AbstractAdenoviruses are responsible for a range of pathologies, including respiratory infections in children, accounting for 5-10% of such cases. Although most adenovirus infections are self-resolving, they can cause serious illness, particularly in immunocompromised individuals. There is currently no approved treatment for adenovirus infections, although various therapeutic approaches are under investigation, including nucleoside analog inhibitors of replication. However, these treatments have shown limited efficacy. In this study, we report on the antiviral activity of LAVR-289, a broad-spectrum acyclonucleoside phosphonate exhibiting potentin vitroefficacy against several adenovirus serotypes, comparable to that of brincidofovir. LAVR-289 specifically inhibits viral replication, blocking the formation of viral replication centers and preventing late protein expression without affecting viral entry or delivery of viral genomes to the nucleus.In vivousing immunocompromised Syrian hamsters infected with HAdV-C6, oral administration of LAVR-289 resulted in 100% animal survival. These results suggest that LAVR-289 holds promise as a potential therapy for adenovirus infections, particularly in immunocompromised patients.HighlightsLAVR-289 is a unique acyclic nucleoside phosphonate prodrug.LAVR-289 displays antiviral activity against Adenovirus with an EC50of about 100 nM against HAdV-C5 ANCHOR.LAVR-289 inhibits viral replication by targeting viral DNA polymerase, preventing DBP clustering and replication center formation.In immunosuppressed Syrian hamsters, LAVR-289 is well tolerated and ensures 100% survival while effectively stopping virus replication.LAVR-289's broad-spectrum activity positions it as a promising treatment for immunocompromised patients facing multiple viral infections.

Adenoviruses are responsible for a range of pathologies, including respiratory infections in children. Although most adenovirus infections are self-resolving, they can cause serious illness, particularly in immunocompromised individuals. There is currently no approved treatment for adenovirus infections. Here, we report on the antiviral activity of LAVR-289, a broad-spectrum acyclonucleoside phosphonate exhibiting potent in vitro efficacy against several adenovirus serotypes, comparable to that of brincidofovir. LAVR-289 specifically inhibits viral replication, blocking the formation of viral replication centers and preventing late protein expression without affecting viral entry or delivery of viral genomes to the nucleus. In vivo, using immunocompromised Syrian hamsters infected with HAdV-C6, oral administration of LAVR-289 resulted in 100% animal survival. These results suggest that LAVR-289 holds promise as a potential therapy for adenovirus infections paving the way for a future treatment of immunocompromised patients.

Poxviruses are double-stranded DNA viruses including relevant zoonotic pathogens with high morbidity and potential biological warfare threats. Although African swine fever virus belongs to the Asfarviridae family and is not strictly classified as a Poxviridae member, both fall within the same class of Pokkesviricetes that replicate in the cytoplasm. Among compounds targeting these viruses, acyclic nucleoside phosphonate (ANP) prodrugs are promising inhibitors of viral DNA polymerases. However, some limitations related to their toxicity and the rapid emergence of resistance highlight the need for new antiviral molecules. In this study, we tested a new ANP called LAVR-289. This product effectively inhibits viral replication by targeting a specific domain in the poxvirus DNA polymerase. Using monkeypox virus models, the subcutaneous or oral administration of LAVR-289 demonstrated protective efficacy in infected animals without toxicity. Its in vivo half-life, long on-the-shelf stability and broad-spectrum efficacy make LAVR-289 a promising candidate for further development and stockpiling as a medical countermeasure against dsDNA virus outbreaks. LAVR-289 can be positioned in the context of recurrent viral epidemics, bioterrorism risk, and the emergence of resistant strains in the population.

