Structure of the Active Nanocomplex of Antiviral and Anti-Infectious Iodine-Containing Drug FS-1

Abstract

Chromatographic analysis shows that the ionic nanostructured complex of the FS-1 drug contains nanocomplexes of α-dextrin with a size of ~40–48 Å. Based on good agreement between the UV spectra of the model structures and the experimental spectrum of the FS-1 drug, the structure of the active FS-1 nanocomplex is proposed. The structure of the active centers of the drug in the dextrin ring was calculated using the quantum-chemical approach DFT/B3PW91. The active centers, i.e., a complex of molecular iodine with lithium halide (I), a binuclear complex of magnesium and lithium containing molecular iodine, triiodide (II), and triiodide (III), are located inside the dextrin helix. The polypeptide outside the dextrin helix forms a hydrogen bond with dextrin in Complex I and coordinates the molecular iodine in Complex II. It is revealed that the active centers of the FS-1drug can be segregated from the dextrin helix and form complexes with DNA nucleotide triplets. The active centers of the FS-1 drug are only segregated on specific sections of DNA. The formation of a complex between the DNA nucleotide and the active center of FS-1 is a key stage in the mechanisms of anti-HIV, anti-coronavirus (Complex I) and antibacterial action (Complex II).

1. Introduction

Molecular iodine has a broad spectrum of antimicrobial and antiviral effects. There are several groups of iodine preparations: (1) containing elemental iodine, (2) inorganic iodides, (3) organic substances—hormones, (4) organic substances eliminating iodine, (5) organic substances that firmly bind iodine.

However, currently, all known molecular iodine containing medications are practically not used for parenteral administration, due to their high toxicity.

A distinctive feature of the FS-1 drug is that its active substance includes not only an iodine-containing polymer complex (a molecular iodine complex with low molecular weight α-dextrins and peptides) but also potassium, lithium and magnesium halides. In the composition of the FS-1 drug, molecular iodine is in such an active form that, when administered parenterally, it minimizes toxic effects in the human body [1].

The use of the antimicrobial properties of iodine in the composition of iodine/polymer complexes began in the second half of the 20th century. Compounds of mobile equilibrium systems of iodine and its salts aqueous solutions with starch, amylase, amylopectin, and polyvinyl alcohol have been well studied. The advantages of such high-polymer iodine complexes as compared to the sulfonamides and antibiotics have been established—a wide spectrum of antimicrobial action both in vivo and in vitro, exclusion of the emergence of new resistant forms of microorganisms, low toxicity in combination with a high chemotherapeutic index and the effectiveness of short-term treatment [2,3].

For the latest three decades, new nanoparticles are being researched for the purpose of developing new drug delivery systems. Macromolecules, such as proteins and nucleic acids, demonstrate low stability in biological matrix. Their efficient delivery to target sites is widely limited in vivo, as these molecules are unlikely to be able to cross various biological barrier, whereas polysaccharides are able to overcome cellular barriers for the delivery of pharmaceutical molecules into cells [4].

Dextrin is an unbranched polysaccharide that exhibits high water solubility and is widely used in food and medicine [5]. The presence of hydroxyl groups makes it possible for dextrin to interact with protein. A hydrogen bond is formed between the OH group of the glucose ring and the oxygen atom of the peptide amido group.

Important tasks that can be achieved with the use of dextrin are drug targeting, improvement of circulation time, stabilization of the therapeutic agent, drug solubilization, reduction of side effects, sustained release action and depot properties [6].

The absorption of dextrin nanoparticles by cellular systems occurs through a process known as endocytosis and is influenced by the physicochemical characteristics of the nanoparticles, such as size, shape and the experimental conditions used [7].

The size of nanoparticles plays a key part in the use of dextrins as carrier systems as well as transmembrane transporters [8].

In this paper, the mass of α-dextrin, which is part of the FS-1 drug, was determined by the chromatographic method. Using the data of X-ray diffraction analysis for the structure of α-dextrins [9], the size of the α-dextrin nanocomplex is ~40–48 Å, which contains the active centers of the drug and determines its main pharmacological properties.

