Biology subjects

Briggs, D. A.

Publications and source records attributed to Briggs, D. A..

4 recordsLinked to original sources

bioRxiv2026

Discovery of stereoselective targeted covalent inhibitors of the RAB27-effector protein-protein interaction

RAB27A and RAB27B are homologous small GTPases that regulate intracellular vesicle trafficking, orchestrating endocytic and exocytic processes that affect cellular communication, immune responses, and dynamics of the cellular microenvironment. Through their interactions with effector proteins, RAB27A/B play roles in tumor metastasis and chronic inflammation. However, pharmacological modulation of their activity faces challenges typical of small GTPases, including a lack of well-defined pockets outside the conserved GTP binding site, and large RAB27-effector protein-protein interaction (PPI) surfaces. Here, we present the discovery and development of the first cell-active, rationally designed covalent inhibitors of the RAB27-effector interaction, targeting a non-conserved cysteine residue flanking the PPI interface. An electrophile-first biochemical screen led to a novel class of acrylamide covalent inhibitors, and X-ray crystallography structure-guided design led to optimized inhibitors and probes that enantioselectively target RAB27A/B-Cys123 in cells. Potency and selectivity were confirmed through biochemical and cellular assays, including chemical proteomics and phenotype recapitulation in melanocytes alongside a matched inactive enantioprobe control. In contrast, a previously reported compound, Nexinhib-20, was found to be toxic and to exert its activity through non-selective reactivity. This work provides the first toolbox of cell-active chemical probes for RAB27 which can be used in future studies to shed light on the function of this protein and its potential as a therapeutic target.

biochemistry↗

bioRxiv2023

A time-resolved Forster resonance energy transfer assay to investigate inhibitor binding to ABCG2

The human ATP-binding cassette (ABC) transporter, ABCG2 is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Forster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps. HighlightsO_LIABCG2 is a multidrug pump which moves between states having low or high affinity for substrates and inhibitors C_LIO_LIWe introduce a time-resolved Forster resonance energy transfer assay to measure interaction of substrates and inhibitors to ABCG2 C_LIO_LIWe confirm that NBD dimerization is associated with a switch from a high to a low affinity site for an ABCG2 inhibitor C_LI

biochemistry↗

bioRxiv2019

Animal evolution coincides with a novel degree of freedom in exocytic transport processes

Exocytic transport of transmembrane receptors and secreted ligands provides the basis for cellular communication in animals. The RAB8/RAB3/RAB27 trafficking regulators function in transport processes towards the cell membrane. The small G-proteins recruit a diversity of effectors that mediate transport along microtubule and actin tracks, as well as membrane tethering and fusion. SPIRE actin nucleators organise local actin networks at exocytic vesicle membranes. By complex formation with class-5 myosins, vesicle transport track generation and motor protein activation are coordinated. Our phylogenetic analysis traced the onset of SPIRE function back to the origin of the Holozoa. We have identified SPIRE in the closest unicellular relatives of animals, the choanoflagellates, and the more distantly related ichthyosporeans. The discovery of a SPIRE-like protein encoding a KIND and tandem-WH2 domains in the amoebozoan Physarum polycephalum suggests that the SPIRE-type actin nucleation mechanism originated even earlier. Choanoflagellate SPIRE interacts with RAB8, the sole choanoflagellate representative of the metazoan RAB8/RAB3/RAB27 family. Major interactions including MYO5, FMN-subgroup formins and vesicle membranes are conserved between the choanoflagellate and mammalian SPIRE proteins and the choanoflagellate Monosiga brevicollis SPIRE protein can rescue mouse SPIRE1/2 function in melanosome transport. Genome duplications generated two mammalian SPIRE genes (SPIRE1 and SPIRE2) and allowed for the separation of SPIRE protein function in terms of tissue expression and RAB GTPase binding. SPIRE1 is highest expressed in the nervous system and interacts with RAB27 and RAB8. SPIRE2 shows high expression in the digestive tract and specifically interacts with RAB8. We propose that at the dawn of the animal kingdom a new transport mechanism came into existence, which bridges microtubule tracks, detached vesicles and the cellular actin cytoskeleton by organising actin/myosin forces directly at exocytic vesicle membranes. The new degree of freedom in transport may reflect the increased demands of the sophisticated cellular communications in animals.

cell biology↗

bioRxiv2018

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

cell biology↗