LAVR-289, a newly described acyclic nucleoside phosphonate prodrug, has previously shown in vitro efficacy against adenovirus (HAdV) species B, C, D, E, and F with EC50 values of 100 nM to 1.2 μM. The compound was efficacious prophylactically against systemic HAdV infection in immunosuppressed Syrian hamsters. Here, we present further, more detailed data on its efficacy against HAdV. We tested the two enantiomers at chiral phosphorus center (Sp and Rp) of LAVR-289 and found that both stereoisomers were equally efficacious against HAdV-C5 and -C6 infecting human foreskin fibroblast primary cells in vitro, indicating that the racemic mixture of the compound could be used as a drug. Following these favorable in vitro results, the antiviral activity of racemic LAVR-289 was evaluated in the immunosuppressed Syrian hamster model, in which the hamsters were challenged with the intravenous LD90 dose of HAdV-C6. Prophylactic administration of LAVR-289 completely prevented mortality at doses of 24 mg/kg p.o. q.d. or higher. At the efficacious dose levels, it significantly inhibited virus replication in the liver and mitigated HAdV-C6-induced liver damage. As prophylactic administration may not be advisable in a clinical setting, we delayed the administration of LAVR-289 and showed that the compound prevented mortality and significantly reduced morbidity even when the drug was withheld for 3 days post challenge. Based on these results, we believe that these data demonstrate in vitro and in vivo potency of LAVR-289 against HAdVs and support its continued development.

The continuous mutational nature of SARS-CoV-2 and its inter-species' similarities emphasize the urgent need to design and develop more direct-acting antiviral agents against highly infectious variants. Herein, we report on the efficient discovery of potent non-covalent non-peptide-derived Mpro inhibitors using miniaturized click chemistry and direct screening. Based on the privileged piperazine scaffold, 68 triazole-containing derivatives were assembled and screened. Notably, representative compound C1N46 (IC50 = 1.87 μM, EC50 = 6.99 μM, CC50 > 100 μM) displayed potent inhibition activity against Mpro and showed promising anti-SARS-CoV-2 properties in vitro. Additionally, C1N46 exhibited improved liver microsome stability compared to lead compound GC-14. Docking studies predicted a multi-site binding mode of the triazole-based compounds. In conclusion, our studies validate the efficacy and feasibility of click chemistry in rapidly discovering antiviral agents.

Human adenoviruses can cause serious, disseminated infections in immunocompromised patients. For pediatric allogeneic stem cell transplant patients, the case fatality rate can reach 80%. Still, there is no available antiviral drug that is specifically approved by the Food and Drug Administration for the treatment of adenovirus infections. To fill this pressing medical need, we have developed NPP-669, a prodrug of cidofovir with broad activity against double-stranded DNA viruses, including adenoviruses. Here, we report on the in vivo anti-adenoviral efficacy of NPP-669. Using the immunosuppressed Syrian hamster as the model, we show that NPP-669 is highly efficacious when dosed orally at 1 mg/kg and 3 mg/kg. In a delayed administration experiment, NPP-669 was more effective than brincidofovir, a similar compound that reached Phase III clinical trials. Furthermore, parenteral administration of NPP-669 increased its efficacy approximately 10-fold compared to oral dosing without apparent toxicity, suggesting that this route may be preferable in a hospital setting. Based on these findings, we believe that NPP-669 is a promising new compound that needs to be further investigated.

Adenovirus infections of immunocompromised patients can cause life-threatening disseminated disease. While there are presently no drugs specifically approved to treat these infections, there are several compounds that showed efficacy against adenovirus in preclinical studies. For any such compound, low toxicity is an essential requirement. As cumulative drug effects can accentuate pathology, especially in patients with other morbidities, it is important to limit antiviral exposure to what is absolutely necessary. This is achievable by monitoring the virus burden of the patients and administering antivirals to suppress virus replication to a non-pathogenic level. We modeled such a system using Syrian hamsters infected with a replication-competent adenovirus vector, in which luciferase expression is coupled to virus replication. We found that virus replication could be followed in vivo in the same animal by repeated measurement of luciferase expression. To test the utility of an interrupted treatment regimen, we used NPP-669 and valganciclovir, two antiviral compounds with high and moderate anti-adenoviral efficacy, respectively. We found that short-term treatment of adenovirus-infected hamsters at times of peak virus replication can prevent virus-associated pathology. Thus, we believe that this animal model can be used to model different treatment regimens for anti-adenoviral compounds.

