Bioactive Alkaloids from Genus Aspergillus: Mechanistic Interpretation of Their Antimicrobial and Potential SARS-CoV-2 Inhibitory Activity Using Molecular Modelling

Genus Aspergillus represents a widely spread genus of fungi that is highly popular for possessing potent medicinal potential comprising mainly antimicrobial, cytotoxic and antioxidant properties. They are highly attributed to its richness by alkaloids, terpenes, steroids and polyketons. This review aimed to comprehensively explore the diverse alkaloids isolated and identified from different species of genus Aspergillus that were found to be associated with different marine organisms regarding their chemistry and biology. Around 174 alkaloid metabolites were reported, 66 of which showed important biological activities with respect to the tested biological activities mainly comprising antiviral, antibacterial, antifungal, cytotoxic, antioxidant and antifouling activities. Besides, in silico studies on different microbial proteins comprising DNA-gyrase, topoisomerase IV, dihydrofolate reductase, transcriptional regulator TcaR (protein), and aminoglycoside nucleotidyl transferase were done for sixteen alkaloids that showed anti-infective potential for better mechanistic interpretation of their probable mode of action. The inhibitory potential of compounds vs. Angiotensin-Converting Enzyme 2 (ACE2) as an important therapeutic target combating COVID-19 infection and its complication was also examined using molecular docking. Fumigatoside E showed the best fitting within the active sites of all the examined proteins. Thus, Aspergillus species isolated from marine organisms could afford bioactive entities combating infectious diseases.


Introduction
Recently, marine-derived fungi have gained significant attention as promising therapeutic approaches for the treatment of a wide array of human ailments and as successful tools for drug discovery [1]. This is mainly attributed to their richness by a diverse array of secondary metabolites comprising terpenoids, alkaloids, peptides, lactones and steroids. These promising activities are represented by antiviral, antibacterial, anti-inflammatory and anticancer activity [2]. The significant diversity in physical and chemical structure of the environment where the marine-derived fungi grow has greatly reflected by the vast structural and functional variation in their produced secondary metabolites and their biological activities [3]. Meanwhile, marine-derived fungal metabolites displayed a promising physico-chemical behavior and oral-bioavailability, constituting a safer therapeutic alternative when compared to synthetic molecules that are considerably important in the process of pharmaceutical dosage form formulation [4,5]. Moreover, many alkaloids were previously isolated from marine fungi and showed a vast array of biological activities [6][7][8][9].
Genus Aspegillus represents a widely spread genus of fungi that are highly popular of possessing a potent medicinal potential comprising mainly antimicrobial, cytotoxic and Moreover, many alkaloids were previously isolated from marine fungi and showed a vast array of biological activities [6][7][8][9].
Genus Aspegillus represents a widely spread genus of fungi that are highly popular of possessing a potent medicinal potential comprising mainly antimicrobial, cytotoxic and antioxidant activities that are highly attributed to its richness by alkaloids, terpenes, steroid and polyketons. These secondary metabolites reflect the considerable importance of genus Aspergillus both in the scientific and pharmaceutical industries levels [10].
Thus, this review aimed to comprehensively explore the diverse alkaloids isolated and identified from different species of genus Aspergillus that were found to be associated with different marine organisms regarding their chemistry and biology. Classification was done on the basis of alphabetical arrangement of species. Around 174 alkaloid metabolites were reported, 66 of which showed important biological activities mainly comprising antiviral, antibacterial, antifungal, cytotoxic, antioxidant and antifouling activities. In addition, data illustrating the bioactive alkaloids obtained from previously mentioned fungal strains, their sources and biological properties are compiled in Table 1 for better representation of the collected data. A pie chart illustrating the different biological activities for the bioactive alkaloids of genus Aspergillus was also provided. Besides, in silico studies on different microbial proteins were done for sixteen alkaloids that showed anti-infective potential for better mechanistic interpretation of their probable mode of action. In addition, the inhibitory potential of these compounds vs. Angiotensin-Converting Enzyme 2 (ACE2) as an important therapeutic target combating COVID-19 infection and its complication was also examined using molecular docking to it can act as a guide for researchers who wish to continue exploring the anti-infectious potential of alkaloid derived from genus Aspergillus.

