Biology subjects

Loppnau, P.

Publications and source records attributed to Loppnau, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Chemically induced USP21-KLHL12 association recruits USP21 to COPII assembly

Ubiquitin-specific protease 21 (USP21) is a deubiquitinase implicated in various biological processes and disease states, including cancer. To better understand USP21 protein interaction networks, we used biotin identification proximity labeling, and uncovered USP21 interactions with the cytoskeleton, endosomal, and transcriptional regulators. Notably, a subset of these interactions was modulated by the USP21 selective inhibitor BAY-805. In cells, BAY-805 promoted proximity between USP21 and Kelch like protein KLHL12, a component of the Cullin3 ligase complex. Structural modeling using AlphaFold3, together with hydrogen deuterium exchange mass spectrometry, identified an orthosteric BAY-805 binding site within USP21. Binding of BAY-805 functionally competes with ubiquitin at USP21, thereby enabling USP21 interaction with KLHL12. This BAY-805 induced proximity elicited relocalization of USP21 to KLHL12 containing coat protein complex II (COPII) vesicles. Induced USP21-KLHL12 association led to enlargement of COPII vesicles and impaired transport of large cargoes such as collagen. Overall, our findings reveal a novel chemically induced proximity mechanism in which the monovalent ligand BAY-805 outcompetes ubiquitin from USP21, thereby recruiting USP21 to KLHL12 associated protein complexes.

cell biology↗

bioRxiv2026

Direct-to-Biology Enables Rapid Identification of Potent FBXO22 Degraders

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that bring a ubiquitin E3 ligase into proximity of a target protein to polyubiquitinate and degrade the target. PROTACs act catalytically, offering distinct advantages over conventional inhibitors and are the subject of intense study. The development of PROTACs involves extensive optimization of the chemical moiety linking two different protein-binding chemotypes, often requiring the synthesis, purification and testing of hundreds of PROTAC candidates. We used this approach to rapidly explore the landscape of targeted degradation of four different targets in parallel, combining and comparing a recently reported FBXO22-recruiting chemical warhead with warheads for the commonly used CRBN and VHL E3 ligases. Using a limited number of compounds (175 compounds in total) we observed no FBXO22-dependent degradation of these four targets. However, our libraries generated potent FBXO22 homo-PROTACs inducing self-degradation, as well as CRBN- and VHL-mediated degraders of FBXO22.

cell biology↗

bioRxiv2026

Hepatocyte growth factor activator inhibitor-2 rapidly inactivates airway-expressed human Type II Transmembrane Serine Proteases

Human Type II Transmembrane Serine Proteases (TTSPs) are essential entry factors for various influenza A and coronaviruses, and drive cancer metastasis when they are overexpressed by tumor cells. However, the natural inhibition mechanisms that regulate these proteases are not well understood. One natural transmembrane protease inhibitor, hepatocyte growth factor activator inhibitor-2 (HAI-2), has been shown to block TMPRSS2 activity and can prevent SARS-CoV-2 infection and reduce TMPRSS2-driven prostate cancer metastasis when overexpressed. In this study, we present biochemical and biophysical evidence showing that HAI-2 effectively inactivates TMPRSS2 and other TTSPs only after they have undergone zymogen activation. Through mutagenesis and ligand binding assays, we demonstrate that Kunitz Domain 1 (KD1) and KD2 can form stable ternary complexes with TMPRSS2 and other TTSPs, but do not employ the typical Laskowski inhibitor mechanism found for other macromolecular serine protease inhibitors. We also show that HAI-2 proteins do not inhibit the coagulation protease thrombin and that multivalent human IgG-tagged HAI-2 proteins are highly potent TMPRSS2 inhibitors. Our findings provide a mechanistic understanding of how TTSP activity is regulated in human airway cells and offer a foundation for developing engineered soluble HAI-2 proteins as anti-TTSP antivirals and anti-cancer therapeutics.

biochemistry↗

bioRxiv2025

Structure-based discovery of inhibitors of Mac1 domain of nonstructural protein-3 of SARS-CoV-2 by machine learning-augmented screening of chemical space

Significant efforts have been recently dedicated to the discovery of small molecule inhibitors against the Macrodomain 1 (Mac1) of nonstructural protein 3 (NSP3) as potential antivirals for SARS-CoV-2. Thus, Mac1 has also been selected as the target for the Critical Assessment of Hit-finding Experiments (CACHE) challenge #3. As contestants in that challenge, we developed a computational strategy that ranked on the top among all 23 participants in the competition and resulted in the discovery of a novel chemical series of non-charged Mac1 inhibitors. Those have been identified through the combination of machine learning-accelerated virtual screening of Enamine REAL Diversity Subset of approximately 25 million compounds and consequent hit expansion into the entire Enamine REAL Space library. In particular, the initially identified hit compound CACHE3-HI_1706_56 (KD = 20 {micro}M) was explored by probing 17 close analogues from a library of 44 billion molecules from the Enamine REAL. All those analogues effectively displaced the Mac1-binding ADP-ribose peptide, and 12 were confirmed to engage with Mac1 by the Surface Plasmon Resonance experiments, revealing a new chemical series of compounds for hit-to-lead optimization. The structure of the CACHE3-HI_1706_56-Mac1 complex was further determined at high resolution with crystallography, confirming initial computational predictions. Our results illustrate the effectiveness of ML-accelerated docking to rapidly identify novel chemical series and provide a strong foundation for the development of SARS-CoV-2 NSP3 Mac1 inhibitors.

