Investigation of HCAR2 antagonists as a potential strategy to modulate bovine leukocytes.

BACKGROUND: Dairy cows experiencing ketosis after calving suffer greater disease incidence and are at greater risk of leaving the herd. In vitro administration of beta-hydroxybutyric acid (BHBA; the primary blood ketone) has inhibitory effects on the function of bovine leukocytes. BHBA is a ligand of HCAR2 and the activation of these receptors promotes an anti-inflammatory response which may be related with immunosuppression observed in transition dairy cattle. The objective of this study was to identify and test antagonists for HCAR2 in bovine immune cells cultured with BHBA. RESULTS: We observed expression of HCAR2 at the protein level within lymphocytes, monocytes, and granulocytes. The proportion of cells expressing HCAR2 tended to be greater in mid-lactation compared to early lactation cows; the increase was a result of increased proportion of T and B cells expressing HCAR2. Stimulation of HCAR2 with niacin or BHBA promoted Ca2+ mobilization in neutrophils and mononuclear cells. Mononuclear cells treated with BHBA had diminished intracellular Ca2+ responses when HCAR2 was knocked down by siRNA silencing, indicating Ca2+ mobilization was mediated by HCAR2 signaling. Two candidate antagonists for HCAR2, synthesized from niacin (NA-1 and NA-5), were tested; monocytes and neutrophils pre-treated with NA-1 and NA-5 had reduced Ca2+ mobilization after incubation with BHBA. Furthermore, NA-5 but not NA-1 prevented BHBA-associated reductions in cyclic AMP. CONCLUSIONS: We demonstrated that HCAR2 is present on bovine leukocytes and has greater expression later in lactation. We confirmed that BHBA and niacin derived HCAR2 antagonists alter bovine leukocyte activity. Our results demonstrate that both BHBA and niacin affect bovine leukocyte Ca2+ mobilization in a HCAR2-dependent manner.

Synthesis of Chiral Tricyclic Pyrone Molecules via Palladium(0)-Catalyzed Displacement Reactions of Chiral Tricyclic Pyrone Acetate With Azide or Amine.

Tricyclic pyrone (TP) molecules have shown protection of MC65 neuroblastoma cells death induced by amyloid-ß proteins through SßC gene, a decrease of amyloid-ß peptide levels, and improvement of motor functions and memory in Alzheimer's disease mouse and rat models. Mechanistic studies suggest TP molecules modulate N-methyl-D-aspartate receptor. A short synthesis of chiral TP analogs was sought using a Pd(0)-catalyzed displacement of TP allylic acetate intermediate with sodium azide or substituted benzylamines. A three-step sequence of reactions by the treatment of 2-{(5aS,7S)-3-methyl-1-oxo-1,5a,6,7,8,9-hexahydropyrano[4,3-b]chromen-7-yl}allyl acetate (9) with (Ph3P)4Pd and sodium azide, followed by reduction with Zn-NH4OCHO and coupling with 3-fluoro-4-hydroxybenzaldehyde and NaCNBH3 was found to give TP coupling molecule, (5aS,7S)-7-(1-(3-fluoro-4-hydroxybenzylamino)prop-2-en-2-yl)-3-methyl-6,7,8,9-tetrahydropyrano[4,3-b]chromen-1(5aH)-one (2), in a good yield. An alternative shorter pathway - a two-step sequence of reactions - by the displacement of 9 by 4-(t-butyldimethylsilyloxy)-3-fluoro-benzylamine with a catalytic amount of (Ph3P)4Pd in THF followed by removal of the silyl ether protecting group gave 2, albeit in a lower chemical yield. The described syntheses should provide general procedures for the synthesis of a library of TP molecules for the discovery of anti-Alzheimer drugs.

Indication of Pd-C or Cu-C Intermediate in Respective Bimetallic Nanoclusters Pd/Au-PVP or Cu/Au-PVP Catalyzed Oxidations of endo-4-Oxatricyclo[5.2.1.02,6]-8-decene and Tetrahydro-γ-carbolines.

