Biology subjects

Bruning, J. B.

Publications and source records attributed to Bruning, J. B..

8 recordsLinked to original sources

bioRxiv2026

Targeting the Mannitol Biosynthesis Pathway in Aspergillus fumigatus: Characterisation and Inhibition of Mannitol-2-Dehydrogenase

Infections caused by the opportunistic fungal pathogen Aspergillus fumigatus pose a serious public health system burden. The inherent limitations in existing antifungal drugs in conjunction with a rising emergence of antifungal resistance emphasizes an urgent need to identify and target alternative pathways crucial to survival and virulence. Targeting the fungal mannitol biosynthesis enzymes provides a promising avenue in the development of new antifungals due to the multifaceted roles mannitol fulfils in the fungal life cycle. However, a distinct lack of available structural information for these enzymes has hindered drug discovery efforts. We report the first crystal structure of mannitol-2-dehydrogenase from A. fumigatus in an unbound monomeric state (1.8 [A]) and bound to its co-factor NADH (2.1 [A]), via. a large, central cavity lined with positively charged residues that readily accommodates NADH. This interaction is further stabilised by a network of hydrogen bond interactions and {pi}-{pi} stacking between Phe45 and the nicotinamide ring of NADH. Furthermore, rigorous kinetic characterisation of A. fumigatus mannitol-2-dehydrogenase demonstrates the dose-dependent inhibitory activity of 1,4-benzoquinone, a cysteine-modifying small molecule inhibitor (IC50 = 1.2 {+/-} 0.2 nM). In addition, intact MS and proteomic analysis further reveal that 1,4-benzoquinone modifies up to five cysteine residues of mannitol-2-dehydrogenase and displays antifungal activity against A. fumigatus, which is enhanced in combination with a front-line antifungal voriconazole. From this work, we have established the foundations for a novel antifungal drug discovery avenue that targets the fungal mannitol biosynthesis pathway to better treat aspergillosis and related pathogenic infections.

biochemistry↗

bioRxiv2026

Structural and Catalytic Architecture of the Cysteine Synthase Cys1a

Antifungal drug resistance in Aspergillus fumigatus is a major global health concern. Due to the increasing frequency of drug-resistant strains, there is a growing need for novel antifungal drugs. Inhibition of the cysteine biosynthesis pathway in A. fumigatus represents a promising strategy for the development of antifungal adjuvants. Within this pathway, the previously uncharacterised enzyme AfuCys1a fulfils several desirable characteristics as an adjuvant target. To enable structure-based inhibitor design, AfuCys1a was structurally and functionally characterised using X-ray crystallography, biochemical assays, and comparative sequence and structural analysis. In the search for effective and viable adjuvants, this research characterises the catalytic mechanism and substrate recognition of AfuCys1a and identifies 2-picolinic acid and related compounds as promising starting points for inhibitor development, working towards the larger goal of combating rising deaths from infection by A. fumigatus.

biochemistry↗

bioRxiv2025

The STAMP inhibitor asciminib is a new treatment option for ABL-rearranged ALL revealing a novel role for the 5' fusion partner in determining drug response

ABL-rearranged (ABLr) acute lymphoblastic leukaemia (ALL) is associated with high rates of treatment failure and relapse and novel treatments are required. We investigated activity of the STAMP inhibitor asciminib in non-BCR::ABL1 ABLr ALL. The most common fusion in ABLr ALL is NUP214::ABL1, which is associated with aggressive disease. For the first time we establish asciminib activity in three pre-clinical patient derived xenograft models of NUP214::ABL1 ALL. Treatment with asciminib reduced NUP214::ABL1 leukaemic burden, splenomegaly and ABL1 kinase activation. We observed significantly increased survival outcomes in asciminib-treated versus control mice. Additionally, site directed mutagenesis, in vitro cell death assays and in silico structural modeling defined a region of the ABL1 SH3 domain critical for asciminib efficacy and necessary for mediation of allosteric inhibition. Our findings establish asciminib as a potential treatment for NUP214::ABL1 ALL, significantly expanding the number of ALL patients who may benefit from asciminib therapy which has an excellent safety and tolerability profile.

cancer biology↗

bioRxiv2025

A ternary switch determines ERα LBD conformation

The transcription factor estrogen receptor (ER) is the primary driver of ER+ breast cancer progression and a target of multiple FDA-approved anticancer drugs. Ligand-dependent activity of ER is determined by the conformation of helix-12 (H12) within the ligand binding domain (LBD), but how H12 transitions from an unliganded (apo) state to active (estrogen-bound) or inactive (SERM/SERD-bound) states remains unresolved. Here, we present the first crystal structure of an apo ER LBD, revealing a third distinct H12 conformation that regulates receptor activity. Structural mass-spectrometry, small-angle X-ray scattering, functional analysis and molecular dynamics simulations reveal that the apo conformation of H12 is stable in the absence of ligand, but is destabilised by Y537S and D538G breast cancer mutations driving constitutive activation. We propose a model in which H12 functions as a ternary molecular switch to determine receptor activity. These findings provide critical insights into the ligand-dependent and -independent regulation of ER and have significant implications for therapeutic intervention.

biochemistry↗

bioRxiv2025

Structural evolution of the estrogen receptor regulatory domain

Estrogen receptors (ERs) , {beta} and {gamma} are ligand-dependent transcription factors that regulate vertebrate reproduction, cell survival and other physiological processes. Here, we report an integrative analysis of mammalian and teleost receptors, including the first structure of an ER{gamma} ligand binding domain (LBD), showing that structural divergence acquired during evolution is accommodated by different strategies within functional regions to preserve intrinsic regulatory mechanisms. Supervised-learning and comparative Markov modelling uncover highly constrained network structures essential for allostery and protein folding, revealing a fundamental regulatory architecture and source of selective pressure in ER+ breast cancer and reproductive disorders. Finally, cross-referencing molecular constraints to genome sequencing reveal evolutionary origins underlying natural genetic variation in humans and widespread disruption of constraints. This work provides structural insights into the conservation of gene regulation by essential transcription factors and has implications for precision medicine.

biochemistry↗

bioRxiv2024

Identification of cysteine metabolism regulator (CymR)-derived pentapeptides as nanomolar inhibitors of Staphylococcus aureus O-acetyl-ʟ-serine sulfhydrylase (CysK)

The conditionally essential pathway of bacterial cysteine biosynthesis is gaining traction for the development of antibiotic adjuvants. Bacterial cysteine biosynthesis is generally facilitated by two enzymes possessing O-acetyl--serine sulfhydrylase (OASS) activity, CysK and CysM. CysK enzymes can also form functional complexes with other proteins that regulate cysteine metabolism. In Staphylococcus aureus there exists a single OASS homologue, herein termed SaCysK. Knockout of SaCysK was found to increase sensitivity to oxidative stress, making it a relevant target for inhibitor development. SaCysK forms two functional complexes via interaction with the preceding enzyme in the pathway serine acetyltransferase (CysE) or the transcriptional regulator of cysteine metabolism (CymR). These interactions occur through the insertion of a C-terminal peptide of CysE or CymR into the active site of SaCysK, inhibiting OASS activity, and therefore represent an excellent starting point for developing SaCysK inhibitors. Here we detail the characterization of CysE and CymR-derived C-terminal peptides as inhibitors of SaCysK. First, interactions between CysE or CymR-derived C-terminal decapeptides and SaCysK were assessed by X-ray crystallography. While both peptides occupied the active site of SaCysK, the alternate sidechains of the CymR decapeptide formed more extensive interactions. Surface plasmon resonance binding assays and SaCysK inhibition assays revealed that the CymR decapeptide bound to SaCysK with nanomolar affinity (KD = 25 nM) and inhibited SaCysK activity (IC50 = 180 nM), making it a promising lead for the development of SaCysK inhibitors. To understand the determinants of this high affinity interaction the structure-activity relationships of 16 rationally designed peptides were also investigated. This identified that the C-terminal pentapeptide of CymR alone facilitates the high affinity interaction with SaCysK, and that subtle structural modification of the pentapeptide is possible without impacting potency. Ultimately, this work has identified CymR pentapeptides as a promising scaffold for the development of antibiotic adjuvants targeting SaCysK. Author summaryThere is increasing interest in the investigation of non-essential pathways including bacterial cysteine metabolism for developing antibiotic adjuvants. Within this pathway the O-acetyl--serine sulfhydrylase (OASS) enzymes CysK and CysM have been a focus. As such, the OASS enzyme of Staphylococcus aureus, SaCysK, gained our interest. Previous efforts to inhibit CysK enzymes have mimicked the interaction between CysK and the C-terminus of serine acetyltransferase (CysE) which occurs inside the CysK active site and inhibits OASS activity. CysE peptides have only moderate potency, typically binding with micromolar affinity. In S. aureus another complex forms between SaCysK and a transcriptional regulator CymR, but the ability of CymR peptides to inhibit CysK enzymes has not been investigated. We noticed there is variation between the C-terminus of CysE and CymR, suggesting that CymR peptides make distinct interactions with SaCysK and may be superior inhibitors. Here we characterized CysE and CymR peptides as SaCysK inhibitors. We found CymR peptides make more extensive molecular interactions with SaCysK and bind with higher affinity, being the most potent peptide inhibitors of a CysK enzyme to date. A CymR pentapeptide is the minimal length required for this potency and provides a promising scaffold for developing antibiotic adjuvants targeting SaCysK.

biochemistry↗

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↗

bioRxiv2022

Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity

The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1, HIF2, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIF-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.

molecular biology↗