Biology subjects

Falke, S.

Publications and source records attributed to Falke, S..

9 recordsLinked to original sources

bioRxiv2026

The structures of two intermediates en route to DNA repair within a (6-4) photolyase

Pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are mutagenic DNA lesions produced when UV light crosslinks two adjacent pyrimidine bases. These lesions must be repaired to maintain genome integrity, a function executed by the (6-4) photolyase enzymes. This repair chemistry is complex, involving the transfer of a hydroxyl group from one base to another, accompanied or followed by cleavage of an interbase bond. It remains unclear how the enzyme orchestrates this reaction. Typically, models invoke a stepwise process in which an oxetane intermediate is transiently populated, but despite over thirty years of investigation, such an intermediate has never been definitively observed. Conducting dose-controlled X-ray crystallography on the (6-4) photolyase from Vibrio cholerae, we observed the transformation of 6-4PP into an oxetane species. Furthermore, we identified a previously unanticipated hemi-cleaved intermediate. The progressive transformation of 6-4PP into oxetane, hemi-cleaved, and fully repaired species requires a reexamination of the existing models of DNA repair by (6-4) photolyases.

biochemistry↗

bioRxiv2026

FoXSight: Facilitating Computational Hit Identification in Crystallographic Fragment Screening Experiments

X-ray crystallographic fragment screening reveals how small molecules bind to proteins, providing important starting points for drug design. Identifying these weak binding events remains difficult when electron density signals are noisy, partially occupied or obscured by solvent, and campaigns may comprise thousands of datasets. Here, we present FoXSight, a computational method for hit identification in crystallographic compound screening. FoXSight applies Difference-of-Gaussian (DoG) filtering to multi-reference subtraction maps to detect recurring, target-specific density features associated with bound ligands. Its output maps remain visually interpretable and support subsequent ligand modeling. We evaluated FoXSight on in-house and public datasets spanning seven protein targets, comparing its maps with PanDDA where suitable comparison data were available. FoXSight was successfully used as the primary hit identification tool in a challenging crystallographic fragment screening campaign for human Cathepsin L, where the output of established methods was impractical to inspect systematically.

bioinformatics↗

bioRxiv2024

SARS-CoV-2 methyltransferase nsp10-16 in complex with natural and drug-like purine analogs for guiding structure-based drug discovery

Non-structural protein 10 (nsp10) and non-structural protein 16 (nsp16) are part of the RNA synthesis complex, which is crucial for the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nsp16 exhibits 2-O-methyltransferase activity during viral messenger RNA capping and is active in a heterodimeric complex with enzymatically inactive nsp10. It has been shown that inactivation of the nsp10-16 protein complex interferes severely with viral replication, making it a highly promising drug target. As information on ligands binding to the nsp10-16 complex (nsp10-16) is still scarce, we screened the active site for potential binding of drug-like and fragment-like compounds using X-ray crystallography. The screened set of 234 compounds consists of derivatives of the natural substrate S-adenosyl methionine (SAM) and adenine derivatives, of which some have been described previously as methyltransferase inhibitors and nsp16 binders. A docking study guided the selection of many of these compounds. Here we report structures of binders to the SAM site of nsp10-16 and for two of them, toyocamycin and sangivamycin, we present additional crystal structures in the presence of a second substrate, Cap0-analog/Cap0-RNA. The identified hits were tested for binding to nsp10-16 in solution and antiviral activity in cell culture. Our data provide important structural information on various molecules that bind to the SAM substrate site which can be used as novel starting points for selective methyltransferase inhibitor designs.

biochemistry↗

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↗

bioRxiv2022

Biomolecular Tau condensation is linked to Tau accumulation at the nuclear envelope

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, this synergy enhances bundling and spatially arranges microtubules. We further show that different Tau condensates efficiently induce pathological Tau in cells, including small accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations, and a condensate-like density for nuclear envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

molecular biology↗

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↗

bioRxiv2020

Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

biophysics↗

bioRxiv2019

Processing of the SARS-CoV pp1a/ab nsp7-10 region

1.1.Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex (RTC), essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsps. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2:2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

biophysics↗