A Non-Active-Site Inhibitor with Selectivity for Zika Virus NS2B-NS3 Protease.

Flavivirus infection usually results in fever accompanied by headache, arthralgia, and, in some cases, rash. Although the symptoms are mild, full recovery can take several months. Flaviviruses encode seven nonstructural proteins that represent potential drug targets for this viral family. Focusing on the Zika virus NS2B-NS3 protease, we uncovered a unique inhibitor, MH1, composed of aminothiazolopyridine and benzofuran moieties. MH1 inhibits ZVP with a biochemical IC50 of 440 nM and effectively blocks cleavage of ZVP substrates in cells. Surprisingly, MH1 inhibits the other flaviviral proteases at least 18-fold more weakly. This same phenomenon was observed in assays of the viral cytopathic effect, where only Zika virus showed sensitivity to MH1. This selectivity was unexpected since flaviviral proteases have high similarity in sequence and protein structure. MH1 binds at an allosteric site, as demonstrated by its ability to stabilize ZVP synergistically with an active site inhibitor. To understand its selectivity, we constructed a series of hybrid proteases composed of select segments of ZVP, which is sensitive to MH1, and dengue virus protease, which is essentially insensitive to MH1. Our results suggest that MH1 binds to the NS3 protease domain, disrupting its interaction with NS2B. These interactions are essential for substrate binding and cleavage. In particular, the unique dynamic properties of NS2B from Zika seem to be required for the function of MH1. Insights into the mechanism of MH1 function will aid us in developing non-active-site-directed, pan-flaviviral inhibitors, by highlighting the importance of evaluating and considering the dynamics of the NS2B regions.

Lactate Dehydrogenase Inhibitors Suppress Borrelia burgdorferi Growth In Vitro.

Borrelia burgdorferi, the causative agent of Lyme disease, has a highly reduced genome and relies heavily on glycolysis for carbon metabolism. As such, established inhibitors of lactate dehydrogenase (LDH) were evaluated in cultures to determine the extent of their impacts on B. burgdorferi growth. Both racemic and enantiopure (AT-101) gossypol, as well as oxamate, galloflavin, and stiripentol, caused the dose-dependent suppression of B. burgdorferi growth in vitro. Racemic gossypol and AT-101 were shown to fully inhibit spirochetal growth at concentrations of 70.5 and 187.5 µM, respectively. Differences between racemic gossypol and AT-101 efficacy may indicate that the dextrorotatory enantiomer of gossypol is a more effective inhibitor of B. burgdorferi growth than the levorotatory enantiomer. As a whole, LDH inhibition appears to be a promising mechanism for suppressing Borrelia growth, particularly with bulky LDH inhibitors like gossypol.

The Action of the Pulsed Electric Field of the Subnanosecond Range on Human Tumor Cells.

The action of the pulsed electric field of the subnanosecond range on Jurkat, HEK 293, and U-87 MG human cell lines was studied. The cells were treated in a waveguide in 0.18 ml electrodeless Teflon cuvettes. The electric field strength in the cell culture medium was ~2 kV/cm, the pulse duration was ~1 ns, the leading edge was 150 ps, the frequency was 100 Hz, and the treatment time was 5 min. According to estimates, the change of the transmembrane potential during the pulse was ~20 mV and we assume that it was insufficient for electroporation. Jurkat and HEK 293 cells appeared to be more resistant to the treatment than U-87 MG cells. We have observed that the impulses with the above-mentioned parameters can cause a noticeable change in the mitochondrial activity of U-87 MG cells. © 2022 Bioelectromagnetics Society.

High-Throughput Screening Assays to Identify Plant Natural Products with Antifungal Properties Against Fusarium oxysporum.

Fusarium oxysporum is a cross-kingdom fungal pathogen that not only causes devastating plant vascular diseases but can also opportunistically infect humans. Here we describe two high-throughput screening assays, a resazurin cell viability assay and an optical density assay, to screen natural products from cultured plant cells with antifungal properties against a clinical isolate of F. oxysporum. After elicitation by applying methyl jasmonate or by co-culture with F. oxysporum, as an abiotic elicitor and a biotic elicitor, respectively, we identified three cell lines that produce materials that inhibit fungal growth. Our procedure validates the powerful potential of combining high-throughput methods for the discovery of novel anti-pathogenic leads.

