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Biology subjects

Cowley, J.

Publications and source records attributed to Cowley, J..

4 recordsLinked to original sources

Reshaping the progranulin/sortilin interaction for targeted degradation of extracellular proteins

Targeted protein degradation (TPD) using PROteolysis TArgeting Chimeras (PROTACs) is a rapidly emerging therapeutic strategy for difficult-to-drug cytosolic proteins. PROTACs are heterobifunctional small molecules that bridge the target with an E3 ubiquitin ligase, destining it for degradation by the proteasome. They have the potential to be orally available and to act catalytically, switching the pharmacology from occupancy-driven to event-driven (1-3). Here we present a strategy for targeted degradation of extracellular proteins by reshaping the interaction between the broadly expressed lysosome sorting receptor sortilin and its ligand progranulin for engineering SORtilin-based lysosome TArgeting Chimeras (SORTACs). SORTACs induce ternary complex formation with the target and sortilin, followed by endocytosis and lysosomal degradation. SORTAC activity can be genetically encoded as demonstrated by converting an IgG binding nanobody to an IgG degrading nanobody or by chemical conjugation, enabling single step conversion of therapeutic antibodies from binding their target to driving its degradation. Importantly, using structure-based design, we generated small molecule SORTACs against the inflammatory cytokine TNFa with nanomolar range potency and with physicochemical properties like PROTACs. Our results demonstrate that SORTACs constitute a versatile and highly modular platform for rapid generation of degraders of in theory any extracellular target and with the potential to have wide impact in drug discovery.

biochemistry↗

A cell-based, spike protein binding assay highlights differences in antibody neutralising capacity for SARS-CoV-2 variants

The engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells and accordingly blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine-elicited serum antibodies. The emergence of SARS-CoV-2 variants necessitates the development of adaptable assays that can be applied to assess the effectiveness of therapeutics. Through testing of a range of recombinant spike proteins, we have developed a cell based, ACE2/spike protein binding assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell bound spike protein fluorescence. Using spike proteins from the original Wuhan SARS-CoV-2 virus, as well as the delta and omicron variants, we identify differential blocking activity of three monoclonal antibodies directed against the spike receptor binding domain. Importantly, biological activity in the spike binding assay translated to efficacy in a SARS-CoV-2 infection assay. Hence, the spike binding assay has utility to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable to quantify impact of antibodies against new and emerging SARS-CoV-2 variants.

cell biology↗

The role of oxidative stress in seed priming to improve germination and vigour

Seed priming improves germination, but responses vary with procedure and plant used, potentially from differential responses in oxidative pathways. This study targeted oxidative pathways in seed priming, using hydrogen peroxide (H2O2), antioxidant-related micronutrients (Zn, Se and Mn), or a combination, to modulate germination and plant growth characteristics of hemp and barley. Hemp tolerated higher H2O2 (1 M vs 0.125 M) and micronutrients (6-fold greater) concentrations than barley, with the combination treatment significantly increasing hemp germination but decreasing barley germination. Seed priming did not improve hemp germination under salt stress (200 mM NaCl), but the micronutrient treatment improved germination of barley under salt stress (100 mM NaCl). Histological staining showed that micronutrient-primed root tips accumulated less O2- in both non-stressed and salt-stressed conditions. We assessed seed priming benefits if grown in soil by measuring plant quality in three-week-old plants potted post-priming, but most quality factors were not significantly improved, except barley where shoot zinc content increased. In summary, seed priming with H2O2 and/or micronutrients increased germination in hemp, but only micronutrients increased barley germination. Future work will continue optimising the priming methodology and further investigate the role of oxidative stress in the observed responses.

plant biology↗

A modified Rayleigh-Plesset equation for a liquid-crystalline shelled microbubble

Premanufactured shelled microbubbles composed of a protein shell are currently licensed as ultrasound imaging contrast agents. Current research is focussing on using the protein shelled microbubbles as transportation mechanisms for localised drug delivery particularly in the treatment of various types of cancer. For the very first time, a theoretical model is developed for an incompressible, gas loaded shelled microbubble with a thin shell composed of a liquid-crystalline material. We show that liquid-crystalline shelled microbubbles exhibit significantly different physical characteristics from commercial protein shelled microbubbles such as Sonovue and Optison. The authors propose that these significantly different physical characteristics may enhance localised drug delivery. We use the technique of linearisation to predict the shelled microbubbles natural frequency and relaxation time. These physical parameters strongly influence sonoporation which is the mechanism that is used for localised drug delivery. The influence of the material properties of the shell on the natural frequency and relaxation time are discussed. We have discovered that liquid-crystalline shelled microbubbles have a relaxation time that is 10 times longer than Sonovue and Optison.

bioengineering↗