Catalytic oxidations of tricyclic endo-norbornene-fused tetrahydrofuran with bimetallic nanoclusters Cu/Au-PVP and H2O2 or t-BuOOH as an oxidant provided C-H bond oxidation adjacent to the ether function and 4-oxa-tricyclo[5.2.1.0]-8,9-exo-epoxydecane (4), however, oxidation with Pd/Au-PVP took place at the C=C function giving epoxide 4 and oxidative three-bond forming dimeric product, dodecahydro-1,4:6,9-dimethanodibenzofurano[2,3-b:7,8-b']bisoxolane (5). Formation of the latter suggests the involvement of a reactive Pd-C intermediate. Similarly, oxidative C-C bond forming reactions were found in cycloaddition reactions of N2-Boc-1,2,3,4-tetrahydro-γ-carbolines and 2,3-dihydroxybenzoic acid with 2 - 5 mol% Cu/Au-PVP and H2O2 at 25 °C, providing two-bond-forming [4+2] cycloadducts. Under similar reaction conditions, Pd/Au-PVP did not produce the cycloadduct, indicating a need of complexation between Cu with the carboxylic acid group of 2,3-dihydroxybenzoic acid and allylic amine function of γ-carbolines in the cyclization reaction. The reported intermolecular coupling reactions using Pd/Au-PVP or Cu/Au-PVP nanocluster catalysts under oxidative conditions at 25 °C are unprecedented.

Chirality-driven self-assembly: application toward renewable/exchangeable resin-immobilized catalysts.

Resin-immobilized catalysts were prepared through chirality-driven self-assembly. The method allows the resin-immobilized catalyst to be regenerated under mild conditions and in situ catalyst exchange to be carried out quantitatively. The uniqueness of the methodology was demonstrated by the preparation of a catalyst for TEMPO oxidation as well as a two-step sequential TEMPO oxidation/aldol condensation sequence enabled by facile catalyst exchange.

Synthesis and Characterization of Bimetallic Nanoclusters Stabilized by Chiral and Achiral Polyvinylpyrrolidinones. Catalytic C(sp3)-H Oxidation.

Second-generation chiral-substituted poly-N-vinylpyrrolidinones (CSPVPs) (-)-1R and (+)-1S were synthesized by free-radical polymerization of (3aR,6aR)- and (3aS,6aS)-5-ethenyl-tetrahydro-2,2-dimethyl-4H-1,3-dioxolo[4,5-c]pyrrol-4-one, respectively, using thermal and photochemical reactions. They were produced from respective d-isoascorbic acid and d-ribose. In addition, chiral polymer (-)-2 was also synthesized from the polymerization of (S)-3-(methoxymethoxy)-1-vinylpyrrolidin-2-one. Molecular weights of these chiral polymers were measured using HRMS, and the polymer chain tacticity was studied using 13C NMR spectroscopy. Chiral polymers (-)-1R, (+)-1S, and (-)-2 along with poly-N-vinylpyrrolidinone (PVP, MW 40K) were separately used in the stabilization of Cu/Au or Pd/Au nanoclusters. CD spectra of the bimetallic nanoclusters stabilized by (-)-1R and (+)-1S showed close to mirror-imaged CD absorption bands at wavelengths 200-300 nm, revealing that bimetallic nanoclusters' chiroptical responses are derived from chiral polymer-encapsulated nanomaterials. Chemo-, regio-, and stereo-selectivity was found in the catalytic C-H group oxidation reactions of complex bioactive natural products, such as ambroxide, menthofuran, boldine, estrone, dehydroabietylamine, 9-allogibberic acid, and sclareolide, and substituted adamantane molecules, when catalyst Cu/Au (3:1) or Pd/Au (3:1) stabilized by CSPVPs or PVP and oxidant H2O2 or t-BuOOH were applied. Oxidation of (+)-boldine N-oxide 23 using NMO as an oxidant yielded 4,5-dehydroboldine 27, and oxidation of (-)-9-allogibberic acid yielded C6,15 lactone 47 and C6-ketone 48.

SYNTHESIS OF PROBE MOLECULES, 6-(DIMETHYLAMINO)-2-PHENYLISOINDOLIN-1-ONES, FOR MECHANISTIC STUDIES OF FIREFLY LUCIFERASE INHIBITION.

Firefly luciferase is used in high-throughput screening based on the detection of chemiluminescence. It catalyzes an esterification reaction of luciferin with adenosine 5'-triphosphate (ATP) followed by decarbonylation with oxygen and concomitance of light. Previously, we reported that firefly luciferase also possesses acyl-CoA synthetase activity and catalyzes an aromatic carboxylic acid group of F-53, using ATP, Mg2+ and coenzyme A (CoA), to produce F-53 covalently attached to active-site lysine-529 residue of firefly luciferase through the formation of an amide group. The amidation of lysine-529 resulted in a deactivation of luciferase. In order to probe firefly luciferase inhibition's mechanism, we synthesized two probe molecules 1 and 2, mimicking F-53. Molecule 1 contains an azido-appended side chain in the aromatic ring of F-53, while 2 possesses an azido and a carboxylic acid group appended side chains. Both synthetic schemes are readily amenable to large-scale syntheses. Molecule 1 was made from 2-allylaniline, which was derived from a thermal-induced aromatic-Claisen rearrangement of N-allylaniline. The azido-appended side chain of 2 was installed from a Horner-Wadsworth-Emmons reaction and the carboxylic acid side chain from a Sonogashira reaction.

Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum.

Proteases are critical signaling molecules and prognostic biomarkers for many diseases including cancer. There is a strong demand for multiplex bioanalytical techniques that can rapidly detect the activity of extracellular proteases with high sensitivity and specificity. This study demonstrates an activity-based electrochemical biosensor of a 3 × 3 gold microelectrode array for the detection of cathepsin B activity in human serum diluted in a neutral buffer. Proteolysis of ferrocene-labeled peptide substrates functionalized on 200 × 200 µm microelectrodes is measured simultaneously over the nine channels by AC voltammetry. The protease activity is represented by the inverse of the exponential decay time constant (1/τ), which equals to (kcat/KM)[CB] based on the Michaelis-Menten model. An enhanced activity of the recombinant human cathepsin B (rhCB) is observed in a low-ionic-strength phosphate buffer at pH = 7.4, giving a very low limit of detection of 8.49 × 10-4 s-1 for activity and 57.1 pM for the active rhCB concentration that is comparable to affinity-based enzyme-linked immunosorbent assay (ELISA). The cathepsin B presented in the human serum sample is validated by ELISA, which mainly detects the inactive proenzyme, while the electrochemical biosensor specifically measures the active cathepsin B and shows significantly higher decay rates when rhCB and human serum are activated. Analyses of the kinetic electrochemical measurements with spiked active cathepsin B in human serum provide further assessment of the protease activity in the complex sample. This study lays the foundation to develop the gold microelectrode array into a multiplex biosensor for rapid detection of the activity of extracellular proteases toward cancer diagnosis and treatment assessment.

Synthesis of 2,24-Diene-12,13,15,16,34,35,37,38-octaphenyl[4.4]-triphenylparacyclophane.

A new octaphenyl[4.4]triphenylparacyclophanediene was readily synthesized in six steps from p-xylene via the installment of bromine atoms, replacement with a vinyl group, carbonylative coupling, intermolecular followed by intramolecular double Grubbs olefin metathesis, Knoevenagel condensation, and Diels-Alder cycloaddition. The belt-shaped structure and trans-stereochemistry of the alkene moieties of the octaphenyl[4.4]triphenylparacyclophane and a synthetic intermediate, 2,21-dioxo-11,30-diene[3.4.3.4]paracyclophane, were determined by X-ray crystallography. The synthetic methodology leading to octaphenyl[4.4]triphenylparacyclophane is applicable for the synthesis of substituted triphenylparacyclophanes and possibly their corresponding bis-hexabenzocoronenylparacyclophanes via a Scholl-Mullen oxidative aryl-aryl coupling reaction.

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis.

BACKGROUND: Targeting insect-specific genes through post-transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double-stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS: Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan-based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION: These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

SYNTHESIS OF 1,3,4-OXADIAZOLES AS SELECTIVE T-TYPE CALCIUM CHANNEL INHIBITORS.

Neuropathic pain, epilepsy, insomnia, and tremor disorder may arrive from an increase of intracellular Ca2+ concentration through a dysfunction of T-type Ca2+ channels. Thus, T-type calcium channels could be a target in drug discovery for the treatments of neuropathic pain and epilepsy. From rational drug design approach, a group of 2,5-disubstituted 1,3,4-oxadiazole molecules was synthesized and their selective T-type channel inhibitions were evaluated. The synthetic strategy consists of a short sequence of three reactions: (i) condensation of thiosemicarbazide with acid chlorides; (ii) ring closing by 1,3-dibromo-5,5- dimethylhydantoin; and (iii) coupling with various acid chlorides. 5-Chloro-N-(5- phenyl-1,3,4-oxadiazol-2-yl)thiophene-2-carboxamide (11) was found to selectively inhibit T-type Ca2+ channel over Na+ and K+ channels in mouse dorsal root ganglion neurons and/or human embryonic kidney (HEK)-293 cells and to suppress seizure-induced death in mouse model. Consequently, compound 11 is a useful probe for investigation of physiologic and pathophysiologic roles of the T-channel, and provides a basis to develop a novel therapeutic to treat chronic neuropathic and inflammatory pains.

