According to recent observations, individuals who are in highly immune-compromised health circumstances following COVID-19 having diabetes or high uncontrolled sugar levels were infected with a disease produced by a "mucormycosis" . The two agents currently approved by the FDA for the primary treatment of mucormycosis are amphotericin B and isavuconazole. Previous research efforts to develop antifungal agents against the Mucorales demonstrated that the inhibition of β-1,3-glucan biosynthesis by using inhibitor drugs like amphotericin/echinocandins inhibited fungal growth, thus abolished replication. In the study conducted in 2014, researchers suggested that CotH3 could be an emerging therapeutic target for mucormycosis as this functions as an invasin that interacts with host cell GRP78 to mediate pathogenic host-cell interactions. Similarly, inhibition of Lanosterol 14 alpha-demethylase interrupts the conversion of lanosterol to ergosterol, which leads to the depletion of ergosterol in the fungal cell membrane and accumulation of aberrant 14-α-methylsterols in fungal cells, thereby causing fungal death . Further, they produce a toxin called mucoricin, which plays a central role in the virulence of Mucorales . Hence, targeting CotH3, Lanosterol 14 alpha-demethylase and Mucoricin may offer a new active antifungal approach to treat mucormycosis. Thus, in our study, we attempted to reveal a novel therapeutic option for treating mucormycosis by the screening of FDA approved drugs, FDA unapproved, investigational-only, natural compounds against our targeted proteins using structure-based virtual screening. To date, no crystal structures were determined for our Rhizopus delemar target proteins. Hence, protein modelling was performed for the prediction of protein structure based on the available sequence data. The 3D-modelled structures were thoroughly investigated and confirmed using the Ramachandran Plot analysis. Moreover, ascertainment of stability can be done by comparing proteins essential dynamics to their normal modes. The protein models were stable and showed some deformability at the molecular level.
Approaches such as virtual screening and de novo drug creation are powerful tools for identifying lead compounds with targeted biological activity. Analysing the interactions of macromolecules and small ligands is an efficient approach to simplify the path of current drug discovery while also reducing the time and expense of the drug development process. Molecular docking using AutoDock Vina results showed that 12,28-Oxamanzamine A, Parsiguine, Haliclonacyclamine B, Vialinin B, 6-Deoxymanzamine X, Natamycin, Olorofim, Deoxytopsentin, Manzamine E and Fascioquinol A were the top leads for CotH3; Pramiconazole, 12,28-Oxamanzamine A, Fascioquinol D, Saperconazole, Nakadomarin A, Plakinamine A, Fascioquinol C, Parsiguine, Hesperidin and Epoxyazadiradione were the top leads for Lanosterol 14 alpha-demethylase; 12,28-Oxamanzamine A, Manzamine A, Parsiguine, Halicyclamine A, Tetrahydrohaliclonacyclamine A, Phaeosphenone 6-Deoxymanzamine X, Goniodomin A, Hesperidin and Stelletin A were the top leads for Mucoricin; and the top leads regarding minimum global binding energy (Tables 4-6). Notably, 12,28-Oxamanzamine A was seen in all three proteins as a lead compound. Further ADME profiling and toxicity analysis were performed to investigate how our lead compounds are processed by a living organism (Tables 7-8). It revealed that most of the lead compounds are highly toxic in nature and possess satisfactory ADME properties. The 12,28-Oxamanzamine and five other compounds were further filtered using these properties for each protein and subjected to bioactivity prediction (Pa>0.7) (Table 9).