A. carneus
Aspergillus species are highly popular due to the presence of a wide variety of alkaloids belonging to diverse classes. Prenylated indole and quinazolinone alkaloids were isolated from A. carneus, a marine associated Aspergillus species, while the former is represented by carneamides A-C (1-3); however, the latter is represented by carnequinazolines A-C (4)(5)(6). Unfortunately, none of the isolated compounds revealed any antimicrobial or cytotoxic activities ( Figure 1) [11].

A. nidulans
Furthermore, four new alkaloids of quinazolinone type which are aniquinazolines A-D (37-40) were isolated from A. nidulans, which was associated with the leaves of Rhizophora stylosa, marine plant. Compounds (37-40) revealed a significant lethal effect on brine shrimp displaying LD50 of 1.27, 2.11, 4.95 and 3.42 µM, in a respective manner which is superior to the positive control colchicine. However, none of these compounds exhibited any antibacterial activity vs. Escherichia coli and S. aureus or any cytotoxic effect on HL-60, BEL-7402, K562 and MDA-MB-231 cancer cells ( Figure 4) [19].
Furthermore, four new alkaloids of quinazolinone type which are aniquinazolines A-D (37)(38)(39)(40) were isolated from A. nidulans, which was associated with the leaves of Rhizophora stylosa, marine plant. Compounds (37-40) revealed a significant lethal effect on brine shrimp displaying LD50 of 1.27, 2.11, 4.95 and 3.42 μΜ, in a respective manner which is superior to the positive control colchicine. However, none of these compounds exhibited any antibacterial activity vs. Escherichia coli and S. aureus or any cytotoxic effect on HL-60, BEL-7402, K562 and MDA-MB-231 cancer cells ( Figure 4) [19].

A. puniceus
Furthermore, A. puniceus is a good source of new alkaloids from which eight new diketopiperazine-type alkaloids were isolated from the extract of its culture broth. Four of these new diketopiperazine alkaloids contain oxepin moiety however the other four contain quinazolinone moiety. The formers are represented by oxepinamides H-K (56-59); meanwhile, the latters were represented by puniceloids A-D (60-63) (Figure 4). Noteworthy to highlight that all the new eight isolated compounds revealed a potent transcriptional stimulation of liver X receptor α displaying EC 50 ranging between 1.7 and 50 µM with puniceloids C and D showed the highest agonist behavior [24]. A. sydowii in which 6-methoxyspirotryprostatin B showed mild cytotoxic activity against HL-60 cells displaying IC50 of 9.71 μM [28].

A. versicolor
A. versicolor is highly popular by the presence of a large number of alkaloids ( Figure  6), which are represented by asperversiamides A-H (80-87), which are indole alkaloids characterized by the presence of a linear fused prenyl groups and cottoquinazoline A (88) [7,31]. Asperversiamide G (86) displayed a significant anti-inflammatory potential evidenced by the pronounced inhibition of iNOS with IC50 value of 5.39 μM [7]. Additionally, ten new alkaloids of diketopiperazine class were isolated from A. versicolor, pyranamides A-D (89-92), secopyranamide C (93), protuboxepin F-J (94-98) in addition to previously isolated compounds which were protuboxepin C (99) and protuboxepin E (100). Protuboxepin G and E displayed mild cytotoxic activity vs. 786-O, OS-RC-2 and ACHN [9]. Further investigation of the coral derived fungus, A. versicolor, resulted in the exploration of six new alkaloids in the polycyclic form, which are versiquinazolines L-Q (101-106). Versiquinazolines P and Q displayed potent prohibition of thioredoxin reductase (TrxR) revealing IC50 of 13.6 and 12.2 μM, respectively being superior in activity relative to curcumin, the positive control, with IC50 of 25 μM accompanied by weak cytotoxic effect. This consequently, provides an evidence on the potential use of both

A. sydowii
A. sydowii was subjected to an intense phytochemical investigation that led to the isolation of many indole alkaloids, fumiquinazoline D and E (69-70) and cyclotryprostatin B (71) in addition to 12,13-dihydroxyfumitremorgin C (14), fumiquinazoline A (18), fumiquinazoline F (19) and fumiquinazoline G (20) ( Figure 5). These compounds were tested for their antifouling activity via assessing their inhibitory effect on the settlement of B. neritina larvae, at a concentration of 25 µg/mL, fumiquinazoline D, fumiquinazoline G and cyclotryprostatins B showed significant antifouling activity [8].

