Abstract 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 Å) and bound to its co-factor NADH (2.1 Å), 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 π-π 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 (IC 50 = 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.

The basic-helix-loop-helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical sensors or actuators of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet-derived metabolites and pollutants) stimuli regulating genes involved in signal adaptation and homeostasis. Despite the importance of this TF family, the mechanisms underlying specificity of DNA binding and target gene regulation by the bHLH-PAS subfamily remain unresolved. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1), revealing large DNA binding footprints (12-15 bp) and unique mechanisms of DNA binding specificity involving preferential DNA sequences flanking the core motif. Flank-encoded DNA binding specificity discerns otherwise identical core sequence binding by SIM1 and the HIFs, mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and core and flank TF binding that allows motif sequence flexibility and underpins multimodal mechanisms for achieving TF binding specificity. Furthermore, novel downstream SIM1 PAS-loop/DNA interactions are associated with AT-rich sequences contributing to DNA binding and transcriptional activity; these interactions are critical for TF biological function underpinning a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model.

ABL-rearranged (ABLr) acute lymphoblastic leukemia (ALL) is associated with treatment failure and relapse and novel treatments are required. We investigated asciminib efficacy against NUP214::ABL1 ALL, the second most common ABL1 rearrangement associated with aggressive disease. Asciminib activity was established in three patient derived xenograft models of NUP214::ABL1 ALL with different ABL1 breakpoints (e31a2, e32a3, e34a3). In all models, treatment with asciminib reduced NUP214::ABL1 leukemic burden and increased survival outcomes compared with control mice. These results contrast with recent in vitro studies in the setting of BCR::ABL1 leukemia, where ABL1 exon 3 breakpoints (e13a3, e14a3) result in asciminib resistance due to incomplete SH3 domain. Conversely, in silico modeling of NUP214::ABL1 e32a3 predicted NUP214 exon 32 mimics the missing ABL1 exon 2, forming a chimeric SH3 domain that rescues asciminib sensitivity. This prediction was supported by viability assays of Ba/F3 cells expressing NUP214::ABL1 with exon 32 present and absent. Additional viability assays and structural modeling of NUP214::ABL1 Ba/F3 cells expressing various ABL1 deletions defined a region of the SH3 domain critical for asciminib efficacy and necessary for allosteric inhibition. Our findings establish asciminib as a potential treatment for NUP214::ABL1 ALL and reveal a critical role for the fusion partner gene. Asciminib efficacy in ABLr leukemia cannot be predicted by evaluating ABL1 exon 2 inclusion/exclusion in isolation. This is of clinical importance as first-line use of STAMP inhibitors such as asciminib becomes more common.

Cytochrome P450 (CYP) enzymes are ubiquitous and important monooxygenases whose archetypal reaction is to insert an oxygen atom from dioxygen into unactivated carbon-hydrogen bonds. They require the orchestrated delivery of electrons as well as protons from the solvent. The latter is controlled through an "acid-alcohol pair" of residues located above the heme though the precise details of proton delivery are unresolved. Here, using variable-temperature X-ray crystallography and all-atom molecular dynamics simulations of the bacterial CYP199A4 enzyme we demonstrate that the conformation of the acidic residue D251, of the "acid-alcohol" pair is allosterically coupled to the heme and the substrate. In general, and in common with other CYPs, the side chain of D251 favours the 'out' of the active site orientation. In this enzyme this overcomes incompatibility with hydrophobic residues. This side chain can rotate into the active site, and this is allosterically coupled to the presence of a distal heme ligand and other structural changes at the E-helix, C-terminal loop and on the proximal side of the heme. These and other structural changes can be related to differences in water molecule access to and egress from the distal side of the heme, which would facilitate proton delivery during the catalytic cycle. Comparison of the different environments of the side chain of D251 in CYP199A4 with the equivalent acidic residue in other diverse CYP enzymes suggest that there may not be a 'universal' model for proton transfer in CYP enzymes, but that allosteric effects and transient interactions are critically important.

Abstract 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 .