Method of Measuring High-LET Particles Dose.

In boron neutron capture therapy, the total absorbed dose is the sum of four dose components with different relative biological effectiveness (RBE): boron dose, "nitrogen" dose, fast neutron dose and γ-ray dose. We present a new approach for measuring the first three doses. In this work, we provide the details of this method of dose measurement and results when this proposed method is employed.

Neutron Source Based on Vacuum Insulated Tandem Accelerator and Lithium Target.

A compact accelerator-based neutron source has been proposed and created at the Budker Institute of Nuclear Physics in Novosibirsk, Russia. An original design tandem accelerator is used to provide a proton beam. The proton beam energy can be varied within a range of 0.6-2.3 MeV, keeping a high-energy stability of 0.1%. The beam current can also be varied in a wide range (from 0.3 mA to 10 mA) with high current stability (0.4%). In the device, neutron flux is generated as a result of the 7Li(p,n)7Be threshold reaction. A beam-shaping assembly is applied to convert this flux into a beam of epithermal neutrons with characteristics suitable for BNCT. A lot of scientific research has been carried out at the facility, including the study of blistering and its effect on the neutron yield. The BNCT technique is being tested in in vitro and in vivo studies, and the methods of dosimetry are being developed. It is planned to certify the neutron source next year and conduct clinical trials on it. The neutron source served as a prototype for a facility created for a clinic in Xiamen (China).

Design and synthesis of C-aryl angular luotonins via a one-pot aza-Nazarov-Friedlander sequence and their Topo-I inhibition studies along with C-aryl vasicinones and luotonins.

A facile one-pot synthesis of C-ring substituted angular luotonins has been realized via a methanesulfonic acid mediated aza-Nazarov-Friedlander condensation sequence on quinazolinonyl enones. Topoisomerase I (topo-I) inhibition studies revealed that the angular luotonin library (7a-7l) and their regioisomeric analogs (linear luotonins, 8a-8l) are weak negative modulators, compared to camptothecin. These results would fare well for the design of topo-I-inert luotonins for non-oncological applications such as anti-fungal and insecticide lead developments. Surprisingly, the tricyclic vasicinones (9h, 9i, and 9j) showed better topo-I inhibition compared to pentacyclic C-aryl luotonins providing a novel pharmacophore for further explorations.

Similarity of angular distribution for THz radiation emitted by laser filament plasma channels of different lengths.

The influence of plasma channel length on an angular terahertz (THz) radiation distribution is experimentally studied for the channel formed under filamentation of an ultrashort laser pulse. It is shown that the angular distribution of the THz emission depends only on laser intensity in the filament and plasma density of the plasma channel and does not depend on the plasma channel length. A qualitative explanation of the THz emission screening by the filament plasma channel is proposed.

Identification of Protein Recognition Elements within Heparin Chains Using Enzymatic Foot-Printing in Solution and Online SEC/MS.

Understanding molecular mechanisms governing interactions of glycosaminoglycans (such as heparin) with proteins remains challenging due to their enormous structural heterogeneity. Commonly accepted approaches seek to reduce the structural complexity by searching for "binding epitopes" within the limited subsets of short heparin oligomers produced either enzymatically or synthetically. A top-down approach presented in this work seeks to preserve the chemical diversity displayed by heparin by allowing the longer and structurally diverse chains to interact with the client protein. Enzymatic lysis of the protein-bound heparin chains followed by the product analysis using size exclusion chromatography with online mass spectrometry detection (SEC/MS) reveals the oligomers that are protected from lysis due to their tight association with the protein, and enables their characterization (both the oligomer length, and the number of incorporated sulfate and acetyl groups). When applied to a paradigmatic heparin/antithrombin system, the new method generates a series of oligomers with surprisingly distinct sulfation levels. The extent of sulfation of the minimal-length binder (hexamer) is relatively modest yet persistent, consistent with the notion of six sulfate groups being both essential and sufficient for antithrombin binding. However, the masses of longer surviving chains indicate complete sulfation of disaccharides beyond the hexasaccharide core. Molecular dynamics simulations confirm the existence of favorable electrostatic interactions between the high charge-density saccharide residues flanking the "canonical" antithrombin-binding hexasaccharide and the positive patch on the surface of the overall negatively charged protein. Furthermore, electrostatics may rescue the heparin/protein interaction in the absence of the canonical binding element.

A study of matrix and admixture elements in fluorine-rich ionic conductors by pulsed glow discharge mass spectrometry.

RATIONALE: Dopants in ionic conductors play a crucial role in achieving the required electrochemical properties. A slight variation in their concentration considerably affects the conductivity of crystals and their applicability as ionic conductors and laser materials. To ensure the growth of high-quality fluoride crystals, adequate approaches for the quantification of matrix and admixture/dopant components are required. METHODS: A panel of SrF2 - and GdF3 -doped LaF3 single crystals was investigated. The electrical conductivity of the crystals was measured using impedance spectroscopy in the frequency range 100 Hz-1 MHz to control for crystal quality. Pulsed glow discharge mass spectrometry (GDMS) was used to simultaneously quantify fluorine, strontium, lanthanum, and gadolinium in the crystals. X-ray fluorescence, scanning electron microscopy-energy dispersive X-ray spectroscopy, and arc optical emission spectrometry were used for validation. RESULTS: Quasiperiodic intensity drifts under sputtering of the ionic conductors were observed and attributed to F- redistribution on the sample surface, affecting surface conductivity and sputtering rate. Several sample preparation protocols were tested to address that effect. Full coating of the sample with a layer of silver several micrometers thick provided stable and effective sputtering. The parameters for the GDMS determination of F, Sr, La, and Gd were optimized. The elements' distribution was studied in different parts of the crystals. CONCLUSIONS: An analytical approach to the direct multi-element analysis of fluoride-containing ionic conductors using pulsed GDMS with La1-x-y Srx Gdy F3-x as an example was designed and tested. Instability effects of ionic conductivity were explained and coped with, providing effective and stable sputtering.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

Ruthenium coordination preferences in imidazole-containing systems revealed by electrospray ionization mass spectrometry and molecular modeling: Possible cues for the surprising stability of the Ru (III)/tris (hydroxymethyl)-aminomethane/imidazole complexes.

Ruthenium is a platinoid that exhibits a range of unique chemical properties in solution, which are exploited in a variety of applications, including luminescent probes, anticancer therapies, and artificial photosynthesis. This paper focuses on a recently demonstrated ability of this metal in its +3 oxidation state to form highly stable complexes with tris (hydroxymethyl)aminomethane (H2 NC(CH2 OH)3 , Tris-base or T) and imidazole (Im) ligands, where a single RuIII cation is coordinated by two molecules of each T and Im. High-resolution electrospray ionization mass spectrometry (ESI MS) is used to characterize RuIII complexes formed by placing a RuII complex [(NH3 )5 RuII Cl]Cl in a Tris buffer under aerobic conditions. The most abundant ionic species in ESI MS represent mononuclear complexes containing an oxidized form of the metal, ie, [Xn RuIII T2 - 2H]+ , where X could be an additional T (n = 1) or NH3 (n = 0-2). Di- and tri-metal complexes also give rise to a series of abundant ions, with the highest mass ion representing a metal complex with an empirical formula Ru3 C24 O21 N6 H66 (interpreted as cyclo(T2 RuO)3 , a cyclic oxo-bridged structure, where the coordination sphere of each metal is completed by two T ligands). The empirical formulae of the binuclear species are consistent with the structures representing acyclic fragments of cyclo(T2 RuO)3 with addition of various combinations of ammonia and dioxygen as ligands. Addition of histidine in large molar excess to this solution results in complete disassembly of poly-nuclear complexes and gives rise to a variety of ionic species in the ESI mass spectrum with a general formula [RuIII Hisk Tm (NH3 )n - 2H]+ , where k = 0 to 2, m = 0 to 3, and n = 0 to 4. Ammonia adducts are present for all observed combinations of k and m, except k = m = 2, suggesting that [His2 RuIII T2 - 2H]+ represents a complex with a fully completed coordination sphere. The observed cornucopia of RuIII complexes formed in the presence of histidine is in stark contrast to the previously reported selective reactivity of imidazole, which interacts with the metal by preserving the RuT2 core and giving rise to a single abundant ruthenium complex (represented by [Im2 RuIII T2 - 2H]+ in ESI mass spectra). Surprisingly, the behavior of a hexa-histidine peptide (HHHHHH) is similar to that of a single imidazole, rather than a single histidine amino acid: The RuT2 core is preserved, with the following ionic species observed in ESI mass spectra: [HHHHHH·(RuIII T2 )m - (3m-1)H]+ (m = 1-3). The remarkable selectivity of the imidazole interaction with the RuIII T2 core is rationalized using energetic considerations at the quantum mechanical level of theory.

