Biology subjects

Thompson, A. P.

Publications and source records attributed to Thompson, A. P..

3 recordsLinked to original sources

bioRxiv2026

Discovery of a molecular glue inhibitor that stabilises a non-productive Kalirin-Rac1 complex

Rho guanosine triphosphatases (GTPases) are molecular-switches implicated in neurodegenerative diseases, yet targeting them through competitive inhibition remains a challenge due to their high affinity for guanine nucleotides. Guanine nucleotide exchange factors (GEFs) catalyse GDP-to-GTP nucleotide exchange to activate GTPases, providing an alternative opportunity for GTPase modulation. Herein, we describe a complex-targeted strategy to inhibit nucleotide exchange with covalent molecular glues that engage the Kalirin-Rac1 GEF-GTPase complex at the nucleotide binding site are sequester the GEF Kalirin. Fragment hits were identified through XChem and in silico screening, and a fragment merging approach resulted in the generation of covalent inhibitors RS-009 and MC-278. Multiple analyses demonstrate our compounds inhibit nucleotide exchange both through competition with the nucleotides and by stabilising a ternary inhibitor-Rac1-Kalirin complex, thereby trapping Kalirin in a non-productive state and reducing GEF turnover. Biochemical selectivity screening and cellular activity-based protein profiling (ABPP) show that selectivity can be achieved across distinct GEF-GTPase complexes, which may result in improved spatiotemporal control over targeting the GTPase alone. This work provides evidence for targeting GTPase signalling via stabilization of the GEF-GTPase complex in a unique covalent molecular glue mechanism and provides the basis of a chemical probe or therapeutic.

biochemistry↗

bioRxiv2025

ProteinDJ: a high-performance and modular protein design pipeline

Leveraging artificial intelligence and deep learning to generate proteins de novo (a.k.a. synthetic proteins) has unlocked new frontiers of protein design. Deep learning models trained on protein structures can generate novel protein designs that explore structural landscapes unseen by evolution. This approach enables the development of bespoke binders that target specific proteins and domains through new protein-protein interactions. However, successful binder generation can suffer from low in silico success rates, often requiring thousands of designs and hundreds of GPU hours to obtain enough hits for experimental testing. While workstation implementations are available for binder design, these are limited in both scalability and throughput. There is a lack of efficient open-source protein design pipelines for high-performance computing (HPC) systems that can maximise hardware resources and parallelise the workflow efficiently. Here, we present ProteinDJ--an implementation of a synthetic protein design workflow that is deployable on HPC systems using the Nextflow portable workflow management system and Apptainer containerisation. It parallelises the workload across both GPUs and CPUs, facilitating generation and testing of hundreds of designs per hour, accelerating the discovery process. ProteinDJ is designed to be modular and includes RoseTTAFold Diffusion (RFdiffusion) or BindCraft for fold generation, ProteinMPNN or Full-Atom MPNN (FAMPNN) for sequence design, and AlphaFold2 or Boltz-2 for prediction and validation of designs and binder-target interfaces, with supporting packages for structural evaluation of designs. ProteinDJ democratises protein binder design through its robust and user-friendly implementation and provides a framework for future protein design pipelines. ProteinDJ is freely available at https://github.com/PapenfussLab/proteindj.

bioinformatics↗

bioRxiv2023

Fortuitous in vitro compound degradation produces a tractable hit against Mycobacterium tuberculosis dethiobiotin synthetase: a cautionary tale of what goes in, does not always come out

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347); here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified by in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when 1 was soaked into MtDTBS crystals, but the density was inconsistent with the structure of 1. Here we show the lactone of 1 hydrolyses to carboxylic acid 2 under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid 3. Crystals soaked directly with authentic 3 produced electron density that matched that of crystals soaked with presumed 1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled subsequent compound development into nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process. SynopsisA molecule flagged in an in silico docking screen against MtDTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme (MtDTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