Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “human activity”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45

Toxicology and Biodegradability of Blendstocks for Mixing Controlled Compression Ignition Combustion: Literature Review of Available Data

The goal of the U.S. Department of Energy’s Co-Optima project is to develop an understanding of potential biomass-derived blendstocks for use in the transportation sector. This report documents the estimated environmental and toxicological impact of potential bioblendstocks for mixing controlled compression ignition (MCCI) engines. The candidate molecules were: 2-nonanol; n-undecane; 2,6,10-trimethyldodecane; 5-ethyl-4-propylnonane; hexylhexanoate; methyldecanoate; dibutoxymethane; 4-butoxyheptane; dipentylether; renewable diesel; and soy biodiesel. To provide a robust comparison, diesel surrogate molecules were also considered: α-methylnaphthalene; decahydronaphthalene; 2,2,4,4,6,8,8-heptamethylnonane; n-butylcyclohexane; n-hexadecane; tetralin; and n-dodecylbenzene. The intent of this work was not to provide absolute answers and remove any molecules from consideration, but to provide more robust data for each molecule that can be used in subsequent evaluations. Little literature data was available for many of the molecules, both potential blendstocks and diesel surrogates, so a quantitative structural activity relationship (QSAR) approach was used to provide input into the relevant models. This assessment included an evaluation of compartment partitioning in the event of an accidental chemical spill, fate and transport indicators, estimated biodegradability, and human and environmental toxicology. The higher molecular weight and longer hydrocarbon chain lengths of these MCCI molecules reduce water solubility and mobility. Generally, the oxygenated molecules improved biodegradability metrics. The hydrocarbons farnesane and 5-Et-4-PrNonane are not predicted to be biodegradable. Human acute toxicity was found to be slightly to non-toxic for the MCCI blendstocks and the diesel surrogates. 2-Nonanol, undecane, MeDecanoate, dibutoxymethane, and dipentylether were likely developmental toxicants. Although poorly water soluble, all the compounds, whether Co-Optima blendstocks or diesel surrogates, showed significant toxicity to D. magna. The oxygenated blendstocks were less likely to bioaccumulate than the non-oxygenated blendstocks, which behaved similar to the diesel surrogates. Overall, this predicted analysis showed that the MCCI blendstocks are very similar to their diesel counterparts. No significant showstoppers were found that would indicate concern moving forward with additional research into these potential fuels. The results presented were based solely on predictive models and serve to provide information going forward for continued evaluation of these molecules.

09 BIOMASS FUELS↗

Quantitative measurements of reactive oxygen species partitioning in electron transfer flavoenzyme magnetic field sensing

Biological magnetic field sensing that gives rise to physiological responses is of considerable importance in quantum biology. The radical pair mechanism (RPM) is a fundamental quantum process that can explain some of the observed biological magnetic effects. In magnetically sensitive radical pair (RP) reactions, coherent spin dynamics between singlet and triplet pairs are modulated by weak magnetic fields. The resulting singlet and triplet reaction products lead to distinct biological signaling channels and cellular outcomes. A prevalent RP in biology is between flavin semiquinone and superoxide (O 2 •–) in the biological activation of molecular oxygen. This RP can result in a partitioning of reactive oxygen species (ROS) products to form either O 2 •– or hydrogen peroxide (H 2 O 2 ). Here, we examine magnetic sensing of recombinant human electron transfer flavoenzyme (ETF) reoxidation by selectively measuring O 2 •– and H 2 O 2 product distributions. ROS partitioning was observed between two static magnetic fields at 20 nT and 50 μT, with a 13% decrease in H 2 O 2 singlet products and a 10% increase in O 2 •– triplet products relative to 50 µT. RPM product yields were calculated for a realistic flavin/superoxide RP across the range of static magnetic fields, in agreement with experimental results. For a triplet born RP, the RPM also predicts about three times more O 2 •– than H 2 O 2 , with experimental results exhibiting about four time more O 2 •– produced by ETF. The method presented here illustrates the potential of a novel magnetic flavoprotein biological sensor that is directly linked to mitochondria bioenergetics and can be used as a target to study cell physiology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

The global anaerobic metabolism regulator fnr is necessary for the degradation of food dyes and drugs by Escherichia coli

ABSTRACT The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made toward identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N = N-R’), found in diverse chemicals in both food and drugs. Surprisingly, the canonical azoR gene in Escherichia coli was dispensable for azo bond reduction. Instead, azoreductase activity was controlled by the fumarate and nitrate reduction ( fnr ) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of the fnr regulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Furthermore, we found that FNR indirectly regulates this process through the small noncoding regulatory RNA fnrS . Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical groups found in both dietary and pharmaceutical compounds. IMPORTANCE This work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context.

