Optimalisatie van de block-and-lock strategie voor een HIV genezing: Het combineren van BRD4 modulatoren met LEDGINs

Abstract

Infection with human immunodeficiency virus (HIV) is currently incurable because the virus integrates into the host genome of long-lived memory CD4+ T cells and can persist in a transcriptionally silent state, known as latency. Antiretroviral therapy (ART) effectively suppresses viral replication, restores CD4+ T cell counts, and prevents disease progression, transforming HIV-1 infection from a death sentence to a manageable chronic disease. However, upon treatment interruption, viral rebound typically occurs within 2-3 weeks due to reactivation of the latent reservoir. Therefore, the development of an HIV cure must address this persistent reservoir. Two main cure strategies are being investigated. First, a sterilizing cure strategy aims to completely eradicate the latently infected cells. Second, a functional cure seeks to permanently suppress transcription in latently infected cells and drive them into a deep latent state in which viral reactivation is no longer possible. The block-and-lock functional cure strategy uses latency-promoting agents (LPAs) to block proviral transcription ("block phenotype") and induce a deep latent state resistant to reactivation ("lock phenotype"). A critical step in the establishment of the latent reservoir is the integration of the provirus into the host genome, a process catalyzed by the viral enzyme integrase. Lens epithelium-derived growth factor p75 (LEDGF/p75) is a key host cofactor that directs the pre-integration complex (PIC) toward transcriptionally active chromatin regions. In this context, LEDGF/p75 serves as a "GPS-system" to redirect the provirus to active chromatin regions. My host lab developed LEDGINs, small molecules that inhibit the interaction between LEDGF/p75 and the viral integrase. By disrupting this interaction, LEDGINs reduce HIV integration and simultaneously disable the proviral "GPS-system", resulting in integration into transcriptionally silent chromatin regions. As a consequence, LEDGIN-retargeted proviruses exhibit minimal to no transcriptional activity and are refractory to reactivation, even after compound withdrawal. Since 2016, multiple orthogonal techniques have been employed to characterize the viral phenotype induced by LEDGIN treatment. In this context, our laboratory used uniquely barcoded viruses to simultaneously track individual proviral DNA and the corresponding RNA transcripts. These studies demonstrated that although LEDGINs efficiently retarget proviruses away from active chromatin marks, such as H3K36me2/3, a subset of individual proviruses still displayed high levels of viral transcription. Interestingly, LEDGINs did not alter the distance between HIV integration sites and enhancers, which are highly acetylated regulatory DNA elements that strongly stimulate gene transcription through the recruitment of transcription factors. Further analysis by our host laboratory revealed that the top 10% highest-expressing barcodes following LEDGIN treatment were preferentially integrated in close proximity to these enhancers. You can compare HIV integration to a car (HIV) looking for a parking spot (open chromatin). LEDGINs effectively shut down the GPS system (LEDGF/p75), so most cars can no longer find their preferred parking spots. However, a small fraction can still enter the parking by chance, despite the loss of navigation. This finding limited the clinical potential of LEDGINs, as these residual transcriptionally active proviruses located near enhancers could contribute to viral rebound after treatment discontinuation. Accordingly, my PhD research focused on further optimizing the LEDGIN-based block-and-lock cure strategy by combining LEDGINs with compounds blocking enhancers. The epigenetic reader, bromodomain-containing protein 4 (BRD4), binds to the acetylated histones on enhancers, contributing to the assembly and stabilization of transcriptional machinery required for enhancer-driven transcription. However, beyond hampering enhancer-mediated transcription, BRD4 also functions as a negative regulator of HIV transcription by competing with the viral trans activator of transcription (Tat) for binding to the positive transcriptional elongation factor b (P-TEFb). In doing so, BRD4 prevents Tat-mediated recruitment of P-TEFb to the viral promoter, thereby restricting transcriptional elongation. Most BRD4-targeting compounds, such as JQ1, act by binding the acetyl-lysine recognition pocket of BRD4, resulting in its displacement from the chromatin. This relieves BRD4-mediated repression of Tat-dependent transcription. In contrast, a research group from Texas (Prof. Dr. Haitao Hu) identified ZL0580, a unique allosteric BRD4 modulator that binds outside the acetyl-lysine pocket of BRD4. Rather than displacing BRD4, ZL0580 stabilizes its chromatin association and further suppresses Tat-mediated transcription. In the first chapter, I confirmed that ZL0580 indeed suppresses HIV-1 transcription and reactivation, while JQ1 stimulates proviral transcription. Furthermore, we showed with fluorescence imaging that ZL0580 enhances the binding of BRD4 to the chromatin, in contrast to JQ1, which releases BRD4 from the chromatin. Next, I combined ZL0580 with LEDGINs in both latency cell lines and CD4+ T cells infected in vitro. Interestingly, ZL0580 and LEDGINs additively blocked HIV-1 transcription and reactivation, with almost a complete suppression of proviral transcription after the addition of both compounds, supporting the hypothesis that BRD4-driven enhancers may steer residual transcription after LEDGIN-treatment. Continuing the parking analogy, where LEDGINs disable the "GPS" system, ZL0580 aims to close the barriers of the remaining parking spots (enhancers), thereby fully limiting access to all parking spots. Unfortunately, ZL0580 displayed high toxicity and a suboptimal selectivity index, hampering its translation into the clinic. Therefore, in the second chapter of my PhD, I collaborated with medicinal chemists from Shandong University in China (Prof. Dr. Zhan Peng). Using structure-based drug design, his research team developed analogues of ZL0580, which were tested by us for their toxicity and inhibitory effect on HIV-1 transcription and toxicity. This resulted in the development of FDE-24, a ZL0580 analog with about a 18-fold higher selectivity in cell lines. Next, I proved that FDE-24 also enhances the binding of BRD4 to the chromatin. Finally, FDE-24 is also shown to suppress HIV-1 reactivation in primary cells, although to a lesser extent as ZL0580. Next, I combined FDE-24 with LEDGINs, also showing additive synergy scores. To conclude, our findings demonstrate that BRD4 can be pharmacologically targeted to either stimulate or suppress HIV-1 transcription. Furthermore, ZL0580 and FDE-24 shows promise in combination with LEDGINs to enhance the efficiency of the block-and-lock cure strategy. Still, they require further mechanistic investigation and lead optimization for future clinical application.