We and others have studied the anti-HIV-1 activity of BA and its derivatives [15
]. In an effort to identify potential cellular targets of these compounds, BA was tested against human 20S proteasomes. The chemical structure of BA is shown in Figure S1 (supplemental data)
. BA did not inhibit the chymotrypsin-like activity of 20S proteasomes (). In fact, the chymotrypsin-like activity was enhanced in the presence of BA. This result raised the possibility that BA could act as a proteasome activator. To test this possibility, the proteasome activator, PA28, was left out of the reaction mixtures. In the absence of PA28, 20S proteasome is inactive. BA was able to activate the chymotrypsin-like activity of 20S proteasome in a dose dependent manner (). The reaction rate reached a plateau with BA at 10 μg/ml. The concentration of BA required to achieve 50% activation (EC50) is approximately 2.5 μg/ml.
BA enhanced the chymotrypsin-like activity of 20S proteasomes
BA preferentially activated chymotrypsin-like proteasome activity
Although BA can activate the proteasome activity, its mode of action is clearly different from that of PA28 or SDS (sodium dodecyl sulfate). PA28 and SDS are two proteasome activators that are commonly used in proteasome assays. Both PA28 and SDS can activate the three main proteasome activities: chymotrypsin-like, trypsin-like, and caspase-like activities. In contrast, BA did not activate the trypsin-like activity and exhibited minimal activation on the caspase-like activity of 20S proteasomes (). PA28 activates proteasome by inducing conformational changes in 20S proteasome, which allows the proteasome substrates to access the proteolytic sites inside the 20S proteasome cylinder. On the other hand, SDS activates proteasomes presumably by partially denaturing the 20S proteasome, and that allows substrates to access the catalytic sites. Therefore, BA appears to activate the chymotrypsin-like activity in a mode distinct from that of PA28 or SDS.
Activation of proteasome by BA raised the possibility that BA derivatives might also activate 20S proteasomes. There are three sites on the BA structure that can be used for chemical modifications: C-3, C-20, and C-28. We have previously shown that the anti-HIV-1 activity of BA derivatives is highly dependent on where their side chain modification resides [21
]. A series of BA derivatives with modification at C-3 and C-28 positions were tested for their effect on 20S proteasomes. Instead of activation of proteasomes, some of the tested BA derivatives inhibited the chymotrypsin-like activity at low μg/ml concentrations ().
Effect of BA derivatives on the chymotrypsin-like activity of 20S proteasome.
A typical example of BA derivatives that inhibited 20S proteasomes is shown in . The BA derivative 3',3'-dimethyl-succinyl BA (DSB, 2)) inhibited the chymotrypsin-like activity of 20S proteasome by 50% at a concentration of approximately 4 μg/ml (). However, DSB with an additional side chain modification at position C-28 (compound LH141, 7) was inactive against proteasomes (). The BA derivative IC9564 (8) with the same C-28 side chain as LH141 was also inactive (). This C-28 side chain also abrogated the ability of BA to activate 20S proteasomes. IC9564 did not activate 20S proteasome in the absence of PA28 (data not shown). These results suggested that the C-3 modifications could transform BA from a proteasome activator into an inhibitor and addition of a side chain at C-28 could nullify the activation activity of BA and inhibitory activity of DSB.
The BA derivative, DSB, preferentially inhibited chymotrypsin-like proteasome activity
The trypsin-like activity of 20S proteasomes was less sensitive to DSB. It took approximately 12 μg/ml of DSB to inhibit the trypsin-like activity by 50% (). On the other hand, DSB did not inhibit the caspase-like activity of proteasomes (). These results suggested that BA derivatives can become a new class of proteasome inhibitors that preferentially inhibit chymotrypsin-like activity of proteasomes.
Inhibition of proteasomes by DSB could be a result of competition with the cellular proteasome activator, PA28, in the reaction mixture. To test this possibility, PA28 was replaced with 0.3% SDS as proteasome activator in the assay mixture. Under this assay condition, DSB inhibited the proteasome activity by 50% at approximately 3 μg/ml (). This inhibitory activity is comparable to that when PA28 was used as the proteasome activator (). Therefore, the data suggest that the inhibitory activity of DSB is not due to the inhibition of PA28 binding to proteasomes.
DSB inhibited SDS-activated chymotrypsin-like proteasome activity
Although DSB could inhibit purified 20S proteasomes, it is not clear whether the compound can effectively inhibit proteasomes in the cells. To determine the effect of DSB on proteasomes in living cells, MT4 cells were treated with DSB or a known proteasome inhibitor Ac-Leu-Leu-Met-CHO (LLM-f). The chymotrypsin-like activity of proteasomes in the cells was analyzed using a Promega cell-based proteasome assay. DSB effectively inhibited the proteasome activity with an IC50 approximately 2 ug/ml (). Thus, DSB is approximately 2-fold more potent in the cell-based assay than in the assays using purified proteasomes. It is not clear why DSB is slightly more potent in the cell-based assay than in the purified proteasome assay. One possible explanation is that DSB might have accumulated to a higher concentration in the cells.
DSB inhibited the chymotrypsin-like proteasome activity in MT4 cells