Development and Characterization of Metalloantibiotics for Pathogen Removal from Water: Insights from Antibacterial and Antiviral Activities

doi:10.21203/rs.3.rs-5053827/v1

Background

In recent efforts to address the critical need for clean and portable water, we have focused on innovative methods to eliminate pathogenic microorganisms. To this aim, the Glycyl-L-leucine peptide ligand was complexed with different transition metal ions [Cu(II), Ni(II), and Cd(II)]. The compounds were characterized and examined using various analytical methods, including elemental analysis (CHN), Fourier transform infrared spectroscopy (FTIR), and assessments of magnetic properties, molar conductivity, and thermogravimetric analysis. An N₂O₂ arrangement of ligand atoms coordinated all metals. The coordination sites were completed with a carbonyl oxygen atom and a water molecule. The complexes showed polymeric structures using bridging carboxylate groups.

Results

Their antibacterial properties were screened using disc diffusion and minimum inhibitory concentrations techniques against the identified bacterial organisms from the water samples collected along the Nile River. Cu(II)-chelate showed the best results for our investigation. The docking results supported and displayed that Cu(II)-chelate exhibited the minimum binding energy as compared to Ni(II), Cd(II), and free peptide which is in agreement with antibacterial results.

Conclusions

our study successfully demonstrated the potential of Glycyl-L-leucine peptide ligands complexed with transition metal ions, particularly Cu(II), in eliminating pathogenic microorganisms from water. Cu(II)-chelate exhibited superior antibacterial properties, as confirmed by both experimental and molecular docking results. This compound not only showed the lowest binding energy but also proved to be the most effective against bacterial and viral targets. These findings highlight the promising application of Cu(II)-chelate in developing advanced water purification systems.

Pollution

AMPs

Metal-Peptide

Bacteria

MIC

COVID-19

Pathogenic microorganisms are transmitted into different water sources that cause “Waterborne diseases”. So, great efforts are being made to find ways of eliminating these bacteria in the water to make it portable(1). Some examples of water-borne pathogens (E.coli, campylobacter jejuni, salmonella, cryptosporidium spp., streptococcus, hepatitis A virus)(2). Peptide antibiotics AMPs are gaining importance as anti-infective agents because they are produced in defense against invasive microbial pathogens by bacterial, mammalian, insect, or plant organisms. we need to search for novel and effective antimicrobial drugs due to increasing bacterial and fungal drug resistance. Various antimicrobial peptides (AMPs) exhibit activity against a range of organisms including fungi, bacteria, viruses, and protozoa, as well as cancerous cells. To date, databases dedicated to AMPs have cataloged over 2900 AMPs that have been experimentally identified and are produced by living entities(3). These peptides, referred to as host defense peptides, are compact molecules ranging from 7 to approximately 100 amino acids in size. They are polycationic and play a role in the innate immune defense common to various life forms. They have been recognized as promising agents in the fight against antibiotic resistance. The myriad of proteases inside the organism can degrade AMPs. Therefore, pairing AMPs with a metal ion could potentially enhance their stability, extend their duration in the body, lower the required dosage, and consequently diminish both the production expenses and the possible toxicity. The high positive charge of the complexes may lead to their higher tighten interaction with DNA and RNA molecules of microbes than uncomplexed compounds. Therefore, Therefore, we can consider the bioinorganic field of chemistry as a powerful weapon to overcome the various challenges encountered in the antibiotic chemistry field. The successful applications of Peptide antibiotics and metallo-antibiotics were discussed(4–6). The target of the present work is to identify the different types of bacterial organisms that exist in the polluted water samples taken from different water sources by synthesizing and characterizing the different metal-peptide complexes that are expected to have antibacterial effects. The biological study involves the evaluation of the inhibition zone and the minimum inhibitory concentration (MIC) techniques. The research also extended to study the interaction of the compounds with the bacterial enzymes by molecular docking system and investigate the antioxidant effect of the synthesized compounds.

Materials and Reagents

N-Glycyl-L-leucine and all metal salts (nickel(II), cadmium(II) and copper(II)) were provided by Aldrich. All biochemical reagents were provided by Merck.

Preparation of metal complexes

0.1 mmol (N-glycyl-L-Leucine) dipeptide was dissolved in the minimum quantity of hot distilled water. To this solution, metal salts were added in 1:1 stoichiometry raising the pH to be slightly alkaline under continuous stirring. The solution was carefully reduced in volume at 80°C until crystals started to form. These crystals were quickly isolated using filtration, rinsed with minimal amounts of a water-acetone solution in equal parts, and then allowed to dry in the ambient air. The suggested structure is pointed out in Figure 1.

Physical Measurements (Supplementary file)

Computational methods (Supplementary file)

Sample collection and Identification

Sampling

Nine water samples were collected from two places along the Nile River at Al Qanater Al Khayreya and Dahab Island. All samples were collected into sterile containers. The place and date were labeled on the containers. For negative control, we used boiled distilled water, when samples were obtained it was stored carefully in ice containers to maintain the samples in a conservative condition (7).

