Totally we docked 40 compounds to 4 COVID-19 targets (3 protease and 1 RNA polymerase). A comparative analysis can be done by referring to (Table 1). As for binding affinities, 20 compounds exhibit good binding affinities to one or more of the COVID-19 targets. Surprisingly, 7 out of 20 compounds exhibit remarkable binding affinities to all the 4 targets (6M0K, 6Y2F, 7BQY or 7bV2). The top 7 hits are Flacourticin (3), Sagerinic acid (16), Hordatine A (23), Hordatine B (24), N-feruloyl tyramine dimer (25), Bisavenanthramides B-5 (29) and Vulnibactins (40) summarized in (Table 2). Docking interactions pattern of the top 7 hits are depicted in (Figure 4). Importantly, Hordatines (23 and 24) were found to interact with both protease and polymerase by exhibiting the highest binding affinity through forming strong hydrogen bonds with some residues of the catalytic site, as well as significant extra interactions with other receptor binding residues.
For protease target 6M0K, Binding interactions of the native ligand (binding score = - 7.1) (Figure 4c`), revealed that there are 2 hydrogen bonds with Glu166. In addition to other interactions with His 41, His 164, His 163 and Cys 145. Whereas in case of Hordatine A binding interactions with 6M0K (binding score = - 8.5) is given in (Figure 4i), six hydrogen bonds were recorded with Glu166 which is assumed to be essential for the activity. Furthermore, extra interactions were observed with Phe144, Asn142 and Gln189. For Hordatine B Binding interactions with 6M0K (binding score = - 8.7) is given in (Figure 4m), six bonds were recorded with Glu166 which is assumed to be essential for the activity. In addition to a hydrophobic interaction was observed with His41. In addition to other interactions with Arg188, val186, Met165 and Asn142.
For protease target 6Y2F, Binding interactions of the native ligand (binding score = - 7.1) (Figure 4d`), revealed that there are 3 hydrogen bonds with Glu166. In addition to other bonds with His 41, His 164, His 163, Cys 145, Ser144 and Gly143. Whereas in case of Hordatine A binding interactions with 6M0K (binding score = - 8.0) is given in (Figure 4j), three hydrogen bonds were recorded with Glu166. Furthermore, extra eight interactions were observed with ser144, Gln192, Glu47 and Met49. Whereas in case of Hordatine B Binding interactions with 6Y2F (binding score = - 8.5) is given in (Figure 4n), 6 bonds were recorded with Glu166. In addition to strong interactions are observed with phe140 and Thr26. Furthermore, Strong hydrogen bond was observed with Asn142. For protease target 7BQY, Binding interactions of the native ligand (binding score = - 7.8) (Figure 4e`), revealed that there are 3 hydrogen bonds with Glu166. In addition to other bonds with Gln189, His163, His164, Cys145, Gln189, Thr190, Ala191, Thr26 and Thr25. Whereas in case of Hordatine A binding interactions with 7BQY (binding score = - 9.0) is given in (Figure 4k), five hydrogen bonds were recorded with Glu166. Furthermore, extra nine interactions were observed with phe140, Gln189, Met49 and Glu47. Whereas in case of Hordatine B Binding interactions with 7BQY (binding score = - 8.5) is given in (Figure 4o), 6 bonds were recorded with Glu166. In addition to other interactions were observed with phe140, Gly143 and Gln189.
