Combination of PD-1 Checkpoint Blockade and Botulinum Toxin Type A1 Improves Antitumor Responses in Mouse Tumor Models of Melanoma and Colon Carcinoma.

BACKGROUND: Tumor innervation has been shown to be utilized by some solid cancers to support tumor initiation, growth, progression, and metastasis, as well as confer resistance to immune checkpoint blockade through suppression of antitumor immunologic responses. Since botulinum neurotoxin type A1 (BoNT/A1) blocks neuronal cholinergic signaling, its potential use as an anticancer drug in combination with anti-PD-1 therapy was investigated in four different syngeneic mouse tumor models. METHODS: Mice implanted with breast (4T1), lung (LLC1), colon (MC38), and melanoma (B16-F10) tumors were administered a single intratumoral injection of 15 U/kg BoNT/A1, repeated intraperitoneal injections of 5 mg/kg anti-PD-1 (RMP1-14), or both. RESULTS: Compared to the single-agent treatments, anti-PD-1 and BoNT/A1 combination treatment elicited significant reduction in tumor growth among B16-F10 and MC38 tumor-bearing mice. The combination treatment also lowered serum exosome levels in these mice compared to the placebo control group. In the B16-F10 syngeneic mouse tumor model, anti-PD-1 + BoNT/A1 combination treatment lowered the proportion of MDSCs, negated the increased proportion of Treg cells, and elicited a higher number of tumor-infiltrating CD4+ and CD8+ T lymphocytes into the tumor microenvironment compared to anti-PD-1 treatment alone. CONCLUSION: Our findings demonstrate the synergistic antitumor effects of BoNT/A1 and PD-1 checkpoint blockade in mouse tumor models of melanoma and colon carcinoma. These findings provide some evidence on the potential application of BoNT/A1 as an anticancer drug in combination with immune checkpoint blockade and should be further explored.

Comparative Pharmacodynamics of Three Different Botulinum Toxin Type A Preparations following Repeated Intramuscular Administration in Mice.

Botulinum neurotoxin type A (BoNT/A) causes muscle paralysis by blocking cholinergic signaling at neuromuscular junctions and is widely used to temporarily correct spasticity-related disorders and deformities. The paralytic effects of BoNT/A are time-limited and require repeated injections at regular intervals to achieve long-term therapeutic benefits. Differences in the level and duration of effectivity among various BoNT/A products can be attributed to their unique manufacturing processes, formulation, and noninterchangeable potency units. Herein, we compared the pharmacodynamics of three BoNT/A formulations, i.e., Botox® (onabotulinumtoxinA), Xeomin® (incobotulinumtoxinA), and Coretox®, following repeated intramuscular (IM) injections in mice. Three IM injections of BoNT/A formulations (12 U/kg per dose), 12-weeks apart, were administered at the right gastrocnemius. Local paresis and chemodenervation efficacy were evaluated over 36 weeks using the digit abduction score (DAS) and compound muscle action potential (CMAP), respectively. One week after administration, all three BoNT/A formulations induced peak DAS and maximal reduction of CMAP amplitudes. Among the three BoNT/A formulations, only Coretox® afforded a significant increase in paretic effects and chemodenervation with a prolonged duration of action after repeated injections. These findings suggest that Coretox® may offer a better overall therapeutic performance in clinical settings.

Comparative Analysis of Hyaluronidase-Mediated Degradation Among Seven Hyaluronic Acid Fillers in Hairless Mice.

PURPOSE: Hyaluronic acid (HA) is the most common injectable dermal filler used for soft-tissue augmentation, and can be removed non-surgically by directly injecting hyaluronidase. In this study, the hyaluronidase-mediated degradation of different types of HA fillers implanted subcutaneously at the back of hairless mice having filler residence time of four days or three months were compared. METHODS: Two sites at the back of female hairless mice were subcutaneously implanted with 0.1-mL of one of the seven HA fillers (NLL, NL, NDL, NVL, and ND, JUVX+, and RESLYFT) and injected with 30 IU or 60 IU hyaluronidase per 0.1-mL filler after reaching a filler residence time of 4 or 91 days, respectively. Filler bolus projection was measured using three-dimensional optical imaging over a 72 h period, and the implantation sites were histologically examined 2 weeks after hyaluronidase injection. RESULTS: Following hyaluronidase injection, all seven HA fillers showed a rapid decrease of filler volume within 24 h, and complete degradation was confirmed by histological examination after 2 weeks. There was no significant difference in filler volume reduction rate among the seven HA fillers, and no evidence of macroscopic or microscopic adverse effects were observed at the implantation sites. CONCLUSION: All seven HA fillers show comparable susceptibility to hyaluronidase-mediated degradation. HA fillers with prolonged filler residence time may require a higher dose of hyaluronidase to achieve efficient degradation owing to tissue integration.