Based on the study of an aqueous solution of the system modeling the specific feature of the drug FS-1, the structure of the active nanocomplex FS-1 is proposed [10,11]. The nanocomplex of the FS-1drug contains a triiodide and two active centers located inside the dextrin helix: molecular iodine coordinated by a lithium halide and a polypeptide (LiI(Cl)I₂, Complex I), a binuclear complex of magnesium and lithium, including molecular iodine (MgI₃LiII₂, Complex II). In these complexes, molecular iodine exhibits acceptor properties with respect to the polypeptide in Complex II or ion I^– in Complex I and donor properties with respect to lithium halide in Complex II or Li⁺ ion in Complex I. The polypeptide coordinates molecular iodine in Complex II and is located outside the dextrin helix.

For the purpose of this paper, the calculation was made using quantum-chemical approach DFT/B3PW91 with full optimization of the geometry of the spatial and electronic structure of the LiI I₂ and MgI₃LiII₂ complexes in the dextrin ring. In the calculations the polypeptide was replaced by an amide. The calculated UV spectra (TD-DFT/B3PW91 approach) of these complexes are in good agreement with the experimental UV spectra of the drug FS-1.

The ability of the FS-1 drug to penetrate the bactericidal cell membrane was proved by the electron spectroscopy method [12]. The active centers are protected from interaction with bioorganic ligands that are part of the cytoplasm of the cell by the α-dextrin helix and interaction with polypeptides. In earlier papers [13,14], we showed that only DNA nucleotides can compete with polypeptides for complexation with molecular iodine, which is part of the active centers of the drug FS-1.

In this paper, it is demonstrated that the active centers of the drug can be segregated from the dextrin helix and form complexes with nucleotide triplets of viral or bacterial DNA.

The interaction of active centers with nucleotide triplets is selective: the active centers of the FS-1 drug are only segregated on specific sections DNA. The mechanism of action of the drug is determined by the structure of the active center, which has formed a complex with a nucleotide triplet. The formation of a complex between the DNA nucleotide and the active center of the FS-1 drug (Complex I) is a key stage in the mechanisms of anti-HIV [14], anti-coronavirus (Complex I, II) [15] and antibacterial action (Complex II) [12].

2. Materials and Methods

The chromatographic method determined the α-dextrin mass, which is part of the FS-1 drug. Using the X-ray structural analysis data for the α-dextrins structure [9], the size of the nanocomplex α-dextrin was found ~40–48Å, containing active centers of the drug and determining its basic pharmacological properties. Chromatographic studies were performed on an Agilent 1200 chromatograph with a refractometric detector on a gel-penetrating column Agilent PL Aquagel Mixed-H 8 μm (size exclusion column).

UV spectra were obtained using a Lambda-35 spectrophotometer (Perkin Elmer Inc., Waltham, MA, USA). Spectral range: 190–1100 nm, wavelength λ ± 0.5 nm. Optical density ± 0.02 units.

An adequate description of the structure and electronic properties of iodine complexes with the transfer of charge and polyiodide ions requires taking into account electron correlation. A detailed comparative analysis of various quantum chemical approaches including CCSD (T) and DFT (BLYP, BPW91, B3LYP, B3PW91) for calculations of dissociation energies, bond lengths, and harmonic frequencies for polyiodide anions and evaluation of the obtained data based on agreement with experimental IR and Raman Spectroscopy studies is presented in [16]. Comparison of the results obtained by DFT-methods with the results obtained by the CCSD (T) and experimental data show that the most reliable results are produced by an approximation of DFT-B3PW91. Based on these conclusions the DFT-B3PW91/6-31G ** for Atom I used basis midi approximation was selected in our investigations of molecular structures and characteristics of the complexes containing lithium and magnesium halides, molecular iodine and iodide anion.

The coincidence of the wavelength of electronic transitions for active centers and a triiodide, calculated using approach TD-DFT/B3PW91/midi, with experimentally obtained UV spectrum for drug FS-1 confirm the structures of active centers.

All calculations were performed on the Fujitsu PRIMERGY BX 920 S1 supercomputer (Fujitsu, Tokyo, Japan) with a performance of 10.9 TFLOPS with the help of the GAUSSIAN 09 program Gaussian 09 (Gaussian, Inc., Wallingford, CT, USA) [17].

