Aotearoa New Zealand emergency ambulance services and the provision of end-of-life care: a short survey.

AIM: To characterise emergency ambulance service (EAS) clinical roles and experiences (including cultural competency and pastoral care) in the delivery of end-of-life (EOL) and palliative care in Aotearoa New Zealand. METHOD: A nine question online survey was distributed to St John and Wellington Free Ambulance clinicians. Four questions enabled voluntary free-text comments to be submitted for thematic analysis. A further opportunity for free-text comments was available at the end of the survey. RESULTS: There were 444 participants, which is 14% of the paid ambulance workforce. 63% reported that they frequently transported EOL care patients to hospital when they could be better managed at home. EAS clinicians depend heavily on informal collegial support for pastoral care as formal debriefs are rarely offered. There were 671 free-text comments. Dominant themes included the importance of seniority, the need for further education, the importance of documented care plans and the need for better integration with community services, including hospice. CONCLUSIONS: More can and should be done to ensure EAS clinicians are supported to deliver quality EOL care for patients alongside other community providers.

Structure Based Discovery of Inhibitors of CYP125 and CYP142 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.

CYP105AS1 is a cytochrome P450 from Amycolatopsis orientalis that catalyzes monooxygenation of compactin to 6-epi-pravastatin. For fermentative production of the cholesterol-lowering drug pravastatin, the stereoselectivity of the enzyme needs to be inverted, which has been partially achieved by error-prone PCR mutagenesis and screening. In the current study, we report further optimization of the stereoselectivity by a computationally aided approach. Using the CoupledMoves protocol of Rosetta, a virtual library of mutants was designed to bind compactin in a pro-pravastatin orientation. By examining the frequency of occurrence of beneficial substitutions and rational inspection of their interactions, a small set of eight mutants was predicted to show the desired selectivity and these variants were tested experimentally. The best CYP105AS1 variant gave >99% stereoselective hydroxylation of compactin to pravastatin, with complete elimination of the unwanted 6-epi-pravastatin diastereomer. The enzyme-substrate complexes were also examined by ultrashort molecular dynamics simulations of 50 × 100 ps and 5 × 22 ns, which revealed that the frequency of occurrence of near-attack conformations agreed with the experimentally observed stereoselectivity. These results show that a combination of computational methods and rational inspection could improve CYP105AS1 stereoselectivity beyond what was obtained by directed evolution. Moreover, the work lays out a general in silico framework for specificity engineering of enzymes of known structure.

Patients treated for acute headache with intranasal droperidol spend less time in the emergency department: A retrospective observational study.

OBJECTIVE: Headache is a common presenting complaint to the ED. Using time from the first provider to discharge as a surrogate for effectiveness, we aimed to determine if intranasal (IN) droperidol is as beneficial as usual treatment for acute headache in the ED. METHODS: There were 1213 consecutive presentations of adults with acute headache over a 42-month period. Electronic records for each event were interrogated, 406 events met pre-determined exclusion criteria. Of the remaining 805 eligible patient events, 139 received IN droperidol, whereas 666 were given usual therapy. RESULTS: There was a 20 min reduction of mean and median ED length of stay (LOS) for the group that got treated with IN droperidol. CONCLUSIONS: IN droperidol reduced LOS in the ED. There are potential cost savings of this effective treatment via this novel route. A prospective multi-centre study of the use of IN droperidol for the treatment of acute headache in the ED is recommended.

Aotearoa New Zealand emergency medicine specialists on the provision of care at or near the end of life: A survey.

OBJECTIVES: The ED is an increasingly important venue for the initiation of palliative care. We sought to characterise the opinions, experience, training and education of ED staff in Aotearoa/New Zealand (NZ) with regard to specific aspects of palliative care in the NZ ED setting. METHODS: All NZ FACEMs were personally emailed a simple unstructured 16-part survey asking questions about initiating palliative care, goals of care, initiation and availability of advance care plans, frailty screening, availability of palliative expertise, training and education, cultural safety and pastoral care of staff. All EDs were contacted and a link provided for non-FACEM ED staff who wished to participate. Free-text comments were analysed for dominant themes. RESULTS: All NZ EDs had at least one participant. There was a high level of senior medical staff engagement with 60% of NZ FACEMs participating. More than 300 free-text comments from this group were available for theme analysis. A total of 93% of NZ FACEM respondents agree that palliative care should be able to be initiated in the ED. Only 25% of this group knew of training in serious illness conversations in the ED while only 34% felt culturally competent when providing end-of-life care for Maori and their whanau (family). Pastoral care for ED staff appears to be ad hoc. Time and privacy limitations were common themes. CONCLUSIONS: There is significant opportunity for quality improvement in the initiation and provision of palliative care from the ED. Attention to how departments provide pastoral care to their staff is needed.