The detailed elucidation on the molecular properties and the interaction profiles of the shortlisted six bioactive compounds against Rhizopus delemar proteins are as follows:
It is isolated from a common Indonesian sponge of the genus Acanthostrongylophora. It has potent anti-inflammatory, antifungal, and anti-HIV-1 activity. It showed a high binding affinity with all our three targeted proteins, CotH3 (-10.2 kcal/mol), Lanosterol 14 alpha-demethylase (-10.9 kcal/mol) and Mucoricin (-8.6 kcal/mol). The molecule has a molecular weight of 546.7 g/mol, 4 H-bond acceptors and 1 H-bond donor, formed three H-bonds with ASN190, TYR142 and ASP199 amino acid residues and five Hydrophobic bonds with PHE180, PHE235, PHE235, ALA145 and VAL231 amino acid residues of CotH3. In contrast, for Lanosterol 14 alpha-demethylase it formed two H-bonds with GLU183 amino acid residues and five hydrophobic interactions with TYR211, ILE186 ALA207 amino acid residues. Similarly, for Mucoricin it formed two electrostatic bonds with GLU87 amino acid residue and four hydrophobic interactions with ALA88, ALA81 and TYR80. ADME analysis revealed that this molecule has poor water solubility and consensus Log Po/w value of 5.3 with high GI absorption while having a poor bioavailability score of 0.17. Toxicity results showed that this molecule was toxic with the predicted LD50 of 4 mg/kg. Although it was fatal, its bioactivity score by molinsipiration revealed that it could probably act as a suitable kinase inhibitor (0.36) and an antineoplastic alkaloid. Since CotH3 is a protein kinase, there might be a chance that 12,28-Oxamanzamine A could act as a CotH3 inhibitor. Also, it displayed a high binding affinity for all our target proteins. So further ADME and toxicity optimisation are needed to evaluate its performance in vitro and in vivo studies.
It is a novel dibenzofuran compound isolated from dry fruiting bodies of an edible mushroom, Thelephora vialis, which potently inhibits TNF-alpha production in RBL-2H3 cells (IC (50) = 0.02nM) and acts as a promising anti-allergic agent . It displayed a binding affinity of -8.9 kcal/mol with CotH3. The molecule has a molecular weight of 576.5 g/mol, 9 H-bond acceptors and 4 H-bond donors, formed 3 H-bonds with SER196, ASN237 and GLY189 amino acid residues and five hydrophobic interactions with TYR197, PHE235, PRO201, VAL195 and ALA145. ADME analysis revealed that this molecule is insoluble in water with the consensus Log Po/w value of 5.08 with low GI absorption and a good bioavailability score of 0.55. Toxicity results showed that this molecule was less toxic with the predicted LD50 of 5000 mg/kg. Bioactivity prediction revealed that this molecule could act as a Histidine kinase inhibitor, Chlordecone reductase inhibitor and HIF1A expression inhibitor. Since it can act as a kinase inhibitor, it might be a possibility to inhibit the CotH3 protein.
It is a naturally occurring sponge metabolite that acts as a bisindole alkaloid inhibitor against the evolutionary conserved MRSA pyruvate kinase (PK). The compound displayed potent low nanomolar inhibitory activity against MRSA PK with significant concomitant selectivity over human PK orthologues. It showed a binding affinity of -8.5 kcal/mol with CotH3. The molecule has a molecular weight of 326.4 g/mol, 2 H-bond acceptors and 3 H-bond donors, formed 2 H-bonds with GLY181 and VAL182 amino acid residues, one electrostatic bond with ASP199 and six hydrophobic interactions with ASP199, TYR197, ALA145 and LYS198 amino acid residues. ADME analysis revealed that this molecule was poorly soluble in water with the consensus Log Po/w value of 3.24 with high GI absorption and a good bioavailability score of 0.55. Toxicity results showed that this molecule has a toxicity class of 4 with the predicted LD50 of 1264 mg/kg. Its bioactivity score by molinsipiration revealed that it could probably act as a suitable kinase inhibitor (0.635) and an antineoplastic alkaloid. So, it might act as an antagonist for CotH3.