Comparative Pharmacodynamics Study of 3 Different Botulinum Toxin Type A Preparations in Mice.

BACKGROUND: A new complexing protein-free botulinum toxin Type A (CBoNT) with the same mechanism of action as the botulinum toxin complex onabotulinumtoxinA (OBoNT) and complexing protein-free incobotulinumtoxinA (IBoNT) was recently developed. OBJECTIVE: To compare the local paresis and chemodenervation efficacy of 3 different botulinum toxin Type A preparations in mice. MATERIALS AND METHODS: Efficacy and duration of action of CBoNT, OBoNT, and IBoNT after a single intramuscular injection to the right gastrocnemius was evaluated by digit abduction score (DAS) and compound muscle action potential (CMAP) assays. RESULTS: Mouse DAS and CMAP responses were comparable between CBoNT and OBoNT, indicating similar paresis and chemodenervation efficacy, as well as duration of action. Both botulinum toxins showed significantly higher efficacy and longer duration of action than IBoNT. Similarly, mean DAS potency of CBoNT (ED50: 3.85 ± 0.34 U/kg) and OBoNT (ED50: 4.13 ± 0.07 U/kg) were significantly higher compared with IBoNT (ED50: 6.70 ± 0.83 U/kg). CONCLUSION: CBoNT displays the same efficacy as OBoNT as shown by their comparable chemodenervation and local paretic effects, and demonstrates superior efficacy and duration of action compared with IBoNT. Likewise, CBoNT has comparable DAS potency to OBoNT and is superior to IBoNT.

Identification of pyrrolo[3,2-c]pyridin-4-amine compounds as a new class of entry inhibitors against influenza viruses in vitro.

Various influenza virus entry inhibitors are being developed as therapeutic antiviral agents in ongoing preparation for emerging influenza viruses, particularly those that may possess drug resistance to the current FDA-approved neuraminidase inhibitors. In this study, small molecules having the pyrrolopyridinamine (PPA), aminothiadiazole (ATD), dihydrofuropyridine carboxamide (HPC), or imidazopyridinamine (IPA) moiety were selected from a target-focused chemical library for their inhibitory activity against influenza A virus by high-throughput screening using the PR8GFP assay. Activity was evaluated by measuring changes the proportion of GFP-expressing cells as a reflection of influenza virus infection. Among them, PPA showed broad-spectrum activity against multiple influenza A viruses and influenza B virus. PPA was found to block the early stages of influenza virus infection using a time-of-addition assay. Using additional phenotypic assays that dissect the virus entry process, it appears that the antiviral activity of PPA against influenza virus can be attributed to interference of the post-fusion process: namely, virus uncoating and nuclear import of viral nucleoprotein complexes. Based on these results, PPA is an attractive chemical moiety that can be used to develop new antiviral drug candidates against influenza viruses.

Drug discovery for human African trypanosomiasis: identification of novel scaffolds by the newly developed HTS SYBR Green assay for Trypanosoma brucei.

Human African trypanosomiasis (HAT) is a vector-transmitted tropical disease caused by the protozoan parasite Trypanosoma brucei. High-throughput screening (HTS) of small-molecule libraries in whole-cell assays is one of the most frequently used approaches in drug discovery for infectious diseases. To aid in drug discovery efforts for HAT, the SYBR Green assay was developed for T. brucei in a 384-well format. This semi-automated assay is cost- and time-effective, robust, and reproducible. The SYBR Green assay was compared to the resazurin assay by screening a library of 4000 putative kinase inhibitors, revealing a superior performance in terms of assay time, sensitivity, simplicity, and reproducibility, and resulting in a higher hit confirmation rate. Although the resazurin assay allows for comparatively improved detection of slow-killing compounds, it also has higher false-positive rates that are likely to arise from the assay experimental conditions. The compounds with the most potent antitrypanosomal activity were selected in both screens and grouped into 13 structural clusters, with 11 new scaffolds as antitrypanosomal agents. Several of the identified compounds had IC50 <1 µM coupled with high selectivity toward the parasite. The core structures of the scaffolds are shown, providing promising new starting points for drug discovery for HAT.

