MULTIPLE LIGAND SIMULTANEOUS DOCKING OF ANTIVIRAL DRUGS AND CYANINE DYES WITH PROTEINS †

Protein nanoparticles are currently regarded as promising biocompatible and biodegradable systems for targeted delivery of different types of pharmacological agents. Prior to fabricating such kind of drug nanocarriers it is reasonable to evaluate the drug-protein binding affinity and possible interaction modes using the computational tools, particularly, the molecular docking technique. The present study was undertaken to evaluate the possibility of creating the protein nanoparticles carrying the antiviral drugs and cyanine dyes as visualizing agents. The components of the examined systems included endogenous functional proteins cytochrome c, serum albumin, lysozyme and insulin, antiviral drugs favipiravir, molnupiravir, nirmatrelvir and ritonavir, mono-and heptamethinecyanine dyes. Using the multiple ligand simultaneous docking technique, it was demonstrated that: i) the drugs and the dyes occupy different binding sites on the protein molecule and do not interfere with each other; ii) the heptamethines AK7-5 and AK7-6 possess the highest affinity for the proteins; iii) among the examined systems the strongest complexes are formed between the heptamethine dyes and serum albumin. Taken together, the results obtained indicate that albumin-based nanoparticles functionalized by the heptamethine cyanine dyes can be used for targeted delivery of the explored antiviral

During the last decades the nanostructured drug delivery systems (DDS) such as liposomes, polymeric, lipidic, protein and inorganic nanoparticles, nanotubes, fullerenes, dendrimers and many others, have become a subject of considerable research interest in biomedical field [1][2][3].Particular attention is devoted to protein nanoparticles which have numerous advantages among which are: i) biocompatibility and biodegradability; ii) a weak or negligible immune response; iii) controlled size and stability; iv) a possibility of drug protection from enzymatic degradation and renal clearance; v) mild preparation and drug encapsulation conditions; vi) an opportunity for surface modification and functionalization with tissue-specific ligands; vii) protein abundance in natural sources; viii) easy synthesis process and cost-effectiveness [4][5][6].The use of proteins makes it possible to deliver the drugs with low aqueous solubility.The first endogenous protein utilized for preparing the nanoparticles was the blood serum albumin.A remarkable ability of human serum albumin to reversibly bind hydrophobic molecules provided a basis for obtaining the albumin-bound formulation of the antitumor low soluble drug paclitaxel (Abraxane) [7].Due to their amphiphilic nature, proteins can interact with both hydrophilic and hydrophobic drugs.Along with albumin, the nanoparticles were fabricated from a variety of animal (gelatin, collagen, casein, silk fibroin) and plant (zein, gliadin, lectin, soy protein) [4,8].Protein nanoparticles are currently used in cancer therapy, lung treatment, vaccine production, etc [9].To create the protein-based drug delivery system it is necessary to ascertain whether the protein can simultaneously interact with a drug and functionalizing molecules, such as targeting, stabilizing or visualizing agents.The cyanine dyes fluorescing in the near-infrared region, in the optical windows of biological samples are among the most promising visualizing agents for DDS.In view of this, it seems of interest to evaluate the possibility of concurrent binding of antiviral drugs and cyanine fluorescent dyes.A series of the examined endogenous proteins included cytochrome c (Ct), serum albumin (SA), lysozyme (Lz), insulin (Ins).These proteins play important roles in the electron transport (Ct), blood circulation (SA), innate immune (Lz) and endocrine (Ins) systems.The antiviral drugs were represented by favipiravir, molnupiravir, nirmatrelvir and ritonavir.The aim of in the present study was to characterize the ternary complexes protein-drug-dye using the technique of multiple ligand simultaneous docking (MLSD).

