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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.

The ability of cross-reactive antibodies to bind multiple related or unrelated targets derived from different species provides not only superior therapeutic efficacy but also a better assessment of treatment toxicity, thereby facilitating the transition from preclinical models to human clinical studies. This chapter provides some guidelines for the directed evolution of cross-reactive antibodies using yeast surface display technology. Cross-reactive antibodies are initially isolated from a naïve library by combining highly avid magnetic bead separations followed by multiple cycles of flow cytometry sorting. Once initial cross-reactive clones are identified, sequential rounds of mutagenesis and two-pressure selection strategies are applied to engineer cross-reactive antibodies with improved affinity and yet retained or superior cross-reactivity.

Human serum albumin (hSA) is the major carrier protein for fatty acids (FAs) in plasma. Its ability to bind multiple FA moieties with moderate to high affinity has inspired the use of FA conjugation as a safe and natural platform to generate long-lasting therapeutics with enhanced pharmacokinetic properties and superior efficacy. In this frame, the choice of the FA is crucial and a comprehensive elucidation of the molecular interactions of FAs with hSA cannot be left out of consideration. To this intent, we report here a comparative analysis of the binding mode of different FA moieties with hSA. The choice among different albumin-binding FAs and how this influence the pharmacokinetics properties of a broad spectrum of therapeutic molecules will be discussed including a critical description of some clinically relevant FA conjugated therapeutics.

Gemfibrozil (GEM) is an orally administered lipid-regulating fibrate derivative drug sold under the brand name Lopid®, among others. Since its approval in the early 80s, GEM has been largely applied to treat hypertriglyceridemia and other disorders of lipid metabolism. Though generally well tolerated, GEM can alter the distribution and the free, active concentration of some co-administered drugs, leading to adverse effects. Most of them appear to be related to the ability of GEM to bind with high affinity human serum albumin (HSA), the major drug-carrier protein in blood plasma. Here, we report the crystal structure of HSA in complex with GEM. Two binding sites have been identified, namely Sudlow’s binding sites I (FA7) and II (FA3–FA4). A comparison of the crystal structure of HSA in complex with GEM with those of other previously described HSA–drug complexes enabled us to appreciate the analogies and differences in their respective binding modes. The elucidation of the molecular interaction between GEM and HSA might offer the basis for the development of novel GEM derivatives that can be safely and synergistically co-administered with other drugs, enabling augmented therapeutic efficacies.

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium-chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA-hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA-binding drugs allowed locating the high affinity binding site in sub-domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.

Peptides and small protein scaffolds are gaining increasing interest as therapeutics. Similarly to full-length antibodies, they can bind a target with a high binding affinity and specificity while remaining small enough to diffuse into tissues. However, despite their numerous advantages, small biotherapeutics often suffer from a relatively short circulating half-life, thus requiring frequent applications that ultimately restrict their ease of use and user compliance. To overcome this limitation, a large variety of half-life extension strategies have been developed in the last decades. Linkage to ligands that non-covalently bind to albumin, the most abundant serum protein with a circulating half-life of similar to 19 days in humans, represents one of the most successful approaches for the generation of long-lasting biotherapeutics with improved pharmacokinetic properties and superior efficacy in the clinic.

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.