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Structure-guided drug design identifies a BRD4-selective small molecule that suppresses HIV
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Qingli Niu,1,2 Zhiqing Liu,3 Edrous Alamer,1,2 Xiuzhen Fan,1,2 Haiying Chen,3 Janice Endsley,1,2 Benjamin B. Gelman,4 Bing Tian,5 Jerome H. Kim,6 Nelson L. Michael,7,8 Merlin L. Robb,7,8 Jintanat Ananworanich,7,8,9 Jia Zhou,3 and Haitao Hu1,2
First published July 22, 2019
We showed that ZL0580 induced HIV suppression in multiple in vitro and ex vivo cell models.
To summarize, we report identification of a small molecule that induces epigenetic suppression of HIV via BRD4. Our study provides a conceptual and translational basis for future development of this class of molecules as tools and/or potential therapeutic agents for HIV epigenetic silencing. Further studies are needed to advance the development of this class of molecules, including improved understanding of MoA, lead optimization, and efficacy testing in vivo in animal models of HIV/SIV infection.
HIV integrates its provirus into the host genome and establishes latent infection. Antiretroviral therapy (ART) can control HIV viremia, but cannot eradicate or cure the virus. Approaches targeting host epigenetic machinery to repress HIV, leading to an aviremic state free of ART, are needed. Bromodomain and extraterminal (BET) family protein BRD4 is an epigenetic reader involved in HIV transcriptional regulation.
Using structure-guided drug design, we identified a small molecule (ZL0580) that induced epigenetic suppression of HIV via BRD4. We showed that ZL0580 induced HIV suppression in multiple in vitro and ex vivo cell models.
Combination treatment of cells of aviremic HIV-infected individuals with ART and ZL0580 revealed that ZL0580 accelerated HIV suppression during ART and delayed viral rebound after ART cessation.
Mechanistically different from the BET/BRD4 pan-inhibitor JQ1, which nonselectively binds to BD1 and BD2 domains of all BET proteins, ZL0580 selectively bound to BD1 domain of BRD4.
We further demonstrate that ZL0580 induced HIV suppression by inhibiting Tat transactivation and transcription elongation as well as by inducing repressive chromatin structure at the HIV promoter. Our findings establish a proof of concept for modulation of BRD4 to epigenetically suppress HIV and provide a promising chemical scaffold for the development of probes and/or therapeutic agents for HIV epigenetic silencing.
Another important finding of our study is that ZL0580 induces significant delay in viral rebound after ART removal ex vivo in aviremic PBMCs (Figure 4, A–C).
We noted that, despite significant delay, viral rebounds eventually occur in all the examined PBMCs after treatment cessation. This could be due to drug decay and loss of activity for ZL0580 after more than several days of drug removal. Another possible explanation is that, in this cell culture, T cells were stimulated and underwent continuous proliferation and turnover; following treatment removal, the newly generated T cells were not exposed to the drugs and might have contributed to viral release.
These data suggest that it may be challenging to “durably” silence HIV by a single “block” approach and that combination approaches may be needed. It is hoped that through comprehensive lead optimization and drug-development efforts (e.g., delivery, dosing, and combination use with other silencers), we can improve the durability and potency of this class of molecules in epigenetically suppressing HIV. Finally, for a block-and-lock HIV cure approach, while therapies targeting host proteins are considered promising and provide advantages (e.g., reduced drug resistance), off-target effects need to be carefully evaluated. To this end, a number of drug candidates targeting BET proteins have been tested in various disease models including HIV infection (35). The data obtained so far in our study support a safe profile of ZL0580 regarding cellular toxicity and off-target effects. Future pharmacokinetics and in vivo toxicity studies are warranted to better evaluate utility of this compound in HIV epigenetic suppression.
Highly consistent with the results in J-Lat cells and human CD4+ T cells infected in vitro, ZL0580 could also induce fairly potent suppression of induced HIV transcription ex vivo in PBMCs of viremic HIV-infected individuals (Figure 3). While the potency of suppression varied among different participants, ZL0580 induced significant suppression in 7 out 8 PBMCs examined (Figure 3).