Molecular docking studies
The set of the investigated pharmaceuticals included favipiravir, molnupiravir, nirmatrelvir and ritonavir [10].The examined drug structures as well as monomethine and heptamethine cyanine dyes (Figure 1) were built in MarvinSketch (version 18.10.0)and optimized in Avogadro (version 1.1.0)using the Universal Force Field with the steepest descent algorithm [11,12].The counterions were not added to the dye structures in order to save the molecular charges.The threedimensional X-ray crystal structures of the examined proteins were obtained from the Protein Data Bank using the PDB IDs 1REX, 3I40, 3ZCF, 6M4R for lysozyme, insulin, cytochrome c and serum albumin, respectively.The proteins were
taken in the native monomeric form.The SA structural model was prepared using the DockPrep module of UCSF Chimera molecular software by removing the water molecules and addition of polar hydrogen atoms and Kollman charges [13].
The blind docking of the drugs with the proteins was performed using the PatchDock server (http://bioinfo3d.cs.tau.ac.il/PatchDock/php.php) in which the molecular docking algorithm is focused on the finding of maximal surface shape complementarity along with the minimal amounts of steric clashes [14].Subsequently, the topscored docked drug-protein complexes were considered as a receptor for docking of the second ligand, one representative of the monomethine or heptamethine cyanine dyes, using the PatchDock server.The control dye-protein systems were also considered.In order to identify the amino acid residues constituting the binding sites and to analyse the types of the drug-protein and the dye-protein contacts, the protein-ligand interaction profiler (PLIP, https://pliptool.biotec.tudresden.de/plip-web/plip/index) was employed [15].The selected docking poses were visualized with the UCSF Chimera software (version 1.14) in which the docking models with the best geometric shape complementary were combined in the same picture to achieve the best visibility of the binding sites [16].2), albumin (Fig. 3), lysozyme (Fig. 4) and insulin (Fig. 5).It can be seen that in the cases of Ct, Lz and Ins the binding sites for the investigated mono-and heptamethines are located rather close to each other in the vicinity of the protein surface.Meanwhile, the drugs penetrate deeper into the interior of Ct, Lz and Ins, occupying the sites different from that of the cyanine dyes.In the case of albumin, the antiviral agents and the dyes reside in the distinct protein pockets.
Table 2.The geometric shape complementarity score and approximate interface area of the complex derived for the albumin-drug-dye systems (F-Favipiravir, M -Molnupiravir, N -Nirmatrelvir, R -Ritonavir).

Dye
Score Approximate interface area of the complex, A 2

Figure 1 .
Figure 1.Structural formulas of the examined antiviral drugs and cyanine dyesRESULTS AND DISCUSSIONShown in Figs 2-5 are the highest-score ternary complexes protein-drug-dye recovered for cytochrome c (Fig.2), albumin (Fig.3), lysozyme (Fig.4) and insulin (Fig.5).It can be seen that in the cases of Ct, Lz and Ins the binding sites for the investigated mono-and heptamethines are located rather close to each other in the vicinity of the protein surface.Meanwhile, the drugs penetrate deeper into the interior of Ct, Lz and Ins, occupying the sites different from that of the cyanine dyes.In the case of albumin, the antiviral agents and the dyes reside in the distinct protein pockets.The analysis of the geometric shape complementarity scores and approximate interface areas derived for the cytochrome c-drug-dye complexes (Table1) revealed that: i) the drugs do not exert influence only on the dye-protein affinity and interface area, only in the cases of AK-1-2-20 for all drugs and AK7-6 for molnupiravir these parameters

Figure 2 .
Figure 2. The highest-score docking positions obtained for cytochrome c using the MLSD in PatchDock.

Figure 3 .
Figure 3.The highest-score docking positions obtained for albumin using the MLSD in PatchDock.

Figure 4 .
Figure 4.The highest-score docking positions obtained for lysozyme using the MLSD in PatchDock.

Figure 5 .
Figure 5.The highest-score docking positions obtained for insulin using the MLSD in PatchDock.

Table 1 .
The geometric shape complementarity score and approximate interface area of the complex derived for the cytochrome cdrug-dye systems (F-Favipiravir, M -Molnupiravir, N -Nirmatrelvir, R -Ritonavir).