59 BASIC BIOLOGICAL SCIENCES↗

Endothelial cells control vascular smooth muscle cell cholesterol levels by regulating 24-dehydrocholesterol reductase expression

Highlights: • Suspended endothelial and vascular smooth muscle cells organize to form spherical vascular organoids in hanging drops. • Vascular smooth muscle cells downregulate the expression of metabolism regulating genes in presence of endothelial cells. • Endothelial cells decrease the levels of 24-dehydrocholesterol reductase and cholesterol in vascular smooth muscle cells. • Diminished cholesterol biosynthesis inhibits vascular smooth muscle cell activity. Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.

60 APPLIED LIFE SCIENCES↗

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated. The Entner-Doudoroff pathway does not have FBP as a pathway intermediate, and ZmPYK is not allosterically activated. In this work, we solved the 2.4 Å X-ray crystallographic structure of ZmPYK. The protein is dimeric in solution as determined by gel filtration chromatography, but crystallizes as a tetramer. The buried surface area of the ZmPYK tetramerization interface is significantly smaller than that of hlPYK, and yet tetramerization using the standard interfaces from higher organisms provides an accessible low energy crystallization pathway. Interestingly, the ZmPYK structure showed a phosphate ion in the analogous location to the 6-phosphate binding site of FBP in hlPYK. Circular Dichroism (CD) was used to measure melting temperatures of hlPYK and ZmPYK in the absence and presence of substrates and effectors. The only significant difference was an additional phase of small amplitude for the ZmPYK melting curves. We conclude that the phosphate ion plays neither a structural or allosteric role in ZmPYK under the conditions tested. In conclusion, we hypothesize that ZmPYK does not have sufficient protein stability for activity to be tuned by allosteric effectors as described for rheostat positions in the allosteric homologues.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Photometric sensing of heavy metal ions using a naphthoquinodimethyl- bis -thioamide dye: Selectivity & photophysics of the metal organic complexes

As Mother Nature is experiencing catastrophic environmental pollutions from both natural sources and anthropogenic activities, many scientists have been working around the clock to develop environmentally benign and cost-effective, yet sensitive, detection techniques for pollutants, especially heavy metal species (e.g. lead, cadmium, mercury, among others) that are deleterious to human health. Herein, we report a novel sulfur-containing small organic dye/sensor naphthoquinodimethyl-bis-thioamide (QDM), which was found to be particularly selective toward mercury ion (Hg 2+ ). Using a combination of UV-vis absorption, photoluminescence, and time-resolved pump-probe techniques, we established that QDM and Hg 2+ can form stable complex(es) due to the strong affinity of sulfur toward mercury. In this investigation, while a higher ratio of QDM:Hg 2+ was necessary to fully quench the fluorescence emission of QDM, only 1 equiv of the Hg 2+ ion was necessary to observe the sensing effect on the excited state photo-behavior(s) of QDM. Furthermore, the present results highlight a synergy between molecular sensors' selectivity/sensitivity and sensor-analytes dynamics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Stimulus-Responsive Modulation of Solvation Environments in Solid Catalysts