ZL0580 treatment did not cause significant toxicity to these cells compared with ART alone or NC
Second, PBMCs of aviremic participants (n = 6) were not activated, but were directly treated with ZL0580 (2 μM) or not treated (without ART). Treatment was given every 3 days for a total of 3 times (day 0, 3, 6) and stopped on day 9 (Figure 4D). This model aimed to determine whether ZL0580 could inhibit spontaneous HIV production and suppress LRA-stimulated latent HIV reactivation. Of interest, while low levels of spontaneous HIV production were readily detectable in some PBMCs of the NC group (1 PBMC on day 3, 2 on day 6, 4 on day 15, and 2 on day 18), ZL0580 treatment inhibited spontaneous HIV production in all 6 PBMCs (Figure 4D). Cell culture was continuously monitored to day 18, and cells remained in good condition. Cells were then stimulated with PHA to reactivate latent HIV. We found that treatment of these PBMCs with ZL0580 led to inhibition of PHA-activated HIV transcription compared with NC (Figure 4E). This finding is consistent with the result in J-Lat cells (Figure 1H) and indicates that ZL0580 displays a repressive effect on latent HIV in aviremic PBMCs.
HIV gene expression is regulated by host cell epigenetic and transcriptional mechanisms (14, 15). Various approaches targeting these mechanisms to suppress integrated HIV have been reported. Small antisense RNAs targeted to the HIV promoter were shown to induce HIV transcriptional suppression and/or silencing (16–20). Chemical inhibition of Tat, a key viral protein in HIV transcription, by an inhibitor (dCA) can suppress Tat transactivation and induce a state of epigenetic repression in the HIV promoter (21, 22). In vivo administration of dCA can reduce residual HIV viremia and delay viral rebound in ART-suppressed, HIV-infected humanized mice (23). Despite substantial efforts to understand mechanisms for epigenetic regulation of HIV, effective approaches, and especially those utilizing small molecules and targeting host epigenetic factors to silence HIV, are limited.
The bromodomain and extraterminal (BET) family proteins, including BRD4, are a group of epigenetic factors characterized by 2 N-terminal bromodomains (BDs) that bind acetylated histones in chromatin (24, 25). As an epigenetic reader, BRD4 is functionally versatile and interacts with a variety of partnering proteins to regulate gene expression (26–29). Accumulating evidence has suggested that BRD4 plays an important role in HIV transcriptional regulation (29–32). It was shown that BRD4 can suppress HIV transcription elongation by competing with Tat for cellular p-TEFb/CDK9 (30–32). Targeted modulation of BET/BRD4 by pan-BET inhibitor JQ1 (33) relieves the competition between BRD4 and Tat and therefore reactivates latent HIV (30, 31). Via its BDs, BRD4 can be recruited to HIV LTR through interactions with acetyl-histone H3 (AcH3) and AcH4. It was recently reported that differential interactions of BRD4 with AcH4 and AcH3 in the HIV promoter are associated with different effects on HIV transcription and latency establishment (34). Together, these studies support the functional versatility of BRD4 in regulation of target gene expression, depending on the partnering proteins it interacts with, and indicate that BRD4 and its associated epigenetic machinery can be potentially modulated to exert positive or negative effects on HIV proviral transcription.
Given the established role of BRD4 in HIV transcription, we were interested in discovering approaches that can modulate this pathway to induce HIV transcriptional activation or repression. Using structure-guided drug design, we have synthesized multiple libraries of BRD4 modulators (35). Screening these compounds led to identification of a small molecule, named ZL0580, that is more selective to the BD1 domain of BRD4 and induces a functional impact on HIV transcription distinct from that of JQ1. We found that, unlike JQ1, ZL0580 induced epigenetic suppression of HIV in multiple in vitro and ex vivo models. Gene KO and overexpression analysis confirmed that BRD4 played a predominant role and is specifically required in ZL0580-induced HIV suppression. We further showed that ZL0580 induced HIV suppression by inhibiting Tat transactivation and transcription elongation as well as by inducing a more repressive chromatin structure at the HIV LTR.

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