Paul Vincent Bernhardt

Cytochrome P450 enzymes form a large superfamily of monooxygenases capable of oxidising diverse substrates. Among them, members of the CYP108 family are recognised for their role in monoterpenoid oxidation. In this study, we report the identification and characterisation of a new CYP108 family member, CYP108D18, from a metagenome‐assembled genome derived from microbial communities in a geothermal hot spring located in northern Queensland, Australia. Taxonomic binning associated CYP108D18 with a Novosphingobium species, a genus well‐known for its bioremediation potential. CYP108D18 was heterologously expressed in Escherichia coli , yielding 640 nmol L ⁻¹ of culture, and purified to homogeneity. The enzyme exhibited moderate thermostability, with a ¹⁵ T ₅₀ value of 47°C ± 1°C. Spectroscopic substrate binding assays revealed that while CYP108D18 bound monoterpenoids similarly to other CYP108 enzymes, it showed higher affinity and pronounced larger heme spin‐state shifts with aromatic hydrocarbons such as phenylcyclohexane and phenanthrene. Subsequent in silico docking and spectral‐binding studies also suggested that CYP108D18 may accommodate larger plant‐derived sesquiterpenes, indicating a potential role in their metabolism. Reconstitution of CYP108D18 with spinach ferredoxin and ferredoxin reductase resulted in the oxidation of phenylcyclohexane to trans ‐4‐phenylcyclohexanol. Molecular dynamics simulations and ONIOM (QM/MM) calculations supported selective hydrogen abstraction at the cyclohexane C4 position, rationalising trans ‐4‐phenylcyclohexanol as the predominant product. The high‐resolution crystal structure of substrate‐free CYP108D18 revealed a bound dioxygen ligand positioned above the heme iron, an unusual feature among P450 structures. Both substrate‐free and phenylcyclohexane‐bound structures revealed a persistent open conformation, suggesting the absence of a canonical open‐to‐closed transition upon ligand binding. Spectroelectrochemical analysis showed a heme redox potential of −451 mV versus NHE for the substrate‐free CYP108D18, shifting subtly to −409 mV upon phenylcyclohexane binding, which contributes to reduced activity with non‐native [2Fe‐2S] ferredoxins.

Novel PPP4HAT analogues with improved solubility and potency reveal structure–activity relationships and selective cysteine reactivity via Michael addition.

Background: Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. The ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the downstream BRN2 signaling cascade driving anoikis resistance remains unknown. Methods: We utilized genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibitors targeting the BRN2 protein were employed as probes for quantitative mass spectrometry analysis, and the functional implication of BRN2 inhibition on cell survival, apoptosis and mitochondrial structure and function assessed in vitro experiments. Results: Inhibition of BRN2 with small molecules in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through increased oxidative phosphorylation and AIFM2 expression. BRN2 inhibition additionally sensitized cells to BRAF-targeted therapy. Conclusions: Our study validated the role of BRN2 in anoikis resistance and identified that BRN2 drives anoikis resistance by promoting mitochondrial dysfunction. For the first time in melanoma models, small molecule inhibitors against BRN2 were demonstrated to reverse anoikis resistance and sensitize cells to BRAF-targeted therapy. Inhibition of BRN2 may prevent the seeding of metastatic disease by reversing anoikis resistance. Graphical Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-κB signaling pathways, resulting in PPARγ dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis.

Disclosed herein is the observation that electrocyclic ring opening of select N-acyl-2-azetines can occur under microwave irradiation or conventional heating to give the corresponding 1-azabutadiene systems, under more mild and accessible conditions than the previously reported flash vacuum pyrolysis conditions. The intermediate 1-azabutadienes, successfully detected by NMR analysis in the case of the 2-naphthoyl system, subsequently undergo intermolecular Diels–Alder reactions with the parent azetine systems to form heterobicyclic dimers, as confirmed by X-ray crystallography. Alternatively, if the 1-azabutadiene is generated in the presence of an electron-rich dienophile (e.g., enol ether or enamine), dimer formation is partially or fully suppressed, giving the corresponding cycloadducts instead. These observations are in agreement with the included theoretical calculations.

Iron complexes of the tetradentate ligand tris(2-pyridylmethyl)amine (TPMA) have been extensively studied as catalysts in oxidative transformations of organic substrates. Herein we have applied them as catalysts in electrochemical atom transfer radical addition (ATRA) of bromoacetonitrile to styrene. A family of Fe-TPMA complexes comprising mononuclear [Fe^II(TPMA)(NCMe)₂]²⁺ and [Fe^III(TPMA)Cl₂]⁺ as well as μ-oxido di-Fe complexes [(L(TPMA)Fe^III)₂O]ⁿ⁺ (L = H₂O (n = 4); Cl^-, Br^- (n = 2)) were studied, which were structurally characterized and electrochemically analyzed in acetonitrile. Yields of ATRA product were dependent on the coligands present and in contrast to their highly reactive Cu-TPMA complexes, radical activation appears to be slower but advantageously this leads to fewer side products.