Pramiconazole from Barrier Therapeutics Inc is a new addition to the triazole antifungal agents that inhibit fungal cell membrane ergosterol synthesis, thereby leading to increased cell permeability and destruction. In preclinical studies, pramiconazole exhibited similar or superior antifungal activity to ketoconazole and itraconazole and selectively inhibited ergosterol synthesis with a broad-spectrum activity. It showed a binding affinity of -11.0 kcal/mol with Lanosterol 14 alpha-demethylase. The molecule has a molecular weight of 659.7 g/mol, 8 H-bond acceptors and 0 H-bond donors, formed five H-bonds with GLN362, GLY60, PHE59, GLY60 and TYR491 amino acid residues and five hydrophobic interactions with PHE59, TYR113, MET494, PRO63 and PRO219 amino acid residues. ADME analysis revealed that this molecule is poorly soluble in water with the consensus Log Po/w value of 4.06 with high GI absorption and a poor bioavailability score of 0.17. Toxicity results showed that this molecule has a toxicity class of 4 with the predicted LD50 of 320 mg/kg. Its bioactivity prediction revealed that this compound is antifungal, and it was experimentally verified by another study to inhibit the lanosterol 14 alpha-demethylase .
The N-1-substituted triazole antifungal, saperconazole, is a potent inhibitor of ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. Fifty % inhibition is already achieved at nanomolar concentrations. The saperconazole induced inhibition of ergosterol synthesis coincides with an accumulation of 14-methylated sterols, such as 24-methylene-dihydro lanosterol, lanosterol, obtusifoliol, 14α-methylfecosterol, 14α-methylergosta-8,24(28)-dien-3, β-6α-diol and 14α-methylergosta-5,7,22,24(28)-tetraenol. It showed a binding affinity of -10.8 kcal/mol with Lanosterol 14 alpha-methylase. The molecule has a molecular weight of 672.7 g/mol, 9 H-bond acceptors and 0 H-bond donors, formed one H-bond with GLN362 amino acid residue, one halogen bond with THR492 and ten hydrophobic interactions with MET494, PHE134, VAL126, LYS138, ILE141, TYR113, PHE134, VAL126, ALA290, PRO63 and PRO219 amino acid residues. ADME analysis revealed that this molecule was poorly soluble in water with the consensus Log Po/w value of 4.28 with high GI absorption and a poor bioavailability score of 0.17. Toxicity results showed that this molecule has a toxicity class of 5 with the predicted LD50 of 4000 mg/kg, and it was experimentally verified by Bossche H Vanden to inhibit the lanosterol 14 alpha-demethylase.
Hesperidin is a flavanone glycoside found in citrus fruits. Its name is derived from "hesperidium", which stands for "fruit from citrus trees". It exhibits various biological properties, including antioxidant, anti-inflammatory and anti-cancer effects. Recent studies indicated that it possesses antimicrobial activity . It displayed a -10.0 kcal/mol binding affinity with Lanosterol 14 alpha-demethylase and -8 kcal/mol with Mucoricin. The molecule has a molecular weight of 610.6 g/mol, 15 H-bond acceptors and 8 H-bond donors, formed seven H-bonds with HIS297, ASP203, ASP214, ARG500 and HIS179 amino acid residues and four hydrophobic interactions with PHE164, ILE186, ALA207 and CYS187 amino acid residues of Lanosterol 14 alpha-demethylase. In contrast, for Mucoricin it formed seven H-bonds with ARG129, GLU97, ASP108, GLU133, MET1 and ALA105 amino acid residues, four electrostatic bonds with ARG129, ASP96, GLU97 and ASP108 amino acid residues and two hydrophobic interactions with ALA94 and CYS103 amino acid residues. ADME analysis revealed that this molecule is soluble in water with the consensus Log Po/w value of -0.72 with low GI absorption and a poor bioavailability score of 0.17. Toxicity results showed that this molecule has a toxicity class of 6 with the predicted LD50 of 12000 mg/kg. Its bioactivity prediction revealed that it could act as a beta-glucuronidase inhibitor and alpha-glucosidase inhibitor. Since Mucoricin comes under the glycosylases, there could be a high chance that hesperidin can inhibit the Mucoricin protein.