Identification of Flavonoids from Scutellaria barbata D. Don as Inhibitors of HIV-1 and Cathepsin L Proteases and Their Structure–Activity Relationships

Scutellaria barbata D. Don (SB, Chinese: Ban Zhi Lian), a well-known medicinal plant used in traditional Chinese medicine, is rich in flavonoids. It possesses antitumor, anti-inflammatory, and antiviral activities. In this study, we evaluated the inhibitory activities of SB extracts and its active components against HIV-1 protease (HIV-1 PR) and SARS-CoV2 viral cathepsin L protease (Cat L PR). UPLC/HRMS was used to identify and quantify the major active flavonoids in different SB extracts, and fluorescence resonance energy transfer (FRET) assays were used to determine HIV-1 PR and Cat L PR inhibitions and identify structure–activity relationships. Molecular docking was also performed, to explore the diversification in bonding patterns of the active flavonoids upon binding to the two PRs. Three SB extracts (SBW, SB30, and SB60) and nine flavonoids inhibited HIV-1 PR with an IC50 range from 0.006 to 0.83 mg/mL. Six of the flavonoids showed 10~37.6% inhibition of Cat L PR at a concentration of 0.1 mg/mL. The results showed that the introduction of the 4′-hydroxyl and 6-hydroxyl/methoxy groups was essential in the 5,6,7-trihydroxyl and 5,7,4′-trihydroxyl flavones, respectively, to enhance their dual anti-PR activities. Hence, the 5,6,7,4′-tetrahydroxyl flavone scutellarein (HIV-1 PR, IC50 = 0.068 mg/mL; Cat L PR, IC50 = 0.43 mg/mL) may serve as a lead compound to develop more effective dual protease inhibitors. The 5,7,3′,4′-tetrahydroxyl flavone luteolin also showed a potent and selective inhibition of HIV-1 PR (IC50 = 0.039 mg/mL).


Introduction
Globally, 766 million confirmed cases of coronavirus disease 2019 (COVID- 19), including 6.93 million deaths, have been reported to the World Health Organization (WHO) [1]. Approximately 10-20% of people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop long COVID-19, with sequelae such as fatigue, shortness of breath, persistent cough, depression and anxiety, brain fog, myocardial inflammation, and myocardial infarction. In particular, cardiovascular health and mortality are emerging as a new epidemic, substantially changing the lives of millions of people globally [2]. To date, 1.33 trillion COVID-19 vaccines have been administered [1]. However, SARS-CoV-2 mutates quickly and is highly infectious, disparities in vaccinations exist, and effective treatments for long COVID-19 are lacking. Current challenges include the development evaluated using renin protease (Table 1) (Please refer to the Supplementary Materials for details). SBW and SB30 had anti-human renin PR activities with IC 50 values of 0.59 and 0.70 mg/mL, respectively, suggesting that these two extracts may present minor toxicity to humans. Three SB extracts (SBW, SB30, and SB60) showed potent activities against HIV-1 PR, with respective IC 50 of 0.006, 0.028 and 0.03 mg/mL. However, no inhibition was observed for any SB extract against Cat L PR. SB85 did not show any activity towards the three PRs. These results indicated that polar SB extracts could potently inhibit HIV-1 PR.