biophysics↗

bioRxiv2025

Identification of Direct-acting nsP2 Helicase Inhibitors with Anti-alphaviral Activity

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/641060v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@856df5org.highwire.dtl.DTLVardef@1f6225borg.highwire.dtl.DTLVardef@4997d5org.highwire.dtl.DTLVardef@18f42f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

ABHD2 activity is not required for the non-genomic action of progesterone on human sperm

Study questionIs the hydrolase ABHD2 required for progesterone-induced Ca2+ influx via CatSper and the resulting motility responses in human sperm? Summary answerProgesterone-induced Ca2+ influx via CatSper and the resulting motility responses in human sperm do not require ABHD2 activity. What is known alreadySperm motility is tightly regulated by signalling pathways that are activated as sperm ascend the female reproductive tract, including progesterone triggering Ca2+ influx via the CatSper channel and inducing hyperactivated motility needed for fertilization. This process is thought to involve ABHD2, which may hydrolyse the endogenous CatSper inhibitor 2-arachidonoylglycerol (2-AG), thereby relieving inhibition and enabling calcium entry into the flagellum. Study design, size, durationPotent small molecule inhibitors of ABHD2 activity were synthesized, characterized, and used as tools to scrutinize the role of ABHD2 in activation of CatSper and regulation of sperm motility. Participants/materials, setting, methodsDerivatives of published ABHD2 inhibitors were optimised for in vitro potency and cellular activity and subsequently tested in human sperm motility and Ca2+ influx assays. Main results and the role of chanceProgesterone does not activate ABHD2 in vitro. In addition, inhibition of ABHD2 in human sperm has no effect on progesterone-induced Ca2+ influx through CatSper nor on basal or progesterone-induced hyperactivated motility. This demonstrates that ABHD2 activity is, in fact, not required for the non-genomic action of progesterone on human sperm. Large scale datanone Limitations, reasons for cautionWe examined the effects of inhibition of the enzymatic activity of ABHD2. We cannot exclude that ABHD2 functions as a part of a larger multiprotein complex, in which it may play a structural role independent of its hydrolase activity. Wider implications of the findingsThis study presents conclusive evidence that ABHD2 does not bind progesterone and that its hydrolase activity is not required for progesterone activation of CatSper and resulting changes in motility of human sperm. These results highlight the need for further research to elucidate the mechanism underlying the non-genomic action of progesterone on human sperm. Study funding/competing interest(s)This publication is based on research funded by the Gates Foundation. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Gates Foundation. LT, CB, and TS were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - project numbers 329621271 (CRU326; CB, TS), and 404595355 (Research Training Group "Chemical biology of ion channels (Chembion); LT, TS). This work was funded by the German Federal Ministry of Education and Research (BMBF) within the framework of Contraception Research, grant number 01GR2501A and 01GR2502A. A.A., K.V., A.T., N.D., A.H., M.W. and R.L. are employees of Nuvisan ICB GMBH, Berlin, Germany. Nuvisan is recipient of a Gates Foundation grant. A.A is associate editor of Human Reproduction Open.

cell biology↗

bioRxiv2024

Identification of Inhibitors of Chikungunya virus nsP2 ATPase

Non-structural protein 2 (nsP2), which plays an essential role in replication of CHIKV, contains a protease, helicase, and methyltransferase-like domain. We executed a simple a screen using malachite green to detect compounds that decreased ATP hydrolysis and tested a library of diverse compounds to find inhibitors of CHIKV nsP2 helicase.

biochemistry↗

bioRxiv2024

A resource to enable chemical biology and drug discovery of WDR Proteins

Protein class-focused drug discovery has a long and successful history in pharmaceutical research, yet most members of druggable protein families remain unliganded, often for practical reasons. Here we combined experiment and computation to enable discovery of ligands for WD40 repeat (WDR) proteins, one of the largest human protein families. This resource includes expression clones, purification protocols, and a comprehensive assessment of the druggability for hundreds of WDR proteins. We solved 21 high resolution crystal structures, and have made available a suite of biophysical, biochemical, and cellular assays to facilitate the discovery and characterization of small molecule ligands. To this end, we use the resource in a hit-finding pilot involving DNA-encoded library (DEL) selection followed by machine learning (ML). This led to the discovery of first-in-class, drug-like ligands for 9 of 20 targets. This result demonstrates the broad ligandability of WDRs. This extensive resource of reagents and knowledge will enable further discovery of chemical tools and potential therapeutics for this important class of proteins.

