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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.

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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a distinctive mode of engaging poly(ADP-ribose)

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Amantadine interactions with phase separated lipid membranes

Amantadine, a small amphilphic organic compound that consists of an adamantane backbone and an amino group, was first recognized as an antiviral in 1963 and received approval for prophylaxis against the type A influenza virus in 1976. Since then, it has also been used to treat Parkinson’s disease-related dyskinesia and is being considered as a treatment for corona viruses. Since amantadine usually targets membrane-bound proteins, its interactions with the membrane are also thought to be important. Biological membranes are now widely understood to be laterally heterogeneous and certain proteins are known to preferentially co-localize within specific lipid domains. Does amantadine, therefore, preferentially localize in certain lipid composition domains? To address this question, here, we studied amantadine’s interactions with phase separating membranes composed of cholesterol, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), as well as single-phase DPhPC (1,2-diphytanoyl-sn-glycero-3-phos-phocholine) membranes. From Langmuir trough and differential scanning calorimetry (DSC) measurements, we determined, respectively, that amantadine preferentially binds to disordered lipids, such as POPC, and lowers the phase transition temperature of POPC/DSPC/cholesterol mixtures, implying that amantadine increases membrane disorder. Further, using droplet interface bilayers (DIBs), we observed that amantadine disrupts DPhPC membranes, consistent with its disordering properties. Finally, we carried out molecular dynamics (MD) simulations on POPC/DSPC/cholesterol membranes with varying amounts of amantadine. Consistent with experiment, MD simulations showed that amantadine prefers to associate with disordered POPC-rich domains, domain boundaries, and lipid glycerol backbones. Since different proteins co-localize with different lipid domains, our results have possible implications as to which classes of proteins may be better targets for amantadine.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Structural insights into RNase H catalytic mechanism from room-temperature X-ray and neutron crystallography of apo- and RNA/DNA hybrid-bound enzyme

RNase H enzymes are sequence-nonspecific endonucleases that cleave RNA strands in RNA/DNA hybrid duplexes, an enzymatic process essential in DNA replication and repair in both prokaryotes and eukaryotes. Also, RNase H activity of the reverse transcriptase in human immunodeficiency viruses (HIV-1 and HIV-2) is indispensable for the viral replication cycle. RNase H enzymes play an central role in the development of gene therapies and are targets for novel antivirals. It is therefore of great importance to gain a detailed understanding of the RNase H catalytic mechanism to improve drug design. We utilized Bacillus halodurans RNase H1 (BhRNase H1) to shed light on its function and catalytic mechanism. Room-temperature neutron crystallography of the wild-type and inactive D132N mutant enzymes revealed that E109, belonging to the catalytic DEDD motif, can change its protonation state, allowing us to propose its role in the protonation of the leaving O3′ hydroxyl group of RNA. X-ray crystallography has demonstrated the ability of the RNA/DNA duplex to slide along the protein surface upon metal ion binding at site M A , transforming a product mimic into a Michaelis-like complex, which confirms an essential role of the M A metal ion in catalysis.

Enzyme mechanisms↗
DOE OSTI2025

Substituent size versus metal binding of inhibitors with variants of influenza endonuclease

The influenza virus causes a significant burden of illness each year. Although vaccination is the most effective method to prevent seasonal influenza infection, viral escape mechanisms make vaccine composition difficult to predict. Antivirals are crucial for decreasing rates of morbidity and mortality from influenza viral infection. The newest anti-influenza drugs target the RNA-dependent RNA polymerase acidic N-terminal (PA N ) endonuclease, a critical component of influenza viral replication machinery. This study examines the structure of inhibitors of PAN that utilize a hydroxypyridinone-based metal-binding pharmacophore (MBP). Specifically, this report explores how the size of substituent groups impacts the binding conformation and affinity of a series of compounds against both wild-type (WT) and resistance mutant strains, I38T and E23K. Co-crystal structures revealed that the distance between compounds and enzyme residue 38 was conserved to maintain strong interactions, resulting in deviations from ideal coordination geometries at the active site metal centers. This suggests the interactions with residue 38 with each compound is important and can impact inhibitor potency as a consequence of distortions in the metal binding geometry of the compounds.