Isolation of bacteria

There are several methods to isolate microorganisms like bacteria from water. The serial dilution-agar plating process is a commonly used procedure. Serial dilution is used to reduce the of colonies number of bacteria.

Identification of bacteria

Using Standard Total Coliform Membrane Filter Procedure using Endo Media

Coliform bacteria definition: Rod-shaped, Gram-negative, non-spore-forming bacteria that can be either non-motile or motile. Acid and gas resulted by the fermentation of lactose after the incubation at 35–37°C. Because some coliform bacteria have a limited capacity to ferment lactose, the definition has been updated to include bacteria that contain the enzyme β-galactosidase. These bacteria are recognized as indicators of the quality and safety of water and foods.

Thermotolerant (Fecal) Coliform Membrane Filter Procedure

A facultatively anaerobic, gram-negative, rod-shaped, non-sporulation bacterium. The term "thermotolerant coliform" is gaining acceptance over "fecal coliform"(8). In general, it originates in the warm-blooded animal’s intestines. It can grow in the presence of bile salts or identical surface agents. It is oxidase-negative. From lactose, it gives an acid and gas through 48 hours at 44 ± 0.5°C. Typical genera include feces (Escherichia) as well as genera not of fecal origin (Enterobacter, Klebsiella, and Citrobacter).

Study the Antimicrobial effect of metal complexes

in vitro, the antimicrobial activity can be evaluated by using various laboratory methods such as broth or agar dilution methods (MIC) and disk diffusion agar.

Zone of inhibition in disk-diffusion Antimicrobial assay:

The agar well diffusion method was used to evaluate the antimicrobial activity. All the compounds were examined in vitro for their antibacterial effectiveness against the separated Enterobacteriaceae group as a whole and the purchased (klebsiella (ATCC:4415) & Escherichia coli (ATCC:3008)) (Gram-negative bacteria). Gentamicin was used as a standard drug. Dimethyl sulfoxide (DMSO) served as the solvent control. The substances were evaluated at a concentration of 1 mg/ml for their effectiveness against various strains of bacteria and fungi (9, 10).

The summary of the procedure is as follows:

1- Each sterilized Petri was filled by pouring the sterilized media (20-25 ml) and allowed to convert to the state of solid form.

2- With the help of a sterilized borer, We created wells measuring of 6 mm diameter in the hardened culture medium.

3- The microbial suspension was distributed evenly by using a sterile swab over the surface of solidified media.

4- With the help of a micropipette, each solution of the tested compounds was added to each well.

5- The assessment of the inhibition zone was conducted using a millimeter scale following a 24-hour incubation period at 37°C, specifically to determine antibacterial activity

Broth or Agar dilution MIC

1- Selection and Transfer: From each bacterial strain, a group of 3-5 separate colonies was carefully picked from a newly cultured agar plate. These colonies were then placed into a test tube filled with 3 to 4 ml of a sterile liquid nutrient medium.

2- Incubation and Mixing: The mixture containing the bacterial cells was thoroughly agitated to ensure even suspension and then kept in an incubator. The temperature was maintained between 35°C and 37°C for a duration ranging from 2 to 6 hours.

3- Turbidity Standard: The cloudiness level of the bacterial mixture was assessed and required to meet or surpass the 0.5 McFarland turbidity standard.

4- Dissolution of the examined compound (1 mg) (antimicrobial agent) in DMSO (1 ml).

5- Dilution Process: Perform a sequential two-fold dilution with the use of a broth medium.

6- Inoculation and Incubation: Introduce a measured amount of the bacterial inoculum into each tube, followed by an incubation period at 37°C for 16 to 20 hours.

7- MIC Determination: Examine for the Minimum Inhibitory Concentration (MIC), which is identified as the smallest concentration of the tested antimicrobial agent that prevents the visible growth of the bacterial isolate, as confirmed by naked-eye observation(11).

Antioxidant activity

The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method was used to investigate the antioxidant behavior of our compounds by preparing different concentrations of every compound. It gives a purple solution in methanol. The resulting mixture’s absorbance, formed from the interaction between DPPH and each compound, was measured spectrophotometrically at a wavelength of 517 nm, utilizing ascorbic acid (Vitamin C) as the standard drug. The radical scavenging activity (DPPH) (%) was calculated (12).

Glycyl-L-leucine peptide ligand was complexed with different transition metals. These metal complexes were investigated using the physicochemical techniques mentioned in the experimental part.

The elemental analyses

The elemental analyses show a satisfactory agreement between the experimental, and theoretical results. All complexes were formed in a stoichiometric ratio (1:1) (Metal: Ligand). The analytical results are concisely presented in Table 1.

The results of the elemental analysis revealed that:

Cu(Gly-Leu) has one coordinated water molecule in its chemical structure.

Ni(Gly-Leu) has two water molecules (hydrated and coordinated) in its chemical structure.

Cd (Gly-Leu) has one coordinated water molecule in its chemical structure.

Table1. Analytical and physical data of metal (Gly-Leu) complexes.