Liquid environments play a crucial role in the biological processes occurring in living organisms as well as in many human-made processes involving electrochemistry, photo-, and thermocatalysis. In the majority of these systems, aqueous phases are ubiquitous due to water’s natural abundance. Water molecules, however, can exert large changes in the chemical environment of catalytically active sites, altering the reaction rates, selectivity, and catalyst stability. These solvation effects induced by water molecules near catalytic sites can drastically change the energy landscape and unlock unique reaction pathways with far more favorable kinetics. In nature, living organisms couple these complex interactions with detection, communication, and actuation mechanisms to induce self-regulatory behavior, ensuring stability of the system and thus long-term durability. Extrapolating this behavior to heterogeneous catalysis is desirable because the resulting “smart materials” can potentially unlock new chemical conversion processes with higher atom efficiency, rates, and stability. The combination of polymer chemistry and heterogeneous catalysis has introduced versatile approaches for creating materials that can respond to cues in the reaction medium that alter the accessibility, intrinsic activity, and selectivity of the catalyst. To achieve this, one could combine stimulus-responsive polymers, which undergo a large volumetric phase transition in response to an external stimulus, with a solid catalyst. This chemo-mechanical response has been employed to create a variety of nanoreactor vessels with stimulus-responsive character that turn on- and off- depending on the reaction conditions. In this Account, we focus on the impact of these polymer coatings on the solvation environment around the active site and the implications of these effects on the reaction energy landscape, molecular arrangement of the solvent, electric fields at the catalyst–liquid interface, binding energy, and mobility of surface reaction intermediates. These seemingly subtle changes in solvent molecules induced by the presence of polymers can have a tremendous impact on the development of bioinspired heterogeneous catalysts, reliable chemical clocks, micro/nanoreactors, and robots. The large library of polymer chemistries offers a plethora of combinations of stimulus-responsive mechanisms (e.g., temperature, pH, light, magnetic field, solvent composition), providing the possibility of creating homeostatic catalysts à la carte.

catalysts↗

The promise and challenge of therapeutic genome editing

Genome editing, which involves the precise manipulation of cellular DNA sequences to alter cell fates and organism traits, has the potential to both improve our understanding of human genetics and cure genetic disease. Here I discuss the scientific, technical and ethical aspects of using CRISPR (clustered regularly interspaced short palindromic repeats) technology for therapeutic applications in humans, focusing on specific examples that highlight both opportunities and challenges. Further, genome editing is—or will soon be—in the clinic for several diseases, with more applications under development. The rapid pace of the field demands active efforts to ensure that this breakthrough technology is used responsibly to treat, cure and prevent genetic disease.

60 APPLIED LIFE SCIENCES↗

Structural insights into the unexpected agonism of tetracyclic antidepressants at serotonin receptors 5-HT 1e R and 5-HT 1F R

Serotonin [5-hydroxytryptamine (5-HT)] acts via 13 different receptors in humans. Of these receptor subtypes, all but 5-HT 1e R have confirmed roles in native tissue and are validated drug targets. Despite 5-HT 1e R’s therapeutic potential and plausible druggability, the mechanisms of its activation remain elusive. To illuminate 5-HT 1e R’s pharmacology in relation to the highly homologous 5-HT 1F R, we screened a library of aminergic receptor ligands at both receptors and observe 5-HT 1e R/5-HT 1F R agonism by multicyclic drugs described as pan-antagonists at 5-HT receptors. Potent agonism by tetracyclic antidepressants mianserin, setiptiline, and mirtazapine suggests a mechanism for their clinically observed antimigraine properties. Using cryo-EM and mutagenesis studies, we uncover and characterize unique agonist-like binding poses of mianserin and setiptiline at 5-HT 1e R distinct from similar drug scaffolds in inactive-state 5-HTR structures. Together with computational studies, our data suggest that these binding poses alongside receptor-specific allosteric coupling in 5-HT 1e R and 5-HT 1F R contribute to the agonist activity of these antidepressants.

59 BASIC BIOLOGICAL SCIENCES↗

Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

59 BASIC BIOLOGICAL SCIENCES↗

Graph Generation with a Focusing Lexicon

Reference datasets are an essential part of developing effective data exploration tools. Many reference datasets are synthetic in nature, seeding real-world properties into generation routines. Others are laboriously curated from original source materials. This is a time-consuming process that requires significant domain expertise all along the process. This paper presents a third path: deriving a network from real-world source materials using automated tools. By properly grounding the tools in a small collection of curated documents, a larger corpus be automatically processed with greater confidence. The end system leverages human knowledge (in the initial document curation), but with much less time commitment enabled by automation. Our ultimate goal is to produce reference datasets, derived from real-world data that capture aspects salient to a specific activity in the context of all of the activities in the data-set. Our reference datasets take the form of graphs.