Identification and Quantitation of Ingredients in SB Extracts Using UPLC-HRMS
In this study, we identified nine compounds inhibiting HIV-1 PR and six compounds inhibiting Cat L PR from SB. The five strongest inhibitors (scutellarin, scutellarein, luteolin, hispidulin, and apigenin) were quantitated (Figures 1 and 2 and Table 2). Calibration curves of the five compounds followed a linear regression (R 2 = 0.9994-0.9998) (Please refer to the Supplementary Materials for details). Scutellarin was the most abundant flavonoid in the SB extracts, with contents of 25.18 µg/mg, 23.53 µg/mg, 30.35 µg/mg, and 22.49 µg/mg in SBW, SB30, SB60, and SB85, respectively. The contents of hispidulin and scutellarein ranged from 1.04 to 1.97 µg/mg, except for scutellarein in SB60 (0.74 µg/mg). The contents of luteolin and apigenin were less than 0.3 µg/mg in the SB extracts.       The anti-Cat L and anti-HIV-1 PR activities of the nine flavonoids isolated from SB were screened using SensoLyte ® 520 Cathepsin L and HIV-1 Assay Kits *Fluorimetric*, respectively, according to a previously reported procedure [20,21]. The nine flavonoid compounds were scutellarin, baicalin, scutellarein, hispidulin, apigenin, luteolin, narigenin, eriodictyol, and wogonin. As shown in Table 3 (Please refer to the Supplementary Materials for details), all nine flavonoids exhibited inhibitory activities against HIV-1 PR (IC 50 = 0.039-0.83 mg/mL), with luteolin as the most potent inhibitor. Towards Cat L PR, apigenin, hispidulin, scutellarin, scutellarein, eriodictyol, narigenin, and baicalin gave an inhibition of 4.95-37.6% at a concentration of 0.1 mg/mL. However, only the IC 50 for Scutellarein was able to be determined, at 0.43 mg/mL. We are developing other experimental protocols to confirm the current results and figure out whether there are any behavioral differences between Scutellarein and the other flavonoids in the experimental system. Nevertheless, these flavonoids had a preference for HIV-1 PR and were much weaker inhibitors than the positive controls.    Figure 3. Structure-activity relationships of flavonoids from SB against HIV-1 and Cat L PRs.  Figure 3. Structure-activity relationships of flavonoids from SB against HIV-1 and Cat L PRs.  Figure 3. Structure-activity relationships of flavonoids from SB against HIV-1 and Cat L PRs.  Figure 3. Structure-activity relationships of flavonoids from SB against HIV-1 and Cat L PRs.

5,7-dihydroxyl-8-methoxy flavone (E type)
Wogonin Figure 3. Structure-activity relationships of flavonoids from SB against HIV-1 and Cat L PRs. We further evaluated the flavonoids and activities in their respective categories (Table 3 and Figure 3). For the 5,6,7-trihydroxyl flavone glycosides (A type), scutellarin exhibited a slightly higher activity against HIV-1 PR than baicalin. However, towards Cat L PR, the inhibitory activity of scutellarin was about three-fold stronger than that of baicalin. This implies that 4 -hydroxyl is critical for inhibition of Cat L PR.    Within the 5,7,4 -trihydroxyl flavone (type B) family, all three compounds, scutellarein, hispidulin, and apigenin, showed potent activities against HIV-1 PR. The stronger activities for scutellarein and hispidulin suggests that C6-subsitution potentiates the inhibitory function. Towards Cat L PR, a moderate inhibition was observed for the compounds. The higher activity of Apigenin implies that C6-substitution is unfavorable for inhibition of Cat L PR. This also illustrates the structural variance and requirements between HIV-1 PR and Cat L PR for binding their respective substrates and inhibitors. Further analysis between type A and type B flavonoids indicated that C7-glycosylation is detrimental to HIV-1 PR inhibition but may be beneficial for Cat L PR inhibition.
For the 5,7,3 ,4 -tetrahydroxyl flavones (type C) and 5,7-dihydroxyl-8-methoxy flavones (type E), only one compound was examined in each category. The type C compound Luteolin and type E compound Wogonin showed good inhibition of HIV-1 PR but no inhibition on Cat L PR. Comparison of these two compounds implied that hydroxylation of the C-ring provides better inhibition of HIV-1 PR; however, further studies are needed to evaluate the contribution of C8-methoxylation to HIV-1 PR inhibition. Further comparison of Luteolin with Apigenin suggested that C3 -hydroxylation provides better inhibition of HIV-1 PR for flavone compounds.
In the 5,7,4 -trihydroxyl flavanones (type D), both compounds, Eriodictyol and Narigenin, exhibited comparable effects towards both HIV-1 PR and Cat L PR, although their inhibitions of HIV-1 PR were much stronger. This implies that C3 -hydroxylation has limited effects on PR inhibition for flavanones. Upon comparing these two flavanone compounds with structurally corresponding flavone compounds, Luteolin and Apigenein, we could conclude that the flavone structure with flat conformation of the B-ring is favorable for HIV-1 PR inhibition. However, we could not reach a conclusion about which type of flavonoid (flavones vs. flavanones) is more potent for Cat L PR inhibition.