For polymerase target 7bv2, Binding interactions of the native ligand Remdesivir (binding score = - 5.9) (Figure 4f`), revealed that there are 5 hydrogen bonds with Arg553. In addition to other bonds with Asp623, Asn691, Ser759 and ser682. For Hordatine A binding interactions with 7bv2 (binding score = - 8.11) is given in (Figure 4l), two hydrogen bonds were recorded with Arg553. Furthermore, extra interactions were observed with Thr680, Asp623, Asp684 and Ser682. Whereas in case of Hordatine B Binding interactions with 7bv2 (binding score = - 8.3) is given in (Figure 4p), Hydrophobic interaction was recorded with Ser682 and one hydrogen bond was recorded with Arg553. In addition to seven interactions were recorded with Asp760. Furthermore, 4 interactions were recorded with Asp618. Also, other interactions were observed with thr680, cys813 and Leu7582. It is worth mentioning that Remdesivir is a nucleotide analogue prodrug that inhibits viral RNA polymerases which has shown in vitro prophylactic and therapeutic efficacy in nonclinical models against COVID-19.[16]
Hordatine A and B could be obtained by extraction from dark-grown barley by several methods as a mixture. One method by homogenization with 2 volume acetic acid followed by centrifugation for 5 min. after being left at 4°C for 20 hrs. The supernatant was decanted and the precipitate washed with 1 volume acetic acid and re- centrifugated. Both acetic acid decanted fractions were combined and evaporated till dryness at 40 °C. The solid residue was re-dissolved in 1 volume 2-5% Trichloroacetic acid and kept for 15min. then centrifuged for 5 min. and the supernatant was stored at -10 °C.[11] Another method was reported by shaking pearled grain flour with 1mL of 75% acetone for 60 min. at room temperature, in three or four replications then centrifugate at 12500 rpm for 10 min. followed by re-extracting the precipitate with 75% acetone twice. Both acetone extracts evaporated under vacuum and dissolved in 3 mL 2.5% acetic acid.[12] Another method was reported by sowing barley seeds in flats containing heat sterilized vermiculite. and incubated in the dark in controlled environment chambers. The developed shoots were extracted by boiling with 100 ml water for 10 min. The filtered extract was shaken with Amberlite IR C 50 (H+) ion-exchange resin (5 g dry weight) for 1 hr. the supernatant liquid was decanted, and the resin rinsed with several portions of water. The adsorbed bases were then eluted by shaking the resin with 100 ml 2N acetic acid for 1 hr and filtering. [13] Hordatine A and B could be obtained by solid-phase extraction[12] or by cationic exchange resin using buffer (0.05 M NaCl/0.13 M NaOH), pH 13 at 95 °C in a mixture form.[17] . Hordatine A is probably synthesized by oxidative dimerization of coumaroylagmatine.[11] Hordatine B could be biosynthesized in two consecutive reactions. In the first, agmatine coumaroyltransferase (ACT) catalyzes the conjugation of agmatine and p-coumaroyl-CoA or feruloyl-CoA. In the second reaction peroxidase catalyzes the oxidative coupling of agmatine conjugates by linking coumaroylagmatine and feruloylagmatine.[12] Identification of Hordatine A and B is achieved using TLC of Avicel (Rf 0.54 for A) and (Rf 0.53 for B) developed with the upper phase of n-butanol - water - acetic acid (4:5:1) in pre-saturated tanks. The Sakaguchi reagent (specific for guanidines), diazotized nitraniline solution, and alcoholic bromocresol green were used as chromogenic sprays.[13]
Since forty natural compounds were subjected to virtual screening molecular docking against 3 protease and one RNA polymerase targets of COVID-19. The compounds exhibited variable degrees of affinities toward COVID-19 targets comparing to the native inhibitor. Seven compounds were found to interact with all COVID- 19 targets by exhibiting the most acceptable binding affinity through forming strong hydrogen bond with the catalytic sites. For RNA polymerase target of COVID-19 (PDB ID: 7bV2), these seven compounds were found to have better binding scores than the native ligand, Remdesivir, the well-known antiviral drug. Importantly, Hordatine (23 and 24) phenolic compounds present in barley, were found to interact with both protease and polymerase by exhibiting the highest binding affinity through forming strong hydrogen bonds with the catalytic residues, as well as significant extra interactions with other receptor-binding residues. Such compounds are recommended to be tested clinically for proposed activity against COVID-19. They may be tested either alone or in combinations. In addition, our results may facilitate the future design and synthesis of new candidates against COVID-19.