Clathrin- and serine proteases-dependent uptake of porcine epidemic diarrhea virus into Vero cells.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, is a causative agent of porcine enteric disease characterized by acute watery diarrhea and dehydration in sucking piglet. Similar to other coronaviruses, PEDV spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. However, the entry mechanism of PEDV is not studied. Here, we determined the entry mechanism of PEDV into Vero cells. Our data confirmed that PEDV entry followed clathrin-mediated endocytosis independence of caveolae-coated pit assembly. The internalized PEDV was co-localized with the clathrin-mediated endocytic marker, but not with the caveolae-mediated endocytic marker. In addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to PEDV. Our data revealed that PEDV entry followed clathrin-mediated endocytosis and was dependent on a low pH and serine proteolysis for successful entry into cells.

Kinome siRNA screen identifies novel cell-type specific dengue host target genes.

Dengue is a global emerging infectious disease, with no specific treatment available. To identify novel human host cell targets important for dengue virus infection and replication, an image-based high-throughput siRNA assay screening of a human kinome siRNA library was conducted using human hepatocyte cell line Huh7 infected with a recent dengue serotype 2 virus isolate BR DEN2 01-01. In the primary siRNA screening of 779 kinase-related genes, knockdown of 22 genes showed a reduction in DENV-2 infection. Conversely, knockdown of 8 genes enhanced viral infection. To assess host cell specificity, the confirmed hits were tested in the DENV-infected monocytic cell line U937. While the expression of EIF2AK3, ETNK2 and SMAD7 was regulated in both cell lines after infection, most kinases were hepatocyte-specific. Monocytic cells represent initial targets of infection and an antiviral treatment targeting these cells is probably most effective to reduce initial viral load. In turn, infection of the liver could contribute to pathogenesis, and the novel hepatocyte-specific human targets identified here could be important for dengue infection and pathogenesis.

Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity--inhibition of virus-induced CPE--likely by targeting kinases involved in apoptosis.

High content screening of a kinase-focused library reveals compounds broadly-active against dengue viruses.

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

Receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion.

Porcine epidemic diarrhea virus (PEDV) infection in Vero cells is facilitated by trypsin through an undefined mechanism. The present study describes the mode of action of trypsin in enhancing PEDV infection in Vero cells during different stage of the virus life cycle. During the viral entry stage, trypsin increased the penetration of Vero-cell-attached PEDV by approximately twofold. However, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon PEDV infection. Trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. Furthermore, we also show that the PEDV spike (S) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. These findings indicate that trypsin affects only cell-attached PEDV and increases infectivity and syncytium formation in PEDV-infected Vero cells by cleavage of the PEDV S protein. These findings strongly suggest that the PEDV S protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion.

Trypsin-induced hemagglutination activity of porcine epidemic diarrhea virus.

The hemagglutination (HA) activity of porcine epidemic diarrhea virus (PEDV) was investigated. Two cell-adapted strains of PEDV (KPEDV-9 and SM98LVec) were subjected to HA test against erythrocytes of various origin. Both strains showed HA activity with rabbit erythrocytes only after treatment with trypsin or neuraminidase. Optimal conditions for inducing HA activity of PEDV were 2 h incubation at 37 degrees C using phosphate-buffered saline containing 0.1% BSA. These results suggest that the HA activity of PEDV is most likely caused by proteolytic action on it, which could be developed as a new diagnostic method to rapidly detect and differentiate PEDV infections from other enteric diseases.

Active reassortment of H9 influenza viruses between wild birds and live-poultry markets in Korea.

