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Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3’-deoxy or 2′-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2′ or 4′ mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. In addition, RSV causes significant morbidity and mortality in hospitalized elderly and immunocompromised patients. Currently, only palivizumab, a monoclonal antibody against the RSV fusion (F) protein, and inhaled ribavirin are approved for the prophylactic and therapeutic treatment of RSV, respectively. Therefore, there is a clinical need for safe and effective therapeutic agents for RSV infections. GS-5806, discovered via chemical optimization of a hit from a high-throughput antiviral-screening campaign, selectively inhibits a diverse set of 75 RSV subtype A and B clinical isolates (mean 50% effective concentration [EC 50 ] = 0.43 nM). The compound maintained potency in primary human airway epithelial cells and exhibited low cytotoxicity in human cell lines and primary cell cultures (selectivity > 23,000-fold). Time-of-addition and temperature shift studies demonstrated that GS-5806 does not block RSV attachment to cells but interferes with virus entry. Follow-up experiments showed potent inhibition of RSV F-mediated cell-to-cell fusion. RSV A and B variants resistant to GS-5806, due to mutations in F protein (RSV A, L138F or F140L/N517I, and RSV B, F488L or F488S), were isolated and showed cross-resistance to other RSV fusion inhibitors, such as VP-14637, but remained fully sensitive to palivizumab and ribavirin. In summary, GS-5806 is a potent and selective RSV fusion inhibitor with antiviral activity against a diverse set of RSV clinical isolates. The compound is currently under clinical investigation for the treatment of RSV infection in pediatric, immunocompromised, and elderly patients.

Introduction: The STING (STimulator of INterferon Genes) protein has been identified as an attractive target for cancer immunotherapy due to its central role in inducing T-cell mediated tumor control. Here we describe the preclinical characteristics of the novel STING agonist, ALG-031048, that demonstrates potent anti-tumoral activity in in vivo mouse models. Methods: Binding to STING was determined using differential scanning fluorimetry (DSF). Cellular activation and cytokine release were assessed using HEK 293T reporter cell lines as well as THP-1 and Raw264.7 cells. In vitro stability was measured in the presence of snake venom phosphodiesterase (SVPD). In vivo anti-tumoral activity was tested in the syngeneic CT26 and B16F10 mouse model. Results: ALG-031048 demonstrated potent binding to the STING R232 protein in the DSF assay with a mean Kd of 3.12 ± 0.12 μM and a thermal shift of 12.8 °C, similar to the natural ligand 2'3'cGAMP (Kd of 2.75 ± 0.78 μM, shift of 16.7°C) or the clinical stage STING agonist, ADU-S100 (Kd of 4.61 ± 0.42 μM, shift of 10.7°C). In the HEK 293T R232 reporter assay, ALG-031048 was more potent than ADU-S100 with a mean EC50 of 55.9 ± 24.1 and 30.3 ± 17.1 nM for the IFNβ and IRF reporter, respectively, compared to ADU-S100 with EC50 values of 233 ± 179 and 88.7 ± 35.6 nM. Cellular activation was confirmed in THP-1 and Raw264.7 cells where ALG-031048 induced the secretion of IFNβ and CXCL-10. ALG-031048 was highly stable and exhibited no degradation for up to 24 hours in the presence of SVPD while ADU-S100 and 2'3'cGAMP were quickly degraded with less than 10% remaining after 24 hours. In vivo, 3 intra-tumoral (IT) doses of 100 μg ALG-031048 q3d caused complete tumor regression in 90% of animals in the CT26 mouse colon carcinoma model, while treatment with 25 μg ALG-031048 IT 3xq3d resulted in survival of 60 % of animals. In contrast, only 10 and 44% of mice showed complete tumor regression when treated IT 3xq3d with 25 or 100 μg ADU-S100, respectively. To study whether induction of a protective immune response was associated with STING agonist treatment, surviving animals were re-challenged with a second inoculation of CT26 cells on the contra-lateral side. All naïve animals formed tumors within 23-30 days. In contrast, 5 out of 5 and 8 out of 9 mice pre-treated with 25 or 100 μg ALG-031048, respectively, did not exhibit any significant tumor growth over 40 days, while 3 out of 4 animals pretreated with 100 μg ADU-S100 showed complete tumor suppression. Initial results using the B16F10 mouse melanoma model confirmed the potent anti-tumoral activity of ALG-031048 observed in the CT26 model. Summary: ALG-031048 is a novel STING agonist combining high stability and excellent in vitro potency. In preclinical models, ALG-031048 demonstrated potent and long-lasting tumor suppression. ALG-031048 is advancing in toxicology studies for further profiling as a clinical candidate. Citation Format: Andreas Jekle, Santosh Thatikonda, Sarah Stevens, Caroline Williams, April Kinkade, Suping Ren, Ruchika Jaisinghani, Qingling Zhang, Dinah Misner, Antitsa Stoycheva, Jerome Deval, Sucheta Mukherjee, Francois Gonzalvez, Sushmita Chanda, David B. Smith, Julian A. Symons, Lawrence M. Blatt, Leonid Beigelman. Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4520.

The salivary glands represent a major site of cytomegalovirus replication and transmission to other hosts. Despite control of viral infection by strong T cell responses in visceral organs cytomegalovirus replication continues in the salivary glands of mice, suggesting that the virus exploits the mucosal microenvironment. Here, we show that T cell immunity in the salivary glands is limited by the induction of CD4 T cells expressing the regulatory cytokine interleukin (IL)-10. Blockade of IL-10 receptor (IL-10R) with an antagonist antibody dramatically reduced viral load in the salivary glands, but not in the spleen. The mucosa-specific protection afforded by IL-10R blockade was associated with an increased accumulation of CD4 T cells expressing interferon γ, suggesting that IL-10R signaling limits effector T cell differentiation. Consistent with this, an agonist antibody targeting the tumor necrosis factor receptor superfamily member OX40 (TNFRSF4) enhanced effector T cell differentiation and increased the number of interferon γ–producing T cells, thus limiting virus replication in the salivary glands. Collectively, the results indicate that modulating effector T cell differentiation can counteract pathogen exploitation of the mucosa, thus limiting persistent virus replication and transmission.