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. Presently, there is no drug specifically approved for the treatment of adenovirus infections by the FDA. The state-of-the-art treatment of such infections is the off-label use of cidofovir, an acyclic nucleotide phosphonate. While cidofovir inhibits adenovirus replication, it has dose-limiting kidney toxicity. There is an apparent need for a better compound to treat adenovirus infections. To this end, we have been developing acyclic nucleotide phosphonate prodrugs that utilize an amino acid scaffold equipped with a lipophilic modifier. Here, we compare the antiviral potential of two prodrugs of HPMPA that differ only in the amino acid-based promoiety: USC-087, based on an N-hexadecyl tyrosinamide, and USC-093, based on an N-hexadecyl serinamide. Oral administration of both compounds was very efficacious against disseminated HAdV-C6 infection in immunosuppressed Syrian hamsters, suppressing virus replication and mitigating pathology even when treatment was withheld until 4 days after challenge. We saw only marginal efficacy after respiratory infection of hamsters, which may reflect suboptimal distribution to the lung. Importantly, neither compound induced intestinal toxicity, which was observed as the major adverse effect in clinical trials of brincidofovir, a prodrug of cidofovir which also contains a C-16 modifier. Notably, we found that there was a significant difference in the nephrotoxicity of the two compounds: USC-087 caused significant kidney toxicity while USC-093 did not, at effective doses. These findings will be valuable guidepoints in the future evolution of this new class of potential prodrugs to treat adenovirus infections.

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. At present, no drug has been approved by FDA for the treatment of adenovirus infections. A current treatment of such infections is off-label use of an antiviral acyclic nucleotide phosphonate, cidofovir (CDV, (S)-HPMPC), which requires i.v. administration and has dose-limiting kidney toxicity. We recently reported that USC-093, a homoserinamide analogue of the tyrosinamide (S)-HPMPA prodrug USC-087, was orally effective at a 10 mg/kg against disseminated human adenovirus infection (HAdV-C6) in a Syrian hamster model, although their efficacy was marginal after respiratory infection. Neither prodrug manifested GI toxicity. Unlike USC-087, USC-093 showed no significant nephrotoxicity at the effective dose. Here, we describe in detail the synthesis of USC-093 and also its D-homoserinamide analogue, USC-093D, in four steps (20-40% overall yield) starting from Boc-protected L-homoserine or D-homoserine lactone, respectively. The two stereoisomeric prodrugs had EC50 30-70 nM vs. Ad5 or 1-6 nM vs. Ad6 in HFF cells, with USC-093D giving the lower values. The prodrugs were 30-59x more potent vs. Ad5 and 82-332x more potent than Ad6 relative to the positive control, CDV. To ascertain whether D-chirality in the promoiety could enhance the performance of the prodrug in vivo, USC-093D and USC-093 were compared in the Syrian hamster model (treated from day 1 q.d at an experimentally determined maximum tolerated oral dose of 20 mg/kg)). In this study, the hamsters were instilled i.n. with vehicle or 4X1010 PFU/kg of HAdV-C6 to promote lung infection. Oral valganciclovir (VGCV) at 200 mg/kg b.i.d. was used as the positive control. The body weights were recorded daily, and at 3 days post challenge, gross pathological observation was performed. Lung samples were collected, and the virus burden was determined by TCID50 assay. The results show that altering homoserine stereochemistry did not markedly improve the efficacy of the orally administered prodrug, consistent with the premise that its mechanism of transport is likely not dependent on stereoselective pathways, such as hPEPT1-mediated uptake.