3. Results

3.1. Determination of the Molecular Weight of α-Dextrin Nanoparticles in the FS-1 Drug by Chromatographic Method

To determine the molar masses of α-dextrins with a low and medium molar mass, oligomeric α-dextrins and α-dextrins with a molar mass of 75,000 g/mol were used as standards. The characteristics of the substances used are presented in

Table 1. Properties of α-dextrins used as standards in calibrations to determine the molar masses of the catalytic reactions products.

Name	Formula	Retention Time, min	Molar Mass, g/mol	lg M	Number of Glucose Units
Maltotriose	C18H32O16	9.4	504	2.70243	3
Maltotriose	C24H42O21	9.356	666	2.82347	4
Maltopentaose	C30H52O26	9.315	831	2.9196	5
Maltohexaose	C36H62O31	9.289	990	2.99564	6
Maltoheptaose	C42H72O36	9.259	1152	3.06145	7
High molecular weight α-dextrin		8.08	75,000	4.87506	417

.

To obtain a calibration, the results were subjected to mathematical treatment. The dependence of the logarithm of the molar mass of the calibration carbohydrates on the retention time was plotted.

Based on the obtained dependence after mathematical treatment, the following equation was obtained:

y = lgM = −1.5968 × x + 17.787

(1)

The correlation coefficient was determined, which was −0.9983. This coefficient indicates the good linearity of the data obtained.

As can be seen in Figure 1 and

Table 2. Peaks and fractions properties in the chromatogram of α-dextrins of a part of the FS-1 drug.

Retention Time, min	Molecular Weight, g/mol	Polymerization Level	Content of the Total Amount of Carbohydrates in Solution, %
	Peaks
5.665	4,800,000	26,667	12.25
8.627	10,260	57	83.09
	Peak Segments 8.627
8.25	41.058	228.1	16.184
8.75	6531	36.28	34.986
9.25	1039	5.772	24.066
9.75	165	0.917	5.708

the high molecular weight peak at 5.665 min is 12 percent of the total α-dextrins. The main peak has a bimodal distribution. However, the average molar mass of the peak is quite high.

The main fraction in the FS-1drug is the fraction with a retention time of 8.75 min, its content in the total amount of α-dextrins was 34.986%. Since this fraction is in the middle of the peak and its share in the segments is maximum, it is reasonable to take its molecular weight as the average for the α-dextrin part of the FS-1drug. It means that the average molecular weight of the main component of α-dextrin in FS-1 with a retention time of 8.75 is 6531 (polymerization level—36.22). The higher molecular weight component of α-dextrins, apparently, should not be considered as capable of forming iodine-polymer complexes, since with such a large molecular weight—4,800,000 Da, the polymer structure is branched and cannot form iodine-polymer complexes.

The chromatographic method determined the average molecular weight of α-dextrin ~6531, which makes it possible to determine the size of α-dextrin nanoparticles. One dextrin ring of α-dextrin includes six glucose rings (the molecular weight of the glucose ring is 173), hence the dextrin nanomplex contains 6–7 rings. It is known from the data of X-ray diffraction analysis that the distance between the dextrin rings is ~8 Å hence the nanoparticle size is ~40–48 Å.

3.2. FS-1 Active Nanocomplex Structure

The FS-1 drug is a chemically complex multicomponent drug containing molecular iodine, potassium and lithium halides, magnesium chloride, α-dextrins, and polypeptides. In terms of its physicochemical properties the drug FS-1 is an ionic nanostructured complex, which contains metal salts (LiCl (I), MgCl₂) of molecular iodine and its ionized forms.

It is not possible to identify active centers in the ionic nanostructured complex of the FS-1drug using physical techniques. We studied the structure of the active centers of the drug FS-1 on a model system, representing an aqueous solution containing potassium and lithium halides, molecular iodine, amino acid (glycine), and ethanol (system (a)) [10,11].

It was shown by UV spectroscopy in [18] that in an aqueous solution of KI—I₂-amylose, the number of units of the dextrin helix can affect the iodine-triiodide equilibrium. With an increase in the number of units (N ≥ 15) by UV spectroscopy, iodine complexes and triiodide complexes can be detected in this system. Dextrin, which is a part of the drug, contains the number of units N ≥ 15, therefore, molecular iodine and triiodide complexes can be present inside the dextrin helix and are identified in the UV spectra.