Simultaneous, multiplex quantification of protease activities using a gold microelectrode array.

Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and monitoring of diseases such as cancers. This study demonstrates the fabrication and characterization of an individually addressable 3 × 3 Au microelectrode array for rapid, multiplex detection of cathepsin B activity based on a simple electrochemical method. The nine individual microelectrodes in the array show highly consistent cyclic voltammetric signals in Au surface cleaning experiments and detecting benchmark redox species in solution. The individual Au microelectrodes are further selectively functionalized with specific ferrocene-labeled peptide molecules which serve as the cognate substrates for the target proteases. Consistent proteolytic kinetics are measured by monitoring the decay of the AC voltammetry signal from the ferrocene label as the peptide molecules are cleaved by cathepsin B. Accurate activity of cathepsin B is derived with an improved fitting algorithm. Simultaneous detection of the proteolysis of cathepsin B on the microelectrode array functionalized with three different hexapeptides is demonstrated, showing the potential of this sensor platform for rapid detection of the activity profiles of multiple proteases in various diseases including many forms of cancer.

Broad-spectrum antifungal activities and mechanism of drimane sesquiterpenoids.

Eight drimane sesquiterpenoids including (-)-drimenol and (+)-albicanol were synthesized from (+)-sclareolide and evaluated for their antifungal activities. Three compounds, (-)-drimenol, (+)-albicanol, and (1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyl-decahydronaphthalene-1-carbaldehyde (4) showed strong activity against C. albicans. (-)-Drimenol, the strongest inhibitor of the three, (at concentrations of 8 - 64 µg/ml, causing 100% death of various fungi), acts not only against C. albicans in a fungicidal manner, but also inhibits other fungi such as Aspergillus, Cryptococcus, Pneumocystis, Blastomyces, Saksenaea and fluconazole resistant strains of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. auris. These observations suggest that drimenol is a broad-spectrum antifungal agent. At a high concentration (100 µg/ml) drimenol caused rupture of the fungal cell wall/membrane. In a nematode model of C. albicans infection, drimenol rescued the worms from C. albicans-mediated death, indicating drimenol is tolerable and bioactive in metazoans. Genome-wide fitness profiling assays of both S. cerevisiae (nonessential homozygous and essential heterozygous) and C. albicans (Tn-insertion mutants) collections revealed putative genes and pathways affected by drimenol. Using a C. albicans mutant spot assay, the Crk1 kinase associated gene products, Ret2, Cdc37, and orf19.759, orf19.1672, and orf19.4382 were revealed to be involved in drimenol's mechanism of action. The three orfs identified in this study are novel and appear to be linked with Crk1 function. Further, computational modeling results suggest possible modifications of the structure of drimenol, including the A ring, for improving the antifungal activity.

Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays.

There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity-based electrochemical method toward this goal. Nanoelectrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis-Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N-(CH2)4-CO-Pro-Leu-Arg-Phe-Gly-Ala-NH-CH2-Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and concentration can reach 2.49 × 10-4 s-1 and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.

Synthesis and biological evaluation of steroidal derivatives bearing a small ring as vitamin D receptor agonists.

A novel series of 3-ketolithocholic acid derivatives as well as estrone derivatives bearing a small ring for the conformational fixation of the side chain were synthesized by using a catalytic [2+2] cycloaddition and a ring-contraction rearrangement. The steroidal derivatives were evaluated for transcriptional activation of vitamin D receptor by luciferase reporter assays. Among them, two estrone derivatives showed a higher efficacy of the transactivation of vitamin D receptor than 3-ketolithocholic acid, and the small ring moieties were found to be important for the efficacy.

The Anti-Amyloid-ß and Neuroprotective Properties of a Novel Tricyclic Pyrone Molecule.

