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. 2024 May 14;14(5):579.
doi: 10.3390/biom14050579.

BSA Binding and Aggregate Formation of a Synthetic Amino Acid with Potential for Promoting Fibroblast Proliferation: An In Silico, CD Spectroscopic, DLS, and Cellular Study

Hayarpi Simonyan  1 , Rosanna Palumbo  2 , Satenik Petrosyan  1 , Anna Mkrtchyan  1 , Armen Galstyan  3 , Ashot Saghyan  1 , Pasqualina Liana Scognamiglio  4 , Caterina Vicidomini  2 , Marta Fik-Jaskólka  5 , Giovanni N Roviello  2
Affiliations

BSA Binding and Aggregate Formation of a Synthetic Amino Acid with Potential for Promoting Fibroblast Proliferation: An In Silico, CD Spectroscopic, DLS, and Cellular Study

Hayarpi Simonyan et al. Biomolecules. .

Abstract

This study presents the chemical synthesis, purification, and characterization of a novel non-natural synthetic amino acid. The compound was synthesized in solution, purified, and characterized using NMR spectroscopy, polarimetry, and melting point determination. Dynamic Light Scattering (DLS) analysis demonstrated its ability to form aggregates with an average size of 391 nm, extending to the low micrometric size range. Furthermore, cellular biological assays revealed its ability to enhance fibroblast cell growth, highlighting its potential for tissue regenerative applications. Circular dichroism (CD) spectroscopy showed the ability of the synthetic amino acid to bind serum albumins (using bovine serum albumin (BSA) as a model), and CD deconvolution provided insights into the changes in the secondary structures of BSA upon interaction with the amino acid ligand. Additionally, molecular docking using HDOCK software elucidated the most likely binding mode of the ligand inside the BSA structure. We also performed in silico oligomerization of the synthetic compound in order to obtain a model of aggregate to investigate computationally. In more detail, the dimer formation achieved by molecular self-docking showed two distinct poses, corresponding to the lowest and comparable energies, with one pose exhibiting a quasi-coplanar arrangement characterized by a close alignment of two aromatic rings from the synthetic amino acids within the dimer, suggesting the presence of π-π stacking interactions. In contrast, the second pose displayed a non-coplanar configuration, with the aromatic rings oriented in a staggered arrangement, indicating distinct modes of interaction. Both poses were further utilized in the self-docking procedure. Notably, iterative molecular docking of amino acid structures resulted in the formation of higher-order aggregates, with a model of a 512-mer aggregate obtained through self-docking procedures. This model of aggregate presented a cavity capable of hosting therapeutic cargoes and biomolecules, rendering it a potential scaffold for cell adhesion and growth in tissue regenerative applications. Overall, our findings highlight the potential of this synthetic amino acid for tissue regenerative therapeutics and provide valuable insights into its molecular interactions and aggregation behavior.

Keywords: fibroblast growth enhancement; in silico; protein binding; self-assembling system; synthetic amino acid; tissue regeneration.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Scheme 1
Scheme 1
Synthetic route to compound 5, depicting the stepwise procedure starting from the chiral NiII complex of Schiff’s base of dehydroalanine (NiII-(S)-BPB-Δ-Ala) (1) with the chiral auxiliary (S)-2-N-(N’-benzylprolyl) aminobenzophenone ((S)-BPB) (4), followed by the addition of substituted 1,3,4-oxadiazole (2) and subsequent isolation and recovery steps to obtain the desired α-amino acid 5 from the main diastereomeric complex 3.
Figure 1
Figure 1
Plot of UV Absorbance against concentration of compound aggregation (a). DLS analysis of compound 5 in PBS 1X pH 7.4 at room temperature (b). Model of an aggregate formed by 512 molecules of compound 5. Note the presence of a cavity within its interior (c). Two 3D pose views of the tetrameric complex formed by the coplanar dimer (yellow) and the ring-staggered dimer (brown), with interaction types highlighted in purple (π-π stacking) and grey (carbon–hydrogen bond). Note how the carbon–hydrogen bond holds together the two dimeric structures into the tetramer and how the stacking also occurs in the staggered dimer (d).
Figure 2
Figure 2
Growth of HDF fibroblasts (%) with 1 nM, 10 nM, and 1 μM concentrations of compound 5. Control corresponds to vehicle-treated cells.
Figure 3
Figure 3
CD binding assay showing complex formation between BSA (0.785 μM) and compound 5 (37.5 nmol) in PBS 1x at pH 7.4 and 22 °C. The blue curve displays the CD spectrum recorded post-mixing of BSA and compound 5 solutions in a tandem dual chamber quartz cell, while the green curve represents the CD spectrum before mixing the two solutions (a). 3D pose view depicting the top-ranked pose predicted by HDOCK software for the molecular docking of BSA with compound 5. The ligand is represented in yellow color within the BSA protein structure (chain A, as indicated by the orange letter ‘A’) (b). 3D diagram illustrating the interaction between BSA and compound 5 for the top-1 pose as visualized by PLIP software. Dotted grey lines represent hydrophobic interactions, while blue lines denote hydrogen bonds. For additional information on the residues involved and other bond characteristics, please refer to Table S1 in the Supporting Information of this work (c).
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Grants and funding

This work was supported by the Science Committee of RA in the frames of the research project № 21T-1D057. G.N. Roviello and H. Simonyan also acknowledge the financial support provided by the Italian National Research Council (CNR) and the Armenian Science Committee of the Ministry of Education and Science (MESRA) for their collaboration within the framework of the Armenian-Italian bilateral research project 23SC-CNR-1D002, as well as the CNR/MESRA Scientific Cooperation (CNR Prot. No. 19140 dated 20230125, 2023-CNR0A00-0019140).