The glycine zwitterions cluster, such as the dextrin helix, creates conditions for the presence of I^-, I₃^- and I₂ ions in an aqueous solution of KI₃; therefore, a system containing glycine, potassium and lithium halides, and molecular iodine (system (a)) can be considered as a model for studying the interaction within the α-dextrin helix of lithium halides, triiodide and molecular iodine. System (a) was investigated by UV-IR spectroscopy and the quantum-chemical approach DFT/B3PW91 [10,11].

Quantum-chemical calculations of the stability of possible complexes in system (a) and spectroscopic studies indicate that lithium chloride interacts only with molecular iodine in system (a). When a complex of molecular iodine with the carboxy group of glycine is formed, a negative charge is transferred to molecular iodine, so I₂ can form a coordination bond with lithium chloride (LiClI₂COO⁻). In such a complex, molecular iodine exhibits acceptor properties with respect to the carboxy group and donor properties with respect to lithium chloride.

The complex of lithium chloride with triiodide, which interacts with the protonated amino group of glycine, is significantly lower in the stability of the complex, in in which triiodide and lithium chloride are spatially separated: triiodide interacts with a protonated amino group, and lithium chloride with a positively charged carboxy group.

Consequently, three complexes are formed in system (a): LiClI₂COO⁻, lithium chloride coordinated by the carboxy group, and triiodide, which forms a coordination bond with the protonated amino group.

These three complexes can also be formed within the α-dextrin helix.

The spatial and electronic structure of the LiI-I₂ complex in a dextrin ring containing six glucose rings and coordinated by an amide was calculated (Complex I). In the calculations, the polypeptide was replaced by an amide.

In Complex I, the lithium ion is not on the same straight line with triiodide; it coordinates two iodine atoms at once (Li-I¹ = 2.69 Å, Li-I² = 2.71 Å). This complex can be interpreted as a complex of molecular iodine (I²-I³ = 2.84 Å) with a lithium ion and an iodine ion I¹. When molecular iodine interacts with an iodine ion, I₂ acts as an acceptor; therefore, a negative charge is transferred to I₂. Negatively charged molecular iodine becomes a donor with respect to the lithium ion. In Complex I molecular iodine is in a special electronic form, not found in drugs containing iodine complexes with bioorganic ligands. The amide forms a hydrogen bond with one of the oxygen atoms of the dextrin ring (O-H = 1.87 Å) and does not interact with the LiI-I₂ complex. (Figure 2).

The structure of the FS-1 drug also contains magnesium halides. Our calculations showed that magnesium ion can penetrate the dextrin ring and form a coordination bond with the OH group of α-dextrin. Inside the α-dextrin helix, the most energetically favorable structure is the one that combines the magnesium ion, triiodide, and LiCl (I)–I₂ (MgI₃LiII₂ Complex II, Figure 3).

A calculation was performed with full optimization of the geometry of the spatial structure of the MgI₃LiII₂ complex in the dextrin ring (Complex II, Figure 3).

The MgI₃LiII₂ complex is a binuclear complex in which a magnesium ion and a lithium ion are bound by an iodine ion (Mg-I⁵ = 2.81 Å, Li-I⁵ = 2.67 Å). Molecular iodine (I¹-I² = 2.85 Å) is located inside the dextrin helix and is coordinated by a lithium ion (O-I² = 2.32 Å, Li-I¹ = 2.72 Å). A peptide located outside the α-dextrin helix. When a magnesium ion interacts with triiodide, triiodide decomposes into an iodine ion and molecular iodine (I⁶-I³ = 3.54 Å, I³-I⁴ = 2.72 Å). However, I³-I⁴ does not possess acceptor properties and cannot interact with either polypeptides or DNA nucleotides. The magnesium ion is coordinated by two iodine ions and three oxygen atoms of the dextrin ring.

Thus, molecular iodine inside active Complexes I and II of the drug is in a special electronic form, not found in other iodine-containing drugs. It is probably this electronic structure of iodine that ensures the low toxicity of the FS-1 drug.

A calculation was performed with full optimization of the geometry of the spatial structure of triiodide in the dextrin ring (complex III, Figure 4). Triiodide is located inside the dextrin helix (I¹-I² = 3.08 Å, I²-I³ = 2.91 Å).

The experimental UV spectrum for the FS-1 drug was obtained for 1/1000 dilution of FS-1 in water.

