Discovery and characterization of novel inhibitors against cathepsin L and exploring their potential as anti Ebola/ SARS virus infection therapeutics [electronic resource].

Wang, Tianhua.
175 p.
Chemical engineering
Local subjects:
Health Sciences, Pharmacology.
Engineering, Chemical.
Biology, Virology.
Penn dissertations -- Chemical and biomolecular engineering.
Chemical and biomolecular engineering -- Penn dissertations.
System Details:
Mode of access: World Wide Web.
Two compounds, oxocarbazate (IC50 = 7 nM) and thiocarbazate (IC50 =56 nM) have been identified and synthesized as potent inhibitors of cathepsin L from past HTS campaign. The mode of action of the two compounds has been characterized both as slowly reversible, competitive inhibition. Through protease activity profiling assay, the two compounds demonstrated high selectivity in inhibiting cathepsin L. Oxocarbazate demonstrated potency in blocking the pseudovirus infection with an IC50 = 193 nM against Ebola and IC50 = 273 nM against SARS pseudovirus. On the contrary, thiocarbazate did not block the infection at concentration as high as 10 µM. Intracellular labeling assay was performed using DCG-04 probe to check for interaction of the two compounds with cathepsin L in HEK 293T cells. The result is consistent with the demonstrated potency in blocking pseutovirus infection.
Microarray screening of a printed chemical library of pharmacologically active compounds against human cathepsin L, in the absence of reducing agents such as dithiothreitol (DTT) or cysteine, allowed the discovery of disulfiram (DSF) as a potent human cathepsin L inhibitor. Reducing agent in cathepsin assay buffer can typically mask the activity of DSF against Cathepsin L. Under an attenuated reducing assay condition with only 20 nM of cysteine present, DSF inhibited Cathepsin L (IC50 = 0.16 µM) and was 6.7, 9.8, and 306-fold more potent against CatL than against cathepsin S, B, and K, respectively. DSF inhibited Ebola pseudovirus (IC50 = 5.3 µM) and SARS pseudovirus (IC50 = 20.1 µM) transduction of HEK 293T cells, while DSF had no effect on transduction by pseudovirus expressing Vesicular Stomatitis Virus glycoprotein which, unlike SARS an Ebola virus, does not require cathepsin L processing for virus entry. For mouse intranasally infected with human SARS-CoV strain Toronto-2, DSF treatment caused significant reduction of virus titer in the lung tissue 2 days post infection compared to saline-treated controls.
87,236 compounds from five compound libraries were screened at 16 µM. From this primary screen, 283 hits have been identified. 57 compounds were later cherry picked from the compound source plate and 25 compounds were confirmed to be active with IC50 below 10 µM. 16 compounds were again selected for repurchase from the vendor and upon confirmation test, 10 compounds were shown to be active Meanwhile, all 16 compounds repurchased from the vendor were subjected to the pseudovirus transduction assay to test its activity in blocking the virus entering 293T cells. PC-1087933, a benzamide and PC-1101019, a diazepane were identified to have virus blocking effect.
In an attempt of virtual screening compound libraries to search for cathepsin L inhibitors, energy optimized pharmacophore (e-pharmacophore) method is used to develop pharmacophore hypotheses derived from protein-ligand complexes generated from docking study. In a validation run, this e-pharmacophore search did successfully identify the hydrazinecarbothioamide series from chembridge10K compound library. Celera library of 40K compounds were also screened using e-pharmacophore approach as described. Experimental confirmation of the top hits is needed to further validate this approach.
Thesis (Ph.D. in Chemical and Biomolecular Engineering) -- University of Pennsylvania, 2012.
Source: Dissertation Abstracts International, Volume: 73-10(E), Section: B.
Adviser: Scott L. Diamond.
Local notes:
School code: 0175.
Greenbaum, Doron committee member
Lazzara, Matthew J. committee member
Radhakrishnan, Ravi committee member
Diamond, Scott L., advisor
University of Pennsylvania. Chemical and Biomolecular Engineering.
Contained In:
Dissertation Abstracts International 73-10B(E).
Access Restriction:
Restricted for use by site license.
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