Identification of hepta-histidine as a candidate drug for Huntington's disease by in silico-in vitro- in vivo-integrated screens of chemical libraries.

We identified drug seeds for treating Huntington's disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD

Human Tridimensional Neuronal Cultures for Phenotypic Drug Screening in Inherited Peripheral Neuropathies

Length-dependent axonal degeneration is the pathologic hallmark of several neurodegenerative disorders, including inherited peripheral neuropathies (Charcot-Marie-Tooth (CMT) disease). CMT is currently an untreatable disorder. This is partially due to lack of translational models suitable for drug discovery. In vitro models of CMT have been hindered by the 2D configuration of neuronal cultures, which limits visualization and orientation of axons. To overcome these limitations, we cultured induced pluripotent stem cell (iPSC)-derived spinal motor neurons as 3D spheroids, which grow axons in a centrifugal fashion when plated. Using these iPSC-derived spinal spheroids, we demonstrate neurofilament deposits in motor neuron axons of three patients with CMT2E, caused by mutations in the NEFL gene. This phenotype is partially reversed by two kinase inhibitors. In summary, we developed a human tridimensional in vitro system that models length-dependent axonopathies, recapitulates key pathophysiologic features of CMT2E, and should facilitate the identification of new therapeutic compounds for CMT

AAV-mediated editing of PMP22 rescues Charcot-Marie-Tooth disease type 1A features in patient-derived iPS Schwann cells

Abstract Background Charcot-Marie-Tooth disease type 1A (CMT1A) is one of the most common hereditary peripheral neuropathies caused by duplication of 1.5 Mb genome region including PMP22 gene. We aimed to correct the duplication in human CMT1A patient-derived iPS cells (CMT1A-iPSCs) by genome editing and intended to analyze the effect on Schwann cells differentiated from CMT1A-iPSCs. Methods We designed multiple gRNAs targeting a unique sequence present at two sites that sandwich only a single copy of duplicated peripheral myelin protein 22 (PMP22) genes, and selected one of them (gRNA3) from screening their efficiencies by T7E1 mismatch detection assay. AAV2-hSaCas9-gRNAedit was generated by subcloning gRNA3 into pX601-AAV-CMV plasmid, and the genome editing AAV vector was infected to CMT1A-iPSCs or CMT1A-iPSC-derived Schwann cell precursors. The effect of the genome editing AAV vector on myelination was evaluated by co-immunostaining of myelin basic protein (MBP), a marker of mature myelin, and microtubule-associated protein  2(MAP2), a marker of neurites or by electron microscopy. Results Here we show that infection of CMT1A-iPS cells (iPSCs) with AAV2-hSaCas9-gRNAedit expressing both hSaCas9 and gRNA targeting the tandem repeat sequence decreased PMP22 gene duplication by 20–40%. Infection of CMT1A-iPSC-derived Schwann cell precursors with AAV2-hSaCas9-gRNAedit normalized PMP22 mRNA and PMP22 protein expression levels, and also ameliorated increased apoptosis and impaired myelination in CMT1A-iPSC-derived Schwann cells. Conclusions In vivo transfer of AAV2-hSaCas9-gRNAedit to peripheral nerves could be a potential therapeutic modality for CMT1A patient after careful examinations of toxicity including off-target mutations

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

<div><p>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)-<i>N</i>-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.</p> </div

Primary screening of the BioFocus kinase inhibitor library.

<p>A collection of 4,000 compounds synthesized on the basis of known kinase binding sites were screened for antiviral activity against DENV1, DENV2, DENV3 and DENV4 infection in Huh-7.5. Scatter-plot distribution shows compound activity based on percent inhibition and percent cell number resulting from the following treatment: 10 µM test compounds (red), 0.5% DMSO carrier (blue), MOCK-infection (green), 500 U/mL IFN-α2A (purple). Area under the dotted squares indicate selected primary hits from each serotype based on the criteria: ≥80% inhbition and <50% cell number.</p

Serotype specificity and virus selective of the hit compounds in the 4-(1-aminoethyl)-N-methylthiazol-2-amine cluster.

<p>SI is determined by the formula: CC₅₀/EC₅₀.</p

Software development for image-based analysis of dengue virus infection.

<p>Acquired confocal images of dengue infection in Huh-7.5 were analyzed using an in-house IM software. The number of cells in the images is determined by detecting the local maxima (B and C) from the DAPI channel (A). The region of a single nucleus (D) is defined using watershed-like method. Afterwards, a weighted distance map (F and G) of Alexa488 signal channel (E) is calculated to direct further watershed from the border of detected nucleus border (D) to define the so-called cell body. Finally, quantitation of dengue-infected cells is done by calculating the number of Alexa488-positive cell cytoplasm (the blue-marked cells in I) divided by the total number of cells (all the colorful-marked cells in H). (All images were taken at 20× magnification, false-color).</p

Chemical structures of the 11 compounds having the 4-(1-aminoethyl)-N-methylthiazol-2-amine core scaffold.

<p>Chemical structures of the 11 compounds having the 4-(1-aminoethyl)-N-methylthiazol-2-amine core scaffold.</p

Assay validation of the image-based dengue HT/HCA.

<p>The first assay validation determined the degree of separation between positive and negative controls (A). <i>Upper left panel</i>: layout of controls in the 384-well plate; <i>Lower left panel</i>: generated heat-map representing percentage of Huh-7.5 infected with DENV2; <i>Right panel</i>: Scatter plot distribution shows the percentage of DENV2-infected cells under the following treatments: 0.5% DMSO carrier (red), 500 U/mL IFN-α2A (orange), 10 pt. DRC of IFN-α2A (green), MOCK-infection (blue). Area under the black and green dotted lines represents the variability of DENV infection and MOCK infection controls, respectively. The arrows represent the degree of separation (Z'-factor) between the two controls. The second assay validation determined the dose-response curves of a reference compound panel (B). <i>Left panel</i>: layout of the reference compounds spotted in designated wells of a 384-well plate; <i>Middle panel</i>: heat-map showing relative percentage of DENV2-infected cells; <i>Right panel</i>: heat-map showing relative percent cell viability based on average cell number. (C) 10 pt. DRC of the reference compound panel. Percent infection was normalized against DENV2-infected Huh-7.5, and percent cell number was normalized against MOCK-infected Huh-7.5.</p