Biology subjects

Seitova, A.

Publications and source records attributed to Seitova, A..

10 recordsLinked to original sources

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

Kinetic characterization of three human DExD/H-box RNA helicases

Human DExD/H-box RNA helicases are ubiquitous molecular motors that unwind and rearrange RNA secondary structures in an ATP-dependent manner. These enzymes play essential roles in nearly all aspects of RNA metabolism. While their biological functions are well-characterized, the kinetic mechanisms remain relatively understudied in vitro. In this study, we describe the development and optimization of a bioluminescence-based assay to kinetically characterize three human RNA helicases: MDA5, LGP2, and DDX1. The assays were conducted using annealed 24-mer RNA (blunt-ended double-stranded RNA) or double-stranded RNA (ds-RNA) with a 25- nt 3' overhang. These findings establish a robust and high-throughput in vitro assay suitable for a 384-well format, enabling the discovery and characterization of inhibitors targeting MDA5, LGP2, and DDX1. This work provides a valuable resource for advancing our understanding of these helicases and their therapeutic potential in Alzheimers disease.

biochemistry↗

bioRxiv2024

Structural basis of TMPRSS11D specificity and autocleavage activation

Transmembrane Protease, Serine-2 (TMPRSS2) and TMPRSS11D are human proteases that enable SARS-CoV-2 and Influenza A/B virus infections, but their biochemical mechanisms for facilitating viral cell entry remain unclear. We demonstrate these proteases can spontaneously and efficiently cleave their own zymogen activation motifs, thereby activating their wider protease activity on other cellular substrates. We determined TMPRSS11D co-crystal structures in complexes with a native TMPRSS11D zymogen activation motif and with an engineered activation motif, providing insights into TMPRSS11D autocleavage activation and revealing unique regions of its substrate binding cleft. We further show that a protease inhibitor that underwent clinical trials for TMPRSS2-targeted COVID-19 therapy, nafamostat mesylate, was rapidly cleaved by TMPRSS11D and converted to low activity derivatives. These insights into human protease viral tropism and into liabilities with existing human serine protease inhibition strategies will guide future drug discovery campaigns for these targets.

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↗

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

Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-CoV-2 activation

Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-coronavirus-2 (SARS-CoV-2). Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) Protein, enabling virus-host membrane fusion and infection of the lungs. We present here an efficient recombinant production strategy for enzymatically active TMPRSS2 ectodomain enabling enzymatic characterization, and the 1.95 [A] X-ray crystal structure. To stabilize the enzyme for co-crystallization, we pre-treated TMPRSS2 with the synthetic protease inhibitor nafamosat to form a stable but slowly reversible (15 hour half-life) phenylguanidino acyl-enzyme complex. Our study provides a structural basis for the potent but non-specific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that will guide future generations of inhibitors to improve selectivity. TMPRSS2 cleaved recombinant SARS-CoV-2 S protein ectodomain at the canonical S1/S2 cleavage site and at least two additional minor sites previously uncharacterized. We established enzymatic activity and inhibition assays that enabled ranking of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.7 nM to 120 M and determination of inhibitor mechanisms of action. These results provide a body of data and reagents to support future drug development efforts to selectively inhibit TMPRSS2 and other type 2 transmembrane serine proteases involved in viral glycoprotein processing, in order to combat current and future viral threats. SUMMARY PARAGRAPHViruses hijack the biochemical activity of host proteins for viral invasion and replication. Transmembrane protease, serine-2 (TMPRSS2) is a surface-expressed protease implicated in the activation of influenza A, influenza B, and coronaviruses, including SARS-CoV-2, to drive efficient infection of the lungs1-5. TMPRSS2 is an attractive target for antiviral therapies, as inhibiting its proteolytic activity blocks efficient viral entry5,6. However, a structural and biochemical understanding of the protease has remained elusive and no selective inhibitors are available. We engineered on-demand activatable TMPRSS2 ectodomain and determined the 1.95 [A] X-ray crystal structure of the stabilized acyl-enzyme after treatment with nafamostat, a protease inhibitor under investigation as a COVID-19 therapeutic. The structure reveals unique features of the TMPRSS2 substrate recognition pocket and domain architecture, and explains the potent, but nonselective inhibition by nafamostat. TMPRSS2 efficiently cleaved the SARS-CoV-2 S protein at the canonical S1/S2 site as well as two minor sites previously uncharacterized. We further established a robust enzymatic assay system and characterized inhibition by two additional clinical protease inhibitors under study for COVID-19, camostat and bromhexine. Our results provide a body of data and reagents to enable ongoing drug development efforts to selectively inhibit TMPRSS2 and other TTSPs involved in viral glycoprotein processing, in order to combat current and future viral threats.

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↗

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↗