Structural Investigation of Diclofenac Binding to Ovine, Caprine, and Leporine Serum Albumins.

Free drug concentration in the blood sera is crucial for its appropriate activity. Serum albumin, the universal blood carrier protein, is responsible for transporting drugs and releasing them into the bloodstream. Therefore, a drug's binding to SA is especially important for its bioavailability and it is a key problem in the drug design process. In this paper, we present crystal structures of three animal serum albumin complexes: ovine, caprine, and leporine, with diclofenac, a popular non-steroidal anti-inflammatory drug that is used in therapy of chronic and acute pain. Details of diclofenac binding mode by the presented serum albumins are compared with analogous complexes of human and equine serum albumins. The analysis of the occupied binding pockets in crystal structures of the investigated serum albumins from different mammals shows that they have two common and a number of unique diclofenac binding sites. The most intriguing is the fact that the albumins from the described species are able to bind different numbers of molecules of this popular anti-inflammatory drug, but none of the binding sites overlap with ones in the human serum albumin.

BH3 mimetics potentiate pro-apoptotic activity of encorafenib in BRAFV600E melanoma cells.

BRAFV600- and MEK1/2-targeting therapies rarely produce durable response in melanoma patients. We investigated five BRAFV600E melanoma cell lines derived from drug-naïve tumor specimens to assess cell death response to encorafenib (Braftovi), a recently FDA-approved BRAFV600 inhibitor. Drug-naïve cell lines (i) did not harbor damaging alterations in genes encoding core apoptotic machinery, but they differed in (ii) mitochondrial priming as demonstrated by whole-cell BH3 profiling, and (iii) levels of selected anti-apoptotic proteins. Encorafenib modulated the balance between apoptosis-regulating proteins as it upregulated BIM and BMF, and attenuated NOXA, but did not affect the levels of pro-survival proteins except for MCL-1 and BCL-XL in selected cell lines. Induction of apoptosis could be predicted using Dynamic BH3 profiling. The extent of apoptosis was dependent on both (i) cell-intrinsic proximity to the apoptotic threshold (initial mitochondrial priming) and (ii) the abundance of encorafenib-induced BIM (iBIM; drug-induced change in priming). While co-inhibition of MCL-1 and BCL-XL/BCL-2 was indispensable for apoptosis in drug-naïve cells, encorafenib altered cell dependence to MCL-1, and reliance on BCL-XL/BCL-2 was additionally found in cell lines that were highly primed to apoptosis by encorafenib. This translated into robust apoptosis when encorafenib was combined with selective BH3 mimetics. Our study provides a mechanistic insight into the role of proteins from the BCL-2 family in melanoma cell response to targeted therapy, and presents preclinical evidence that (i) MCL-1 is a druggable target to potentiate encorafenib activity, whereas (ii) pharmacological inhibition of BCL-XL/BCL-2 might be relevant but only for a narrow group of encorafenib-treated patients.

Crystal structures of serum albumins from domesticated ruminants and their complexes with 3,5-diiodosalicylic acid.

Serum albumin (SA) is the most abundant protein in plasma and is the main transporter of molecules in the circulatory system of all vertebrates, with applications in medicine, the pharmaceutical industry and molecular biology. It is known that albumins from different organisms vary in sequence; thus, it is important to know the impact of the amino-acid sequence on the three-dimensional structure and ligand-binding properties. Here, crystal structures of ovine (OSA) and caprine (CSA) serum albumins, isolated from sheep and goat blood, are described, as well those of their complexes with 3,5-diiodosalicylic acid (DIS): OSA-DIS (2.20 Šresolution) and CSA-DIS (1.78 Šresolution). The ligand-free OSA structure was determined in the trigonal space group P3221 at 2.30 Šresolution, while that of CSA in the orthorhombic space group P212121 was determined at 1.94 Šresolution. Both albumins are also capable of crystallizing in the triclinic space group P1, giving isostructural crystals that diffract to around 2.5 Šresolution. A comparison of OSA and CSA with the closely related bovine serum albumin (BSA) shows both similarities and differences in the distribution of DIS binding sites. The investigated serum albumins from domesticated ruminants in their complexes with DIS are also compared with the analogous structures of equine and human serum albumins (ESA-DIS and HSA-DIS). Surprisingly, despite 98% sequence similarity, OSA binds only two molecules of DIS, whereas CSA binds six molecules of this ligand. Moreover, the binding of DIS to OSA and CSA introduced changes in the overall architecture of the proteins, causing not only different conformations of the amino-acid side chains in the binding pockets, but also a significant shift of the whole helices, changing the volume of the binding cavities.