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Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry–based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 A crystal structure of SF-1 bound to dioleoyl PIP3(18:1/18:1) to compare it with a matched structure of SF-1 bound to dipalmitoyl PIP3(16:0/16:0). The dioleoyl-bound structure was severely disordered in a specific SF-1 region associated with pathogenic human polymorphisms and within the coactivator-binding region critical for SF-1 function while inducing increased sensitivity to protease digestion in solution. Validating these structural observations, in vitro functional studies showed dioleoyl PIP3 induced 6-fold poorer affinity of a peroxisome proliferator-activated receptor gamma coactivator 1-alpha coactivator peptide for SF-1 compared with dipalmitoyl PIP3. Together, these data suggest the chemical nature of the phosphoinositide acyl chains controls the ordered state of specific, clinically important structural regions in SF-1, regulating SF-1 function in vitro.

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C28- and C29-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC50 values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼Ki 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.

Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8-Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a "switch" between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8-Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity.

A set of sterol methyl transferases (SMT) from amoeba, 2 synthesized in Acanthamoeba castellanii (Ac), and 1 in Naegleria gruberi (Ng), were cloned, functionally expressed in Escherichia coli and their substrate specificities determined. Bioinformatic analysis of the amino acid sequences for the protozoan-distinct SMT’s revealed that they possessed relatedness more to plants than to fungi. Enzyme-generated substrate and product specificities were determined by a combination of GC/MS, NMR, HPLC, and isotopic labelling with D3-SAM. The favored substrate for Ac24SMT was cycloartenol, Ac-28SMT was 24-methylenelophenol, and Ng24SMT was zymosterol, which corresponds to their natural distribution in the respective amoebas. Ac24SMT and Ng24SMT catalyzed sterol methylation through the Δ24(28) pathway affording a single product. Alternatively, Ac28SMT catalyzed sterol methylation through the Δ25(27) pathway, affording 4α-methyl-24βethyl-stigmasta-7,25(27)-dienol and other minor products. Inhibition of the three SMTs was demonstrated using high-affinity transition state analogs (TSA) of the sterol methylation reaction; the TSA were equally potent against the Ac and Ng SMTs and exhibited Ki’s in the low nanomolar range, consistent with their mechanism of action. These results show that a heretofore unrecognized substrate preference and product outcome of SMT catalyzed reactions that could influence ergosterol biosynthesis and growth of these parasitic organisms. SMT is a clear target enzyme that warrants further study in rational drug design targeting pathogenic amoeba.

In this paper the acoustic stability of a premixed turbulent natural gas flame confined in a combustor is investigated. Specifically when the flame is operated in a lean premixed mode, the thermoacoustic system is known to exhibit instabilities. These arise from a feedback mechanism between the oscillatory flow and heat release rate perturbations in the flame and often lead to large amplitude pressure and velocity perturbations in the combustor. The acoustics of the combustor are described with a one-dimensional transfer matrix method. The feedback mechanisms that can cause instabilities are included in this method. The (complex) frequency for which the determinant of the transfer matrix goes to zero indicates an instability. An important factor in the one-dimensional acoustic model is the transfer function between the oscillatory flow and heat release rate perturbations. This transfer function is obtained from a well-stirred reactor dynamic combustion model. Results show that the one-dimensional acoustic model in combination with a well-stirred reactor model is able to describe realistic gas turbine stability behaviour.

The relation between harmonic structural vibrations and the corresponding acoustic radiation is given by the Helmholtz integral equation (HIE). To solve this integral equation a new solver (BEMSYS) based on the boundary element method (BEM) has been implemented. This numerical tool can be used for both sound radiation and nearfield acoustic source localization purposes. After validation of the solver with analytic solutions of simple test problems, a well-defined experimental setup has been designed. The setup consists of a thick walled aluminum box covered with a flexible plate. Inside the box a speaker generates a sound field which is transmitted through the flexible plate into the surrounding medium. This paper describes this experimental setup and briefly explains the properties of the boundary element solver. A comparison is made between the computed and measured sound pressures and particle velocities surrounding the acoustic source. In order to obtain a boundary condition for the BEM computation, the structural vibrations of the flexible plate are measured with a laser Doppler vibrometer. The acoustic pressures are measured with a condenser microphone. The particle velocities are measured with a novel Microflown sensor, which directly measures particle velocity. From the comparison between numerical and measured results, it becomes evident that a good agreement is obtained and thus that the BEMSYS solver is successfully validated.

To decrease NOx emissions from a combustion system lean premixed combustion in combination with an annular combustor is used. The disadvantage is that sound pressure levels in the combustion system become higher which excite the liner (the surrounding structure). This limits the life of the combustor, because it will fail earlier due to fatigue. This problem is studied in the European project DESIRE (Design and demonstration of highly reliable low NOx combustion systems for gas turbines). A simplified model is studied first. This model consists of a rigid rectangular box which has a flexible plate on one of the sides. Sound is injected into the box using a tube coming from a rectangular box. A loudspeaker generates sound inside this box. A fully coupled finite element model has been made of the structure and the acoustic cavity. The results of the model are compared to the measurements performed on the actual setup. Both the structural mode shapes (measured using a laser vibrometer) and the transfer function from pressure to displacement are used for this purpose. The results match very well.

An important cause for interior noise in vehicles is structure-borne sound from the engine. The vibrations of the source (engine) are transmitted to the receiver structure (the vehicle) causing interior noise in the vehicle. For this reason the engine is supported by rubber isolators for passive isolation in especially the high-frequency region. To make a good judgment of the characteristics of a vibration isolator in the design process, it is useful to use numerical models. In this paper a cylindrical vibration isolator is modelled numerically with the Finite Element package ABAQUS. The investigation is split in two parts: first a nonlinear analysis is performed for different pre-deformations of the mount. After that, a linear harmonic analysis is superimposed on the pre-deformed isolator. The structure-borne sound is transmitted by the isolator by six degrees of freedom, so the harmonic analysis must be performed for different excitations. With the results the behavior of the isolator can be represented by dynamic stiffness matrices as function of the frequency and predeformation. These matrices can be used to model the passive isolation components as part of numerical models of hybrid isolation systems. These isolation systems describe a combination of active and passive isolation to reduce the structure-borne sound transmission to receiver structures.