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Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC 50 ) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro . The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRβ agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRβ-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRβ and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1 . In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRβ agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

The nucleotide analog AL-335 is a pangenotypic hepatitis C virus (HCV) nonstructural protein (NS)5B inhibitor being evaluated as treatment for chronic HCV infection. This three-part randomized, double-blind study evaluated the safety and pharmacokinetics of single and multiple ascending oral doses of AL-335. Healthy volunteers (HVs) received single doses of AL-335 (100-1,200 mg) or placebo in a fasted or fed (400 mg) state. Non-cirrhotic subjects (HCV genotype [GT]1-4) and GT1-infected subjects with Child Pugh A cirrhosis received multiple doses of AL-335 (400, 800, 1,200 mg) or placebo once daily (QD) for 7 days. Forty-eight HVs and 64 subjects with HCV GT1-4 were randomized and received treatment. AL-335 was well tolerated in HVs and HCV-infected subjects with/without cirrhosis. AL-335 was rapidly absorbed and converted to the metabolites ALS-022399 and ALS-022227. ALS-022227 exposure increased less than dose-proportionally and was unaffected by food, while AL-335 and ALS-022399 exposure increased with food by 85% and 50%, respectively, in HVs. Rapid and dose-dependent reductions in HCV-RNA were observed in GT1-infected subjects. In non-cirrhotic, GT1-4-infected subjects receiving AL-335 800 mg QD, potent antiviral activity was observed, regardless of genotype (mean maximum reductions in HCV-RNA of 4.0-4.8 log10 IU/mL). The same dose in GT1-infected cirrhotic subjects resulted in a 3.5 log10 IU/mL mean maximum reduction in HCV-RNA. AL-335 was well tolerated when administered as single and multiple doses, with an acceptable pharmacokinetic profile. The drug also demonstrated potent antiviral activity in HCV GT1-4-infected subjects, including GT1-infected subjects with cirrhosis.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

There are limited treatment options for infection with the respiratory syncytial virus. In this human challenge model, a new oral nucleoside analogue, ALS-008176, showed modest antiviral activity. Respiratory syncytial virus (RSV) infections are a cause of substantial morbidity and mortality in various patient populations worldwide, including children. Globally, RSV infections were estimated to cause 3.4 million hospitalizations and 66,000 to 199,000 deaths in 2005 in children younger than 5 years of age, primarily in the developing world. 1 In U.S. infants, RSV infection causes substantial outpatient disease 2 and is a common cause of hospitalization. 3 The risk of death from respiratory causes is nine times as high among U.S. infants with RSV infection as the risk among infants with influenza. 4 Immunocompromised patients and elderly patients, especially those with coexisting . . .

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THR[beta] agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THR[beta]-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THR[beta] and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THR[beta] agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

BackgroundPD-1/PD-L1 antibody-based therapies have demonstrated tremendous success in the treatment of a variety of cancers. However, these antibody drugs are associated with several disadvantages, such as weak tumor penetration, immune-related adverse events (irAEs) due to their long half-life and development of anti-drug antibodies. Here, we report the discovery of ALG-093989, a highly potent and orally bioavailable PD-L1 small molecule inhibitor, that may overcome the limitation of PD-1/PD-L1 antibodies.MethodsThe biochemical interaction of PD-1/PD-L1 and PD-L1 dimerization was assessed by AlphaLISA®. Cellular activity was measured using a co-culture assay of PD-1 expressing Jurkat NFAT luciferase T cells with PD-L1 expressing CHO cells. Pharmacokinetic (PK) studies were performed in rat. In vivo PD-L1 target occupancy was assessed at 6 hours following a single oral dose in a humanized-PD-L1 MC38 subcutaneous mouse model.ResultsALG-093989 inhibits PD-1/PD-L1 interaction with an IC50 of 14 pM and induces PD-L1 dimerization with an EC50 of 13 nM. ALG-093989 has similar T-cell activation potency as durvalumab, and approximately 10-fold improved T-cell activation potency vs. INCB086550, a PD-L1 small molecule inhibitor that demonstrated clinical response in a phase I study. Oral bioavailability of ALG-093989 in rat following a single dose at 15 mg/kg dosing was 40%. In the in vivo humanized-PD-L1 MC38 mouse model, a single oral dose of ALG-093989 at 5 mg/kg demonstrated similar PD-L1 target occupancy to that of 150 mg/kg orally-administered INCB086550 and 5 mg/kg intravenously-administered durvalumab.ConclusionsWe have discovered ALG-093989, a highly potent PD-L1 small molecule inhibitor, which shows similar T-cell activation potency as durvalumab, and ~ 10-fold improved T-cell activation potency vs. INCB086550. ALG-093989 has the same target occupancy in mice following oral dosing at a 30-fold lower dose than INCB03989. The properties of ALG-093989 warrant further evaluation as a potential candidate for drug development.