The wavelengths of electronic transitions in Complexes I–III calculated using the quantum-chemical approach TD-DFT/B3PW91/midi (

Table 3. The wavelengths of electronic transitions (nm) in Complexes I–III.

Complex I
293	1A → 1A (I3 → I1-I2-I3)
263	1A → 1A (I2 → I1-I2-I3)
307	1A → 1A (I3 → I1-I2-I3)
Complex II
339	1A → 1A (I6-I5-I1 → I1-I2-I5-Mg-Li)
	1A → 1A (I6,I5 → I1-I2-I5-Mg-Li)
Complex III
267	1A → 1A (I1-I2-I3 → I1-I2-I3)

).

The wavelengths of electronic transitions in Complexes I–II fall within the range in the experimental spectrum of 270–310 nm. The maximum of this interval is well described by the 293 nm band in Complex I, which corresponds to the transition between the occupied orbital with the main contribution of ion I³ and the unoccupied orbital with the main contribution of triiodide. The 263 nm and 307 nm transitions in Complex I can also be attributed to transitions between the occupied and unoccupied triiodide orbitals. The 339 nm transition in Complex II is due to the orbital interaction between iodine ions (I¹, I², I⁵, I⁶) and magnesium and lithium ions. Transition 267 in Complex III is the transition between the occupied and unoccupied orbital of triiodide in the dextrin ring.

The wavelengths of electronic transitions in Complexes I–III, within the error of the method (~10 nm), are in good agreement of experimentally obtained UV spectrum of the FS-1 drug (Figure 5).

Thus, nanocomplex of the FS-1 drug includes a triiodide and two active centers: a complex of iodine with lithium halide and a binuclear complex of magnesium and lithium with molecular iodine and triiodide.

3.3. Interaction of Active Centers of the FS-1 Drug with Nucleotide Triplets of Bacterial and Viral DNA

In earlier paper, using electron spectroscopy, we demonstrated that the FS-1 drug penetrates the bactericidal cell membrane [12].

The active centers of the FS-1 drug are protected from interaction with bioorganic ligands that are part of the cytoplasm of the cell by the α-dextrin helix and interaction with polypeptides. Only DNA nucleotides can compete with polypeptides for complexation with molecular iodine, which is part of the active centers of the FS-1 drug [13,14].

The structures of complexes in which of the active centers of the FS-1 drug interact with the AAG nucleotide triplet are shown in Figure 6.

We calculated the stability of complexes MgI₃LiII₂ and LiII₂ with nucleotide triplets and compared the results of calculations with the stability of these complexes with a dextrin ring.

The energy ΔE was calculated, which characterizes the difference in the stability of the complexes MgI₃LiII₂ and LiII₂ with a dextrin helix and with nucleotide triplets.

ΔE is calculated as follows:

ΔE = (E^tot(IV or V) + E^tot(dex) + E^tot(amid)) − (E^tot(I or II) + E^tot(nucleotide trip))

(2)

where:

E^tot(IV or V)—total energy of complex IV or V;

E^tot(dex)—total energy of the dextrin ring;

E^tot(amid)—total energy of amide;

E^tot(amid)—total energy of amide;

E^tot(nucleotide trip)—total energy of nucleotide triplet.

If ΔE is less than zero, when nanocomplex approaches bacterial or viral DNA, one or two active centers are segregated from the dextrin ring and form a complex with a nucleotide triplet (

Table 4. Interaction energies (kcal/mol) of nucleotide triplets with active centers of the FS-1 drug.

	LiII2	MgI3LiII2
GGT	−12.17	−10.72
GAA	−12.07	+19.02
GGG	+11.94	−13.61
CGG	−7.22	−17.60
CTG	+17.30	+26.40
ACC	+8.50	−6.30
GCG	1.26	32.47
CGC	2.89	52.91

).

Calculations have shown that ΔE depends on the nucleotide triplet structure. The active centers of the FS-1 drug are only segregated on specific sections of bacterial or viral DNA. The mechanism of action of the drug is determined by the structure of the active center, which has formed a complex with a nucleotide triplet.