A. versicolor
A. versicolor is highly popular by the presence of a large number of alkaloids ( Figure 6), which are represented by asperversiamides A-H (80-87), which are indole alkaloids characterized by the presence of a linear fused prenyl groups and cottoquinazoline A (88) [7,31]. Asperversiamide G (86) displayed a significant anti-inflammatory potential evidenced by the pronounced inhibition of iNOS with IC 50 value of 5.39 µM [7]. Additionally, ten new alkaloids of diketopiperazine class were isolated from A. versicolor, pyranamides A-D (89-92), secopyranamide C (93), protuboxepin F-J (94-98) in addition to previously isolated compounds which were protuboxepin C (99) and protuboxepin E (100). Protuboxepin G and E displayed mild cytotoxic activity vs. 786-O, OS-RC-2 and ACHN [9]. Further investigation of the coral derived fungus, A. versicolor, resulted in the exploration of six new alkaloids in the polycyclic form, which are versiquinazolines L-Q (101-106). Versiquinazolines P and Q displayed potent prohibition of thioredoxin reductase (TrxR) revealing IC 50 of 13.6 and 12.2 µM, respectively being superior in activity relative to curcumin, the positive control, with IC 50 of 25 µM accompanied by weak cytotoxic effect. This consequently, provides an evidence on the potential use of both compounds in the control of microenvironment of tumor progression and metastasis [32]. In addition, versicoloid A and B (107-108), 3,6-O-dimethylviridicatin (109) and 3-O-methylviridicatol (110) were also isolated from A. versicolor, in which versicoloid A and B displayed a potent anti-fungal activity with MIC of 1.6 µg/mL against Colletotrichum acutatum approaching cycloheximide, the positive control drug, that showed MIC of 6.4 µg/mL. [33].

Miscellaneous Aspergillus Species
Besides, a plethora of alkaloid compounds were isolated from miscellaneous Aspergillus species such as fumiquinazoline S (119), fumiquinazolines F (29) and L (120), isochaetominines A-C (121-123), 14-epi-isochaetominine C (124) (Figure 8). All these compounds revealed a mild inhibitory effect on Na(+)/K(+)-ATPase [35]. Additionally, asperginine (125), an alkaloid with a rare skeleton, and misszrtine A (126), an indole alkaloid with novel skeleton, possess phenylpropanoic amide arm attached to N-isopentenyl tryptophan methyl ester were isolated from two different Aspergillus species. The cytotoxic activity of the former was evaluated using MTT assay against human HCT116 and PC3 (prostate cancer cells) but it revealed no activity against the previously mentioned cell lines [36]. However the latter was assessed for its cytotoxic activity on HL60 and LNCaP and revealed a promising activity with IC50 value of 3.1 and 4.9 μM, respectively owing to the presence of indole nitrogen [37].
Besides, asperindoles A-D (127-130), new jndole alkaloids possessing diterpene structure, were also isolated from Aspergillus species. Asperindoles C and D possess a 2-hydroxyisobutyric acid moiety; however, asperindole A revealed a potent cytotoxic activity on both hormone therapy-resistant and sensitive PC-3 (Human prostate cancer cells) in addition to 22Rv1 cancer cells (human prostate carcinoma epithelial cell line) at low concentrations calculated in micromolar [38]. Golmaenone (131), new alkaloid with diketopiperazine skeleton, and neoechinulin A (132) were also isolated from Aspergillus species (Figure 8). Both compounds revealed a potent antioxidant activity evidenced by their IC50 values which are 20 and 24 μM, respectively in 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity assay comparable to that of ascorbic acid (IC50 = 20 μM). Their

