Niemann-Pick disease, type C (NPC) is a rare autosomal recessive genetic disease caused by mutations in either NPC1 or NPC2, which encodes an intracellular cholesterol-binding protein in lysosome. Deficiency of either NPC1 or NPC2 protein results in malfunction of intracellular cholesterol trafficking and lysosomal accumulation of unesterified cholesterols. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a male patient that has a homozygous p.I1061T missense mutation in NPC1 using a non-integrating Sendai virus technique. This NPC1 iPSC line offers a useful resource for disease modeling and drug development.

Antibiotic-resistant nosocomial infections are an emerging public health issue; carbapenem-resistant gram-negative bacteria such as Acinetobacter baumannii are among the pathogens against which new therapeutic agents are desperately needed. Drug repurposing has recently emerged as an alternative approach to rapidly identifying effective drugs and drug combinations to combat drug resistant bacteria. We performed a drug repurposing screen against a highly virulent, multidrug resistant, Acinetobacter baumannii strain AB5075. This strain, isolated from a patient, is resistant to 25 first-line antibiotics for gram-negative bacteria. A compound screen using a bacterial growth assay led to identification and confirmation of 43 active compounds. Among these confirmed compounds, seven are approved drugs or pharmacologically active compounds for non-antimicrobial indications. Three of these drugs, 5-fluorouracil, fluspirilene, and Bay 11-7082 resensitized strain AB5075 to azithromycin and colistin in a two-drug combination format. The approach using a drug repurposing screen with a pathogen sample isolated from a patient and a high throughput bacterial growth assay led to the successful identification of new drug combinations to overcome a multidrug resistant bacterial infection.

NGLY1 deficiency is a rare genetic disease caused by mutations in the NGLY1 gene that encodes N-glycanase 1. The disease phenotype in patient cells is unclear. A human induced pluripotent stem cell (iPSC) line was generated from skin dermal fibroblasts of a patient with NGLY1 deficiency that has compound heterozygous mutations of a p.Q208X variant (c.622C > T) in exon 4 and a p.G310G variant (c.930C > T) in exon 6 of the NGLY1 gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development to treat NGLY1 deficiency.

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

BACKGROUND: Infantile and late infantile neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases affecting the central nervous system (CNS). The infantile NCL (INCL) is caused by mutations in the PPT1 gene and late-infantile NCL (LINCL) is due to mutations in the TPP1 gene. Deficiency in PPT1 or TPP1 enzyme function results in lysosomal accumulation of pathological lipofuscin-like material in the patient cells. There is currently no small-molecular drug treatment for NCLs. RESULTS: We have generated induced pluripotent stem cells (iPSC) from three patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). Using these new disease models, we evaluated the effect of δ-tocopherol (DT) and hydroxypropyl-β-cyclodextrin (HPBCD) with the enzyme replacement therapy as the control. Treatment with the relevant recombinant enzyme or DT significantly ameliorated the lipid accumulation and lysosomal enlargement in the disease cells. A combination therapy of δ-tocopherol and HPBCD further improved the effect compared to that of either drug used as a single therapy. CONCLUSION: The results demonstrate that these patient iPSC derived NCL NSCs are valid cell- based disease models with characteristic disease phenotypes that can be used for study of disease pathophysiology and drug development.

The Ebola virus (EBOV) causes severe human infection that lacks effective treatment. A recent screen identified a series of compounds that block EBOV-like particle entry into human cells. Using data from this screen, quantitative structure-activity relationship models were built and employed for virtual screening of a ∼17 million compound library. Experimental testing of 102 hits yielded 14 compounds with IC50 values under 10 μM, including several sub-micromolar inhibitors, and more than 10-fold selectivity against host cytotoxicity. These confirmed hits include FDA-approved drugs and clinical candidates with non-antiviral indications, as well as compounds with novel scaffolds and no previously known bioactivity. Five selected hits inhibited BSL-4 live-EBOV infection in a dose-dependent manner, including vindesine (0.34 μM). Additional studies of these novel anti-EBOV compounds revealed their mechanisms of action, including the inhibition of NPC1 protein, cathepsin B/L, and lysosomal function. Compounds identified in this study are among the most potent and well-characterized anti-EBOV inhibitors reported to date.

Although a group of FDA-approved drugs were previously identified with activity against Ebola virus (EBOV), most of them are not clinically useful because their human blood concentrations are not high enough to inhibit EBOV infection. We screened 795 unique three-drug combinations in an EBOV entry assay. Two sets of three-drug combinations, toremifene-mefloquine-posaconazole and toremifene-clarithromycin-posaconazole, were identified that effectively blocked EBOV entry and were further validated for inhibition of live EBOV infection. The individual drug concentrations in the combinations were reduced to clinically relevant levels. We identified mechanisms of action of these drugs: functional inhibitions of Niemann-Pick C1, acid sphingomyelinase, and lysosomal calcium release. Our findings identify the drug combinations with potential to treat EBOV infection.

Current antimicrobial susceptibility testing has limited screening capability for identifying empirical antibiotic combinations to treat severe bacterial infections with multidrug-resistant (MDR) organisms. We developed a new antimicrobial susceptibility assay using automated ultra-high-throughput screen technology in combination with a simple bacterial growth assay. A rapid screening of 5170 approved drugs and other compounds identified 25 compounds with activities against MDR Klebsiella pneumoniae. To further improve the efficacy and reduce the effective drug concentrations, we applied a targeted drug combination approach that integrates drugs' clinical antimicrobial susceptibility breakpoints, achievable plasma concentrations, clinical toxicities and mechanisms of action to identify optimal drug combinations. Three sets of three-drug combinations were identified with broad-spectrum activities against 10 MDR clinical isolates including K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Citrobacter freundii, Enterobacter cloacae and Escherichia coli. Colistin-auranofin-ceftazidime and colistin-auranofin-rifabutin suppressed >80% growth of all 10 MDR strains; while rifabutin-colistin-imipenem inhibited >75% of these strains except two Acinetobacter baumannii isolates. The results demonstrate this new assay has potential as a real-time method to identify new drugs and effective drug combinations to combat severe clinical infections with MDR organisms.

Glycolate oxidase (GO) and alanine:glyoxylate aminotransferase (AGT) are both involved in the peroxisomal glyoxylate pathway. Deficiency in AGT function causes the accumulation of intracellular oxalate and the primary hyperoxaluria type 1 (PH1). AGT enhancers or GO inhibitors may restore the abnormal peroxisomal glyoxylate pathway in PH1 patients. With stably transformed cells which mimic the glyoxylate metabolic pathway, we developed an indirect glycolate cytotoxicity assay in a 1,536-well plate format for high throughput screening. This assay can be used to identify compounds that reduce indirect glycolate-induced cytotoxicity by either enhancing AGT activity or inhibiting GO. A pilot screen of 4,096 known compounds identified two membrane permeable GO inhibitors: dichromate salt and colistimethate. We also developed a GO enzyme assay using the hydrogen peroxide-Amplex red reporter system. The IC50 values of potassium dichromate, sodium dichromate, and colistimethate sodium were 0.096, 0.108, and 2.3 μM in the GO enzyme assay, respectively. Further enzyme kinetic study revealed that both types of compounds inhibit GO activity by the mixed linear inhibition. Our results demonstrate that the cell-based assay and GO enzyme assay developed in this study are useful for further screening of large compound libraries for drug development to treat PH1.

Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKV(C), African ZIKV(M), and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKV(C), but not ZIKV(M), induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKV(C) and ZIKV(M) in hNPCs, with higher potency against ZIKV(C)-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV.