Enolase Inhibitors As Therapeutic Leads for Naegleria fowleri Infection

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains \u3e95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 μM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 μM) while the reported CC50 was \u3e300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM

Is the observable Universe consistent with the cosmological principle?

The cosmological principle (CP)—the notion that the Universe is spatially isotropic and homogeneous on large scales—underlies a century of progress in cosmology. It is conventionally formulated through the Friedmann-Lemaître-Robertson-Walker (FLRW) cosmologies as the spacetime metric, and culminates in the successful and highly predictive Λ-Cold-Dark-Matter (ΛCDM) model. Yet, tensions have emerged within the ΛCDM model, most notably a statistically significant discrepancy in the value of the Hubble constant, H0. Since the notion of cosmic expansion determined by a single parameter is intimately tied to the CP, implications of the H0 tension may extend beyond ΛCDM to the CP itself. This review surveys current observational hints for deviations from the expectations of the CP, highlighting synergies and disagreements that warrant further study. Setting aside the debate about individual large structures, potential deviations from the CP include variations of cosmological parameters on the sky, discrepancies in the cosmic dipoles, and mysterious alignments in quasar polarizations and galaxy spins. While it is possible that a host of observational systematics are impacting results, it is equally plausible that precision cosmology may have outgrown the FLRW paradigm, an extremely pragmatic but non-fundamental symmetry assumption

Potential, pitfalls, and prospects of food allergy diagnostics with recombinant allergens or synthetic sequential epitopes

This article aims to critically review developments in food allergy diagnostics with regard to the verification of specific IgE antibodies and the identification of the responsible allergens. Results of IgE-binding tests with food extracts are hampered by cross-reactive proteins, low-quality test agents, or both. Specificity can be increased by defining adequate cutoff values, whereas sensitivity can be improved by using high-quality test agents. IgE-binding tests with purified allergens enabled reliable quantification of allergen-specific IgE titers, with higher levels found in individuals with food allergy compared with individuals without food allergy. However, the overlap in individual test reactivity between allergic and nonallergic subjects complicates interpretation. Recombinant allergens and synthetic sequential epitopes enabled detection of sensitization profiles, with IgE specific to several allergens and substructures now being suggested as markers of severity, persistence, or both. However, high-power quantitative studies with larger numbers of patients are required to confirm these markers. IgE-binding tests merely indicate sensitization, whereas the final proof of clinical relevance still relies on family/case history, physical examinations, and provocation tests. Novel technologies promise superior diagnostics. Microarray technology permits simultaneous measurement of multiple IgE reactivities regarding specificity, abundance, reactivity, or interaction. Improved functional tests might enable reliable estimation of the clinical relevance of IgE sensitizations at justifiable expenses

Structure of Klebsiella pneumoniae adenosine monophosphate nucleosidase.

Klebsiella pneumoniae is a bacterial pathogen that is increasingly responsible for hospital-acquired pneumonia and sepsis. Progressive development of antibiotic resistance has led to higher mortality rates and creates a need for novel treatments. Because of the essential role that nucleotides play in many bacterial processes, enzymes involved in purine and pyrimidine metabolism and transport are ideal targets for the development of novel antibiotics. Herein we describe the structure of K. pneumoniae adenosine monophosphate nucleosidase (KpAmn), a purine salvage enzyme unique to bacteria, as determined by cryoelectron microscopy. The data detail a well conserved fold with a hexameric overall structure and clear density for the putative active site residues. Comparison to the crystal structures of homologous prokaryotic proteins confirms the presence of many of the conserved structural features of this protein yet reveals differences in distal loops in the absence of crystal contacts. This first cryo-EM structure of an Amn enzyme provides a basis for future structure-guided drug development and extends the accuracy of structural characterization of this family of proteins beyond this clinically relevant organism

Structural Determinants for Substrate Selectivity in Guanine Deaminase Enzymes of the Amidohydrolase Superfamily

Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of Escherichia coli and Saccharomyces cerevisiae guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity

Conservation of KpAmn by residue.

The 150-sequence alignment generated by ConSurf is represented in logo format using WebLogo [38], in which letter height corresponds to strength of individual residue conservation. The N-terminal unresolved residues and C-terminal five residues following a series of unresolved residues are omitted. Active site residues are marked with boxes. (TIF)</p

<i>K</i>. <i>pneumoniae</i> AMP nucleosidase chemistry.

In K. pneumoniae, AMP is broken down into adenine and ribose 5-phosphate by the enzyme AMP nucleosidase (Amn). In many other organisms, including mammals, AMP is deaminated by AMP deaminase (AD) to generate inosine monophosphate.</p