Compound (empirical formula)	Color	Yield %	Found (Calc.) %
Compound (empirical formula)	Color	Yield %	C	H	N
[Cu(Gly-Leu)( H₂O)] C₈H₁₆CuN₂O₄	Brown	70.0	34.99 (35.88)	5.30 (6.02)	10.01 (10.46)
[Ni(Gly-Leu)(H₂O)] .H₂O C8H18N2NiO5	Green	82.0	33.79 (34.20)	6.20 (6.46)	9.42 (9.97)
[Cd(Gly-Leu)( H₂O)] C₈H₁₆CdN₂O₄	White	90.0	29.99 (30.35)	4.40 (5.09)	8.56 (8.85)

Infrared analysis

The infrared spectra are, as expected, Interestingly, they exhibit considerable similarity in the spectral ranges where the vibrational bands of the coordinating groups appear. These vibration positions are compared in Table 2, and the proposed assignment is based on several references (13-15). The interpretation of spectra is based on the comparison between the chelated peptide with its free form. These assignments were pointed out as:

After coordination, a clear shift to higher wavenumbers values was observed for the amino NH₂ groups stretching vibrations with some broadening related to the coordinated or hydrated water molecules stretching vibrations.
After coordination, the disappearance of the N–H stretching of the amido moiety, because of the deprotonation of this group.
After coordination, there was a noticeable shift in the carbonyl group (C=O) stretch within the amide linkage towards lower energy levels, which aligns with its participation in the bonding.
After coordination, the stretching vibrations of the two carboxylate groups underwent minimal alterations, a characteristic typically associated with bridging carboxylate groups (16).
Within all the complexes, the coordination environment is completed by the inclusion of water molecules. This is substantiated by the detection of OH bending vibrations from the water molecules that are coordinated(5).
The bands assigned to metal-ligand vibrations are tabulated in Table 2 (5, 17)

Table 2. The most important IR frequencies bands of complexes (200- 4000 cm⁻¹).

Compound	_υ(NH₂)	_υ(COO^- )_as	_υ (M-O)	_υ (M-N)
Cu-(Gly-Leu)	3410	1558,1442	447, 401	331,308
Ni-(Gly-Leu)	3410	1581, 1504	493, 416	339,308
Cd-(Gly-Leu)	3410	1558, 1512	450,408	345,321

¹HNMR analysis

The ¹HNMR spectra of the free peptide and its Cd (II) complex as a representative example from the synthesized complexes were screened in DMSO-d₆ against TMS as internal reference is obtained.

For ligand: ¹HNMR gave the aliphatic protons at 0.84, 0.89, 1.4, and 3.7 for (CH₃) groups, (CH)_A, (CH₂)_B, and (CH₂)_C respectively. NH₂ (amino), NH (amide) and OH protons are given as sharp singlet peaks at 3.3, 4.2, and 8.2 ppm respectively (D₂O exchangeable). After coordination, all (A, B & C) and NH₂(amino) signals were shifted to lower values with the absence of the protons corresponding to each NH(amide) and OH carboxylate group. The suggested structures are seen in Figure 1.

Molar conductance and magnetic susceptibility studies

Conductivity measurements indicate that values below 50 Ώ ^-1 ohm^-1cm² in DMSO solution are for nonelectrolytes (18). The low molar conductance readings of the synthesized complexes suggest their non-electrolytic nature. This observation corroborates the complexes’ coordination with the carboxylate anion and the subsequent ionization of the amide proton ( C=NH ), resulting in the full neutralization of the metal’s cationic charge. These findings are in line with prior elemental analysis, (IR), and (¹H NMR) studies.

We have two observations about the values of our Cu(II) magnetic moment (1.81 B.M.): First, the value is present in the values registered for the hexa-coordinate Cu(II)complex (19). Second, the measured value is slightly more than the registered value for one unpaired electron (1.73 B.M.) (spin-only value). So, it suggested mainly distorted octahedral geometry(20).

The magnetic moment reading of our Ni(II) chelate was 3.40 BM. It presents within the octahedral geometry structure range of 2.8-3.5 BM for Ni(II) complexes (21). For the Cd (II) complex, the magnetic moment has a diamagnetic nature. It corresponds to octahedral geometry due to its electronic configuration (d¹⁰).

