Thomas Helleday

T cells are central drivers of inflammation across autoimmune and inflammatory diseases, yet current therapies inadequately target pathogenic T-cell pathways, limiting durable disease control. Here, we identified a novel, targetable transcriptional-metabolic axis that sustains inflammatory T-cell responses, characterized by NFATc1-regulated activation of MTHFD2-dependent one-carbon metabolism. We demonstrate that NFATc1 directly binds the MTHFD2 promoter region, driving metabolic reprogramming in activated T cells from rheumatoid arthritis (RA) patients as well as in experimental arthritis models. Pharmacological inhibition of MTHFD1/2 using the novel small molecule TH9619 suppresses proinflammatory cytokine production, expands Foxp3⁺ regulatory T cells and protects against cartilage and bone damage in vivo. Proteomic profiling reveals that TH9619 elicits a distinct molecular response in patients’ T cells, divergent from the currently used anti-folate therapy, particularly in inadequate responders. These findings use RA as the proving ground to establish NFATc1-mediated MTHFD2 activation as a critical regulator of sustained T-cell-driven inflammation and support selective MTHFD1/2 inhibition as a novel, mechanism-based therapeutic strategy for RA.

Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.

Mitochondrial dysfunction is a major component or driver of neurodegeneration and metabolic disease. The mitochondrial genome is prone to oxidative damage and thus repair proteins are important to maintain mitochondrial DNA integrity. To initiate base excision repair, 8-oxoguanine DNA glycosylase 1 recognizes and binds 8-oxoguanine with high affinity in double-stranded DNA in nuclear and mitochondrial genomes. OGG1 has been proposed as a therapeutic target in conditions of mitochondrial dysfunction. Herein, we report the discovery of mitochondrial selective organocatalytic switches, or short ORCAs, of OGG1, that bind the active site of the enzyme and stimulate its intrinsic, but weak, AP-lyase activity. We demonstrate that ORCAs protect mitochondrial function and morphology under oxidative stress, specifically affecting mitochondrial membrane potential and mitochondria fragment number in the cell whilst attenuating metabolic stress. Mechanistically, ORCAs significantly improved NAD levels in mouse dissociated cortical neurons and provided neuroprotection in a retina axotomy model. Together, the presented findings position OGG1-ORCAs as potential novel therapeutic compounds for the treatment of age-related diseases and neurodegeneration.

The base excision repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) plays a central role in maintaining genome integrity and mediating cellular responses to oxidative stress. As such, it represents an attractive target for pharmaceutical modulation. Small-molecule organocatalytic switches (ORCAs) greatly enhance the rate of OGG1-catalyzed cleavage of DNA abasic sites, thereby accelerating DNA repair. Here, we present the discovery and hit-to-lead optimization of a novel class of highly potent serotonin-derived ORCAs with greatly improved pharmacokinetic properties. Biochemical assays, X-ray crystallography, and molecular dynamics simulations point toward a water-mediated mechanism of activation, distinct from previously proposed Brønsted base-assisted models. These findings establish serotonin-based ORCAs as promising chemical probes and potential leads for therapeutic modulation of OGG1 in oxidative stress-driven diseases.

Noncanonical amino acids (NCAAs) expand the chemical diversity of peptides beyond the twenty standard residues, offering new opportunities for designing binders with novel interaction modes and functional activity. G protein-coupled receptors (GPCRs) are central to cellular signalling and represent one of the largest classes of therapeutic targets, yet their functional modulation remains challenging. Here, we present RareFoldGPCR (RFG), a GPCR-specialised AI model for structure prediction and design that supports NCAAs. By applying transfer learning on high-resolution GPCR structures, RFG accurately models and rationally designs both linear and cyclic peptides that incorporate NCAAs and modulate GPCR activity. This is achieved without the model ever being trained on NCAA-based GPCR modulators. We showcase the capability of RFG by designing peptide agonists for the glucagon-like peptide-1 receptor (GLP1R) and validating their functional activity experimentally in cell-based assays. We investigate the precise capabilities of generating active agonists by expanding different regions of the native GLP-1 hormone, and further demonstrate the design of cyclic peptide agonists with entirely novel sequences and topologies, creating new agonist modes. We analyse how design metrics relate to pathway specificity, enabling precise modulation of pathway activity, such as activating the cAMP response without recruiting β-arrestin to reduce receptor desensitisation. RFG shows how transfer learning on specific target classes enables generalisation to new chemistry and molecular topology, providing a broadly applicable strategy for designing functional ligands beyond the constraints of natural amino acid chemistry. RFG is freely available: https://github.com/patrickbryant1/RareFoldGPCR

Human MutT homolog 1 (hMTH1) removes damaged nucleotides from the nucleotide pool, preventing their incorporation into DNA. Due to its potential as an anticancer drug target, hMTH1 has been the focus of several inhibitor development studies. Unexpectedly, we show that the anabolic steroid stanozolol (Stz) is a potent nanomolar inhibitor of hMTH1. We present the structure of hMTH1 in complex with Stz, which indicates a unique core scaffold that could be exploited for future inhibitor development. Comparison with human protein structures bound with dihydrotestosterone (DHT) shows hMTH1 is entirely unrelated in terms of its structure. As these DHT binding proteins are all involved in steroid regulation, this makes the identification of Stz as a potent hMTH1 inhibitor all the more unusual.

Agonists of the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP1R) are key to treating metabolic diseases like type 2 diabetes and obesity, but often limited by being linear. Cyclic peptides offer greater stability and potential oral delivery, but their de novo design with computational methods as agonists is unproven. Here, we used the EvoBind AI platform to design cyclic peptide agonists for GCGR from sequence alone, without any structural templates or prior binding site information. EvoBind consistently identified the receptor’s activation surface, producing peptides that also activated GLP1R. Of three synthesised peptides, two showed potent dual activation of GCGR and GLP1R in cAMP assays, while all three activate GLP1R, with the 18-residue design achieving EC ₅₀ values of 32 nM (GLP1R) and 542 nM (GCGR), rivalling natural hormones. The designed sequences show no similarity to known agonists, and structural modelling indicates that they adopt novel binding modes compatible with active-state stabilisation. This is made possible by EvoBind’s purely sequence-based, template-free approach, which enables the discovery of alternative solutions that are inaccessible to structure-guided methods. This study presents the first successful blind de novo design of a cyclic peptide agonist for a G protein-coupled receptor (GPCR), using only the receptor sequence as input. The findings highlight EvoBind’s capacity to go beyond static binding, offering a generalisable strategy for therapeutic discovery through target-agnostic, sequence-driven peptide design.

The base excision repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) plays a central role in maintaining genome integrity and mediating cellular responses to oxidative stress. As such, it represents an attractive target for pharmaceutical modulation. Small-molecule organocatalytic switches (ORCAs) greatly enhance the rate of OGG1-catalyzed cleavage of DNA abasic sites, thereby accelerating DNA repair. Here, we present the discovery and hit-to-lead optimization of a novel class of highly potent serotonin-derived ORCAs with greatly improved pharmacokinetic properties. Biochemical assays, X-ray crystallography, and molecular dynamics simulations point toward a water-mediated mechanism of activation, distinct from previously proposed Brønsted base-assisted models. These findings establish serotonin-based ORCAs as promising chemical probes and potential leads for therapeutic modulation of OGG1 in oxidative stress-driven diseases.