NPP-669, a prodrug of cidofovir, is highly efficacious against human adenovirus infection in the permissive Syrian hamster model.

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.

Targeting Lin28 axis enhances glypican-3-CAR T cell efficacy against hepatic tumor initiating cell population.

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.

NPP-669, a Novel Broad-Spectrum Antiviral Therapeutic with Excellent Cellular Uptake, Antiviral Potency, Oral Bioavailability, Preclinical Efficacy, and a Promising Safety Margin.

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 ß-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.

Identification of Ebselen derivatives as novel SARS-CoV-2 main protease inhibitors: Design, synthesis, biological evaluation, and structure-activity relationships exploration.

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.

Filociclovir Is a Potent In Vitro and In Vivo Inhibitor of Human Adenoviruses.

Human adenovirus (HAdV) infection is common in the general population and can cause a range of clinical manifestations, among which pneumonia and keratoconjunctivitis are the most common. Although HAdV infections are mostly self-limiting, infections in immunocompromised individuals can be severe. No antiviral drug has been approved for treating adenoviruses. Filociclovir (FCV) is a nucleoside analogue which has successfully completed phase I human clinical safety studies and is now being developed for treatment of human cytomegalovirus (HCMV)-related disease in immunocompromised patients. In this report, we show that FCV is a potent broad-spectrum inhibitor of HAdV types 4 to 8, with 50% effective concentrations (EC50s) ranging between 1.24 and 3.6 µM and a 50% cytotoxic concentration (CC50) of 100 to 150 µM in human foreskin fibroblasts (HFFs). We also show that the prophylactic oral administration of FCV (10 mg/kg of body weight) 1 day prior to virus challenge and then daily for 14 days to immunosuppressed Syrian hamsters infected intravenously with HAdV6 was sufficient to prevent morbidity and mortality. FCV also mitigated tissue damage and inhibited virus replication in the liver. The 10-mg/kg dose had similar effects even when the treatment was started on day 4 after virus challenge. Furthermore, FCV administered at the same dose after intranasal challenge with HAdV6 partially mitigated body weight loss but significantly reduced pathology and virus replication in the lung. These findings suggest that FCV could potentially be developed as a pan-adenoviral inhibitor.

Pathology in Permissive Syrian Hamsters after Infection with Species C Human Adenovirus (HAdV-C) Is the Result of Virus Replication: HAdV-C6 Replicates More and Causes More Pathology than HAdV-C5.

Syrian hamsters are permissive for the replication of species C human adenoviruses (HAdV-C). The virus replicates to high titers in the liver of these animals after intravenous infection, while respiratory infection results in virus replication in the lung. Here we show that two types belonging to species C, HAdV-C5 and HAdV-C6, replicate to significantly different extents and cause pathology with significantly different severities, with HAdV-C6 replicating better and inducing more severe and more widespread lesions. The virus burdens in the livers of HAdV-C6-infected hamsters are higher than the virus burdens in HAdV-C5-infected ones because more of the permissive hepatocytes get infected. Furthermore, when hamsters are infected intravenously with HAdV-C6, live, infectious virus can be isolated from the lung and the kidney, which is not seen with HAdV-C5. Similarly to mouse models, in hamsters, HAdV-C6 is sequestered by macrophages to a lesser degree than HAdV-C5. Depletion of Kupffer cells from the liver greatly increases the replication of HAdV-C5 in the liver, while it has only a modest effect on the replication of HAdV-C6. Elimination of Kupffer cells also dramatically increases the pathology induced by HAdV-C5. These findings indicate that in hamsters, pathology resulting from intravenous infection with adenoviruses is caused mostly by replication in hepatocytes and not by the abortive infection of Kupffer cells and the following cytokine storm.IMPORTANCE Immunocompromised human patients can develop severe, often lethal adenovirus infections. Respiratory adenovirus infection among military recruits is a serious problem, in some cases requiring hospitalization of the patient. Furthermore, adenovirus-based vectors are frequently used as experimental viral therapeutic agents. Thus, it is imperative that we investigate the pathogenesis of adenoviruses in a permissive animal model. Syrian hamsters are susceptible to infection with certain human adenoviruses, and the pathology accompanying these infections is similar to what is observed with adenovirus-infected human patients. We demonstrate that replication in permissive cells in a susceptible host animal is a major part of the mechanism by which systemic adenovirus infection induces pathology, as opposed to the chiefly immune-mediated pathology observed in nonsusceptible hosts. These findings support the use of compounds inhibiting adenovirus replication as a means to block adenovirus-induced pathology.

