Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “SARS”

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 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MPro WT ) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is ~400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (T m ), respectively, relative to MPro WT . The H41A mutation results in a 20-fold increase in the dissociation constant (K d ), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and T m , respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MPro H41A , whereas no significant change is observed in binding to MPro WT . Structures of the four inhibitor complexes with MPro 1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MPro WT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MPro WT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MPro C145A also results in a significant, but smaller, increase in K d and decrease in ΔG and T m , relative to NMV.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ~7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ~6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor

We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T m ) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [ (-6) MPro H41A and (-6*) MPro H41A , respectively], and its corresponding mature MPro H41A were systematically examined. While the H41A mutation exerts negligible effect on T m and dimer dissociation constant (K dimer ) of MPro H41A , relative to the wild type MPro, both miniprecursors show a 4–5 °C decrease in T m and > 85-fold increase in K dimer as compared to MPro H41A . The K d for the binding of the covalent inhibitor GC373 to (-6*) MPro H41A increases ~12-fold, relative to MPro H41A , concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers’ oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Finally, disorder of the terminal residues 1–2 and 302–306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Here, through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 3 10 -helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Vaccination compartmental epidemiological models for the delta and omicron SARS-CoV-2 variants

We explore the inclusion of vaccination in compartmental epidemiological models concerning the delta and omicron variants of the SARS-CoV-2 virus that caused the COVID-19 pandemic. We expand on our earlier compartmental-model work by incorporating vaccinated populations. We present two classes of models that differ depending on the immunological properties of the variant. The first one is for the delta variant, where we do not follow the dynamics of the vaccinated individuals since infections of vaccinated individuals were rare. The second one for the far more contagious omicron variant incorporates the evolution of the infections within the vaccinated cohort. We explore comparisons with available data involving two possible classes of counts, fatalities and hospitalizations. We present our results for two regions, Andalusia and Switzerland (including the Principality of Liechtenstein), where the necessary data are available. In the majority of the considered cases, the models are found to yield good agreement with the data and have a reasonable predictive capability beyond their training window, rendering them potentially useful tools for the interpretation of the COVID-19 and further pandemic waves, and for the design of intervention strategies during these waves.

60 APPLIED LIFE 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 OSTI2023

Neutralization escape by SARS-CoV-2 Omicron subvariant BA.2.86

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variant BA.2.86 has over 30 mutations in spike compared with BA.2 and XBB.1.5, which raised the possibility that BA.2.86 might evade neutralizing antibodies (NAbs) induced by vaccination or infection. In this study, we show that NAb titers are substantially lower to BA.2.86 compared with BA.2 but are similar or slightly higher than to other current circulating variants, including XBB.1.5, EG.5.1, and FL.1.5.1. Moreover, NAb titers against all these variants were higher in vaccinated individuals with a history of XBB.1.5 infection compared with vaccinated individuals with no history of XBB.1.5 infection, suggesting the potential utility of the monovalent XBB.1.5 mRNA boosters.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

AI-Accelerated Design of Targeted Covalent Inhibitors for SARS-CoV-2

Direct-acting antivirals for the treatment of the COVID-19 pandemic caused by the SARS-CoV-2 virus are needed to complement vaccination efforts. Given the ongoing emergence of new variants, automated experimentation, and active learning based fast workflows for antiviral lead discovery remain critical to our ability to address the pandemic’s evolution in a timely manner. While several such pipelines have been introduced to discover candidates with noncovalent interactions with the main protease (M pro ), here we developed a closed-loop artificial intelligence pipeline to design electrophilic warhead-based covalent candidates. Here, this work introduces a deep learning-assisted automated computational workflow to introduce linkers and an electrophilic “warhead” to design covalent candidates and incorporates cutting-edge experimental techniques for validation. Using this process, promising candidates in the library were screened, and several potential hits were identified and tested experimentally using native mass spectrometry and fluorescence resonance energy transfer (FRET)-based screening assays. We identified four chloroacetamide-based covalent inhibitors of M pro with micromolar affinities (K I of 5.27 μM) using our pipeline. Experimentally resolved binding modes for each compound were determined using room-temperature X-ray crystallography, which is consistent with the predicted poses. The induced conformational changes based on molecular dynamics simulations further suggest that the dynamics may be an important factor to further improve selectivity, thereby effectively lowering KI and reducing toxicity. These results demonstrate the utility of our modular and data-driven approach for potent and selective covalent inhibitor discovery and provide a platform to apply it to other emerging targets.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Tuning Proton Transfer Thermodynamics in SARS-CoV-2 Main Protease: Implications for Catalysis and Inhibitor Design

The catalytic reaction in SARS-CoV-2 main protease is activated by a proton transfer (PT) from Cys145 to His41. The same PT is likely also required for the covalent binding of some inhibitors. Here we use a multiscale computational approach to investigate the PT thermodynamics in the apo enzyme and in complex with two potent inhibitors, N3 and the α-ketoamide 13b. We show that with the inhibitors the free energy cost to reach the charge-separated state of the active-site dyad is lower, with N3 inducing the most significant reduction. We also show that a few key sites (including specific water molecules) significantly enhance or reduce the thermodynamic feasibility of the PT reaction, with selective desolvation of the active site playing a crucial role. The approach presented is a cost-effective procedure to identify the enzyme regions that control the activation of the catalytic reaction and is thus also useful to guide the design of inhibitors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
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 OSTI2022

An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog ( m7 GpppA m2'-O ). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase: from the apo form to ligand complexes

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07–2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Room-temperature neutron and X-ray data collection of 3CL M pro from SARS-CoV-2

The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL M pro ) into a series of smaller functional proteins. At the heart of 3CL M pro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-binding cavity, and to visualize the hydrogen-bonding networks throughout the enzyme, room-temperature neutron and X-ray data were collected from a large H/D-exchanged crystal of ligand-free (apo) 3CL M pro .

3CL Mpro↗
DOE OSTI2022

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The SARS-CoV-2 Conserved Macrodomain Is a Mono-ADP-Ribosylhydrolase

SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed.

59 BASIC BIOLOGICAL SCIENCES↗