Identification of plant-derived natural products as potential inhibitors of the Mycobacterium tuberculosis proteasome

Abstract

Background: The Mycobacterium tuberculosis (Mtb) proteasome has been established as a viable target for the development of anti-tuberculosis agents. In this study, the inhibitory activities of 100 plant-derived natural products on the Mtb proteasome were analyzed to identify novel potential inhibitors.

Methods: The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC can be hydrolyzed by the proteasome to release free AMC, the fluorescence of which is proportional to the proteasome activity. The inhibitory activities of 100 natural products (each at a final concentration of 200 μM) were detected by this method using MG132 as a positive control.

Results: Twelve of these natural products (10 of which were flavonoids) inhibited the activity of the Mtb proteasome by more than 65%. Comparison of the structural differences between the flavonoids with good inhibitory activity and those without inhibitory activity revealed that the hydroxyl at the flavonoid C ring C-3 or the hydroxyl/methoxyl at the flavonoid A ring C-6 were critical for the inhibition of proteasomal activity.

Conclusions: These data indicate that flavonoids represent a basis for rational structural design in the process of novel anti-tuberculosis drug discovery.

Figure 1

Mtb proteasome inhibitory activities of 12 natural products (200 μM). The inhibitory activities of 12 natural products (200 μM) were: hispidulin 82.06%, baicalein 84.30%, pectolinarin 88.69%, myricetin 84.50%, quercetin 74.40%, kaempferol 68.29%, isoliquiritigenin 83.60%, icariin 74.30%, baicalin 78.47%, curcumin 69.53%, celastrol 69.50% and emodin 73.09%, the inhibitory activity of MG132 (100 μM) is 79.66%. Data are shown as mean ± SD of three independent experiments.

Figure 2

Dose response curves of inhibitory activities of MG132, baicalein, pectolinarin, myricetin, hispidulin, isoliquiritigenin and quercetin. The IC₅₀ values were calculated by fitting with the four parameter logistic (4-PL) model: A. MG132 IC₅₀=27.97 μM, R²=0.99. B. baicalein IC₅₀=45.65 μM, R²=0.97. C. pectolinarin IC₅₀=49.96 μM, R²=0.97. D. myricetin IC₅₀=55.39 μM, R²=0.96. E. hispidulin IC₅₀=58.31 μM, R²=0.97. F. isoliquiritigenin IC₅₀=63.90 μM, R²=0.99. G. quercetin IC₅₀=71.29 μM, R²=0.96. R², adjust R square values. Dose response curves of inhibitory activity were presented. Data are shown as mean ± SD of three independent experiments.

Figure 3

Dose response curves of inhibitory activities of emodin, baicalin, icariin, kaempferol, celastrol and curcumin. The IC₅₀ values were calculated by fitting with the four parameter logistic (4-PL) model: A. emodin IC₅₀=81.05 μM, R²=0.97. B. baicalin IC₅₀=83.52 μM, R²=0.99. C. icariin IC₅₀=88.67 μM, R²=0.99. D. kaempferol IC₅₀=106.74 μM, R²=0.99. E. celastrol IC₅₀=114.02 μM, R²=0.98. F. curcumin IC₅₀=114.27 μM, R²=0.99. R², adjust R square values. Dose response curves of inhibitory activity were presented. Data are shown as mean ± SD of three independent experiments.

Figure 4

Basic structures of flavonoids.

Figure 5

Six natural products of the flavonoids with good inhibitory activity on the Mtb proteasome. Inhibitory activities of six flavonoids (each 200 μM) were as follows: baicalein 84.30%, pectolinarin 88.69%, hispidulin 82.06%, myricetin 84.50%, quercetin 74.40% and kaempferol 68.29%. The common structural features are the hydroxyl at the C ring C-3 (myricetin, quercetin and kaempferol), or the hydroxyl (baicalein) or the methoxyl (pectolinarin and hispidulin) at the A ring C-6.

Figure 6

Six natural products of the flavonoids with little inhibitory activity on the Mtb proteasome. Inhibitory activities of six flavonoids (at 200 μM) were zero. Compared with Figure  5, these compounds are without the hydroxyl at the C ring C-3 and the hydroxyl or methoxyl at the A ring C-6.