Thermal study

Thermogravimetric analysis, which tracks weight changes, was performed for synthesized complexes using the TG technique. This analysis spanned from ambient temperature up to 800°C. The primary purpose of these methods is to deduce details about the water molecules’ existence and their location—whether they are part of the inner or outer coordination sphere surrounding the central metal ion. Additionally, these procedures assess the thermal stability of the complexes. The data regarding the percentage of mass loss are detailed in Table 3. The initial stage of weight loss corresponds to the water molecules that are either directly bonded to the metal ions or incorporated within the crystal lattice (5). As represented in Figure 2, all complexes showed the evaporation of one water molecule at a temperature of more than 200^oC corresponding to the coordinated water molecule. The values of their weight loss % are 6.20, 6.02, and 6.0 for Cu-(Gly-Leu), Ni-(GlyLeu) and Cd-(Gly-Leu) complexes respectively. Ni-chelate only showed evaporation for one hydrated water molecule at a temperature equal to 100 ^oC. Upon additional heating, the complexes display a decline in mass, which is attributed to the decomposition of the organic components. Cu (II), Ni (II) and Cd (II) complexes showed the breaking of the organic ligand in one step with the mass loss [Cu 4.45 %( 63.58%)], [Ni 65.7% (56.0%)] and [Cd 53.89% (53.76%)] for the experimental and the calculated values respectively. In conclusion, the segment of the thermogram that aligns with the x-axis (indicative of temperature) represents the remaining residue. It was anticipated that this residue would constitute metal oxide for every type of complex analyzed. The metal residue percentages are [Cu 29.29 %( 29.68%), Ni 24.30 %( 24.98%) and Cd 40.11 %( 40.55%)] for the experimental and the calculated values respectively. It was observed that Cu-(Gly-Leu) chelate has higher stability because it decomposed at a higher temperature than the other chelates. Finally, from all previous studies, we can deduce that the coordination is carried out through the N₂O₂ arrangement of ligand atoms. The coordination sites were completed with a carbonyl oxygen atom and a water molecule. The complexes showed polymeric structures using bridging carboxylate groups.

Table 3. The thermal decompositions of metal complexes.

Compound

DTG, Experimental (Calculated)

[Cu(Gly-Leu)( H₂O)]

Coordinated water = 6.2% (6.7%)

Organic compound =64.45% (63.58%)

Residue CuO= 29.29% (29.68%)

[Ni(Gly-Leu)( H₂O )].H₂O

Hydrated water + Coordinated water = 12.5% (12.04%)

Organic compound =65.7% (65.0%)

Residue NiO= 24.3% (24.98%)

[Cd(Gly-Leu)( H₂O)]

Coordinated water = 6.0% (5.6%)

Organic compound =53.89% (53.76%)

Residue CdO= 40.11% (40.55%)

Antibacterial investigation

Antimicrobial peptides are potent agents with diverse structural and antimicrobial peptides, which represent one of the hopeful future drugs for combating infections and microbial drug resistance. So, the following work represents the bacterial investigation of the (Gly-Leu) peptide ligand and its modified compounds by its chelation with Copper (II), Cadmium (II), and Nickel (II) metal ions. Water culture is discussed in the experimental part revealing the presence of Gram-negative (Enterobacteriaceae group). Enterobacteriaceae are Gram-negative bacteria of a large family that includes Escherichia coli, Klebsiella, Salmonella, Shigella, Yersinia pestis8 (22) and Gram-negative (fecal coliform). Klebsiella pneumonia . and Escherichia coli hurt humans as Klebsiella pneumonia is a considerable pulmonary pathogen leading to severe pneumonia and sepsis, mainly in immunocompromised patients. Escherichia coli is the major common reason for acute urinary tract infections as well as urinary tract sepsis. Escherichia coli is a usual cause of ‘traveler's diarrhea, a dysentery-like disease affecting humans and may raise acute enteritis in humans as well as animals (23) so the antibacterial investigation was done against Klebsiella and Escherichia Coli strains. Also, it performed against the Enterobacteriaceae group as a whole. The investigation involved the inhibition zone technique by measuring the diameters of inhibition zone values presented in Figures 3(a, b) and Table 4. The antibacterial effect of the peptide ligand appeared after its coordination with the metal ions. Cd(II) and Ni(II) complexes were found to possess significant antibacterial activity against Klebsiella pneumonia and Escherichia coli bacteria respectively when compared to the standard drug (Gentamicin). Cu-chelate showed moderate activity against the Enterobacter organism. Klebsiella pneumonia effectively died by using Cu-chelate rather than the standard Gentamicin drug. The metal complexes exhibited greater antimicrobial properties compared to their ligands This enhanced activity is likely due to the structural alterations that occur upon coordination. Within these complexes, the polarity of the metal ions is diminished as a result of the partial sharing of the negative charge from the heterocyclic ligands. Consequently, this reduction in polarity augments the lipophilicity of the metal ions, enabling them to more effectively infiltrate and traverse the lipid membranes of microorganisms, leading to their destruction. additionally, the activity index is shown in Fig.4(a)(17). In addition, the antimicrobial activity of Cu-complex was significantly greater than others, which may be mostly because of the relatively strong lipophilic property and its higher stability as mentioned before in the thermal study. The results revealed that the antibacterial assay against gram-negative bacteria was effective. It may be due to the composition of the gram-negative bacteria having a thin peptidoglycan layer that facilitates the penetration of the compound into the bacteria and kills it. MIC was performed for the best zone of inhibition results, Fig. 4(b). It was tested for Cd(II) and Cu(II) chelates

against Klebsiella pneumonia organism but Ni-chelate against Escherichia coli. The MIC values were tabulated in Table 5. The Cu-chelate demonstrated strong activity against Klebsiella, with a MIC value of 62.5 μg/ml, comparable to that of the standard drug gentamicin, whereas the Cd-complex showed a moderate effect (MIC=125 μg/ml) equal to the double effect of Cu and gentamicin compounds. By comparing MIC values of gentamicin and Ni-complex against Escherichia coli, Ni complex showed a mild effect (31.25μg/ml).