Peroxisome proliferator-activated receptor gamma (PPARγ) is a validated therapeutic target for type 2 diabetes (T2D), but current FDA-approved agonists are limited by adverse effects. SR10171, a non-covalent partial inverse agonist with modest binding potency, improves insulin sensitivity in mice without bone loss or marrow adiposity. Here, we characterize a series of SR10171 analogs to define structure-function relationships using biochemical assays, hydrogen-deuterium exchange (HDX), and computational modeling. Analogs featuring flipped indole scaffolds with N-alkyl substitutions exhibited 10- to 100-fold enhanced binding to PPARγ while retaining inverse agonist activity. HDX and molecular dynamic simulations revealed that ligand-induced dynamics within ligand-binding pocket and AF2 domain correlate with enhanced receptor binding and differential repression. Lead analogs restored receptor activity in loss-of-function PPARγ variants and improved insulin sensitivity in adipocytes from a diabetic patient. These findings elucidate mechanisms of non-covalent PPARγ modulation establishing a framework for developing safer, next-generation insulin sensitizers for metabolic disease therapy.

Estrogen receptors (ERs) alpha, 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 ERgamma 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. ### Competing Interest Statement The authors have declared no competing interest.

The bifunctional enzyme N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a promising antibiotic drug target, as it facilitates the biosynthesis of uridine 5'-diphospho-N-acetylglucosamine, an essential precursor of cell wall constituents. We identified that Staphylococcus aureus GlmU ( Sa GlmU), which was previously targeted for inhibitor development, possesses a dual-cysteine variation (C379/C404) within the acetyltransferase active site. Enzyme assays performed under reducing and non-reducing conditions revealed that the acetyltransferase activity of Sa GlmU is redox-sensitive, displaying ~15-fold lower turnover and ~3-fold higher K M value for the acetyl CoA substrate under non-reducing conditions. This sensitivity was absent in a C379A Sa GlmU mutant. Analysis of Sa GlmU by mass spectrometry, x-ray crystallography, and in silico modeling support that C379 and C404 act as a reversible, redox-sensitive switch by forming a disulfide under non-reducing conditions that impedes acetyl CoA recognition and turnover. Therefore, we recommend that future in vitro screening and characterization of Sa GlmU inhibitors consider both reducing and non-reducing conditions.

Cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyze oxidation reactions. The binding of substrates and inhibitors can be assessed using a set of standard techniques, but less is known about how the products bind within the active site. Although substrate binding and product removal from the active site are generally not rate-determining steps, they are important components of the multistep catalytic cycle and the selectivity of the enzyme. The bacterial P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, catalyzes highly selective oxidation reactions on para-substituted benzoic acids such as the oxidative O-demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and the hydroxylation of 4-methylbenzoic acid to 4-(hydroxymethyl)benzoic acid. Here, we examine the binding of the products of these reactions to this enzyme using UV-visible absorbance spectroscopy, biochemical assays, X-ray crystallography, and molecular dynamics (MD) simulations. Experimental results show that the sixth aqua ligand is not displaced on addition of either product ligand and they bind less tightly than their respective substrates. Structural changes included an increase in the number of active site water molecules present, and changes in the position of several hydrophobic amino acid residues were observed. These experimental findings were compared with computational studies simulating both the 4-methoxybenzoic acid substrate and 4-hydroxybenzoic acid product bound to CYP199A4. Combining experimental and theoretical analyses, this study provides a detailed molecular rationale on how this enzyme can bind its substrates tightly yet effectively release the products, facilitating efficient catalysis with solvent molecules playing an important role in the process of product release.

ABL -rearranged ( ABL r) 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 ABL r ALL. The most common fusion in ABL r 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. ### Competing Interest Statement D.T.Y. receives research support from BMS and Novartis, and Honoraria from Novartis, Pfizer, Ascentage and Amgen. T.P.H. receives research support from BMS and Novartis, and Honoraria from BMS, Novartis, and Fusion Pharma. D.L.W. receives research support from BMS and Honoraria from BMS and Amgen. All other authors have no conflicts to declare. None of these pharmaceutical companies had roles in the design of the study, collection and analysis of the data or the decision to publish. Cancer Council SA's Peter Nelson Leukaemia Research Fellowship, 022212 Cancer Council SA's Beat Cancer Project, MCR2257 SAHMRI Early and Mid-Career Researcher Seed Funding Grant, 023130

