Biology subjects

Spring, D. R.

Publications and source records attributed to Spring, D. R..

7 recordsLinked to original sources

bioRxiv2025

Targeting a Pleckstrin Homology Domain with a Lysine-Reactive Cova-lent Binder

Brutons Tyrosine Kinase (BTK) is a validated target for haematological malignancies, with numerous FDA approved inhibitors on the market. Current therapies target the highly conserved ATP binding site and hence limit the therapeutic index given the sites highly conserved nature across the kinome. We explore a novel approach for BTK inhibition, by targeting the PH domain-mediated membrane recruitment and activation of BTK. We have identified a fragment which covalently labels a lysine in the inositol phosphate (PIP3) binding site. Fragment growth and an extensive structure-binding relationship study uncovered 27 crystal structures and a best-in-class analog, 24. Evaluation of pKa values of the targeted lysine in BTK and other PH domains suggests this as a more general approach to PH domain inhibition.

biochemistry↗

bioRxiv2023

Selective inhibitors of the Aurora A-TPX2 protein-protein interaction exhibit in vivo efficacy as targeted anti-mitotic agents

The protein kinase Aurora A, and its close relative, Aurora B, regulate human cell division. Aurora A is frequently overexpressed in cancers of the breast, ovary, pancreas and blood, provoking genome instability and resistance to anti-mitotic chemotherapy. Intracellular localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. Here, we have used fragment-based, structure-guided lead discovery to develop small-molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). These compounds act by novel mechanism compared to existing Aurora A inhibitors and they are highly specific to Aurora A over Aurora B. Our biophysically, structurally and phenotypically validated lead compound, CAM2602, exhibits oral bioavailability, favourable pharmacokinetics, pharmacodynamic biomarker modulation, and arrest of growth in tumour xenografts. Consistent with our original finding that Aurora A overexpression drives taxane-resistance in cancer cells, inhibition of Aurora A-TPX2 PPI synergizes with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

biochemistry↗

bioRxiv2023

A Recombinant Approach For Stapled Peptide Discovery YieldsInhibitors of the RAD51 Recombinase

Stapling is a macrocyclisation method that connects amino acid side chains of a peptide to improve its pharmacological properties. We describe an approach for stapled peptide preparation and biochemical evaluation that combines recombinant expression of fusion constructs of target peptides and cysteine-reactive divinyl-heteroaryl chemistry, as an alternative to solid-phase synthesis. We then employ this workflow to prepare and evaluate BRC-repeat-derived inhibitors of the RAD51 recombinase, showing that a diverse range of secondary structure elements in the BRC repeat can be stapled without compromising binding and function. Using X-ray crystallography, we elucidate the atomic-level features of the staple moieties. We then demonstrate that BRC-repeat-derived stapled peptides can disrupt RAD51 function in cells following ionising radiation treatment.

biochemistry↗

bioRxiv2022

LGR5 targeting molecules as therapeutic agents for multiple cancer types

Leucine-rich repeat-containing G-protein receptor 5 (LGR5) has been characterised as a stem cell and cancer stem cell marker. Previous analyses of LGR5 transcript levels indicate high level expression discriminates malignancies such as colorectal cancer (CRC) and pre-B acute lymphoblastic leukaemia (pre-B ALL) from healthy tissues suggesting LGR5 protein expression may provide a molecular handle for prognosis and treatment. We have developed highly specific, high affinity antibodies to the extracellular domain of human LGR5 (-LGR5) that detect high LGR5 protein levels in colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL. In contrast, there is low to undetectable levels of LGR5 protein in normal colon and rectal epithelia, liver, ovarian tissues, brain and immune cell types. LGR5 is rapidly internalised from the plasma membrane and trafficked to intracellular vesicular compartments including lysosomes. Treatment of high LGR5-expressing CRC and pre-B ALL cancer cell lines with an antibody-drug conjugate version of -LGR5 (-LGR5-ADC) lead to effective cell killing at nanomolar concentrations. Interventional treatment of pre-B ALL tumours with -LGR5-ADC in vivo led to rapid tumour attrition. We further demonstrated the therapeutic utility of humanised -LGR5 by using the corresponding scFv fragment for the generation of -LGR5 chimeric antigen receptors (CARs) and a Bispecific T cell Engager (BiTE). -LGR5-CAR-NK cells were effective at killing LGR5-expressing cells while -LGR5/-CD3 BiTEs induce T cell activation and killing of NALM6 cells by cytotoxic CD8+ T cells. Taken together, this study establishes -LGR5-based therapeutic modalities that effectively discriminate and target CRC, HCC and pre-B ALL tumour cells. One Sentence SummaryWe generated novel antibodies against the cancer cell marker LGR5, validated diagnostic use in prioritizing specific cancer types for targeting, and developed antibody-based therapeutics.

cell biology↗

bioRxiv2022

A unified in vitro to in vivo fluorescence lifetime screening platform yields amyloid β aggregation inhibitors

Inhibiting the aggregation of amyloid {beta} (1-42) is a promising strategy for the development of disease-modifying Alzheimers disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of >200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic "nanoFLIM" assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid {beta} self-assembly in vitro as well as in cellular and whole organism disease models.

molecular biology↗

bioRxiv2020

Formation of flavone-based wooly fibres by glandular trichomes of Dionysia tapetodes.

Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "wooly" farina. This contrasts with some related Primula which instead possess a powdery farina. Using a combination of cell biology and analytical chemical techniques, we provide a detailed insight of wooly farina formation by glandular trichomes that produce a mixture of flavone and substituted flavone derivatives, including hydroxyflavones. Conversely, our analysis show that the powdery form consist almost entirely of flavone. The wooly farina in D. tapetodes is extruded through specific sites at the surface of the glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle. The data is consistent with formation and thread elongation occurring from within the cell. The putative mechanism of wool thread formation and its stability is discussed.

plant biology↗

bioRxiv2019

GLP-1R is downregulated in beta cells of NOD mice and T1D patients

Glucagon-like peptide 1 (GLP-1) is produced by L cells in the small intestine in response to ingested glucose and increases insulin release from pancreatic beta cells by activation of its cognate receptor (GLP-1R). Stimulation of this receptor also contributes to increased beta cell survival and regeneration. We have found that pancreatic beta cells from Non Obese Diabetic (NOD) mice express significantly lower levels of GLP-1R than C57BL/6 mice, leaving the NOD beta cells with an impaired response to GLP-1 stimulation. The lower expression appears to be caused by accelerated degradation of GLP-1R in the beta cells, a process that can be reversed by inhibiting trafficking to the lysosome. Importantly, our results appear to translate to the human disease since we also observed significantly lower expression of the GLP-1R in pancreatic islets from donors with type 1 diabetes. These results suggest that beta cell physiology may play a role in susceptibility to autoimmune inflammation.

physiology↗