The main protease (Mpro) represents one of the most effective and attractive targets for designing anti-SARS-CoV-2 drugs. In this study, we designed and synthesized a novel series of Ebselen derivatives by incorporating privileged fragments from different pockets of the Mpro active site. Among these compounds, 11 compounds showed submicromolar activity in the FRET-based SARS-CoV-2 Mpro inhibition assay, with IC50 values ranging from 233 nM to 550 nM. Notably, compound 3a displayed submicromolar Mpro activity (IC50 = 364 nM) and low micromolar antiviral activity (EC50 = 8.01 µM), comparable to that of Ebselen (IC50 = 339 nM, EC50 = 3.78 µM). Time-dependent inhibition assay confirmed that these compounds acted as covalent inhibitors. Taken together, our optimization campaigns thoroughly explored the structural diversity of Ebselen and verified the impact of specific modifications on potency against Mpro.

PDF file, 4006K, Neutralization of IP-10 does not affect the trafficking and distribution of tumor-specific effector CD8+ TIL586 cells into tumor sites in tumor-bearing mice.

Morquio A disease is a genetic disorder resulting in N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, and patients are currently treated with enzyme replacement therapy via weekly intravenous enzyme infusions. A means of sustained enzyme delivery could improve patient quality of life by reducing the administration time, frequency of hospital visits, and treatment cost. In this study, we investigated poly(ethylene-glycol) (PEG) hydrogels as a tunable, hydrolytically degradable drug delivery system for the encapsulation and sustained release of recombinant human GALNS (rhGALNS). We evaluated hydrogel formulations that optimized hydrogel gelation and degradation time while retaining rhGALNS activity and sustaining rhGALNS release. We observed the release of active rhGALNS for up to 28 days in vitro from the optimized formulation. rhGALNS activity was preserved in the hydrogel relative to buffer over the release window, and encapsulation was found to have no impact on the rhGALNS structure when measured by intrinsic fluorescence, circular dichroism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vivo, we monitored the retention of fluorescently labeled rhGALNS in C57BL/6 albino mice when administered via subcutaneous injection and observed rhGALNS present for up to 20 days when delivered in a hydrogel versus 7 days in the buffer control. These results indicate that PEG hydrogels are suitable for the encapsulation, preservation, and sustained release of recombinant enzymes and may present an alternative method of delivering enzyme replacement therapies that improve patient quality of life.

PDF file, 944K, IP-10 is the only functional chemokine inducing the migration of Treg cells.

Overexpression of Lin28 is detected in various cancers with involvement in the self-renewal process and cancer stem cell generation. In the present study, we evaluated how the Lin28 axis plays an immune-protective role for tumor-initiating cancer cells in hepatocellular carcinoma (HCC). Our result using HCC patient samples showed a positive correlation between indoleamine 2,3-dioxygenase-1 (IDO1), a kynurenine-producing enzyme with effects on tumor immune escape, and Lin28B. Using in silico prediction, we identified a Sox2/Oct4 transcriptional motif acting as an enhancer for IDO1. Knockdown of Lin28B reduced Sox2/Oct4 and downregulated IDO1 in tumor-initiating hepatic cancer cells. We further observed that inhibition of Lin28 by a small-molecule inhibitor (C1632) suppressed IDO1 expression. Suppression of IDO1 resulted in a decline in kynurenine production from tumor-initiating cells. Inhibition of the Lin28 axis also impaired PD-L1 expression in HCC cells. Consequently, modulating Lin28B enhanced in vitro cytotoxicity of glypican-3 (GPC3)-chimeric antigen receptor (CAR) T and NK cells. Next, we observed that GPC3-CAR T cell treatment together with C1632 in a HCC xenograft mouse model led to enhanced anti-tumor activity. In conclusion, our results suggest that inhibition of Lin28B reduces IDO1 and PD-L1 expression and enhances immunotherapeutic potential of GPC3-CART cells against HCC.

SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) is considered an attractive target for the development of anti-COVID-19 agents due to its vital function. The N-substituted isatin derivative L-26 is a potential SARS-CoV-2 3CLpro inhibitor, but it has poor cell-based antiviral activity and high cytotoxicity. With L-26 as the lead compound, 58 isatin derivatives were prepared using click-chemistry-based miniaturized synthesis and their 3CLpro inhibitory activities were determined by a fluorescence resonance energy transfer-based enzymatic assay. Compounds D1N8 (IC50 = 0.44 ± 0.12 μM) and D1N52 (IC50 = 0.53 ± 0.21 μM) displayed excellent inhibitory potency against SARS-CoV-2 3CLpro, being equivalent to that of L-26 (IC50 = 0.30 ± 0.14 μM). In addition, the cytotoxicity of D1N8 (CC50 >20 μM) and D1N52 (CC50 >20 μM) decreased significantly compared with L-26 (CC50 <2.6 μM). Further molecular dynamics simulations revealed the potential binding interactions between D1N52 and SARS-CoV-2 3CLpro. These efforts lay a solid foundation for the research of novel anti-SARS-CoV-2 agents targeting 3CLpro.

The ongoing transmission of SARS-CoV-2 necessitates the development of additional potent antiviral agents capable of combating the current highly infectious variants and future coronaviruses. Here, we present the discovery of potent nonpeptide main protease (Mpro) inhibitors with prominent antiviral activity and improved pharmacokinetic properties. Three series of 1,2,4-trisubstituted piperazine derivatives were designed and synthesized, and the optimal GC-78-HCl demonstrated high enzyme-inhibitory potency (IC50 = 0.19 μM) and exhibited excellent antiviral activity (EC50 = 0.40 μM), reaching the same level as Nirmatrelvir (EC50 = 0.38 μM). Additionally, GC-78-HCl displayed potent antiviral activities against various SARS-CoV-2 variants as well as HCoV-OC43 and HCoV-229E, indicating its potential broad-spectrum anticoronaviral activity. Notably, the pharmacokinetic properties of GC-78-HCl were somewhat enhanced compared to those of the lead compound. Furthermore, the cocrystal and molecular docking elucidated the mechanism of action. In conclusion, we discovered a novel nonpeptidic Mpro inhibitor with promising antiviral activity and a favorable pharmacokinetic profile.

DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize β-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC50 for 24 h. These experiments identified a novel lead candidate, NPP-669. NPP-669 demonstrated efficacy against CMV infections in mice and AdV infections in hamsters following oral (p.o.) dosing at a dose of 1 mg/kg BID and 0.1 mg/kg QD, respectively. We further showed that NPP-669 at 30 mg/kg QD did not exhibit histological signs of toxicity in mice or hamsters. These data suggest that NPP-669 is a promising lead candidate for a broad-spectrum antiviral compound.

PDF file, 2822K, Correlation analyses of tumor-infiltrating T cells and tumor IP-10 expression levels in human breast cancer tissues.

The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 μM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 μM), similar to that of remdesivir (EC50 = 2.27 μM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.

Clusters of acute non HepA-E hepatitis cases in previously healthy children have been reported globally. At least, 1010 cases were identified in 35 countries, 5% of those cases required liver transplantation and 2% died. The exact cause is not yet known, but there is circumstantial evidence suggesting that human adenovirus F41 (HAdV-F41) might be playing a role. No antiviral drug has been approved for treating human adenovirus infections. Furthermore, HAdV-F41 is notoriously difficult to grow in cell culture, which hindered studying the efficacy of an antiviral compound against this virus. Here, we show that filociclovir (FCV), a nucleoside analog, is a potent inhibitor of HAdV-F41 in cell culture using 2 approaches, namely immunostaining of infected cells and virus yield reduction assay. The activity of FCV was compared to 3 other known antivirals: cidofovir (CDV), ganciclovir (GCV) and valganciclovir (VGCV). Among the 4 compounds examined in this study, FCV was the most potent, with an EC50 of 3.5 μM. These compounds can be ranked by potency as follows: FCV > CDV > GCV ≥ VGCV. In addition, FCV was 10-fold more potent than CDV in a virus yield reduction assay. This report provides timely and valuable methodologies to the research community for testing antivirals against HAdV-F41. Our findings also support the continued development of FCV for various therapeutic applications, including pediatric hepatitis, if a causal relationship is firmly established in the future.