Research paperOptimization of 5-substituted thiazolyl ureas and 6-substituted imidazopyridines as potential HIV-1 latency reversing agents
Introduction
The emergence of antiretroviral therapies (ART) 25 years ago has allowed for the control of HIV-1 by suppressing plasma viral loads to undetectable levels [1,2]. However, discontinuation of treatment results in rebound of the virus, constraining patients to a lifelong drug treatment in order to subdue HIV replication and consequently decrease the severity of opportunistic pathogenic and viral infections [3,4]. This in itself poses many challenges including the need for long term compliance, the emergence of multidrug resistant viruses and drug toxicities resulting from life-long therapy [5]. The virus can rebound after cessation of ART due to long lived and proliferating latently infected CD4+ T cells that harbour replication-competent virus [6,7]. These latently infected cells are established early after infection, prior to the detection of viremia [8,9], and persist on ART, presenting a major barrier to the eradication of the virus [10,11].
One strategy being investigated to eliminate latently infected CD4+ T cells is known as “shock and kill”. The aim of this approach is the pharmacological activation of proviral transcription and subsequent virion production. Infected cells would then be susceptible to immune clearance and viral cytopathic effects, allowing for their elimination. Used in combination with ART, this approach could potentially eradicate or reduce the number of latently infected cells allowing for enhanced virological control when ART is stopped [12,13].
A diverse range of epigenetic proteins have been investigated as latency reversing agents (LRAs) for the “shock and kill” approach and include histone deacetylase (HDACs), bromodomain and extra-terminal motif (BET) proteins, histone methyltransferases (HMT) and protein kinase C (PKC) activators [14,15]. Several inhibitors of these proteins have progressed to human clinical trials. One example is the HDAC inhibitor, Vorinostat. In this clinical trial, people living with HIV (PLWH) on suppressive ART were treated with Vorinostat (Fig. 1) for 14 days and showed increased levels of CD4+ T cell associated unspliced (CA-US) HIV RNA [16], however, plasma HIV RNA, concentration of HIV DNA, integrated DNA and inducible virus in CD4+ T-cells remained unchanged. Significant changes in host gene expression were also observed, questioning the safety and long-term use of Vorinostat. Similar findings with preclinical and clinical trials on other HDAC inhibitors, including panobinostat [17,18] and romidepsin [19,20] and the BET inhibitor, JQ1 [21,22] (Fig. 1). In general, epigenetic modulators demonstrate poor efficacy in ex vivo models and do not induce substantial increases in intracellular HIV-1 mRNA or free virions in patient cells [22]. Therefore, new LRAs are needed with a novel mechanism of action.
We recently reported on the design of a FlpIn.FM luciferase reporter cellular assay capable of detecting small molecules that activate HIV-1 gene expression [23]. The FlpIn.FM reporter cell line will detect both transcriptional and post-transcriptional steps of gene expression, including effects on RNA capping, RNA splicing, RNA transport, RNA-modifications, RNA stability and translation of protein. The HEK293 derived reporter cell line expresses a HIV-1 Nef – click beetle red (CBR) fusion protein from a spliced mRNA expressed under direction from the long terminal repeat (LTR) HIV promoter, allowing for detection of early viral gene expression. The cell line also contains a click beetle green (CBG) luciferase reporter driven from a cytomegalovirus (CMV) promoter allowing for the detection of non-specific host cell protein expression.
We used the HEK293 FlpIn.FM cell line in a high throughput screen and the identified the 2-acylaminothiazole class (Fig. 2) [23]. Our initial optimization efforts concentrated on defining structure activity relationship (SAR) of the 2-acylaminothiazole class and resulted in compounds with enhanced HIV gene expression (LTR) in both HEK293 FlpIn.FM and the Jurkat 10.6 latent HIV-GFP reporter cell line. The optimized compounds also increased CA-U.S. HIV RNA in resting CD4+ T cells isolated from PLWH on ART. While a modest enhancement in HIV gene expression was demonstrated with optimized analogues, the selectivity window between activation of HIV and non-specific (CMV) gene expression was limited. This is comparable to the level of selectivity observed with epigenetic modulators used as LRAs in clinical trials [23].
In this report we describe further optimization of the 5-substituted 2-acylaminothiazole class focused on rigidifying the central oxy-carbon chain and bioisosteric substitution of the 2-acylaminothiazole moiety to improve LTR specific activity and physicochemical properties. We show the 5-substituted thiazolyl urea and 6-substituted imidazopyridine analogues derived from this study have enhanced HIV-1 LTR activity in both the FlpIn.FM cell line and J.Lat10.6 cell lines and improved in vitro metabolic stability.
Section snippets
Results and discussion
The initial focus of the research reported here was to rigidify the central aliphatic chain, linking the thiazole and the pendant aryl group (Fig. 2), by replacement with a variety of 4-, 5-, 6- and 7- membered unsaturated heterocyclic systems. It was proposed that this change could improve HIV-1 LTR activity by reducing conformational energy required for the substituted thiazolyl and aryl motifs to engage with pockets of the unknown cellular target. In addition, it was hypothesized, the
Conclusions
The FlpIn.FM HEK293 cellular assay was previously utilized to identify the 2-acylaminothiazole hit class. Exploration of the structure activity relationship afforded compounds with modest activity in HIV cellular models (Fig. 2). Building on our previous work, we applied two design strategies to further enhance physicochemical properties and improve activity in HIV gene expression models. The first approach explored rigidification of the central oxy-carbon linker with a variety of 4-, 5-, 6-
FlpIn.FM HEK293 cellular assay
The FlpIn.FM HEK293 cellular assay was performed according to previously described protocol [23]. Briefly, FlpIn.FM HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. A total of 1500 cells in 25 μL of DME media with 5% FCS were transferred to each well of the assay plate using a Multidrop reagent dispenser. Plates were left at room temperature in a single layer for 1 h to allow adhesion to commence and were then incubated at 37 °C and 5% CO2 for
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was funded by the National Health and Medical Research Council of Australia (Development Grant 1113712 to D.F.P., B.E.S. and S.R.L.; Project Grant 1129320 to D.F.P. and B.E.S.; Program Grant 1052979 to D.F.P. and S.R.L.; Practitioner Fellowship to S.R.L.), the Australian Centre for HIV and Hepatitis Virology Research, the Australian Cancer Research Foundation, the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. B.E.S. is a Corin
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