Chandra Shekar, Mayanka↗

YAP1 Dysfunction Promotes Molecular Properties Linked to Breast Cancer Susceptibility

YAP1 is a cotranscription factor that promotes malignant and stem cell properties in cancer. We previously found that YAP1 dysregulation is associated with aging in human mammary epithelia. With increased age, YAP1 expression changes in luminal epithelial cells, the prospective breast cancer cell of origin. Because age is a significant risk factor for breast cancer, we tested whether YAP1 dysregulation acted early in cancer progression by conferring cellular states associated with increased cancer susceptibility. In this study, we find that with increased age and genetic risk for developing cancer, human breast tissues showed significantly increased YAP1 expression, and cultured primary human mammary epithelial cells (HMEC) showed significantly increased expression of both YAP1 and its transcriptional targets. Increased YAP1 expression in cultured HMEC induced gene expression changes associated with increased cancer susceptibility, such as genes associated with stem cell states, increased telomerase activity, breast cancer progression, and increased age and genetic breast cancer risk. Furthermore, overexpression of YAP1 in post-stasis HMEC—finite lifespan cells that have bypassed a retinoblastoma-mediated senescence barrier—promoted properties related to increased growth potential. We found that YAP1 dysregulation in finite epithelial cells allows for access to gene programs and functions that are typically thought to be restricted to stem cells. We hypothesize that YAP1 acts early in breast cancer progression, long before the development of a tumor, to impose cancer-susceptible molecular states.

YAP1↗

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency.

59 BASIC BIOLOGICAL SCIENCES↗

Optimization of Species-Selective Reversible Proteasome Inhibitors for the Treatment of Malaria

Abstract Malaria remains a critical global health challenge, with increasing resistance to frontline therapies necessitating novel drug targets. The proteasome has emerged as a promising target for antimalarial drug discovery. This study describes efforts to optimize a series of species-selective reversible inhibitors targeting the Plasmodium falciparum 20S proteasome. Starting from the carboxypiperidine scaffold identified through a high-throughput viability screen, we conducted iterative structure–activity relationship studies, leading to the development of highly potent and selective inhibitors with good oral bioavailability. Lead compounds demonstrated nanomolar potency against P. falciparum blood-stage parasites and selective inhibition of the parasite proteasome over the human counterpart. Cryo-EM structural studies confirmed binding at the β5 subunit, while in vivo pharmacokinetic studies identified promising candidates for further development. These findings support proteasome inhibition as a viable strategy for novel antimalarial drug development.

Gahalawat, Suraksha [UT Southwestern Medical Cente↗

Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

59 BASIC BIOLOGICAL SCIENCES↗

Genotype-Specific Neutralization of Norovirus Is Mediated by Antibodies Against the Protruding Domain of the Major Capsid Protein

Abstract Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural shell and protruding (P) domains, from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design.

Ford-Siltz, Lauren A.↗

Radsource Mr: Mixed Reality Planning Tool For Radioactive Recovery

The RadSource MR system leverages Meta Quest 3's advanced mixed reality capabilities to create a comprehensive spatial planning platform for end-of-life sealed radioactive source recovery operations. The application utilizes the Quest 3's high-resolution passthrough cameras and spatial mapping algorithms to generate accurate 3D environmental models. Core technical components include: (1) Real-time spatial measurement algorithms calculating distances, angles, slopes, and surface areas with sub-centimeter accuracy; (2) Virtual object placement system allowing users to position digital representations of recovery equipment (trailers, containment vessels, protective barriers) within the real environment; (3) Voice recording and annotation system for hands-free documentation in protective equipment; (4) 3D mesh capture and storage capabilities for post-operation analysis and regulatory documentation. (5) Procedure documentation is available for viewing in Mixed Reality, providing an innovative and convenient way to access the information during pre-visit and inspection activities. (6) Support for screen capture for the view for real world and virtual objects together to use it later for planning. The system integrates computer vision techniques for environmental understanding, spatial mathematics for precise measurements, and human-computer interaction principles optimized for hazardous environment operations. Data persistence allows teams to save and share planning sessions across multiple stakeholders while maintaining operational security requirements.

Khadka, Rajiv [Idaho National Laboratory (INL), Id↗

A non-traditional crystal-based compound screening method targeting the ATP binding site of Plasmodium falciparum GRP78 for identification of novel nucleoside analogues

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight ‘hit’ compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti-Plasmodium specific compounds.

59 BASIC BIOLOGICAL SCIENCES↗