Miscellaneous Aspergillus Species
Besides, a plethora of alkaloid compounds were isolated from miscellaneous Aspergillus species such as fumiquinazoline S (119), fumiquinazolines F (29) and L (120), isochaetominines A-C (121-123), 14-epi-isochaetominine C (124) (Figure 8). All these compounds revealed a mild inhibitory effect on Na(+)/K(+)-ATPase [35]. Additionally, asperginine (125), an alkaloid with a rare skeleton, and misszrtine A (126), an indole alkaloid with novel skeleton, possess phenylpropanoic amide arm attached to N-isopentenyl tryptophan methyl ester were isolated from two different Aspergillus species. The cytotoxic activity of the former was evaluated using MTT assay against human HCT116 and PC3 (prostate cancer cells) but it revealed no activity against the previously mentioned cell lines [36]. However the latter was assessed for its cytotoxic activity on HL60 and LNCaP and revealed a promising activity with IC 50 value of 3.1 and 4.9 µM, respectively owing to the presence of indole nitrogen [37].
Additionally, versicolamide B and notoamides L-N were isolated from a marine derived Aspergillus species [41].

Interpretation of the Antimicrobial Activity of Bioactive Alkaloids Using In Silico Studies
Many mechanisms explained the antimicrobial behavior of many anti-infective drugs such as prevention of nucleic acid, protein and cell wall synthesis, inhibition of functional cell membrane, as well as interfering with many metabolic processes [61][62][63]. Herein, molecular modelling was performed on six proteins which were downloaded from the protein data bank and are considered essential for growth, division, the survival of microbes and in the development of resistance using C-docker protocol [64][65][66] Table 2). The tight fitting of fumigatoside E can be interpreted by the virtue of formation of many tight bonds and interactions within the active sites ( Figure 11). • Mild cytotoxic activity against HL-60 cells [28]

Interpretation of the Antimicrobial Activity of Bioactive Alkaloids Using in Silico Studies
Many mechanisms explained the antimicrobial behavior of many anti-infective drugs such as prevention of nucleic acid, protein and cell wall synthesis, inhibition of functional cell membrane, as well as interfering with many metabolic processes [61][62][63]. Herein, molecular modelling was performed on six proteins which were downloaded from the protein data bank and are considered essential for growth, division, the survival of microbes and in the development of resistance using C-docker protocol [64][65][66]. Among all the examined compounds only fumigatoside E (28) showed the best fitting within the active sites of all examined proteins as evidenced by its free binding energies (∆G) that are equal to −14.18, −18.16, -5.02, −20.31, −10.84 and −17.59 Kcal/mol for DNAgyrase, topoisomerase IV, dihydrofolate reductase, β-lactamase, transcriptional regulator TcaR and aminoglycoside nucleotidyl transferase, respectively. It showed in this aspect a superior activity comparable to levofloxacin and moxifloxacin, the potent DNA-gyrase, topoisomerase IV inhibitors, respectively with ∆G = −9.89 Kcal/mol for levofloxacin and −10.19 Kcal/mol for moxifloxacin, respectively whereas aspergicin (150) showed slight fitting. All of the other tested compounds showed unfavorable interaction within the active sites of the examined proteins manifested by the positive values of their free binding energies (∆G) ( Table 2). The tight fitting of fumigatoside E can be interpreted by the virtue of formation of many tight bonds and interactions within the active sites ( Figure 11).   Within the active site of DNA-gyrase, fumigatoside E formed two conventional Hbonds, a π-π bond, three π-alkyl bonds in addition to one C-H interaction and many Van der Waals interactions ( Figure 11A). Regarding topoisomerase IV, fumigatoside E forms one conventional H-bond, three π-π bonds, two π-alkyl bonds in addition to many Van der Waals interactions and π-cation interaction with the amino acid residues at the active site ( Figure 11B). Meanwhile, it forms five conventional H-bonds with Gly75, Ala76 and Ala73, two πsulphur and one alkyl interactions with Met72 in addition to many Van der Waals interactions at dihydrofolate reductase active site ( Figure 11C). Besides, fumigatoside E forms two H-bonds with Lys87 and Asp255, two π-cation interactions with Arg187 and four π-alkyl bonds with Ile117 at β-lactamase active site ( Figure 11D). Concerning transcriptional regulator TcaR (protein), fumigatoside E forms two H-bonds with Gln 61 and His 42, and five πalkyl bonds with Ala38, Ala24 and His42 and many Van der Waals interactions with the amino acid existing at the active site ( Figure 11E). Three H-bonds with Asp46, Asp86 and four π-π interactions with Tyr74, Tyr132 and Tyr134 are formed between fumigatoside E and active site of aminoglycoside nucleotidyl transferase ( Figure 11F). The notable binding of fumigatoside E with DNA-gyrase and topoisomerase active sites IV could greatly interpret its mode of antimicrobial via potent inhibition of both enzymes.

