Supplementary data for the article: Selaković, Z.; Soloveva, V.; Gharaibeh, D. N.; Wells, J.; Šegan, S.; Panchal, R. G.; Šolaja, B. A. Anti-Ebola Activity of Diazachrysene Small Molecules. ACS Infectious Diseases 2016, 1 (6), 264–271. https://doi.org/10.1021/acsinfecdis.5b00028

Supporting information for: [https://doi.org/10.1021/acsinfecdis.5b00028]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2035

Supplementary data for the article: Selaković, Z.; Soloveva, V.; Gharaibeh, D. N.; Wells, J.; Šegan, S.; Panchal, R. G.; Šolaja, B. A. Anti-Ebola Activity of Diazachrysene Small Molecules. ACS Infectious Diseases 2016, 1 (6), 264–271. https://doi.org/10.1021/acsinfecdis.5b00028

Supporting information for: [https://doi.org/10.1021/acsinfecdis.5b00028]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2035

Phenotypic MicroRNA Microarrays

Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models

Anti-Ebola Activity of Diazachrysene Small Molecules

Herein we report on a diazachrysene class of small molecules that exhibit potent antiviral activity against the Ebola (EBOV) virus. The antiviral compounds are easily synthesized, and the most active compounds have excellent in vitro activity (0.34–0.70 μM) and are significantly less lipophilic than their predecessors. The three most potent diazachrysene antivirals do not exhibit any toxicity in vivo and protected 70–90% of the mice at 10 mg/kg following EBOV challenge. Together, these studies suggest that diazachrysenes are a promising class of compounds for hit to lead optimization and as potential Ebola therapeutics

Identification of Potent Ebola Virus Entry Inhibitors with Suitable Properties for in Vivo Studies

Previous studies identified an adamantane dipeptide piperazine <b>3.47</b> that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann–Pick C1 (NPC1). The physicochemical properties of <b>3.47</b> limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified <b>3.47</b> derivatives with improved in vitro ADME properties that are also highly active against EBOV infection, including when tested in the presence of 50% normal human serum (NHS). In addition, 3A4 was identified as the major cytochrome P450 isoform that metabolizes these compounds, and accordingly, mouse microsome stability was significantly improved when tested in the presence of the CYP3A4 inhibitor ritonavir that is approved for clinical use as a booster of anti-HIV drugs. Oral administration of the EBOV inhibitors with ritonavir resulted in a pharmacokinetic profile that supports a b.i.d. dosing regimen for efficacy studies in mice

Identification of Potent Ebola Virus Entry Inhibitors with Suitable Properties for in Vivo Studies

Previous studies identified an adamantane dipeptide piperazine <b>3.47</b> that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann–Pick C1 (NPC1). The physicochemical properties of <b>3.47</b> limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified <b>3.47</b> derivatives with improved in vitro ADME properties that are also highly active against EBOV infection, including when tested in the presence of 50% normal human serum (NHS). In addition, 3A4 was identified as the major cytochrome P450 isoform that metabolizes these compounds, and accordingly, mouse microsome stability was significantly improved when tested in the presence of the CYP3A4 inhibitor ritonavir that is approved for clinical use as a booster of anti-HIV drugs. Oral administration of the EBOV inhibitors with ritonavir resulted in a pharmacokinetic profile that supports a b.i.d. dosing regimen for efficacy studies in mice

Second generation of diazachrysenes: Protection of Ebola virus infected mice and mechanism of action

Ebola virus (EBOV) causes a deadly hemorrhagic fever in humans and non-human primates. There is currently no FDA-approved vaccine or medication to counter this disease. Here, we report on the design, synthesis and anti-viral activities of two classes of compounds which show high potency against EBOV in both in vitro cell culture assays and in vivo mouse models Ebola viral disease. These compounds incorporate the structural features of cationic amphiphilic drugs (CAD), i.e they possess both a hydrophobic domain and a hydrophilic domain consisting of an ionizable amine functional group. These structural features enable easily diffusion into cells but once inside an acidic compartment their amine groups became protonated, ionized and remain trapped inside the acidic compartments such as late endosomes and lysosomes. These compounds, by virtue of their lysomotrophic functions, blocked EBOV entry. However, unlike other drugs containing a CAD moiety including chloroquine and amodiaquine, compounds reported in this study display faster kinetics of accumulation in the lysosomes, robust expansion of late endosome/lysosomes, relatively more potent suppression of lysosome fusion with other vesicular compartments and inhibition of cathepsins activities, all of which play a vital role in anti-EBOV activity. Furthermore, the diazachrysene 2 (ZSML08) that showed most potent activity against EBOV in in vitro cell culture assays also showed significant survival benefit with 100% protection in mouse models of Ebola virus disease, at a low dose of 10 mg/kg/day. Lastly, toxicity studies in vivo using zebrafish models suggest no developmental defects or toxicity associated with these compounds. Overall, these studies describe two new pharmacophores that by virtue of being potent lysosomotrophs, display potent anti-EBOV activities both in vitro and in vivo animal models of EBOV disease