Surveillance of H9 avian influenza viruses in Korean live-poultry markets from September 2004 through October 2007 was carried out to investigate active reassortment between wild migratory birds and domestic poultry in Korea. Antigenic and phylogenetic analyses showed that most of the isolates belong to the previous Korean H9N2-like lineage and differ from the southeastern Chinese strains. Interestingly, the Ck/Korea/LPM77/06 group (genotype B) and Dk/Korea/LPM248/07 group (genotype C) showed unique properties distinct from those of other Korean H9N2 strains. Although the HA genes of these two groups belong to Korean H9N2-like lineage, the PA genes closely resemble those of the Chinese Y280-like lineage. In addition, the PB2 genes of the Dk/Korea/LPM248/07 group were closely related to those isolated from migratory birds. Several other isolates also clustered within the H9N2 B genotype, an indication that there are at least two predominant H9N2 influenza genotypes in Korea. Another isolate, Dk/Korea/LPM71/06, was identified as an H9N1 subtype, the first ever discovered in Korean live-poultry markets. These findings reveal that reassortment of Korean H9 influenza viruses has occurred frequently in live-poultry markets and may have been mediated by introduction of genetic material from viruses circulating among migratory wild birds to domestic birds. Consequently, the new dominant H9N2 genotypes have become established in Korean live-poultry markets through continued reassortment.

The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus.

The spike protein of Porcine epidemic diarrhea virus is the main surface glycoprotein involved in virus attachment and entry and therefore is the target of neutralizing antibodies. Here, the immunogenicity of a novel antigenic domain found on the carboxy-terminal of the spike protein characterized by the peptide motif GPRLQPY, was evaluated. A synthetic peptide whose linear sequence is identical to the 24a.a. carboxy-terminal portion of the spike protein (S-CT24) elicited a strong antibody response in BALB/c mice that had specific reactivity against the S-CT24 and PEDV. These antibodies were shown to have a specific affinity to the GPRLQPY motif, as demonstrated by non-reactivity with a peptide that lacks this motif. In addition, antiS-CT24 antibodies exhibited neutralizing activities against KPEDV-9 in focus reduction neutralization tests suggesting that the GPRLQPY motif induces neutralizing antibodies against PEDV.

Application of a focus formation assay for detection and titration of porcine epidemic diarrhea virus.

A focus formation assay (FFA) for detection and titration of porcine epidemic diarrhea virus (PEDV) in a micro-culture system using Vero cells and PAP staining technique was evaluated. A linear correlation between the virus dilution and virus titer determined by FFA was observed between the range of 10 and 30 foci per well. Comparative analysis between FFA and plaque assay showed no significant difference in estimating the titer of cell adapted PEDV. However, the culture time required for detecting the virus was considerably shorter for FFA. In addition, FFA had higher sensitivity for detecting field isolates of PEDV as well as positive identification of the virus with the antibody specific reaction. A broader range of dilutions and number of replicates may be used for titration. A FFA may be applied as an alternative method for detection and titration of PEDV.

Phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (PEDV) neutralizing epitopes.

Seven-mer phage random peptide libraries were panned against 2C10, a monoclonal antibody that showed neutralizing activities against PEDV. Recombinant M13 phages displaying the peptides SHRLP(Y/Q)(P/V) or GPRPVTH on the g3p minor coat protein showed strong binding affinity with 2C10 (70% and 30% of recovered phages, respectively) after multiple panning. Sequence analysis suggested that these peptides are similar with (1368)GPRLQPY(1374) found at the carboxy-terminal of the S protein. In neutralization inhibition assays, the two peptide motifs and a 24-mer synthetic peptide corresponding to the C-terminal endodomain of PEDV S protein were observed to compete for the antigen binding site of 2C10, as demonstrated by the loss or reduction of neutralizing activity of the monoclonal antibody. This new finding suggests that the newly discovered peptide motifs mimic a neutralizing epitope PEDV.

Comparison of clinical features and hematologic abnormalities between dengue fever and dengue hemorrhagic fever among children in the Philippines.

To demonstrate the differences of clinical features and hematologic abnormalities between dengue fever (DF) and dengue hemorrhagic fever (DHF), 359 pediatric patients admitted St. Luke's Medical Center in Quezon City, between 1999 and 2001 in Metro Manila, and adjoining provinces the Philippines, with a laboratory-confirmed dengue virus infection were evaluated. One third of the patients had DHF, and most of these patients were without shock. Restlessness, epistaxis, and abdominal pain were more associated with DHF. The platelet count was significantly lower in the DHF group than in the DF group before and after defervescence. In the DHF patients, the hematocrit was significantly increased before defervescence, and decreased the day after due to administration of intravenous fluid. Coagulation abnormalities associated with most DHF patients were thrombocytopenia and an increased fibrinolysis, but not disseminated intravascular coagulation. We present recent data on readily obtained clinical and laboratory data that can be used for early diagnosis and consequently earlier appropriate treatment of dengue virus infections.