Table 4. Zone inhibition (mm.) of (Gly-Leu) ligand and its chelates.

Table 5. Minimum Inhibitory Concentration (MIC) of (Gly-Leu) ligand and its chelates.

Molecular Docking Investigation:

Molecular docking plays an important essential role in the rational design of drugs. Molecular docking can be described as the “best-fit” orientation of a ligand that binds to a certain protein of interest (24, 25). Antimicrobial peptides (AMPs), as emerging therapeutic agents, offer a strategy to counteract the development of pathogens. Therefore, our objective is to develop a potential AMP that can control bacterial growth by focusing on various targets, allowing us to forecast and analyze its antibacterial impact.

Killing bacteria may be done in different ways.

One of these ways is perhaps due to the inhibition of bacterial cell wall synthesis. We can target penicillin-binding proteins (PBP) and glucosamine-6-phosphate enzymes as key examples involved in bacterial cell wall synthesis. (PBP) plays a pivotal role in the synthesis of the bacterial cell wall, primarily through its transglycosylation and transpeptidation activities (26, 27). It’s a recognized fact that alterations in PBP can result in the emergence of strains of pathogenic bacteria that are resistant to antibiotics (28). The altered version of the penicillin-binding protein 5’s crystal structure was sourced from the Protein Data Bank, under the identifier 1NJ4. This variant of PBP5’s structure was ascertained through X-ray crystallography and documented with a resolution of 1.9 Ångströms (29). Furthermore, the resistance of bacteria to Gly-Leu compounds was investigated through molecular docking studies with the altered PBP5.

Interaction analyses of our compounds are seen in Table 6 and Figure S1-S4. S1-S4 showed that the Cu-chelate forms a high interaction with the mutated version of PBP5, exhibiting a binding energy of −9.3 kcal/mol. This strong bond is facilitated by three hydrogen bonds, each less than 3 Ångströms in length, with the mtPBP5. Hydrogen bonds were found between Asp113 with (amino position) of cu-chelate of 1.81 Å bond length and (Asp105 & Asn112) with coordinated water molecules of (2.02 & 1.82) Å bond lengths respectively. Cu-chelate showed side chain acceptor and backbone donor types of interactions towards the selected protein with a higher number than the other investigated compounds.

The computational forecast aligns closely with the outcomes of antibacterial tests, as demonstrated in the subsequent section on antibacterial analysis. This correlation underscores the considerable influence of molecular docking research.

Glucosamine-6-phosphate (GlcN-6-P) was synthesized from d-l-glutamine and fructose-6-phosphate by using the glucosamine-6-phosphate synthase enzyme. Synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) depends initially on (GlcN-6-P). UDP-GlcNAc is present in all types of organisms (30). The GlcN-6-P synthase inactivation time has a different effect between the microbes and the human cells. GlcN-6-P disappearance in bacteria is fatal for microbes but vice versa in mammals (31). So, Glucosamine-6-phosphate (GlcN-6-P) synthase is a compelling target for protein interaction studies due to its critical function in safeguarding the cell walls of a wide range of microorganisms and human cells alike (32).

Consequently, a substantial body of research on molecular docking targeting GlcN-6-P synthase has been conducted, emphasizing its role as a key enzyme in the development of antibacterial agents. (33, 34). The potential inhibitor is expected to bind with the enzyme’s active site, resulting in the formation of a complex between the ligand and the enzyme. This complex is a key element in the mechanism that leads to inhibition. (35).

The process of docking synthesized molecules with enzymes demonstrated that the expected inhibitor compounds exhibited hydrogen bonding interactions with multiple amino acid residues within the active pockets of GlcN-6-P synthase, as illustrated in Figures S5-S8. In the chemical structure of the synthesized compounds, it was observed that the Gly-Leu ligand interacts with Met-89 at a distance of (3.0 Ǻ). Gly-Leu ligand forms interactions through the (NH₂) amino, carbonyl oxygen (COOH) and (NH) amide oxygen atoms with Val567, Pro521 and Asp548 active moieties of enzyme respectively. All three synthesized chelates form hydrogen bonds with Asp474 and Ser316 residues. Glu569 active amino acid was observed in both Cd and Ni chelates. Furthermore, based on the molecular docking prediction in Table 7. The three chelates exhibited high performance against the target enzyme as supported by their lower binding energies (-6.7, -6.25 and -6.16 kcal mol-1) for Ni-, Cd- and Cu- chelates respectively) with comparing to Gly-Leu peptide as the Previous studies indicated that a low binding energy is necessary for enzyme inhibition, as reported by various research works (36). The activation energy of the inhibition reaction prefers the more negative binding energy which leads to tight binding of the enzyme-inhibitor complex (35). The backbone acceptor type of interaction was the most widespread type with all molecules against the selected protein.

Another way for killing bacteria is perhaps due to inhibition of bacterial DNA replication, transcription, repair, and recombination by targeting DNA gyrase enzymes as examples.