The mechanism of anti-HIV action of the FS-1 drug was proposed by us earlier [14]. The complex of the active center LiII₂ with the nucleotide triplet (Complex IV) inhibits the active center of the HIV enzyme: integrase. The formation of complex IV destroys the pre-integration complex of the viral DNA with the active center of the integrase and forms a new nucleoprotein complex, where Complex IV binds both the viral DNA and the active center of the integrase.

In the recent work we proposed the mechanism of anti-coronavirus action of the FS-1 drug [15]. Active centers of the FS-1drug destroy the complex formed by the phosphate group of the viral RNA and the catalytic fragment of the ExoN domain of exoribonuclease and create a new nucleoprotein complex in which active centers bind both viral RNA and magnesium ions of the catalytic fragment.

The active complex MgI₃LiII₂ with a nucleotide triplet (Complex V) can inhibit the active site of the DNA-dependent RNA polymerase of bacteria [12].

For DNA S aureus analysis of epigenetic modifications in nucleotide triplets (

Table 5. Energy interactions (kcal/mol) of nucleotide triplets with active centers of the FS-1 drug and the frequency of epigenetic modifications in nucleotide triplets Staphylococcus aureus under the action of the FS-1 drug (N).

	LiII2	MgI3LiII2	N
GGT	−12.17	−10.72	3645
GAA	−12.07	+19.02	2021
GGG	+11.94	−13.61	1866
ACC	+8.50	−6.30	298
CTG	+17.30	+26.40	234
CGC	2.89	52.91	276

) [19].

Epigenetic modifications are any modifications to DNA that involve chemical modifications, such as methylation, halogenation, and DNA binding to proteins or other possible complexes.

As can be seen from Table 5. Between the stability of the complexes of active centers with nucleotide triplets and the frequency of epigenetic modifications in these nucleotide triplets (N) a qualitative correlation is observed.

The largest number of epigenetic modifications is observed in the GGT triplet. This triplet forms stable complexes immediately with two active centers. Triplet GAA and GGG form a complex only with one active center and the frequency of epigenetic modifications is significantly reduced. The energy of the ACC triplet interaction with the active center MgI₃LiII₂ is less than with the triplet GGG and N is also less. CTG and CGC triplets do not form complexes with active centers and in them N has further decreased.

It can be assumed that one of the reasons for epigenetic modifications in DNA under the influence of the FS-1drug is the formation of complexes of active centers of the FS-1 drug with nucleotide triplets Staphylococcus aureus.

This result allows to simulate the antibacterial efficacy of the FS-1drug. If the active center MgI₃LiII₂ interacts with nucleotide triplets, which are most common in the genome of bacteria, then the FS-1 drug has antibacterial properties.

4. Conclusions

An ionic nanostructured complex of the FS-1 drug contains nanocomplexes of α-dextrin ~40–48 Å in size. Active centers: molecular iodine complex with lithium halide, binuclear complex of magnesium and lithium, containing molecular iodine and triiodide, are located inside the dextrin helix. The polypeptide, which is located outside the dextrin helix, forms a hydrogen bond with dextrin in Complex I and coordinates molecular iodine, which is part of Complex II.

Molecular iodine inside the active Complexes I and II of the drug is found in a special electronic form, not found in other iodine-containing drugs. In these complexes molecular iodine exhibits acceptor properties in relation to polypeptide in Complex II or ion I^- in Complex I and donor properties—in relation to lithium halide in Complex II or ion Li⁺ in Complex I. Probably, it is such a special electronic form of iodine that ensures low toxicity of the FS-1 drug.

It has been shown that the active centers of the drug can be segregated from dextrin helixes and form complexes with nucleotide triplets.

The interaction of active centers with nucleotide triplets carries a selective character: the active centers of the FS-1 drug are only segregated on specific sections of DNA. The formation of a complex between the nucleotide DNA and the active center of the FS-1 drug is a key stage in the anti-HIV mechanisms (Complex I), anti-coronavirus (Complex I, II) and antibacterial action (Complex II).

For S aureus DNA, the epigenetic modifications analysis in triplets was carried out. A qualitative correlation is observed between the stability of active sites complexes with nucleotide triplets and epigenetic modifications frequency in these nucleotide triplets (N). This result makes it possible to simulate the antibacterial efficacy of FS-1. If the active center of the MgI₃LiII₂ drug interacts with nucleotide triplets that are most often found in the bacterial genome, then it has antibacterial properties.