The results of cur-IONP docking with muc5AC and muc2 are represented in Table 2. The cur-IONPs binding affinity was − 6.015 kcal/mol and − 6.58 kcal/mol for muc 5AC and muc 2, respectively. RMSD scores were 1.08A and 2.60A, as shown in Fig. 3. The cur-IONPs bounded to muc5AC with one H-bond with GLU 3576 (A) amino acid. In contrast, they bounded to muc 2 with three bonds, a metallic bond between the Fe atom and ASP 1320 (A), and two pi-H bonds with PRO 1375 (A) and PHE 1376 (A) residues. This could reflect the greater stability of cur-IONPs/muc2 than cur-IONPs/muc 5AC due to more bonds between the ligand and the protein. To investigate the toxicity of our model, its smiles code was delivered to SWISS-ADME and PRO-TOX-3 servers, six physicochemical properties were considered. From SWISS-ADME results, the bioavailability radar shows that our model falls entirely in the pink area as recommended, which means it can be regarded as oral bioavailable and drug-like. The pink area represents the optimal range for each property, as represented in detail in the SWISS-ADME user interface16.Table 4 shows the physicochemical properties of the modeled cur-IONPs. In this section of the SWISS-ADME user interface, the general characteristic of our compound reveals that cur-IONPs molecular weight exceeds 500 Dalton. A molecular weight below 500 Dalton is a prime property of drug-likeness22.The lipophilicity of our IONPs is shown in Table 5; SWISS-ADME provides five free models to evaluate the compound’s lipophilicity characteristic, namely iLOGP, XLOGP3, WLOGP, MLOGP, and Silicos-IT Log P. The consensus Log P is the average of these predictions. The best range of Log P of a drug, according to the sources provided, is typically between 0 and 5, and 90% of marketed drugs are within this Log P range; having a Log P > 5 means that the drug is highly lipophilic, and it tends to accumulate in lipoidal areas of tissues and to have poor solubility. As presented in Table 5, we can see that cur-IONPs got a Log P score of 1.1, which is accepted, because this score means it will be optimal for oral administration due to the balance of solubility and permeability. (Table 6) shows that water solubility characteristics of modeled cur-IONPs were moderately soluble in adopted models.Regarding drug pharmacokinetics, (Table 7) represents the pharmacokinetics parameters and bioavailability of our modeled IONPs and illustrates the boiled egg model for each of our compounds. According to boiled egg analysis in Fig. 5, cur-IONPs represented high HIA (human intestinal absorption).P-glycoprotein (P-gp), also known as multidrug resistance protein 1 (MDR1), is an efflux transporter protein, Being a substrate or non-substrate of the permeability glycoprotein (P-gp) is crucial to appraising active efflux through biological membranes17, For instance, from the gastrointestinal wall to the lumen, despite cur-IONPs are represented as P-gp substrates it still has high HIA score, this means it may not being negatively affected by being a substrate to P-gp.being P-gp substrate also could influence bioavailability and bio distribution of drug, it also could increase renal clearance18, this finding extremely stress on the importance of further in vivo studies focusing on dosage, efficacy, and rate of clearance of cur-IONPs. Despite that it was reported that coating IONPs with curcumin enhanced the bioavailability and clearance rate significantly as serum iron level began to decline slowly returning to its normal level after 3 weeks19.Moreover, as illustrated in Table 7, cur-IONPs show an inhibition activity with one of the CYP 450 superfamily isoenzymes (CYP3A4). These enzymes are the key players in the detoxification and elimination through metabolic biotransformation. The inhibition activity of a drug against this category of enzymes could alter the clearance rate of the drug and increase its bioavailability and residency in the body. Furthermore, the best range of log Kp for a drug to be orally bioavailable and GI absorbable is between − 8 and − 120. This means that cur-IONPs fall in the best range of this value for a designed drug for oral administration.Regarding drug-likeness properties as shown in Table 8, the Lipinski filter (Pfizer) characterizes small molecules based on physicochemical property profiles such as MLOGP ≤ 4.15, Molecular Weight (MW) less than 500, NH or OH ≤ 5, N or O ≤ 10,21. Table 8 shows a drug-likeness rule score according to Lipinski Ghose, Veber, Egan, and Muegge. We found that cur-IONPs met four of the five drug-likeness rules, except the Ghose rule, with one violation due to its high molecular weight MW > 480. Despite the molecule fitting Lipinski’s rule, it shows one violation due to the high MW, MW > 500, however our molecule has MW = 530 g/mol which exceeds the desired upper limit of MW slightly, it is not an absolute requirement for efficacy or approval as according to the rule of five a drug can have one violation of these criteria and still be considered likely to be an effective oral drug, in fact many FDA approved drugs exceeded 500 g/mol. Finally, regarding bioavailability, cur-IONPs get a score of 0.55, which is considered good and accepted22. Our SWISS-ADME results conclude that cur-IONP is a good candidate for oral administration in treating IDA and deserves focus and further investigations.Protox-3 server analyzes and predicts oral acute toxicity, organ toxicity, toxicological and genotoxicological endpoints, and stress response pathways. Figure 6 indicates the rat’s oral toxicity lethal dose of 50 (LD50) as mg/Kg, cur-IONPs obtained LD50 value (3000 mg/Kg), which reflects the safety of cur-IONPs if it is administered orally23. The oral toxicity assessment of cur-IONPs indicates that they are generally safe when administered orally. The