DNA gyrase enzyme is required for bacterial DNA replication, transcription, repair, and recombination. the target is to evaluate the possible relationship between the docking activity of the synthesized compounds with the crystal structure of the DNA-gyrase receptor (PDB Code: 5BS8). The results of molecular docking were investigated (Table 8) based on energy score. Cu-chelate was docked with a better binding energy score of -18.1 kcal/mol in comparison to other synthesized compounds. So, Cu-chelate showed a higher antibacterial potency. The results were compatible with the previous antimicrobial assay. The hydrogen bonding was found to be the prominent interaction of amino groups with DNA-gyrase Figures. S9-S12. The amino acid residues interacted are ArgA39, Asp-B536, Asp-B534, and GluB459 with side chain donor, Metal contact, side chain acceptor, and side chain donor interaction types respectively.

Finally, we found docking DNA gyrase and penicillin-binding protein enzymes results were in good agreement with the experimental results that revealed that Cu-chelate is the best one as an antibacterial drug.

Table 6. Molecular Docking Results with Mutant PBP5 (mtPBP5) (PDB Code: 1nj4)

	Docking 1nj4
Compounds	Involved amino acids	Type of interaction	Scoring Energy (kcal/mol) (RMSD)
Gly-Leu	Met-89 (3.0 Å)	Backbone acceptor	-4.139(0.99)
Cu-(Gly-Leu)	Asp-113 and Asp-105 Asn-112	Side chain acceptor backbone donor	-9.373(1.85)
Cd-(Gly-Leu)	Gln-195 (2.09 Å), (2.14 Å)	Side chain acceptor and side chain donor	-3.148(2.49)
Ni-(Gly-Leu)	Ligand exposure	Ligand exposure	-5.049(2.48)

Table 7. Molecular docking parameters towards GlcN-6-P synthase (PDB Code: 1moq)

	docking 1moq
Compound	Involved amino acids	Type of interaction	Scoring Energy (kcal/mol) (RMSD)
Gly-Leu	Val-567 Pro-521 (2.85 Ǻ) Asp-548	Backbone acceptor Backbone acceptor Side chain acceptor	-2.7(1.03)
Cu-(Gly-Leu)	Asp-474 (1.69 Ǻ) Ser-316(2.29 Ǻ)	Side chain acceptor Side chain acceptor	-6.16(2.90)
Cd-(Gly-Leu)	Glu-569 (2.15 Ǻ) Asp-474 (2.44 Ǻ) Ser-316 (3.0 Ǻ)	Side chain acceptor Side chain acceptor Side chain donor	-6.25(1.80)
Ni-(Gly-Leu)	Glu-569 (2.6 Ǻ) Asp-474 (2.01 Ǻ) Ser-316 (2.99 Ǻ)	Metal contact Side chain acceptor Side chain donor	-6.7(3.20)

Table 8. Binding affinity of complexes against DNA-gyrase receptor (PDB Code: 5BS8)

Antitumor docking 5BS8
Compound	Involved amino acids	Type of interaction	Scoring Energy (kcal/mol) (RMSD)
Gly-Leu	Gln-C113 (2.32 Ǻ) Ser-C69 (3 Ǻ) Asp-A157 (2.86) His-C70 (2.43)	side-chain donor side-chain donor side-chain acceptor backbone acceptor	-3.88(0.841)
Cu-(Gly-Leu)	Arg-A39 (2.02 Ǻ) (2.077 Ǻ) Asp-B536 Asp-B534 (2.05 Ǻ) Glu-B459 (2.05 Ǻ)	side-chain donor Metal contact side-chain acceptor side-chain acceptor	-18.13(2.11)
Cd-(Gly-Leu)	Arg-C128 (2.21 Ǻ) Ser-A91 (3.0 Ǻ) His-A87 (1.81 Ǻ)	side chain donor side-chain donor backbone acceptor	-6.649(1.89
Ni-(Gly-Leu)	Gly-A88 (1.96 Ǻ) Asp-B532 (2.1 Ǻ) Glu-B459 (2.01 Ǻ),(2.25 Ǻ)	side-chain donor side-chain acceptor side-chain acceptor	-10.88(2.77)

Antioxidant Activity

The attack of reactive species such as free radicals on cellular structures leads to the onset of numerous diseases associated with oxidative damage. In the last decades, antioxidants become important because of the high exposure to free radical pollution (37). Newly, the investigation and seeking for metal-derived antioxidants become very interesting to identify new compounds having a high capacity in scavenging free radical pollution. Due to synthetic antioxidants being more effective and cheaper than natural antioxidants, they become widely used. Consequently, a specific criterion exists for evaluating the antioxidant activity of metal complexes.

DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable nitrogen radical and a widely known free radical used in vitro for testing antioxidant activity. It has an odd electron and exhibits a strong absorption band at 517 nm. When this electron is paired off, the absorption decreases stoichiometrically relative to the number of electrons accepted(38). Due to the variation in absorbance readings, DPPH has been extensively used to assess the ability of different substances to act as free radical scavengers. On the other hand, DPPH also is decolorized by reducing agents as well as H transfer, which also contributes to inaccurate interpretations of antioxidant capacity. So, DPPH is usually used as a suitable material to evaluate the antioxidant activity of other antioxidants. We employed ascorbic acid (commonly known as Vitamin C) as our reference standard.