A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen).

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism.

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3's high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.

Variation in resources and impact on performance: results of the emergency department benchmarking survey.

AIM: The resources and capacity of New Zealand's emergency departments (EDs) to cope with surges in demand are unknown. The aims were to describe the current resources and capacity of New Zealand EDs and explore how these relate to ED performance. METHODS: A survey of EDs in New Zealand was conducted to capture elements of governance, staffing and structure of the EDs in the calendar year 2018. These were linked to processes and outcomes of care. RESULTS: Eighteen of 26 EDs responded. These were representative of the range of EDs nationally. There was wide variability between the EDs across all the surveyed elements. Although no single element was strongly related to performance measures, combinations of elements were. When there was a lack of doctors and available ED or hospital beds relative to the workload, then performance was worse. The correlations were: for time to assessment r=0.728, p=0.001, for ED length of stay r=0.759, p<0.001, for patients who did not wait r=0.619, p=0.006 and for deaths in the ED r=0.649, p=0.004. CONCLUSION: There is marked variation among New Zealand hospitals with respect to structure, staffing and workload, which may be impacting negatively on ED performance and limit the ability of some hospitals to cope with surges in demand for acute care.

Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate.

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

Clobetasol Propionate Is a Heme-Mediated Selective Inhibitor of Human Cytochrome P450 3A5.

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV-visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme-clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.

Structure-Activity Relationships of cyclo(l-Tyrosyl-l-tyrosine) Derivatives Binding to Mycobacterium tuberculosis CYP121: Iodinated Analogues Promote Shift to High-Spin Adduct.

A series of analogues of cyclo(l-tyrosyl-l-tyrosine), the substrate of the Mycobacterium tuberculosis enzyme CYP121, have been synthesized and analyzed by UV-vis and electron paramagnetic resonance spectroscopy and by X-ray crystallography. The introduction of iodine substituents onto cyclo(l-tyrosyl-l-tyrosine) results in sub-µM binding affinity for the CYP121 enzyme and a complete shift to the high-spin state of the heme FeIII. The introduction of halogens that are able to interact with heme groups is thus a feasible approach to the development of next-generation, tight binding inhibitors of the CYP121 enzyme, in the search for novel antitubercular compounds.

MhuD from Mycobacterium tuberculosis: Probing a Dual Role in Heme Storage and Degradation.

The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD-diheme dissociation is far slower than monoheme dissociation at ∼0.12 min-1 and ∼0.25 s-1, respectively, indicating that MhuD has a strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

Design and Synthesis of Imidazole and Triazole Pyrazoles as Mycobacterium Tuberculosis CYP121A1 Inhibitors.

The emergence of untreatable drug-resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (K D). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert-butyl (10 g) compounds displaying optimal activity (MIC 1.562 µg/mL, K D 0.22 µM (10 f) and 4.81 µM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H-bond acceptors/H-bond donors 4/0, number of rotatable bonds 5-6, molecular volume >340 Å3, topological polar surface area <40 Å2.

Synthesis and biological evaluation of novel cYY analogues targeting Mycobacterium tuberculosis CYP121A1.

The rise in multidrug resistant (MDR) cases of tuberculosis (TB) has led to the need for the development of TB drugs with different mechanisms of action. The genome sequence of Mycobacterium tuberculosis (Mtb) revealed twenty different genes coding for cytochrome P450s. CYP121A1 catalyzes a CC crosslinking reaction of dicyclotyrosine (cYY) producing mycocyclosin and current research suggests that either mycocyclosin is essential or the overproduction of cYY is toxic to Mtb. A series of 1,4-dibenzyl-2-imidazol-1-yl-methylpiperazine derivatives were designed and synthesised as cYY mimics. The derivatives substituted in the 4-position of the phenyl rings with halides or alkyl group showed promising antimycobacterial activity (MIC 6.25 µg/mL), with the more lipophilic branched alkyl derivatives displaying optimal binding affinity with CYP121A1 (iPr KD = 1.6 µM; tBu KD = 1.2 µM). Computational studies revealed two possible binding modes within the CYP121A1 active site both of which would effectively block cYY from binding.