BackgroundPD-1/PD-L1 antibody-based therapies have demonstrated success in the treatment of liver cancers. Most systemic immune-related adverse events (irAEs) associated with PD-1/PD-L1 antibodies are mild to moderate, but severe irAEs can be life threatening, due to the long half-lives of antibodies. Here, we report the discovery of an orally bioavailable PD-L1 small molecule inhibitor, ALG-094103, which preferentially partitions into the liver and may thereby mitigate extra-hepatic irAEs.MethodsThe biochemical interaction of PD-1/PD-L1 and PD-L1 dimerization was assessed by AlphaLISA®. Cellular activity was measured using a co-culture assay of PD-1 expressing Jurkat NFAT luciferase T-cells with PD-L1 expressing CHO cells. Pharmacokinetic (PK) and tissue distribution studies were performed in C57BL/6 mice and PK in Wistar Han rats. In vivo PD-L1 target occupancy was assessed at 6 hours following a single dose in the humanized-PD-L1 MC38 subcutaneous mouse model.ResultsALG-094103 inhibits the PD-1/PD-L1 axis with similar potency to PD-1/PD-L1 antibodies. In addition, ALG-094103 has in vitro potency similar to a non-liver targeted PD-L1 small molecule inhibitor, INCB086550, which demonstrated clinical response in a phase I study. The oral bioavailability of ALG-094193 in rats following a single dose at 15 mg/kg dosing was 40%. ALG-094103 demonstrated significantly higher liver concentrations vs. other tested tissues (liver/lung ratio of 17) in mice at 12 hours after a single oral dose. In the in vivo PD-L1 target occupancy model, oral dosing of ALG-094103 at 50 mg/kg demonstrated higher PD-L1 target occupancy than oral dosing of INCB086550 at 150 mg/kg and IV dosing of durvalumab at 5 mg/kg.ConclusionsWe have discovered a novel liver-targeted and orally bioavailable PD-L1 small molecule inhibitor, ALG-094103, with comparable in vitro potency to INCB086550. The properties of ALG-094103 will be further evaluated as a potential candidate for drug development.

Type I and type II interferons (IFNs) bind to different cell surface receptors but activate overlapping signal transduction pathways. We examined the effects of a type I IFN (IFN-αcon1) and a type II IFN (IFN-γ1b) on gene expression in A549 cells and demonstrate that there is a common set of genes modulated by both IFNs as well as a set of genes specifically regulated by each, reflecting the activation of different signaling pathways. In particular, IFN-γ induced many more genes of the signaling pathways, apoptosis, and cytokine interactions than did IFN-α. Even with genes induced by both IFNs there were distinctive quantitative differences in expression. IFN-γ1b plays a major role in the induction and regulation of the complement pathway. Previous work has shown a synergistic antiviral and antiproliferative effect of type I and type II IFNs in cell culture and in the treatment of tumors in mice. We demonstrate that a majority of genes showed an additive effect of IFN-αcon1 and IFN-γ1b, but a subset of genes is synergistically induced; these include ISG20, MX2, OAS2, and other genes known to be involved in the antiviral response, TRAIL (TNFSF10) and caspases involved in apoptosis and chemokine genes RANTES, CXCL10, and CXCL11. Greater than additive transcription of some of these genes in the presence of both IFNs was confirmed by real-time kinetic RT-PCR. Elevated induction of many of these genes may be sufficient to explain the synergistic antiviral and antitumor effects of this combination of IFNs in vivo.