compounds known as new-generation PFASs. A key aspect to be clarified, to address the adverse health effects associated with the exposure to PFASs, is their binding mode to human serum albumin (hSA), the most abundant protein in plasma. In this study, we investigated the interaction between hSA and two representative branched short-chain PFASs, namely, HPFO-DA and C6O4. In-solution studies revealed that both compounds bind hSA with affinities and stoichiometries lower than that of the legacy long-chain perfluoroalkyl compound PFOA. Competition experiments using hSA-binding drugs with known site-selectivity revealed that both HPFO-DA and C6O4 bound to pockets located in subdomain IIIA. The crystal structure of hSA in complex with HPFO-DA unveiled the presence of two binding sites. The characterization and direct comparison of hSA interactions with new-generation PFASs may be key elements for the understanding of the toxicological impact of these compounds.

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (K_i = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (K_d MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target.

Genetically encoded peptides possess unique properties, such as a small molecular weight and ease of synthesis and modification, that make them suitable to a large variety of applications. However, despite these favorable qualities, naturally occurring peptides are often limited by intrinsic weak binding affinities, poor selectivity and low stability that ultimately restrain their final use. To overcome these limitations, a large variety of in vitro display methodologies have been developed over the past few decades to evolve genetically encoded peptide molecules with superior properties. Phage display, mRNA display, ribosome display, bacteria display, and yeast display are among the most commonly used methods to engineer peptides. While most of these in vitro methodologies have already been described in detail elsewhere, this review describes solely the yeast surface display technology and its valuable use for the evolution of a wide range of peptide formats.