Cancer cells exhibit accelerated protein production to accommodate their high rates of growth and proliferation. Elevated protein synthesis creates a dependency on endoplasmic reticulum (ER)-resident proteins and chaperones, which are required to maintain proteostasis. In this study, we identified the protein disulfide isomerases (PDIs) PDIA1 and PDIA5, which play a critical role in folding of client proteins in the ER, as important regulators of prostate cancer growth and response to therapy. PDIA1 and PDIA5 are upregulated in prostate cancer and induced by the androgen receptor (AR) signaling axis. Genetic or pharmacological disabling of PDIA1/PDIA5 caused redox stress, mitochondrial dysfunction, growth inhibition, and death of prostate cancer cells in vitro and in vivo. The critical functions of these enzymes in redox homeostasis and cell survival were observed in both AR-driven and AR-independent models of prostate cancer. Loss of PDIA1/PDIA5 activity led to ubiquitination and degradation of the AR, revealing a feedback loop between these chaperones and the AR pathway. Mechanistically, PDIA1/PDIA5 regulated AR stability by mediating disulfide bond formation, an activity that required cysteines 669 and 844 in AR's ligand-binding domain. Importantly, targeting PDIAs sensitized prostate cancer cells to the AR antagonist, enzalutamide. This study reveals a mechanism governing AR proteostasis in prostate cancer and positions PDIA1/5 as viable therapeutic targets.

The transcription factor estrogen receptor α (ERα) is the primary driver of ER+ breast cancer progression and a target of multiple FDA-approved anticancer drugs. Ligand-dependent activity of ERα 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 ERα 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 ERα and have significant implications for therapeutic intervention. ### Competing Interest Statement The authors have declared no competing interest.

ABL2 rearranged (ABL2r) acute lymphoblastic leukemia (ALL) is a subtype of high-risk Philadelphia chromosome-like ALL. Patients with ABL2r ALL are treated with high-dose multiagent chemotherapy, and the addition of tyrosine kinase inhibitors to their treatment regimen is currently being explored. We have previously demonstrated the in vitro sensitivity of cells harboring the ZC3HAV1::ABL2 fusion to asciminib, the first inhibitor that specifically targets the Abl myristate pocket (STAMP). In this study, we extended these in vitro findings to demonstrate similar sensitivity to the second-generation STAMP inhibitor, TERN-701, using ZC3HAV1::ABL2 ALL cells. In addition, using truncated ZC3HAV1::ABL2 isoforms, we identified that exon 3 of Abl2 (encoded by ABL2) is essential for the efficacy of both STAMP inhibitors. In an in silico model, we further demonstrated that different myristate pocket residues impact the effective binding of asciminib to Abl2 compared to Abl. Importantly, this suggests that, in the clinical setting, different asciminib binding site mutations may be anticipated with STAMP treatment for ABL2r ALL. Finally, we demonstrated the efficacy of both STAMP inhibitors against cells from patients with ZC3HAV1::ABL2 and asciminib as a novel treatment for ZC3HAV1::ABL2 disease in a preclinical in vivo study.

Modern agricultural practices rely on herbicides to reduce yield losses. Herbicide-resistant weeds threaten herbicide utility and, hence, food security. New herbicide modes of action and integrated pest-management practices are vital to mitigate this threat. As the antimalarials that target the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been shown to be herbicidal, DHFR-TS might represent a mode-of-action target for the development of herbicides. Here, we present the crystal structure of a DHFR-TS (AtDHFR-TS1) from the model dicot Arabidopsis thaliana. It shows a divergent DHFR active site and a linker domain that challenges previous classifications of bifunctional DHFR-TS proteins. This plant-conserved architecture enabled us to develop highly selective herbicidal inhibitors of AtDHFR-TS1 over human DHFR and identify inhibitors with unique scaffolds via a large-library virtual screen. These results suggest that DHFR-TS is a viable herbicide target.

The transcription factor estrogen receptor α is the primary driver of ER+ breast cancer progression and a target of multiple FDA-approved anticancer drugs. Ligand-dependent activity of ERα is determined by helix-12 conformation within the ligand binding domain. However, how helix-12 transitions from an unliganded (apo) state to active (estrogen-bound) or inactive (SERM/SERD-bound) states remains unresolved. Here, we present the crystal structure of an apo estrogen receptor α ligand binding domain from the teleost Melanotaenia fluviatilis, revealing a third distinct helix-12 conformation. Structural mass spectrometry and molecular dynamics simulations reveal that apo helix-12 is maintained in a stable and distinct conformation prior to ligand binding. Clashes between ligand and evolutionarily conserved residues L525, L536 and L540 displace helix-12, to promote activation or inactivation of the receptor. The crystal structure further reveals that breast cancer-associated mutations, Y537S and D538G, disrupt residue contacts critical for stabilising apo helix-12 conformation. We propose a model whereby helix-12 functions as a ternary molecular switch to determine receptor activity. These findings provide critical insights into the ligand-dependent and -independent regulation of estrogen receptor α and have significant implications for therapeutic intervention.