Xanthine oxidase plays a central role in purine catabolism in animals, plants and bacteria where it oxidises hypoxanthine to xanthine and xanthine to uric acid. In this work we have immobilised bovine xanthine oxidase on a glassy carbon electrode within a glutaraldehyde film and demonstrated hypoxanthine and xanthine oxidase electrocatalysis using mediator methylene blue as a mediator. The role of xanthine oxidase in the reduction of nitrite to nitric oxide has been probed in two ways. Using the electrochemically reduced mediator methyl viologen, nitrite reduction by xanthine oxidase is complicated by non-enzymatic reduction of nitrite by the mediator. Using hypoxanthine as reductant, nitrite reduction occurs but the product nitric oxide rapidly inactivates the enzyme. Xanthine oxidase can be restored to its functional form by redox cycling on an electrode in the presence of sulfide showing that nitric oxide leads to desulfuration of the molybdenum active site. Graphical abstract The alternative text for this image may have been generated using AI.

Anthrasteroids were first encountered as acid rearrangement products of the fungal steroid ergosterol and then as geomarkers in deep-sea shale deposits, and while 3α-hydroxyanthrasteroids have been reported as fungal metabolites, 2β-hydroxyanthrasteroids remain rare. Our investigation of Australian soil-derived fungi yielded the 2β-hydroxyanthrasteroid pullenvasterols A-G (1-7), with structures assigned by spectroscopic and chemical analysis and biosynthetic considerations. Of note, 4 and 5 selectively inhibit the growth of coisolated fungi, suggestive of an ecological antifungal defense.

A detailed chemical investigation of the Australian pasture soil-derived Actinomadura sp. S4S-00069B10, utilizing optimized cultivation on four different solid-phase media (M1, IMA, D400, and YEME), yielded the new isoindolinone goondicones I-K (1-3) and anthra-γ-pyrone cratermycins A-D (4-7). Structures were assigned to 1-7 on the basis of detailed spectroscopic, ECD, and X-ray analyses as well as chemical transformations and biosynthetic considerations. The goondicones 1-3 are especially noteworthy in that they feature an unprecedented carbo-heterocyclic framework, with 1 and 2 undergoing a facile keto-enol-mediated equilibration to a 1:3 mixture (of C-13 epimers) that exhibits promising anthelmintic properties against the dog heartworm Dirofilaria immitis microfilariae (EC₅₀ 1.1 μM) and L4 larvae (EC₅₀ 1.3 μM). Notably, this activity is accompanied by measurable mammalian cytotoxicity, indicating a narrow therapeutic window. Together, these findings highlight the chemical novelty of Actinomadura-derived metabolites, and their value as structurally unique anthelmintics leads to further optimization.

The synthesis and characterization of complexes of the macrobicyclic ligand Me₈tricosane (1,5,5,9,13,13,20,20-octamethyl-3,7,11,15,18,22-hexaazabicyclo-[7.7.7]tricosane) with Ni(II) and Mn(II) are reported, along with further details of the synthesis and characterization of complexes of Mn(II) and Cr(III) with the related ligand Me₅tricosane (1,5,9,13,20-pentamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane). X-ray structural analysis of (H₃O)[Cr(Me₅tricosane)]Cl₄·3H₂O revealed the presence of longer Cr-N bonds than in comparable Cr(III) hexaamine complexes, but this was accompanied by modest alterations to electronic spectroscopy and electrochemical behavior. Both Me₅tricosane and Me₈tricosane formed Mn(II) complexes which exhibited magnetic and electronic properties typical of high spin d⁵ systems. The solid-state structures of [Mn(Me₅tricosane)](NO₃)₂·1.5H₂O and [Mn(Me₈tricosane)]Cl₂·3H₂O revealed a distorted trigonal prismatic MnN₆ chromophore with longer Mn-N bonds than in similar Mn(II) complexes, and both [Mn(Me₅tricosane)]²⁺ and [Mn(Me₈tricosane)]²⁺ exhibited irreversible redox chemistry in cyclic voltammetry experiments. X-ray crystallographic analysis of [Ni(Me₈tricosane)](NO₃)₂·H₂O revealed remarkably long Ni(II)-N bonds, resulting in an unusual blue color for a Ni(II)N₆ complex.

Designing ligands for cancer has traditionally overlooked complex dissociation and transmetalation in enhancing efficacy. Another neglected criterion is the ligands' ability to intercept the labile Fe(II) pool released after transferrin endocytosis and reduction of transferrin-bound Fe(III). Given iron's essential role in cancer proliferation, disrupting metal homeostasis offers a promising therapeutic strategy. Herein, we introduce a new class of Fe(II)-selective ligands and their Ga(III) complexes for cancer therapy, guided by insights into their dissociative dynamics and transmetalation behavior. Unlike prior approaches focused on static metal coordination, this work integrates dissociation and transmetalation as design features, enabling selective interception of intracellular Fe(II) trafficking. Relative to the ligand, Ga(III) complexation led to a pronounced (p < 0.001–0.0001) enhancement in anti-proliferative activity, with up to a 70-fold increase in potency. This result was in contrast to the modest increase in potency (up to 2.4-fold) observed for the Cu(II) or Zn(II) complexes. Mechanistic dissection demonstrated that, unlike the complete dissociation of the Ga(III) complexes, the relative Zn(II) and Cu(II) complexes underwent only partial dissociation. This difference facilitates complete ligand and Ga(III) release from the complex and may account for the superior cytotoxicity of the Ga(III) complexes versus their Zn(II) and Cu(II) counterparts. Furthermore, their potency was linked to Fe(II) ligation rather than Fe(III), despite electronic similarity to Ga(III). This study introduces three underexplored design principles for anti-cancer ligand engineering: (i) dynamic complex dissociation; (ii) selective intracellular transmetalation using NNO-containing ligands; and (iii) interception of labile Fe(II) generated after endosomal Fe(III) reduction.