Novel insights into P450 BM3 interactions with FDA-approved antifungal azole drugs.

Flavocytochrome P450 BM3 is a natural fusion protein constructed of cytochrome P450 and NADPH-cytochrome P450 reductase domains. P450 BM3 binds and oxidizes several mid- to long-chain fatty acids, typically hydroxylating these lipids at the ω-1, ω-2 and ω-3 positions. However, protein engineering has led to variants of this enzyme that are able to bind and oxidize diverse compounds, including steroids, terpenes and various human drugs. The wild-type P450 BM3 enzyme binds inefficiently to many azole antifungal drugs. However, we show that the BM3 A82F/F87V double mutant (DM) variant binds substantially tighter to numerous azole drugs than does the wild-type BM3, and that their binding occurs with more extensive heme spectral shifts indicative of complete binding of several azoles to the BM3 DM heme iron. We report here the first crystal structures of P450 BM3 bound to azole antifungal drugs - with the BM3 DM heme domain bound to the imidazole drugs clotrimazole and tioconazole, and to the triazole drugs fluconazole and voriconazole. This is the first report of any protein structure bound to the azole drug tioconazole, as well as the first example of voriconazole heme iron ligation through a pyrimidine nitrogen from its 5-fluoropyrimidine ring.

Cytochrome P450 1A1 opens up to new substrates.

The cytochromes P450 (CYPs) oxidatively transform a huge number of substrates in both prokaryotic and eukaryotic organisms, but the mechanisms by which they accommodate these diverse molecules remain unclear. A new study by Bart and Scott reports two co-crystal structures of CYP1A1 that reveal structural rearrangements and flexible interaction networks that explain how the active site cavity shapes itself around new ligands. These data open the door to an increased understanding of fundamental enzyme behavior and improved searches for anti-cancer compounds.

Characterization of Cytochrome P450 Enzymes and Their Applications in Synthetic Biology.

The cytochrome P450 monooxygenase enzymes (P450s) catalyze a diverse array of chemical transformations, most originating from the insertion of an oxygen atom into a substrate that binds close to the P450 heme. The oxygen is delivered by a highly reactive heme iron-oxo species (compound I) and, according to the chemical nature of the substrate and its position in the active site, the P450 can catalyze a wide range of reactions including, e.g., hydroxylation, reduction, decarboxylation, sulfoxidation, N- and O-demethylation, epoxidation, deamination, CC bond formation and breakage, nitration, and dehalogenation. In this chapter, we describe the structural, biochemical, and catalytic properties of the P450s, along with spectroscopic and analytical methods used to characterize P450 enzymes and their redox partners. Important uses of P450 enzymes are highlighted, including how various P450s have been exploited for applications in synthetic biology.

Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus.

The CYP152 family of cytochrome P450 enzymes (P450s or CYPs) are bacterial peroxygenases that use hydrogen peroxide to drive hydroxylation and decarboxylation of fatty acid substrates. We have expressed and purified a novel CYP152 family member - CYP152K6 from the methylotroph Bacillus methanolicus MGA3. CYP152K6 was characterized using spectroscopic, analytical and structural methods. CYP152K6, like its peroxygenase counterpart P450SPα (CYP152B1) from Sphingomonas paucimobilis, does not undergo significant fatty acid-induced perturbation to the heme spectrum, with the exception of a minor Soret shift observed on binding dodecanoic acid. However, CYP152K6 purified from an E. coli expression system was crystallized and its structure was determined to 1.3 Šwith tetradecanoic acid bound. No lipids were present in conditions used for crystallogenesis, and thus CYP152K6 must form a complex by incorporating the fatty acid from E. coli cells. Turnover studies with dodecanoic acid revealed several products, with 2-hydroxydodecanoic acid as the major product and much smaller quantities of 3-hydroxydodecanoic acid. Secondary turnover products were undec-1-en-1-ol, 2-hydroxydodec-2-enoic acid and 2,3-dihydroxydodecanoic acid. This is the first report of a 2,3-hydroxylated fatty acid product made by a peroxygenase P450, with the dihydroxylated product formed by CYP152K6-catalyzed 3-hydroxylation of 2-hydroxydodecanoic acid, but not by 2-hydroxylation of 3-hydroxydodecanoic acid.