biochemistry↗

bioRxiv2023

Discovery of a first-in-class small molecule ligand for WDR91 using DNA-encoded chemical library selection followed by machine learning

WD40 repeat-containing protein 91 regulates endosomal phosphatidylinositol 3-phosphate levels at the critical stage of endosome maturation and plays vital roles in endosome fusion, recycling, and transport by mediating protein-protein interactions. Due to its various roles in endocytic pathways, WDR91 has recently been identified as a potential host factor responsible for viral infection. We employed DNA-Encoded Chemical Library (DEL) selection against the WDR domain of WDR91, followed by machine learning to generate a model that was then used to predict ligands from the synthetically accessible Enamine REAL database. Screening of predicted compounds enabled us to identify the hit compound 1, which binds selectively to WDR91 with a KD of 6 {+/-} 2 M by surface plasmon resonance. The co-crystal structure confirmed the binding of 1 to the WDR91 side pocket, in proximity to cysteine 487. Machine learning-assisted structure activity relationship-by-catalog validated the chemotype of 1 and led to the discovery of covalent analogs 18 and 19. Intact mass LC-MS and differential scanning fluorimetry confirmed the formation of a covalent adduct, and thermal stabilization, respectively. The discovery of 1, 18, 19, accompanying SAR, and co-crystal structures will provide valuable insights for designing more potent and selective compounds against WDR91, thus accelerating the development of novel chemical tools to evaluate the therapeutic potential of WDR91 in disease.

molecular biology↗

bioRxiv2023

Probing the DCAF12 interactions with MAGEA3 and CCT5 C-terminal degrons

DCAF12 is the substrate recognition component of the CRL4 E3 ligase complex that can recognize C-terminal double-glutamic acid degrons to promote degradation of its cognate substrates via the ubiquitin proteasome system. MAGEA3 and CCT5 proteins were reported to be cellular targets of DCAF12. To further characterize the DCAF12 interactions with both MAGEA3 and CCT5, we developed a suite of biophysical and a proximity-based cellular NanoBRET assays showing that both MAGEA3 and CCT5 C-terminal degron peptides interact with DCAF12 in nanomolar affinity in vitro and in cells. Furthermore, we report here the 3.17 [A] cryo-EM structure of DCAF12-DDB1-MAGEA3 complex revealing the key DCAF12 residues involved in C-terminal degron recognition and binding. Our study provides new tools and resources to enable the discovery of small molecule handles targeting the WDR domain of DCAF12 for future PROTAC design and development.

biochemistry↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

bioRxiv2022

Expanding the Huntingtons disease research toolbox; validated huntingtin subdomain constructs for biochemical and structural investigation of the huntingtin protein

Huntingtons disease is characterised by CAG expansion in the huntingtin gene above a critical threshold of ~35 repeats, resulting in polyglutamine expansion of the huntingtin protein (HTT). The biological role of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part, due to challenges characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of rationally designed, high-quality recombinant HTT subdomains; one spanning the N-terminal HEAT and bridge domains (NTD) and the second spanning the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with 40 kDa HTT-associated protein (HAP40). We report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD-HAP40 complex binds the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validate the interaction between the CTD and HAP40 using a luciferase two-hybrid assay and use subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will enable the wider HD community to study fundamental HTT biology, discover new macromolecular or small-molecule binding partners and map interaction sites across this very large protein.

biochemistry↗

bioRxiv2021

HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Huntingtons disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 [A] cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntingtons disease.

biochemistry↗

bioRxiv2021

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 {+/-} 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 {+/-} 0.2 M showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

biochemistry↗

bioRxiv2020

A high throughput RNA displacement assay for screening SARS-CoV-2 nsp10-nsp16 complex towards developing therapeutics for COVID-19

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in 384-well format with a Z'-Factor of 0.6, suitable for high throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, product of the reaction, SAH, and a common methyltransferase inhibitor, sinefungin using Isothermal Titration Calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high throughput method for screening nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.

biochemistry↗

bioRxiv2019

Structural basis for EPC1-mediated recruitment of MBTD1 into the NuA4/TIP60 acetyltransferase complex

MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.

biochemistry↗

bioRxiv2018

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

The gene mutated in Huntingtons disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.

biochemistry↗

bioRxiv2018

Structural and histone binding studies of the chromo barrel domain of TIP60

TIP60 consists of an N-terminal chromo barrel domain (TIP60-CB) and a C-terminal acetyltransferase domain and acetylates histone and non-histone proteins within diverse cellular processes. Whereas the TIP60-CB is thought to recognize histone tails, molecular details of this interaction remain unclear. Here we attempted a quantitative analysis of the interaction between the TIP60-CB and histone peptides, but did not observe any binding through either fluorescence polarization or isothermal titration calorimetry. We solved a crystal structure of the TIP60-CB alone. Analysis of the crystal structure demonstrates a putative peptide binding site that may be occluded by the basic side chain of a residue in a unique {beta} hairpin between the two N-terminal strands of the {beta} barrel.

biochemistry↗