As the most abundant flavonoid in the SB extracts, scutellarin exhibited only moderate inhibition of both PRs. Interestingly, as an aglycone and one of the metabolites of scutellarin [22], scutellarein showed potent inhibition of HIV-1 and Cat L PRs. This indicates that free 5,6,7-hydroxyl groups are likely critical for the inhibitions and provide better bonding affinity within the active sites of both PRs. The introduction of 4′-hydroxyl and 6-hydroxyl/methoxy groups was shown to be essential in 5,6,7-trihydroxyl and 5,7,4′trihydroxyl flavones, respectively, to enhance dual anti-PR activities. Hence, among the 5,6,7,4′-tetrahydroxyl flavones, scutellarein may serve as a leading molecule for developing more effective dual PR inhibitors. The 5,7,3′,4′-tetrahydroxyl flavone luteolin was a selective and potent inhibitor of HIV-1 PR. Quite a few flavonoids, such as quercetin , and hispidulin (c) to HIV-1 PR. Carbon is colored green, while oxygen, hydrogen, nitrogen, and sulfur atoms are colored red, white, blue, and yellow, respectively. The blue dotted lines represent π-π interactions, the green dotted line shows a π-cation interaction and the hydrogen bond is depicted as a yellow dotted line.
As the most abundant flavonoid in the SB extracts, scutellarin exhibited only moderate inhibition of both PRs. Interestingly, as an aglycone and one of the metabolites of scutellarin [22], scutellarein showed potent inhibition of HIV-1 and Cat L PRs. This indicates that free 5,6,7-hydroxyl groups are likely critical for the inhibitions and provide better bonding affinity within the active sites of both PRs. The introduction of 4′-hydroxyl and 6-hydroxyl/methoxy groups was shown to be essential in 5,6,7-trihydroxyl and 5,7,4′trihydroxyl flavones, respectively, to enhance dual anti-PR activities. Hence, among the 5,6,7,4′-tetrahydroxyl flavones, scutellarein may serve as a leading molecule for developing more effective dual PR inhibitors. The 5,7,3′,4′-tetrahydroxyl flavone luteolin was a selective and potent inhibitor of HIV-1 PR. Quite a few flavonoids, such as quercetin , and hispidulin (c) to HIV-1 PR. Carbon is colored green, while oxygen, hydrogen, nitrogen, and sulfur atoms are colored red, white, blue, and yellow, respectively. The blue dotted lines represent π-π interactions, the green dotted line shows a π-cation interaction and the hydrogen bond is depicted as a yellow dotted line.
As the most abundant flavonoid in the SB extracts, scutellarin exhibited only moderate inhibition of both PRs. Interestingly, as an aglycone and one of the metabolites of scutellarin [22], scutellarein showed potent inhibition of HIV-1 and Cat L PRs. This indicates that free 5,6,7-hydroxyl groups are likely critical for the inhibitions and provide better bonding affinity within the active sites of both PRs. The introduction of 4 -hydroxyl and 6-hydroxyl/methoxy groups was shown to be essential in 5,6,7-trihydroxyl and 5,7,4trihydroxyl flavones, respectively, to enhance dual anti-PR activities. Hence, among the 5,6,7,4 -tetrahydroxyl flavones, scutellarein may serve as a leading molecule for developing more effective dual PR inhibitors. The 5,7,3 ,4 -tetrahydroxyl flavone luteolin was a selective and potent inhibitor of HIV-1 PR. Quite a few flavonoids, such as quercetin and kaempferol, demonstrated potent inhibitions of HIV-1 PR and deserve further studies to develop more potent PR inhibitors [23,24]. Our current study also revealed that three SB extracts (SBW, SB30, and SB60) and their respective active components, including luteolin and scutellarein, exhibited potent inhibitory activities against HIV-1 PR. However, the selected phytochemical samples are poorly soluble in a neutral assay buffer, and this may lower the bio-availability towards Cat L PR. To develop more dual-and even pan-viral protease inhibitors from medicinal plants, further studies are warranted, to explore potential protease inhibitors based on these active components, for the prevention and/or treatment of COVID-19.