Endonuclease↗
DOE OSTI2021

Development of a rapid viability RT-PCR (RV-RT-PCR) method to detect infectious SARS-CoV-2 from swabs

Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, here we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (C T ) difference (ΔC T ). An algorithm based on ΔC T ≥ 6 representing ~ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 h (for a batch of 12 swabs), compared to days typically required by the cell-culture-based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser

NendoU from SARS-CoV-2 is responsible for the virus’s ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evidence that Maackia amurensis seed lectin (MASL) exerts pleiotropic actions on oral squamous cells with potential to inhibit SARS-CoV-2 infection and COVID-19 disease progression

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 125 million confirmed COVID-19 cases that have caused over 2.7 million deaths worldwide as of March 2021. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface “spike” protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. In addition, we report that MASL also inhibits SARS-CoV-2 infection of kidney epithelial cells in culture. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Antitumor CD8 T cell responses in glioma patients are effectively suppressed by T follicular regulatory cells

Regulatory T (Treg) cells are thought to contribute to tumor pathogenesis by suppressing tumor immunosurveillance and antitumor immunity. T follicular regulatory (Tfr) cells are a recently characterized Treg subset that expresses both the Treg transcription factor (TF) Foxp3 and the T follicular helper (Tfh) TF Bcl-6. The role of Tfr cells in glioma patients remains unclear. In this study, we found that the level of Tfr cells, identified as Foxp3{sup +}Bcl-6{sup +} CD4 T cells, was significantly elevated in tumor-infiltrating CD4 T cells from resected glioma tumors. Both Tfr cells and Treg cells significantly suppressed the proliferation and the cytotoxic capacity of CD8 T cells toward glioma tumor cells, and the suppression was positively associated with the proportion of Tfr cells and Treg cells, respectively. Tfr and Treg cells from glioma tumor samples demonstrated higher suppression potency than those from healthy blood samples and glioma blood samples. Interestingly, canonical CXCR5{sup -} Treg cells could suppress both CXCR5{sup +} and CXCR5{sup -} CD8 T cells, albeit with stronger potency toward CXCR5{sup -} CD8 T cells. However, Tfr cells presented much higher suppression potency toward CXCR5{sup +} CD8 T cells, whereas CXCR5{sup +} CD8 T cells are a potent CD8 T cell subset previously described to have antiviral and antitumor roles. Overall, these data indicate that Tfr cells are enriched in glioma tumors and have suppressive capacity toward CD8 T cell-mediated effector functions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2)

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here we use structure-based drug prototyping to identify two classes of promising leads that non-competitively inhibit UCK2 activity. In the process we have identified a hitherto unknown allosteric site at the inter-subunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Impact of Remdesivir Incorporation along the Primer Strand on SARS-CoV-2 RNA-Dependent RNA Polymerase

Remdesivir was the first antiviral drug that received emergency use authorization from the United States Food and Drug Administration and is now formally approved to treat COVID-19. Remdesivir is a nucleotide analogue that targets the RNA-dependent RNA polymerase (RdRp) of coronaviruses, including SARS-CoV-2. The solution of multiple RdRp structures has been one of the main axes of research in the race against the SARS-CoV-2 virus. Several hypotheses of the mechanism of inhibition of RdRp by remdesivir have been proposed, although open questions remain. This work uses molecular dynamics simulations to explore the impact of remdesivir and two analogues as incoming nucleotides and of up to four incorporations of remdesivir along the primer strand on RdRp. The simulation results suggest that the overall structure and the dynamical behavior of RdRp are destabilized by remdesivir and the two analogues in the incoming position. The incorporation of remdesivir along the primer strand impacts specific non-bonded interactions between the nascent RNA and the polymerase subunit, as well as the overall dynamical networks on RdRp. The strongest impact on the structure and dynamics are observed after three incorporations, when remdesivir is located at position -A3, in agreement with previously reported experimental and computational results. Our results provide atomic-level details of the role played by remdesivir on the disruption of RNA synthesis by RdRp and the main drivers of these disruptions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure–activity relationship (SAR) study of noncovalent compounds that bind in the enzyme’s substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor’s P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

3D-Scaffold: A Deep Learning Framework to Generate 3D Coordinates of Drug-like Molecules with Desired Scaffolds

The prerequisite of therapeutic drug design is to identify novel molecules with desired biophysical and biochemical properties. Deep generative models have demonstrated their ability to find such molecules by exploring a huge chemical space efficiently. An effective way to obtain molecules with desired target properties is the preservation of critical scaffolds in the generation process. To this end, we propose a domain aware generative framework called 3D-Scaffold that takes 3D coordinates of a desired scaffold as an input and generates 3D coordinates of novel therapeutic candidates as an output while always preserving the desired scaffolds in generated structures. We show that our framework generates predominantly valid, unique, novel, and experimentally synthesizable molecules that have drug-like properties similar to the molecules in the training set. Using domain specific datasets, we generate covalent and non-covalent antiviral inhibitors. Therefore, to measure the success of our framework in generating therapeutic candidates, generated structures were subjected to high throughput virtual screening via docking simulations, which shows favorable interaction against SARS-CoV-2 main protease and non-structural protein endoribonuclease (NSP15) targets. Most importantly, our model performs well with relatively small volumes of training data and generalizes to new scaffolds, making it applicable to other domain.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Human Coronavirus-229E Hijacks Key Host-Cell RNA-Processing Complexes for Replication