Probable SARS-CoV-2 Inhibitory Potential of Bioactive Antimicrobial Alkaloids Using in Silico Studies
COVID-19 infection relies upon host cell factors as Angiotensin-Converting Enzyme 2 (ACE2). The entrance of coronaviruses within the host cell is accomplished by the effective binding of the viral spike (S) proteins to cellular receptors that facilitate their cell entrance, viral attachment to the surface of target cells with subsequent infection triggering. SARS-S engages angiotensin-converting enzyme 2 (ACE2) as the entry receptor in which SARS-S/ACE2 interface was previously elucidated at the atomic level, and the effectiveness to bind with ACE2 was found to be a key determinant of SARS-CoV transmissibility. Thus the prohibition of ACE2 catalytic pocket by bioactive entities could alters the conformation of ACE2 in a manner that it could prohibit SARS-CoV-2 entrance within the host cells through ACE2 [67,68]. Thus, molecular modelling was performed for the sixteen alkaloids that previously displayed antimicrobial potential on Angiotensin-Converting Enzyme 2 (PDB ID 1R4L; 3.00 A • ) which was downloaded from the protein data bank. Fumigatoside E (28) showed the most fitting within the active sites of ACE2 followed by aspergicin (150) displaying ∆G of −21.17 and −17.66 Kcal/mole, respectively (Table 3). Table 3. Free binding energies (∆G) in Kcal/mole of alkaloids isolated from Aspergillus and showed anti-infective potential using in silico studies on Angiotensin-Converting Enzyme 2 (ACE2). Fumigatoside E (forms many tight interactions with the amino acid moieties at the active pocket of ACE2 represented by one H-bond with Arg273, π-π bond with His 379, three π-alkyl interactions with Pro346 and Phe274 in addition to the formation of two π-cation interactions with Lys363 and Ar273 ( Figure 12A). Meanwhile, aspergicin forms one H-bond with Arg518, π-π bond with Phe 274, two π-alkyl interactions with His345 and Pro346, C-H bonds with Asn149 and Thr 371( Figure 12B).

Conclusions
Around 174 alkaloid metabolites were reported from genus Aspergillus, 66 of which showed important biological activities with respect to the tested biological activities mainly comprising antiviral, antibacterial, antifungal, cytotoxic, antioxidant and antifouling activities. Besides, in silico studies on different microbial proteins were done for sixteen alkaloids that showed anti-infective potential for better mechanistic interpretation for their probable mode of action. Fumigatoside E showed the best fitting within the active sites of all examined proteins as evidenced by its free binding energies. Additionally, fumigatoside E showed the most fitting within the active sites of ACE2 followed by aspergicin and thus could serve as bioactive candidates for combating SARS-CoV-2 infection. Further studies are to be conducted to examine the biological activities of the additional alkaloids that displayed no activity meanwhile in vitro followed by in vivo studies are to be performed to ascertain the results of molecular modelling.

Conclusions
Around 174 alkaloid metabolites were reported from genus Aspergillus, 66 of which showed important biological activities with respect to the tested biological activities mainly comprising antiviral, antibacterial, antifungal, cytotoxic, antioxidant and antifouling activities. Besides, in silico studies on different microbial proteins were done for sixteen alkaloids that showed anti-infective potential for better mechanistic interpretation for their probable mode of action. Fumigatoside E showed the best fitting within the active sites of all examined proteins as evidenced by its free binding energies. Additionally, fumigatoside E showed the most fitting within the active sites of ACE2 followed by aspergicin and thus could serve as bioactive candidates for combating SARS-CoV-2 infection. Further studies are to be conducted to examine the biological activities of the additional alkaloids that displayed no activity meanwhile in vitro followed by in vivo studies are to be performed to ascertain the results of molecular modelling.