Biology subjects

Hubbard, R. E.

Publications and source records attributed to Hubbard, R. E..

4 recordsLinked to original sources

bioRxiv2025

P-Rex1 Limits the Agonist-Induced Internalisation of GPCRs Independently of its Rac-GEF Activity

The Rac-GEF P-Rex1 mediates GPCR signalling by activating the small GTPase Rac. We show here that P-Rex1 also controls GPCR trafficking. P-Rex1 inhibits the agonist-stimulated internalisation of the GPCR S1PR1 independently of its Rac-GEF activity, through its PDZ, DEP and IP4P domains. P-Rex1 also limits the agonist-induced trafficking of CXCR4, PAR4, and GLP1R, but does not control steady-state GPCR levels, nor the agonist-induced internalisation of the RTKs PDGFR and EGFR. P-Rex1 blocks the phosphorylation required for GPCR internalisation. P-Rex1 binds Grk2, both in vitro and in cells, but does not appear to regulate Grk2 activity. We propose that P-Rex1 limits the agonist-induced internalisation of GPCRs through its interaction with Grk2 to maintain high levels of active GPCR at the plasma membrane. Therefore, P-Rex1 plays a dual role in promoting GPCR responses, by controlling GPCR trafficking through an adaptor function as well as by mediating GPCR signalling through its Rac-GEF activity. HighlightsO_LIP-Rex1 controls GPCR trafficking, independently of its Rac-GEF activity C_LIO_LIP-Rex1 limits the agonist-induced internalisation of S1PR1, CXCR4, PAR4 and GLP1R C_LIO_LIP-Rex1 does not control steady-state GPCR levels, or PDGFR and EGFR trafficking C_LIO_LIP-Rex1 binds Grk2 and inhibits the phosphorylation required for GPCR internalisation C_LI eTOC blurbP-Rex1 activates Rac downstream of GPCRs to regulate processes ranging from innate immunity to neuronal plasticity, its deregulation contributing to cancer. Here, Baker et al. show that P-Rex1 also controls GPCR trafficking, limiting agonist-induced GPCR internalisation through an adaptor function. Thus, P-Rex1 promotes GPCR responses in a dual manner. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/648762v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@20e1e2org.highwire.dtl.DTLVardef@cdd416org.highwire.dtl.DTLVardef@11f2a35org.highwire.dtl.DTLVardef@10c2b49_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Expanding the scope of a catalogue search to bioisosteric fragment merges using a graph database approach

The efficiency of fragment-to-lead optimization could be improved by automated workflows for the design of follow-up compounds. Pipelines that are able to fully exploit the interaction opportunities identified from the crystal structures of bound fragments would greatly aid this goal. To do so, these pipelines need to require minimal intervention from the user and be computationally efficient. In this work, we describe an updated version of our fragment merging methodology, which provides several feature enhancements, primarily by expanding the chemical space searched, allowing the identification of more diverse follow-up compounds, thus maximizing the chances of finding successful hits. While the original method focused on finding perfect merges, meaning compounds that directly incorporate substructures from the original fragments, here we expand the search to what we term bioisosteric merges, involving the incorporation of substructures that replicate the pharmacophoric features of the original fragments but may not be exactly identical. Unlike existing pharmacophore and shape-based descriptors used for virtual screening, this approach combines the search for these properties with the incorporation of novelty, which is necessary when searching for ways to link together distinct substructures. Compared with perfect merging, our new approach is able to find compounds that are directly informed by structures within the original fragments but are more chemically diverse. We contrast our approach with the use of a pharmacophore-constrained docking pipeline, run in parallel for select fragment pairs, and show that our method requires between 1.1-45.9-fold less computational time for conformer generation per merging hit identified, referring to compounds that show a favourable degree of shape and colour overlap and recapitulation of original fragment interactions. Overall, our results show that our method has potential to be used to generate designs inspired by all fragments within a given pocket.

bioinformatics↗

bioRxiv2022

The use of a graph database is a complementary approach to a classical similarity search for identifying commercially available fragment merges

AO_SCPLOWBSTRACTC_SCPLOWFragment screening using X-ray crystallography can yield rich structural data to help guide the optimization of low-molecular-weight compounds into more potent binders. Fragment merging, whereby substructural motifs from partially overlapping fragments are incorporated into a single larger compound, represents a potentially powerful and efficient approach for increasing potency. Searching commercial catalogues provides one useful way to quickly and cheaply identify follow-up compounds for purchase and further screening, and circumvents the challenge of synthetic accessibility. The Fragment Network is a graph database that provides a novel way to explore the chemical space surrounding fragment hits. We use an iteration of the database containing >120 million catalogue compounds to find fragment merges for four XChem fragment screening campaigns. Retrieved molecules were filtered using a pipeline of 2D and 3D filters and contrasted against a traditional fingerprint-based similarity search. The two search techniques were found to have complementary results, identifying merges in different regions of chemical space. Both techniques were able to identify merges that are predicted to replicate the interactions made by the parent fragments. This work demonstrates the use of the Fragment Network to increase the yield of fragment merges beyond that of a classical catalogue search, thus increasing the likelihood of finding promising follow-up compounds. We present a pipeline that is able to systematically exploit all known fragment hits by performing large-scale enumeration of all possible fragment pairs for merging.

bioinformatics↗

bioRxiv2020

Multiplexed experimental strategies for fragment library screening using SPR biosensors

Surface plasmon resonance biosensor technology (SPR) is ideally suited for fragment-based lead discovery. However, generally suitable experimental procedures or detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are lacking. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. By adopting a multiplexed strategy, the range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded. We here illustrate innovative strategies using five challenging targets and variants thereof. Two libraries of 90 and 1056 fragments were screened using two different flow-based SPR biosensor systems, allowing different experimental approaches. Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted.

biophysics↗