The recent rise in zoonotic coronavirus outbreaks underscores the urgency to understand virus-host interactions and develop potent antiviral therapeutics. Systems biology approaches, particularly proteomics have been invaluable in providing a global overview of such interactions. However, these conventional approaches rely on measuring protein abundance changes which don’t reflect functional shifts. In this study, we employed a high-throughput structural proteomics approach called limited proteolysis-based mass spectrometry (LiP-MS) to capture conformational changes, which we demonstrate are better proxies for functional alterations. We applied this tool to both immortalized and primary human lung cells following human coronavirus 229E (HCoV-229E) infection. We identified significant infection-induced structural changes within RNA processing complexes such as the spliceosome-C and NOP56-associated complex. These observations emphasize that HCoV-229E infection propagates a multi-pronged effort to obstruct the house keeping RNA processing functions in the host. Finally, we show that HCoV-229E replication can be attenuated by the targeted disruption of these complexes, indicating that the identified cellular factories are viable targets to prevent coronavirus infection.

coronavirus↗
DOE OSTI2021

Potent Noncovalent Inhibitors of the Main Protease of SARS-CoV-2 from Molecular Sculpting of the Drug Perampanel Guided by Free Energy Perturbation Calculations

Starting from our previous finding of 14 known drugs as inhibitors of the main protease (M pro ) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC 50 values in a kinetic assay. Free-energy perturbation (FEP) calculations for M pro -ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to M pro . Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment of COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2025

Influence of Steric and Electronic Properties of P2 Groups on Covalent Inhibitor Binding to SARS-CoV-2 Main Protease

The main protease (MPro) of SARS-CoV-2 is a critical enzyme required for viral replication, making it a prime target for antiviral drug development. Covalent inhibitors, which form a stable interaction with the catalytic C145, have demonstrated strong inhibition of MPro, but the influence of steric and electronic properties of P2 substituents, designed to engage the S2 substrate-binding subsite within the MPro active site, on inhibitor binding affinity remains underexplored. Here, in this study, we design and characterize two hybrid covalent inhibitors, BBH-3 and BBH-4, and present their X-ray crystallographic structures in complex with MPro, providing molecular insights into how their distinct P2 groups, a dichlorobenzyl moiety in BBH-3 and an adamantyl substituent in BBH-4, affect binding conformation and active site adaptability. Comparative structural analyses with previously characterized inhibitors, including BBH-2 and Mcule-5948770040, reveal how the P2 bulkiness and electronic properties influence active site dynamics, particularly through interactions with the S2 and S5 subsites. The P2 group of BBH-3 induces conformational shifts in the S2 helix and the S5 loop, while BBH-4 displaces M49, stabilizing its binding through hydrophobic interactions. Isothermal titration calorimetry further elucidates the impact of P2 modifications on inhibitor affinity, revealing a delicate balance between enthalpic and entropic contributions. The data demonstrate that BBH-3 exhibits less favorable binding, affirming that dichlorobenzyl substitution at the P2 position has a more negative impact on the affinity for MPro than bulky saturated cyclic groups. This underscores the feature that MPro active site malleability may be accompanied by a conformational strain.

SARS-CoV-2↗
DOE OSTI2021

Diagnostic, Prognostic, and Therapeutic Use of Radiopharmaceuticals in the Context of SARS-CoV-2

The coronavirus disease 2019 (COVID-19) outbreak has devastated the healthcare systems and economies of over 200 countries in just a few months. The etiological agent of COVID-19, SARS-CoV-2, is a highly contagious virus that can be transmitted by asymptomatic and symptomatic carriers alike. While in vitro testing techniques have allowed for population-wide screening, prognostic tools are required to assess the disease severity and therapeutic response, contributing to improve the patient clinical outcomes. Moreover, no specific antiviral against COVID-19 exists at the time of publication, severely limiting treatment against the infection. Hence, there is an urgent clinical need for innovative therapeutic strategies that may contribute to manage the COVID-19 outbreak and prevent future pandemics. In this work, we critically examine recent diagnostic, prognostic, and therapeutic advancements for COVID-19 in the field of radiopharmaceuticals. First, we summarize the gold standard techniques used to diagnose COVID-19, including in vitro assays and imaging techniques, and then discuss how radionuclide-based nuclear imaging provides complementary information for prognosis and treatment management of infected patients. Second, we introduce new emerging types of radiotherapies that employ radioimmunoconjugates, which have shown selective cytotoxic response in oncological studies, and critically analyze how these compounds could be used as therapeutic agents against SARS-CoV-2. Finally, this Perspective further discusses the emerging applications of radionuclides to study the behavior of pulmonary SARS-CoV-2 aerosol particles.

62 RADIOLOGY AND NUCLEAR MEDICINE↗