Optogenetic Regulation of Integrated Stress Responses: Developing Novel Broad-Spectrum Antiviral Strategies

Abstract

In a recent publication in Cell, Wong et al. presented an optogenetic system for screening compounds that specifically modulate the integrated stress response (ISR) [1]. The authors identified eight non-cytotoxic ISR enhancers as broad-spectrum antiviral agents and revealed their key mechanism: the selective targeting of general control nonderepressible 2 (GCN2) to upregulate activating transcription factor 4 (ATF4) expression, thereby sensitizing cells to stress and apoptosis [1].

Optogenetics enables precise spatiotemporal control of cellular activity by conferring light sensitivity via heterologous expression of photosensitive proteins. This approach is increasingly being integrated with synthetic biology to facilitate novel paradigms in phenotypic drug discovery [2]. Meanwhile, the ISR pathway represents a conserved signaling pathway activated by four stress sensor kinases—heme-regulated inhibitor (HRI), protein kinase R (PKR), protein kinase R-like ER kinase (PERK), and GCN2—which respond to diverse stressors such as viral double-stranded RNA [3]. Upon activation, these kinases phosphorylate the eukaryotic translation initiation factor 2 subunit alpha (eIF2α), leading to the selective translation of ISR-related proteins, such as ATF4, C/EBP homologous protein (CHOP), and growth arrest and DNA damage-inducible protein 34 (GADD34), thereby modulating cell survival and function. Given this regulatory capacity, ISR-enhancing compounds represent a novel strategy for the development of broad-spectrum antiviral therapeutics [3]. The combination of optogenetics-driven precise manipulation and deeper exploration of the ISR network—particularly its crosstalk with other signaling pathways—may open breakthrough directions for future broad-spectrum antiviral research.

To achieve precise control of the ISR pathway, Taivan et al. developed an optogenetic platform that dynamically stimulates the ISR signaling using a light-activated optogenetic PKR (opto-PKR) [4]. The dsRBM1 and dsRBM2 regions of PKR were replaced with an optimized mutant of the Arabidopsis blue light receptor Cry, namely Cry2Olig (E490G). Upon transduction of opto-PKR into cells, exposure to blue light prompted Cry2 aggregation, inducing in PKR oligomerization, kinase activation, and subsequent initiation of ISR. This light-controlled system simulates PKR-mediated ISR activation as observed during viral infection, while avoiding off-target cytotoxicity [4]. The platform's efficacy was validated through both pharmacological activators and inhibitors of the ISR pathway (Figure 1a) [1]. Crucially, unlike traditional small-molecule stressors that often cause cross-pathway interference, this approach minimizes such off-target effects and offers immediate deactivation in darkness, enabling transient response unattainable with conventional small-molecule activators. Collectively, these features underscore the spatiotemporal precision and specificity advantages of the optogenetic platform, providing a novel tool for the dynamic study between viruses and hosts. It not only enables high-throughput screening of specific ISR modulators but also permits real-time observation of key mechanisms that viruses evade ISR defenses, thereby establishing a foundation for developing effective antiviral therapies.

In this study, a high-throughput screen of 370,830 compounds identified eight non-cytotoxic small molecules (IBX-200 to IBX-207) that effectively decreased the viability of opto-PKR cells in light. These compounds were not acutely cytotoxic to opto-PKR cells in the dark at concentrations up to 50 μM but exhibited half-maximal effective concentration values (EC₅₀) ∼0.1 to ∼1 μM in light. Based on their ability to selectively enhance cell death induced by diverse ISR pathways, these compounds were classified as ISR enhancers [1]. Mechanistic investigation revealed that representative compounds IBX-200, IBX-202, and IBX-204 induced ISR activation, including substantial elevation of ATF4 and CHOP expression, as well as increased ATP consumption, yet differentially regulated eIF2α phosphorylation. Further analysis showed that IBX-200 and IBX-204 specifically bound to GCN2 with dissociation constants (K_d) of 25.2 and 3.2 μM, respectively, without affecting the activity of HRI, PKR, or PERK kinases. Computational and cellular assays confirmed that both compounds promote GCN2 phosphorylation at Thr899, particularly under light exposure. Notably, chemical conversion of IBX-202 into IBX-200-like analogues family compounds resulted in similar effects to those of IBX-200 (Figure 1b).

Cellular assays demonstrated that all three compounds (IBX-200, IBX-202, and IBX-204) inhibited HSV-1, ZIKV, and RSV, with half-maximal inhibitory concentrations (IC₅₀) values ranging from 1 to 100 μM. Furthermore, IBX-200 significantly reduced HSV-1 replication in ocular tissues of infected mice [1]. Collectively, these GCN2-targeting compounds exhibit broad-spectrum antiviral activity and represent promising candidates for novel antiviral therapies. This study represents a significant advancement in ISR-targeted therapies by facilitating a transition from traditional “activation/inhibition” binary regulation to a novel paradigm of “precision regulation” [1].

In summary, this study confirms optogenetics as an effective platform for the identification of ISR-modulating compounds, with the screened ISR enhancers demonstrating potential as candidate drugs for the development of broad-spectrum antiviral agents. Importantly, this optogenetic strategy can be extended to screen modulators of other critical pathways, such as Wnt/β-catenin and RAS/ERK signaling, offering specificity and precise spatiotemporal control compared with traditional activation methods reliant on multitarget compounds. Nevertheless, the system utilized in this study also possesses certain limitations, including the risk of excessive ISR activation and apoptosis under continuous presence of ISR enhancers, as well as the limited tissue penetration of blue light, which currently restricts its application in organoids and in situ tissues. Therefore, clinical translation requires precise regulation of ISR activation to balance antiviral efficacy against apoptotic risk, indicating that ISR enhancers may be unsuitable for long-term maintenance therapies. To improve clinical applicability, targeted or localized delivery approaches should be explored to concentrate drug effects at viral infection sites and minimize systemic damage. Additionally, intermittent dosing regimens or combination therapies with agents that inhibit excessive apoptosis and inflammatory responses might contribute to potential therapeutic benefits. Future research may focus on developing light-controlled small molecule screening tools for additional pathways, advancing mechanistic studies, and facilitating the discovery of next-generation paradigm-shifting drugs.

Bixia Hong designed the research; Shizhan Cui and Zehan Pang read the papers and analyzed the data; Shizhan Cui, Zehan Pang and Bixia Hong wrote and revised the manuscript. All authors have read and approved the final manuscript.

The authors have nothing to report.

The authors declare no conflicts of interest.

The authors have nothing to report.