Structural Heterogeneity in the Preamyloid Oligomers of ß-2-Microglobulin.

In dialysis patients, the protein ß2-microglobulin (ß2m) forms amyloid fibrils in a condition known as dialysis-related amyloidosis. To understand the early stages of the amyloid assembly process, we have used native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble preamyloid oligomers. ESI-IM-MS reveals the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, results which are distinct from ß2m oligomers formed at low pH. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers-one that is compact and another that is more expanded. Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems in which conformational heterogeneity is often associated with different amyloid morphologies or off-pathway events, conformational heterogeneity in the tetramer seems to be a necessary aspect of Cu(II)-induced amyloid formation by ß2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of Cu(II) release in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies.

Synthesis of C-Ring-Substituted Vasicinones and Luotonins via Regioselective Aza-Nazarov Cyclization of Quinazolinonyl Enones.

A facile synthesis of C-ring substituted luotonins and vasicinones has been realized via a super-acid-mediated aza-Nazarov cyclization of quinazolinonyl enones. The regioselectivity of the cyclization is highly dependent on proton availability in the reaction medium.

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

Modulation of Amyloid-ß42 Conformation by Small Molecules Through Nonspecific Binding.

Aggregation of amyloid-ß (Aß) peptides is a crucial step in the progression of Alzheimer's disease (AD). Identifying aggregation inhibitors against AD has been a great challenge. We report an atomistic simulation study of the inhibition mechanism of two small molecules, homotaurine and scyllo-inositol, which are AD drug candidates currently under investigation. We show that both small molecules promote a conformational change of the Aß42 monomer toward a more collapsed phase through a nonspecific binding mechanism. This finding provides atomistic-level insights into designing potential drug candidates for future AD treatments.

Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes.

Soil-transmitted nematodes (STNs), namely hookworms, whipworms, and ascarids, are extremely common parasites, infecting 1-2 billion of the poorest people worldwide. Two benzimidazoles, albendazole and mebendazole, are currently used in STN mass drug administration, with many instances of low/reduced activity reported. New drugs against STNs are urgently needed. We tested various models for STN drug screening with the aim of identifying the most effective tactics for the discovery of potent, safe and broad-spectrum agents. We screened a 1280-compound library of approved drugs to completion against late larval/adult stages and egg/larval stages of both the human hookworm parasite Ancylostoma ceylanicum and the free-living nematode Caenorhabditis elegans, which is often used as a surrogate for STNs in screens. The quality of positives was further evaluated based on cheminformatics/data mining analyses and activity against evolutionarily distant Trichuris muris whipworm adults. From these data, two pairs of positives, sulconazole/econazole and pararosaniline/cetylpyridinium, predicted to target nematode CYP-450 and HSP-90 respectively, were prioritized for in vivo evaluation against A. ceylanicum infections in hamsters. One of these positives, pararosaniline, showed a significant impact on hookworm fecundity in vivo. Taken together, our results suggest that anthelmintic screening with A. ceylanicum larval stages is superior to C. elegans based on both reduced false negative rate and superior overall quality of actives. Our results also highlight two potentially important targets for the discovery of broad-spectrum human STN drugs.

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6.

Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.

Facile synthesis of 3-substituted imidazo[1,2-a]pyridines through formimidamide chemistry.

A facile entry to 3-aryl/alkenyl/alkynyl substituted imidazo[1,2-a]pyridines (3a-p, 6a-d & 9a-9e) has been developed from readily available benzyl/allyl/propargyl halides and 2-amino pyridines as substrates via formimidamide chemistry that is devoid of caustic or expensive reagents, such as transition metal complexes. Quantum chemical calculations performed to understand the underlying mechanism of the transformation revealed a preference for intramolecular Mannich-type addition over pericyclic 1,5-electrocyclization for the systems reported herein that enable a Baldwin allowed 5-exo-trig cyclization instead of a formally anti-Baldwin 5-endo-trig process.