We evaluated the antioxidant activity of both the free peptide (Glycyl-L-leucine) and its three metal chelates [Cu(II), Cd(II), & Ni(II)], alongside ascorbic acid serving as the standard reference drug. This assessment was based on their ability to scavenge stable DPPH free radicals. Additionally, IC50 values were calculated to determine the potency of the tested samples, representing the concentration required to reduce a specific activity by half. The variations in the free radical scavenging efficacy of the test compounds, expressed as percent inhibition, are depicted in Figure 5. From these data, it is clear that most of the chelates showed excellent antioxidant activity than the parent ligand, especially Ni(II) and Cu(II) chelates. Both complexes have IC₅₀ are very near to the IC₅₀ value of the standard (ascorbic acid). The IC ₅₀ values are (14.4, 15.5, 18, 44.9 and 78) for (Vit. C, Ni, Cu, peptide and Cd) respectively. The increased antioxidant potency observed in the metal complexes compared to the original ligand might stem from the electron-withdrawing influence of the metals, which hastens hydrogen removal to counteract DPPH. Notably, our compounds contain numerous proton sources. Consequently, hydrogen atoms are susceptible to abstraction by free radicals, leading to the formation of stable radicals. This is evidenced by the transition of the solution from purple to yellow, indicating the scavenging of DPPH radicals through hydrogen donation (38).

Antiviral Molecular Docking:

In this paragraph, we asked if our AMP-derived compounds can serve as anti-viral drugs against the COVID-19 virus. Focusing on the main protease (PDB id: 6LU7) is becoming increasingly crucial in the development of anti-CoV medications. The Protein-ligand interaction of the stable docked AMPs-based compounds with the main protease complex was visualized in Figure 6. All AMPs based compounds showed hydrogen bond interactions with the active amino acids mentioned in Table 9 as Gln-C113, Ser-C69, Asp-A157, His-C70, Arg-A39, Asp-B534, Glu-B459, Arg-C128, Ser-A91, His-A87, Gly-A88 and Asp-B532. According to the lower scoring energy value, Cu-chelate provides the more successfully docked interactions. Cu-chelate molecular docking proved that the better antibacterial drug was the better antiviral one also. But finally, we can’t say that, in general, the antibacterial drug should help as an antiviral drug.

Table 9. Binding affinity of complexes with main protease (6LU7).

docking 6LU7
Compound	Involved amino acids	Type of interaction	Scoring Energy (kcal/mol) (RMSD)
Gly-Leu	Gln-C113 (2.32 Ǻ), Ser-C69 (3.0 Ǻ) Asp-A157 (2.86 Ǻ) His-C70 (2.43 Ǻ)	Side chain donor Side chain acceptor Backbone acceptor	-2.46(1.28)
Cu-(Gly-Leu)	Arg-A39 (2.02 Ǻ) (2.077 Ǻ) Asp-B536 Asp-B534 (2.05 Ǻ), Glu-B459 (2.05 Ǻ)	Side chain donor Metal contact Side chain acceptor	-9.22(2.49)
Cd-(Gly-Leu)	Arg-C128 (2.21 Ǻ), Ser-A91 (3.0 Ǻ) His-A87 (1.81 Ǻ)	Side chain donor Backbone acceptor	-5.85(1.82)
Ni-(Gly-Leu)	Gly-A88 (1.96 Ǻ) Asp-B532 (2.1 Ǻ), Glu-B459 (2.01 Ǻ)	Side chain donor Side chain acceptor	-7.67(1.51)

The ¹HNMR and IR spectra provide clear evidence that the peptide ligands coordinate in an N₂O₂ configuration, involving carboxylate, carbonyl, and amino groups in a bridging mode. The deprotonation of the amide group and the involvement of the carbonyl oxygen in metal-ligand interactions are confirmed by shifts observed in the IR spectra after coordination.

The ¹H NMR spectra also support this coordination mode, as the disappearance of NH and OH proton signals suggests their participation in the complexation process. Shifts in aliphatic proton signals further show that coordination affects the peptide backbone (39, 40).

Molar conductance data indicate the non-electrolytic nature of the complexes, suggesting complete neutralization of the metal’s cationic charge. Magnetic susceptibility studies are consistent with the proposed octahedral geometries for the Cu(II) and Ni(II) complexes, while the diamagnetic nature of the Cd(II) complex confirms its octahedral structure.

Thermal investigations have revealed that all of the complexes are thermally stable, with the Cu-(Gly-Leu) complex being the most stable. When the coordinated water molecules evaporate at relatively high temperatures, strong metal-water interactions are demonstrated. When the organic ligand breaks down and metal oxides are produced as residues, it indicates that the complexes' high-temperature structural integrity is intact (41).

