tosylphenylalanyl-chloromethyl-ketone and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

Topics: Animals; Behavior, Animal; Cell-Free System; Dermatitis, Contact; Disease Models, Animal; Ganglia, Spinal; Humans; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pruritus; Receptors, Atrial Natriuretic Factor; Reproducibility of Results; Signal Transduction; Small Molecule Libraries

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Topics: Animals; Antifungal Agents; Candida parapsilosis; Candidiasis; Cell Adhesion; Disease Models, Animal; Larva; Lepidoptera; Oxidative Stress; Serine Proteinase Inhibitors; Survival Analysis; Tosylphenylalanyl Chloromethyl Ketone; Treatment Outcome; Virulence

Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit.

Topics: Animals; Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Camptothecin; Caspase 6; Cerebral Cortex; COS Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Down Syndrome; Gene Expression Regulation; Humans; Huntingtin Protein; Mice; Mice, Transgenic; Mutation; Neurons; Protein Synthesis Inhibitors; Quinolones; Time Factors; Tosylphenylalanyl Chloromethyl Ketone

N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) suppresses apoptosis and protects neurons from damage in animal models. TPCK is thought to act by inhibiting ceramide production by sphingomyelinase. Ceramide is a proapoptotic intracellular signal that is involved in the cerebral ischemia. We wished to see whether ceramide contributes to TPCK's neuroprotective effects in vivo. Seven-day-old rat pups had the right carotid arteries permanently ligated followed by 2.5 h of hypoxia (8% oxygen). TPCK (10 mg/kg, n=62) or vehicle (n=63) was administered by i.p. 5 min prior to hypoxia. The level of ceramide in brain cortex both in lesioned and unlesioned hemispheres was measured at 8 h, 18 h, 24 h, 2 and 5 days after hypoxia-ischemia using reversed phase high performance liquid chromatography. The level of ceramide significantly increased due to hypoxic-ischemia at 18, 24 h and 2 days after hypoxia (P<0.05 or P<0.01) but not at 8 h or 5 days after hypoxia as compared to the contralateral hemisphere or a sham group. Pretreatment with TPCK reduced this increase. We also examined the level of sphingomyelin and the activities of the ceramide synthesizing sphingomyelinase enzymes by thin layer chromatography. The activities of acidic and neutral sphingomyelinase significantly increased due to hypoxic ischemia at 24 h after hypoxia. TPCK significantly reduced this increase (P<0.05 vs. vehicle) but did not affect the level of sphingomyelin. The results are consistent with the hypothesis that ceramide is involved in TPCK's neuroprotective effects in hypoxic-ischemic brain injury in the newborn rat.

Topics: Animals; Animals, Newborn; Brain Ischemia; Ceramides; Cerebral Cortex; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Disease Models, Animal; Enzyme Inhibitors; Hypoxia-Ischemia, Brain; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Sphingomyelin Phosphodiesterase; Time Factors; Tosylphenylalanyl Chloromethyl Ketone

N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K(+) deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 microM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death.

Topics: Animals; Apoptosis; Behavior, Animal; Brain Injuries; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Coumarins; Culture Media, Serum-Free; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Intraventricular; Isocoumarins; Male; Motor Activity; Neurons; Nucleic Acid Synthesis Inhibitors; Potassium; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Serine Proteinase Inhibitors; Spatial Behavior; Staurosporine; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Wounds, Nonpenetrating