Structural evidence of the species-dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties

Abstract

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein-serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 μM after 24 h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins.

Keywords: antiproliferative action; crystal structure; cyclic phosphatidic acid (cPA); lysophospholipid transport; modified lysophospholipids; serum albumin.

Figure 1. Chemical structures of studied lysophospholipids

(A) Lysophosphatidic acid, (B) cyclic phosphatidic acid, (C) phosphorothioate and (D) phosphorodithioate analogues of cyclic phosphatidic acid. R indicates the aliphatic chain of fatty acid residue.

Figure 2. Arrangement of the helices in serum albumin

The molecule is shown in two orientations for better visibility of its secondary elements. Domains I (h1-I–h10-I), II- (h1-II–h10-II) and III (h1-III–h10-III) are colored blue, pink and green, respectively.

Figure 3. Effects of LPA and cPA analogues on the viability of PC-3 cells

(A) Effects of cPA, cPA analogues (20 μM) and LPA (10 μM) on PC-3 cells viability after 24 or 48 h incubation; ###, significantly different from methanol-treated cells (CNT) (P < 0.001). (B) Effects of cPA analogs concentration on PC-3 cell viability after 24 h; IC50 was extrapolated from the inhibition curve. (C) Effects of cPA analogues on LPA-dependent proliferation of PC-3 cells; cell viability was tested after 24 or 48 h incubation with 20 μM cPA or its analogues in the presence of 10 μM LPA; results significantly different from cells treated only with LPA were marked with *, P < 0.05 and **, P < 0.01.

Figure 4. Myr-2S-cPA binding sites identified in HSA and ESA

(A) Superposition of HSA (light pink) and ESA (light blue) complexes with Myr-2S-cPA. (B) Conformation of the Myr-2S-cPA/1 in site 1 of HSA. (C) Conformation of the Myr-2S-cPA/2 in site 2 of HSA. (D) Conformation of the Myr-2S-cPA/3 in site 3 of HSA. (E) Conformation of the Myr-2S-cPA bound in ESA. 2Fo–Fc electron density maps for the bound ligands are contoured at 1 σ level. Atoms of the Myr-2S-cPA are colored as follows: carbon (cyan), oxygen (red), sulfur (yellow) and phosphorus (violet); the color coding is consistent in all figures.

Figure 5. Myr-2S-cPA/1 binding site of HSA

(A) Amino acids residues involved in the interactions with ligand via hydrogen bonds [Å]. (B) Comparison of the Myr-2S-cPA/1 (cyan) binding site with the same pocket of HSA (PDB ID: 1E7G) in which two myristic acid molecules (gray) are bound.

Figure 6. Myr-2S-cPA/2 binding site of HSA

(A) Amino acids residues involved in the interactions with ligand via hydrogen bonds [Å]. (B) Comparison of the Myr-2S-cPA/2 binding site with the same pocket of HSA (PDB ID: 1E7G) where the myristic acid residue (gray) is bound.

Figure 7. Myr-2S-cPA/3 binding site of HSA

(A) Amino acids residues interacting with ligand via hydrogen bonds [Å]. (B) Comparison of the Myr-2S-cPA/3 binding site with the place of Palm-LPE (green) bound in HSA (PDB ID: 3CX9).

Figure 8. Myr-2S-cPA binding site of ESA

(A) Amino acids residues interacting with ligand via hydrogen bonds [Å]. (B) Comparison of the Myr-2S-cPA binding site in ESA (light blue) with the analogical pocket in HSA where myristic acid (gray) is bound (light pink; PDB ID: 1E7G).