Correction: STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

[This corrects the article DOI: 10.1371/journal.ppat.1005084.].

STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.

[Long term results after invasive treatment of critical limb ischemia]. / A krónikus kritikus végtagischaemia invazív kezelésének hosszú távú eredményei.

INTRODUCTION AND AIM: Surgical tibial bypass for critical limb ischemia is associated with significant morbidity, mortality, and graft failure, whereas percutaneous angioplasty and stenting has promising results. The objective of this study was the investigation of the long term results of below-knee percutaneous angioplasty for restoring straight inline arterial flow in patients with critical limb ischemia. METHOD: The clinical and angiographic data of 281 consecutive patients with critical limb ischemia treated by PTA between 2008 and 2011 was evaluated in a prospective register. The aim of the revascularization was to achieve a straight inline flow to the wound with balloon angioplasty. Stent implantation was done in the case of recoil and flow limiting dissection. Primary end points were clinical success (relief of resting pain, healing of ulceration, limb survival) and major adverse events (death, myocardial infarction, major unplanned amputation, need for surgical revascularization, or major bleeding). Secondary end points were the angiographic result of the intervention, procedural data and consumption of angioplasty equipment. The impact of diabetic leg syndrome and the result of the angioplasty on the limb salvage was also investigated. We have analysed the impact of major amputation on long term mortality. RESULTS: Mean age of patients was 72.5 ± 10.6 years and the follow-up period was 40.8 ± 9.7 months. Technical success was reached in 255 (90.7%) of the patient's: 255 limbs straight inline flow with good angiographic result was restored to at least one tibial vessel. Balloon angioplasty, stent implantation and rotational atherectomy was performed in 278 (98.9%), 74 (26.3%) and 2 patients (0.7%). From clinical end points the rest pain was ceased in 56.6%, the ulcer and the gangrena was healed in 73.5% and 46.5%. The long term limb survival was 73.5%; 65.8% in diabetic and 89.6% in non-diabetic leg syndrome (p = 0.001). The major adverse events at long-term follow-up occured in 122 (43.8%) patients. Death occured in 57 (20.3%) of the patients during the long-term follow-up: 38 (13.5%) vs. 19 (6.8%) in diabetic vs. non-diabetic leg subgroup, respectively (p = 0.932). Long-term limb saving occured in 72.3% vs. 84.6% of the patients dependening the procedure was successful or unsuccessful (p = 0.225). CONCLUSION: Below-knee stent angioplasty for critical limb ischemia results in good clinical outcome, but the major adverse event rate is high. Diabetes mellitus is associated with a high rate of mortality and amputation. Orv. Hetil., 2017, 158(11), 418-425.

Investigation of hydrophobically derivatized hyperbranched polyglycerol with PEGylated shell as a nanocarrier for systemic delivery of chemotherapeutics.

We report the synthesis and characterization of a polymeric nanoparticle (NP) based on hyperbranched polyglycerol (HPG) containing a hydrophobic core and a hydrophilic shell, and assessed its suitability to be developed as a systemic anticancer drug carrier. HPG NP displayed low toxicity to primary cell cultures and were well-tolerated in mice after intravenous administration. When tested in mice tumor xenograft models, HPG NP accumulated significantly in the tumors with low accumulation in the liver and the spleen. In vitro studies demonstrated that HPG NP was capable of hydrophobically binding docetaxel and releasing it in a controlled manner. The HPG NP formulation of docetaxel conferred a preferential protective effect on primary non-cancerous cells while effectively killing cancer cells, indicating great potential for widening its therapeutic index. Taken together, these data indicate that HPG NP is a highly promising nanocarrier platform for systemic delivery of anticancer drugs. FROM THE CLINICAL EDITOR: The use of polyethylene glycol on nano-carriers as "stealth" to deliver intravenous drugs is well known. Here, the authors developed polymeric nanoparticle (NP) with hyperbranched polyglycerol (HPG) and tested its efficacy in delivering docetaxel. The results showed that this formulation could preferentially killed cancer cells with a high therapeutic index. It seems that this platform could have a great potential in cancer therapy.

Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

Transpedal access after failed anterograde recanalization of complex below-the-knee and femoropoliteal occlusions in critical limb ischemia.

BACKGROUND: Successful angioplasty is one of the main factor of limb salvage during critical limb ischemia. In complex femoropopliteal to infrapopliteal occlusions, an anterograde recanalization attempt can fail in up to 20% of the cases. The purpose of this dual center pilot study was to evaluate the acute success and clinical impact of retrograde transpedal access for retrograde below-the-knee and femoropopliteal chronic total occlusions after failed anterograde attempt and to access the late complications at the puncture site. METHODS: The clinical and angiographic data of 51 consecutive patients with CLI treated by retrograde transpedal recanalization between 2010 and 2011 were evaluated in a pilot study. We have examined the 2-month and 1 year major adverse events (MAEs) and clinical success. In all cases after failure of the anterograde recanalization of occluded below-the-knee segments due to unsuccessful penetration or failed re-entry, the anterior tibial or posterior tibial artery was punctured under fluoroscopic guidance and retrograde recanalization was performed. Direct revascularization was tried firstly following the angiographic zones, but in failed cases indirect revascularization was carried out with increasing the collateral flow to the wound. RESULTS: Successful direct retrograde revascularization was achieved successfully in 40 patients (78.4%) and indirect revascularization was done in 10 patients (19.6%). Revascularization was failed in one patient (2%). MAE at 2 and 12 months follow-up was 6 (11.7%) and 11 (24%). Limb salvage at 2 and 12 months was 93% and 82.3%, respectively. Balloon angioplasty was performed in all interventions and provisional stenting was done in 34 patients (66.7%). One major and three minor vascular complications occurred after the procedure. The mean basal and control creatinine level was 120.9 ± 133.4 and 123.8 ± 131.3 µmol/L (P = 0.83) after the procedure. CONCLUSION: Failed antegrade attempts to recanalize CTO-s of femoropopliteal and infrapopliteal vessels can be salvaged using a retrograde transpedal access, with a low acute and late complication rate. This technique could be valuable for patients with critical limb ischemia due to femoropopliteal and infrapopliteal occlusions.

The Clinical Impact of Access Site Selection for Successful Thrombolysis and Intervention in Acute Critical Lower Limb Ischaemia (RAD-ALI Registry).