In stable polymeric complexes, the coordination of metal ions is achieved by both carboxylate and peptide groups, and the coordination environment is completed by additional water molecules, as indicated by the combined data from thermal analysis, 1H NMR, peptide studies, and IR. These data support the suggested N₂O₂ coordination mode and demonstrate the produced complexes are thermally stable, especially for copper-based structures.

The antibacterial investigation results show that coordinating the Gly-Leu peptide with metal ions improves its antimicrobial properties. The Cd(II) and Ni(II) complexes exhibited significant antibacterial activity, while the Cu(II) complex demonstrated moderate to strong effectiveness against Klebsiella pneumonia and Enterobacter. Increased lipophilicity after complexation allows metal complexes to easily pass through bacterial cell membranes, leading to improved antibacterial activity.

(MIC) values verify the Cu(II) complex is greater efficacy against Klebsiella pneumonia, matching that of the conventional antibiotic Gentamicin. Strong activity was also shown by the Ni(II) complex against Escherichia coli. These results are in line with the structural alterations observed in the IR and NMR data, wherein interaction with metal ions modifies the electronic environment of the ligand and increases biological activity.

The antibacterial mechanism of the metal complexes can be better understood through molecular docking investigations. High binding interactions between the Cu(II) complex and DNA gyrase, two crucial bacterial proteins involved in DNA replication and cell wall formation, are observed. The robust antibacterial activity of the Cu(II) complex is supported by its high binding energies, indicating that it has potential as a therapeutic agent. The mild antibacterial activities of the Ni(II) and Cd(II) complexes were also explained by their positive interactions with bacterial enzymes.

The results of the antioxidant activity demonstrate how metal coordination increases the peptide ligand's capacity to eliminate free radicals. The Cu(II) and Ni(II) complexes showed IC50 values that were comparable to those of ascorbic acid, suggesting that they could be useful antioxidants. Free radicals are neutralized by the complexes' enhanced capacity to donate hydrogen atoms due to the metal ions' electron-withdrawing properties.

The best docking results between the Cu(II) complex and the COVID-19 primary protease indicate that the complex has antiviral potential and may be able to effectively limit viral replication. These findings need more experimental research to determine whether antibacterial metal complexes can also function as antiviral medicines. All things considered, the results emphasize the significance of a multi-targeted drug design strategy, taking into account a compound's capacity to inhibit both bacterial and viral.

Finally, as mentioned before in the biological assay and all docked protein results, the complexation increased the reactivity of the (Gly-Leu) ligand. The experimental and molecular docking studies supported the superior antibacterial effect of Cu(II) chelate than others. The Cu(II) chelate exhibited intriguing interactions with the amino acids located in the active site of the lethal bacterial proteins. The superior efficiency of Cu(II) chelate was extended to the antioxidant effect against DPPH free radicals. Also, Ni(II) chelate had a comparable IC₅₀ value to the standard ascorbic acid based on the concept of the metal-induced electron withdrawal effect, which speeds up the elimination of hydrogen to reduce DPPH.

Gly-Leu

Glycyl-L-leucine

AMPs

Antimicrobial peptides

E. coli

Escherichia coli

DNA

Deoxyribonucleic Acid

RNA

Ribonucleic Acid

MIC

the minimum inhibitory concentration

DPPH

The 1, 1-diphenyl-2-picrylhydrazyl

DTG

differential thermal gravimetric

RMSD

Root Mean Square Deviation

PBP

penicillin-binding proteins

PDB

Protein Data Bank

(GlcN-6-P)

Glucosamine-6-phosphate

Met

Methionine

Gly

Glycine

Asp

Aspartic acid

Gln

Glutamine

Asn

Asparagine

Val

Valine

Pro

Proline

Ser

Serine

Glu

Glutamic acid

His

Histidine

Arg

Arginine

Ethics approval and consent to participate

Not applicable.

Funding

Not applicable.

Author Contribution

Safaa S Hassan, Eman F. Mohamed, Aml M. Saleh, and Samar A. Aly: Testing, Validation, Supervision, Data Curation and Writing—review, and editing. Kirolos Maged, Salma Hassan, Alaa Omran, and Shahinda Nasr: Conceptualization, Investigation, and Writing—review, and editing. Salma Reda, Paula Nabil, Andrew George: methodology, software. Mohamed M Shoukry: Supervision, review, and editing.

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No competing interests reported.

supplementary.docx

Development and Characterization of Metalloantibiotics for Pathogen Removal from Water: Insights from Antibacterial and Antiviral Activities

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Materials and Method

Materials and Reagents

Preparation of metal complexes

Physical Measurements (Supplementary file)

Computational methods (Supplementary file)

Sampling

Isolation of bacteria

Results

¹HNMR analysis

Molar conductance and magnetic susceptibility studies

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Funding

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Development and Characterization of Metalloantibiotics for Pathogen Removal from Water: Insights from Antibacterial and Antiviral Activities

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Materials and Method

Materials and Reagents

Preparation of metal complexes

Physical Measurements (Supplementary file)

Computational methods (Supplementary file)

Sampling

Isolation of bacteria

Results

1HNMR analysis

Molar conductance and magnetic susceptibility studies

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Funding

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

¹HNMR analysis