Biology subjects

Ewert, W.

Publications and source records attributed to Ewert, W..

4 recordsLinked to original sources

bioRxiv2023

Structural elucidation and antiviral activity of cathepsin L inhibitors with carbonyl and epoxide warheads

Emerging RNA viruses including SARS-CoV-2 continue to be a major threat around the globe. The cell entry of SARS-CoV-2 particles via the endosomal pathway involves the cysteine protease cathepsin L (CatL) among other proteases. CatL is rendered as a promising drug target in the context of different viral and lysosome-related diseases. Hence, drug discovery and structure-based optimization of inhibitors is of high pharmaceutical interest. We herein verified and compared the anti-SARS-CoV-2 activity of a set of carbonyl and succinyl-epoxide-based inhibitors, which have previously been identified as cathepsin inhibitors. Calpain inhibitor XII (CI-XII), MG-101 and CatL inhibitor IV (CLI-IV) possess antiviral activity in the very low nanomolar IC50 range in Vero E6 cells. Experimental structural data on how these and related compounds bind to CatL are however notably lacking, despite their therapeutic potential. Consequently, we present and compare crystal structures of CatL in complex with 14 compounds, namely BOCA (N-BOC-2-aminoacetaldehyde), CLI-IV, CI-III, CI-VI, CI-XII, the main protease -ketoamide inhibitor 13b, MG-101, MG-132 as well as E-64d (aloxistatin), E-64, CLIK148, CAA0225, TC-I (CID 16725315) and TPCK at resolutions better than 2 [A]. Overall, the presented data comprise a broad and solid basis for structure-guided understanding and optimization of CatL inhibitors towards protease drug development.

biochemistry↗

bioRxiv2021

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗

bioRxiv2020

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

biophysics↗

bioRxiv2019

Miswired enhancer logic drives translocation positive rhabdomyosarcoma

Core regularity transcription factors (CR TFs) define cell identity and lineage through an exquisitely precise and logical order during embryogenesis and development. These CR TFs regulate one another in three-dimensional space via distal enhancers that serve as logic gates embedded in their TF recognition sequences. Aberrant chromatin organization resulting in miswired circuitry of enhancer logic is a newly recognized feature in many cancers. Here, we report that PAX3-FOXO1 expression is driven by a translocated FOXO1 distal super enhancer (SE). Using 4C-seq, a technique detecting all genomic regions that interact with the translocated FOXO1 SE, we demonstrate its physical interaction with the PAX3 promotor only in the presence of the oncogenic translocation. Furthermore, RNA-seq and ChIP-seq in tumors bearing rare PAX translocations implicate enhancer miswiring is a pervasive feature across all FP-RMS tumors. HiChIP of enhancer mark H3K27ac showed extended connectivity between the distal FOXO1 SE and additional intra-domain enhancers and the PAX3 promoter. We show by CRISPR-paired-ChIP-Rx that PAX3-FOXO1 transcription is diminished when this network of enhancers is selectively ablated. Therefore, our data reveal a mechanism of a translocated hijacked enhancer which disrupts the normal CR TF logic during skeletal muscle development (PAX3 to MYOD to MYOG), replacing it with an infinite loop logic that locks rhabdomyosarcoma cells in an undifferentiated proliferating stage.

cancer biology↗