There is an urgent unmet need for new therapeutics for Alzheimer's disease (AD), the most common cause of dementia in the elderly. Therapeutic approaches targeting amyloid-ß (Aß) and its downstream toxicities have become major strategies in AD drug development. We have taken a rational design approach and synthesized a class of tricyclic pyrone (TP) compounds that show anti-Aß and other neuroprotective actions. The in vivo efficacy of a lead TP named CP2 to ameliorate AD-like pathologies has been shown in mouse models. Here we report the selection and initial characterization of a new lead TP70, which exhibited an anti-Aß therapeutic index even higher than CP2. Moreover, TP70 was able to reduce oxidative stress, inhibit acyl-coenzyme A:cholesterol acyltransferase (ACAT), and upregulate the expression of ATP-binding cassette subfamily A, member 1 (ABCA1), actions considered neuroprotective in AD. TP70 further showed excellent pharmacokinetic properties, including brain penetration and oral availability. When administered to 5xFAD mice via intraperitoneal or oral route, TP70 enhanced the overall solubility and decreased the level of cerebral Aß, including both fibrillary and soluble Aß species. Interestingly, TP70 enhanced N-methyl-D-aspartate (NMDA) receptor-mediated excitatory post-synaptic potential (EPSP) in the hippocampal CA1 area, increased the magnitude of NMDA-dependent hippocampal long-term potentiation (LTP), a cellular model of learning and memory, and prevented the Aß oligomer-impaired LTP. Significantly, a single dose of TP70 administered to aged 5xFAD mice was effective in mitigating the impaired LTP induction, recorded at 24 h after administration. Our results support a potential of TP70 in clinical development for AD in view of its synergistic neuroprotective actions, ability to positively modulate NMDA receptor-mediated hippocampal plasticity, and favorable pharmacokinetic properties in rodents.

Chiral-Substituted Poly-N-vinylpyrrolidinones and Bimetallic Nanoclusters in Catalytic Asymmetric Oxidation Reactions.

A new class of poly-N-vinylpyrrolidinones containing an asymmetric center at C5 of the pyrrolidinone ring were synthesized from l-amino acids. The polymers, particularly 17, were used to stabilize nanoclusters such as Pd/Au for the catalytic asymmetric oxidations of 1,3- and 1,2-cycloalkanediols and alkenes, and Cu/Au was used for C-H oxidation of cycloalkanes. It was found that the bulkier the C5 substituent in the pyrrolidinone ring, the greater the optical yields produced. Both oxidative kinetic resolution of (±)-1,3- and 1,2-trans-cycloalkanediols and desymmetrization of meso cis-diols took place with 0.15 mol % Pd/Au (3:1)-17 under oxygen atmosphere in water to give excellent chemical and optical yields of (S)-hydroxy ketones. Various alkenes were oxidized with 0.5 mol % Pd/Au (3:1)-17 under 30 psi of oxygen in water to give the dihydroxylated products in >93% ee. Oxidation of (R)-limonene at 25 °C occurred at the C-1,2-cyclic alkene function yielding (1S,2R,4R)-dihydroxylimonene 49 in 92% yield. Importantly, cycloalkanes were oxidized with 1 mol % Cu/Au (3:1)-17 and 30% H2O2 in acetonitrile to afford chiral ketones in very good to excellent chemical and optical yields. Alkene function was not oxidized under the reaction conditions. Mechanisms were proposed for the oxidation reactions, and observed stereo- and regio-chemistry were summarized.

Correction: Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

[This corrects the article DOI: 10.1371/journal.ppat.1005531.].

Current tools for norovirus drug discovery.

INTRODUCTION: Rapid transmission of norovirus often occurs due to its low infectious dosage, high genetic diversity and its short incubation time. The viruses cause acute gastroenteritis and may lead to death. Presently, no effective vaccine or selective drugs accepted by the United States Food and Drug Administration (FDA) are available for the treatment of norovirus. Advances in the development of norovirus replicon cell lines, GII.4-Sydney HuNoV strain human B cells, and murine and gnotobiotic pig norovirus models have facilitated the discovery of effective small molecule inhibitors in vitro and in vivo. AREAS COVERED: This review gives a brief discussion of the biology and replication of norovirus before highlighting the discovery of anti-norovirus molecules. The article coverage includes: an overview of the current state of norovirus drug discovery, the targeting of the norovirus life cycle, the inhibition of structural and nonstructural proteins of norovirus such as proteases and polymerase, and the blockage of virus entry into host cells. Finally, anti-norovirus drugs in the clinical development stage are described. EXPERT OPINION: The current approach for the counteraction of norovirus focuses on the inhibition of viral RNA polymerase, norovirus 3C-like protease and the structural proteins VP1 as well as the blockade of norovirus entry. Broad-spectrum anti-norovirus molecules, based on the inhibition of 3C-like protease, have been developed. Other host factors and ways to overcome the development of resistance through mutation are also being examined. A dual approach in targeting viral and host factors may lead to an effective counteraction of norovirus infection. Current successes in developing norovirus replicon harboring cells and norovirus infected human cells, as well as murine norovirus models and other animal models such as piglets have facilitated the discovery of effective drugs and helped our understanding of its mechanism of action.

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.