study on male BALB/c mice, where 6 doses of 5 mg/kg Cur-IONPs were given on alternating days for two weeks, showed promising results regarding toxicity and biodistribution. Additionally, another study reported the long-term safety and stability of cur-IONPs after a single dose administration in mice19.The predicted toxicity class of cur-IONPs was 5, respectively, as shown in Fig. 6. Regarding organ toxicity, all data are represented in detail for our compound in Table 9. Cur-IONPs exhibit hepatotoxic-inactive or non-hepatotoxic with a probability score of 0.71, cardiotoxicity, immune toxicity, and clinical toxicity with prediction scores of 0.60, 0.99, and 0.50, respectively. Cur-IONPs were investigated in vitro and in vivo and many studies assure its safety as reported in a study involving male BALB/c mice treated with multiple doses of Cur-IONPs showed no significant differences in liver enzyme levels (AST, ALT) compared to control groups, in the same study The biodistribution of Cur-IONPs revealed that they predominantly accumulated in the liver, spleen, and brain. Notably, histopathological examinations showed no abnormalities in these organs, indicating that the presence of Cur-IONPs did not lead to visible tissue damage19. Regarding suspected cardio toxicity, immune toxicity, and clinical toxicity of our molecule, to the best of our knowledge we found no previous study reported any related observations, on contrary cur-IONPs coating IONPs with curcumin was reported to enhance biocompatibility and safety in comparison to uncoated IONPs19,24,25.This suggests that Cur-IONPs do not induce notable organ toxicity under the tested conditions and more investigations could be very beneficial in this point.Furthermore, it is predicted to be able to cross the blood–brain barrier with prediction scores of 0.56, this comes in agreement with previous studies which reported the ability of cur-IONPs to cross BBB19. Regarding genotoxicity, our compound is not suspected to be carcinogenic, mutagenic, or cytotoxic, and it is predicted not to activate stress pathways as presented in the Table 9, in fact the role of curcumin as antioxidant26, anticancer27,28, and anti-inflammatory agent29 is well known. Based on the results, cur-IONPs show a favorable safety profile and promising results for further application as therapeutic nanoparticles. From our docking results, in addition to ADMET studies, we conclude that cur-IONPs would be potential for further investigations, so cur-IONPs/protein complexes interred to molecular dynamic simulation to assess the stability of the complex in more reliable ways, mimicking the real interaction environment in our body.To assess the ligand-induced alteration in protein structure, CABS-FLEX was used to generate root mean square fluctuations (RMSF) profiles as illustrated in Fig. 7. CABS-FLEX can generate RMSF profiles in addition to 10 models of structures after submitting the docked protein–ligand complex. RMSF value is usually calculated to indicate the stability of the complex, and the best RMSF value should be 1–3 A for optimum stable binding conditions. CABS-FLEX can generate RMSF from 10 ns simulations of all proteins/ligand complexes. Cur-IONPs/muc 5AC showed a max RMSF of 8.19 at residue 3516, a min RMSF was 0.195 at residue 3613, and an average RMSF was 1.49, while cur-IONPs/muc 2 showed a max RMSF of 4.028 at residue 1375, min RMSF of 0.333 at residue 1338, and average RMSF equals 1.567. Our results from the CABS-FLEX dynamic simulation showed that cur-IONPs/protein complexes are stable. Most complex residues show RMSF values below 3A, which is the main indicator of structure stability30. A lower value of RMSF specifies restricted movements from the average position throughout the simulation, while a higher value of RMSF demonstrates more movement flexibility31.To evaluate the physical movement and stability of our ligand-proteins complexes, we performed MD simulation using IMODS. (NMA) normal mode analysis was conducted to investigate the stability of our docked complexes. NMA of our docked complexes is illustrated in Figs. 9 and 10. The deformability and B-factor of complexes display the peaks corresponding to the regions with deformability in the protein, the highest peaks the highest deformability. The docked complexes’ significant mobility was shown by the NMA analysis, demonstrating the structural flexibility of the proteins with cur-IONPs. Furthermore, the deformability of proteins is demonstrated by our data; most of the proteins had several peaks that showed deformability. The mobility of the protein is related to the B-factor, and the energy required to deform the structure is directly correlated with the eigenvalues produced for the docked proteins. It represents the protein–ligand complex’s motion stiffness. The complex has stronger stability and easier deformability with a lower eigenvalue.The bound proteins’ MD analysis showed that the complexes have a notable degree of deformability. Additionally, the cur-IONPs/muc 5AC and cur-IONPs/muc 2 complexes had eigenvalues of 3.87 e−04 and 6.32 e−04, respectively, demonstrating the good flexibility and stability of the molecular motion of the docked complexes. The complexes’ covariance matrix shows the correlations between the residues in each complex. In the matrix, the red color denotes a reasonable degree of correlation between residues, while the white color denotes uncorrelated motion. Based on the plausible interactions of the selected proteins with cur-IONPs, we speculate that it can serve as a potential drug candidate for a highly bioavailable and long-resident IDA treatment. More in-vitro and in-vivo studies should be conducted to clearly reveal the toxicity of cur-IONPs and assess their efficacy as a potential drug for IDA.
