Kyungae Lee

Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann–Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

Development of Improved Inhibitors of Wall Teichoic Acid Biosynthesis with Potent Activity Against Staphylococcus aureus

A small molecule (1835F03) that inhibits Staphylococcus aureus wall teichoic acid biosynthesis, a proposed antibiotic target, has been discovered. Rapid, parallel, solution-phase synthesis was employed to generate a focused library of analogs, providing detailed information about structure-activity relationships and leading to the identification of targocil, a potent antibiotic.

Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease

West Nile virus (WNV) is a mosquito-borne member of flaviviruses that causes significant morbidity and mortality especially among children. There is currently no approved vaccine or antiviral therapeutic for human use. In a previous study, we described compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of WNV serine protease (NS2B/NS3pro) in a high throughput screen (HTS) using the purified WNV NS2B/NS3pro as the target. In this study, we analyzed potencies of some commercially available as well as chemically synthesized derivatives of 8-HQ by biochemical assays. An insight into the contribution of various substitutions of 8-HQ moiety for inhibition of the protease activity was revealed. Most importantly, the substitution of the N1 of the 8-HQ ring by -CH- in compound 26 significantly reduced the inhibition of the viral protease by this naphthalen-1-ol derivative. The kinetic constant (K(i)) for the most potent 8-HQ inhibitor (compound 14) with an IC(50) value of 2.01 ± 0.08 μM using the tetra-peptide substrate was determined to be 5.8 μM. This compound inhibits the WNV NS2B/NS3pro by a competitive mode of inhibition which is supported by molecular modeling.

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

Previous studies identified an adamantane dipeptide piperazine 3.47 that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann-Pick C1 (NPC1). The physicochemical properties of 3.47 limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified 3.47 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.

Critical role for cholesterol in Lassa fever virus entry identified by a novel small molecule inhibitor targeting the viral receptor LAMP1

Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.