Biology subjects

Stokes, L.

Publications and source records attributed to Stokes, L..

4 recordsLinked to original sources

bioRxiv2025

Microtubule end stabilisation by cooperative oligomers of Ska and Ndc80 complexes

During mitosis, properly aligned chromosomes stabilise microtubule ends with the help of kinetochores to ensure timely segregation of chromosomes. Microtubule-binding components of the human outer kinetochore, such as Ndc80 and Ska complexes, are present in multiple copies and together bind several microtubule ends, creating a highly multivalent binding interface. Whereas Ndc80:Ndc80 and Ndc80:microtubule binding is crucial for interface stability, Ndc80 alone in absence of Ska is unable to support stable kinetochore-attachments. Using cryoET, we demonstrate that oligomeric Ndc80:Ska assemblies stabilise microtubule ends against shortening by strengthening lateral contacts between tubulin protofilaments at microtubule plus-ends. We further identify a point mutation within the SKA1 microtubule-binding domain that does not affect microtubule-binding of individual Ska molecules, but does abolish Ska:Ska interactions. Finally, we report that oligomerisation of Ska, in a cooperative fashion together with the Ndc80, is necessary to maintain stable microtubule attachments both in vivo and in vitro.

biochemistry↗

bioRxiv2025

On-cell Saturation Transfer Difference (STD) NMR on ion channels: characterizing negative allosteric modulator binding interactions of P2X7.

P2X7 receptors are important drug targets involved in pathologies ranging from psychiatric disorders to cancer. Being membrane embedded receptors, they are challenging for structural characterisation and at present we only have a small number of X-ray and cryoEM structures for P2X7 bound to antagonists. We demonstrate that Saturation Transfer Difference (STD) NMR on live mammalian cells (on-cell STD NMR) overexpressing P2X7 receptors allows further structural insight on the complexes of P2X7 with two potent negative allosteric modulators, namely AZ10606120 and JNJ-47465567, via the determination of the binding epitope mapping of the interactions e.g. the main region of contact between the ligand and the binding pocket. This approach, reported for the first time on membrane-embedded ion channels, in combination with molecular docking, allows us to propose the first NMR-validated 3D molecular models for two antagonists as bound to human P2X7 receptors, and to correlate the structural knowledge acquired with the pharmacology data. We highlight the transformative potential of this application to aid drug design efforts in a less resource-demanding fashion than X-ray crystallography and cryo-EM and we envisage on-cell STD NMR to fast become an asset for structure-activity-relationship studies helping knowledge-based development of efficient drugs targeting P2X7 and other ion channels/membrane-embedded proteins.

pharmacology and toxicology↗

bioRxiv2023

Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

bioengineering↗

bioRxiv2022

Modeling protected species distributions and habitats to inform siting and management of pioneering ocean industries: A case study for Gulf of Mexico aquaculture

Marine Spatial Planning (MSP) provides a process that uses spatial data and models to evaluate environmental, social, economic, cultural, and management trade-offs when siting ocean industries. Aquaculture is the fastest-growing food sector in the world. The U.S. has substantial opportunity for offshore aquaculture development given the size of its exclusive economic zone, habitat diversity, and variety of candidate species for cultivation. However, many protected species rely upon habitats that overlap with promising aquaculture areas. Siting surveys, farm construction, operations, and decommissioning can alter the habitat and behavior of animals in the vicinity of these activities. Vessel activity, underwater noise, and physical interactions between protected species and farms can potentially increase the risk of injury or cause direct mortality. In 2020, the U.S. Gulf of Mexico was identified as one of the first regions to be evaluated for offshore aquaculture opportunities as directed by a Presidential Executive Order. We developed a generalized scoring model for protected species data layers that captures vulnerability using species conservation status and demographic information. We applied this approach to data layers for eight species listed under the Endangered Species Act, including five species of sea turtles, Rices Whale, Smalltooth Sawfish, and Giant Manta Ray. We evaluated several methods for scoring (e.g., arithmetic mean, geometric mean, product, lowest scoring layer) and created a combined protected species data layer that was used within a multi-criteria decision-making modeling framework for MSP. The product approach for scoring provided the most logical ordering of and the greatest contrast in site suitability scores. This approach provides a transparent and repeatable method to identify aquaculture site alternatives with the least conflict with protected species. These modeling methods are transferable to other regions, to other sensitive or protected species, and for spatial planning for other ocean-uses.

scientific communication and education↗