Background: Acute limb ischaemia (ALI) is of great clinical importance due to its consequent serious complications and high comorbidity and mortality rates. The purpose of this study was to compare the acute success and complication rates of CDT performed via transradial, transbrachial, and transfemoral access sites in patients with acute lower limb vascular occlusion and to investigate the 1-year outcomes of CDT and MT for ALI. Methods: Between 2008 and 2019, 84 consecutive patients with ALI were treated with CDT in a large community hospital. Data were collected and retrospectively analysed. The primary ("safety") endpoints encompassed major adverse events (MAEs), major adverse limb events (MALEs), and the occurrence of complications related to the access site. Secondary ("efficacy") endpoints included both technical and clinical achievements, treatment success, fluoroscopy time, radiation dose, procedure time, and the crossover rate to an alternative puncture site. Results: CDT was started with radial (n = 17), brachial (n = 9), or femoral (n = 58) access. CDT was technically successful in 74/84 patients (88%), but additional MT and angioplasty and/or stent implantation was necessary in 17 (20.2%) and 45 cases (53.6%), respectively. Clinical success was achieved in 74/84 cases (88%). The mortality rate at 1 year was 14.3%. The cumulative incidence of MAEs and MALEs at 12 months was 50% and 40.5%, respectively. After conducting multivariate analysis, history of Rutherford stage IIB (hazard ratio [HR], 3.64; 95% confidence interval [CI], 1.58-8.41; p = 0.0025), occlusion of the external iliac artery (HR, 27.52; 95% CI, 2.83-267.33; p = 0.0043), being a case of clinically unsuccessful thrombolysis (HR, 7.72; 95% CI, 2.48-23.10; p = 0.0004), and the presence of diabetes mellitus (HR, 2.18; 95% CI, 1.01-4.71; p = 0.047) were independent predictors of a high MAE mortality rate at 12 months. For MALEs, statistically significant differences were detected with the variables history of Rutherford stage IIB (HR, 4.30; 95% CI, 1.99-9.31; p = 0.0002) and external iliac artery occlusion (HR, 31.27; 95% CI, 3.47-282.23; p = 0.0022). Conclusions: Based on the short-term results of CDT, acute limb ischaemia can be successfully, safely, and effectively treated with catheter-directed thrombolytic therapy with radial, brachial, or femoral access. However, radial access is associated with fewer access site complications. A history of Rutherford stage IIB, occlusion of external iliac artery, unsuccessful thrombolysis, and the presence of diabetes mellitus were independently associated with an increased risk of MAEs. A history of Rutherford stage IIB and external iliac artery occlusion are independent predictors of MALEs.

Oral USC-093, a novel homoserinamide analogue of the tyrosinamide (S)-HPMPA prodrug USC-087 has decreased nephrotoxicity while maintaining antiviral efficacy against human adenovirus infection of Syrian hamsters.

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.

Hydrogel Delivery Device for the In Vitro and In Vivo Sustained Release of Active rhGALNS Enzyme.

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.

Identification of novel 1,2,3-triazole isatin derivatives as potent SARS-CoV-2 3CLpro inhibitors via click-chemistry-based rapid screening.

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.

Design, Synthesis, and Biological Evaluation of Trisubstituted Piperazine Derivatives as Noncovalent Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors with Improved Antiviral Activity and Favorable Druggability.

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.

Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of the cases of renal cell carcinoma. In ccRCC deactivation of Von-Hippel-Lindau (VHL) gene contributes to the constitutive expression of hypoxia inducible factors 1 and 2 alpha (HIF-α), transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1α by Se-Methylselenocysteine (MSC) via stabilization of prolyl hydroxylases 2 and 3 (PHDs) and a significant therapeutic synergy when combined with chemotherapy. This study was initiated to investigate the expression of PHDs, HIF-α, and VEGF-A in selected solid cancers, the mechanism of HIF-α inhibition by MSC, and to document antitumor activity of MSC against human ccRCC xenografts. METHODS: Tissue microarrays of primary human cancer specimens (ccRCC, head & neck and colon) were utilized to determine the incidence of PHD2/3, HIF-α, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s) of HIF-α inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1α positive), 786-0 (HIF-2α positive) and VHL wild type head & neck cancer cells FaDu (HIF-1α) were utilized. PHD2 and VHL gene specific siRNA knockdown and inhibitors of PHD2 and proteasome were used to determine their role in the degradation of HIF-1α by MSC. RESULTS: We have demonstrated that ccRCC cells express low incidence of PHD2 (32%), undetectable PHD3, high incidence of HIF-α (92%), and low incidence of VEGF-A compared to head & neck and colon cancers. This laboratory was the first to identify MSC as a highly effective inhibitor of constitutively expressed HIF-α in ccRCC tumors. MSC did not inhibit HIF-1α protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1α was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-α were associated with significant antitumor activity against ccRCC xenograft. CONCLUSIONS: Our results show the role of PHD2/3 in stable expression of HIF-α in human ccRCC. Furthermore, HIF-1α degradation by MSC is achieved through PHD2 dependent and VHL independent pathway which is unique for HIF-α regulation. These data provide the basis for combining MSC with currently used agents for ccRCC.

Filociclovir is a potent inhibitor of human adenovirus F41.

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.