The cytochrome P450 (CYP) enzyme CYP125A1 is a crucial enzyme for the long-term survival and pathogenicity of Mycobacterium tuberculosis. CYP125 genes are found not only in pathogenic mycobacteria but are also widely dispersed within the Actinobacteria phylum, with many species possessing multiple copies of CYP125 encoding genes. Their primary function is the catalytic hydroxylation of the terminal methyl group of cholesterol and phytosterols. We have previously shown that CYP125 enzymes from distinct mycobacteria have substrate selectivity preferences for animal versus plant steroid oxidation. An evolutionary understanding of this selectivity is not known. Here, we use Ancestral Sequence Reconstruction (ASR), to support the hypothesis that some CYP125 enzymes evolved in a manner reflective of their adaptation to a pathogenic niche. We constructed a maximum-likelihood, most-recent common ancestor of the CYP125 clade (CYP125MRCA). We were then able to produce and characterise this enzyme both functionally and structurally. We found that CYP125MRCA was able to catalyse the terminal hydroxylation of cholesterol, phytosterols, and vitamin D3 (cholecalciferol); the latter was hydroxylated at both C-25 and C-26. This is the first example to date of vitamin D3 oxidation by a CYP125 enzyme, thereby demonstrating an increased substrate range of CYP125MRCA relative to its characterised extant relatives. The X-ray crystal structures of CYP125MRCA bound with sitosterol and vitamin D3 were determined, providing important insight into the changes that enable the expanded substrate range.

Cytochromes P450 (P450s) commonly catalyze hydroxylation but can also be responsible for dehydrogenation reactions, important in drug metabolism and biosynthesis; the mechanism of the latter transformation remains poorly understood. The well-characterized bacterial CYP199A4 catalyzes both hydroxylation and dehydrogenation of p-alkylbenzoic acids and thus provides an ideal model system in which to investigate the mechanism of P450-catalyzed aliphatic dehydrogenation. Through use of enantioselectively deuterated probes, metabolite analysis, protein crystallography, molecular dynamics simulations and QM/MM (ONIOM) modeling, CYP199A4-catalyzed dehydrogenation was found to be completely enantioselective and postulated to occur through an asynchronous proton coupled electron transfer. No definitive evidence of a cationic intermediate was uncovered but instead, the positioning of the substrate was postulated to be key in directing the chemoselectivity of the reaction i.e., dehydrogenation versus hydroxylation. This knowledge could be exploited to control dehydrogenation in other P450s and helps explain the common occurrence of P450-desaturated drug metabolites alongside hydroxylated ones.

Estrogen receptors α (ERα) and β (ERβ) are ligand-regulated transcription factors that control important biological processes in humans. The endogenous steroid androstenediol possesses estrogenic activity, despite being a precursor of the primary androgen, testosterone. While androstenediol is an agonist of both ERs, it is ∼ 3-fold selective for ERβ. Additionally, it has been reported that androstenediol can repress proinflammatory responses of the central nervous system (CNS) in an ERβ-dependent manner, but the primary estrogen, estradiol (E2), cannot. As no structural characterization of the interaction between ERα or ERβ with androstenediol has been reported, the basis of ERβ selectivity, and whether this is responsible for the anti-inflammatory effects of androstenediol, remains unclear. To address these gaps in knowledge we determined crystal structures of the human ER LBDs (hERα and hERβ) complexed with androstenediol and coactivator-derived peptide. This revealed that androstenediol stabilizes the active conformation of both receptors in the same manner as E2. The binding mode of androstenediol between the hERα and hERβ LBDs is extremely similar, suggesting that subtle differences in the van der Waals interactions mediated by non-conserved residues of the ligand binding pocket confer selectivity toward hERβ. Finally, in both receptors the coactivator-derived peptide occupied the activation function 2 (AF2) surface, as observed for previous agonist-bound hER structures. Therefore, as androstenediol does not induce any distinct structural changes within the hERβ LBD compared to E2, this suggests that the hERβ-dependent anti-inflammatory effects of androstenediol on the CNS are mediated by other factors.