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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 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Abstract Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with k inact /K I = 9,600 M −1 s −1 , achieves sub-μM EC 50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (M pro also known as 3CL pro ) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the M pro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the M pro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors

A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MPro WT ) from precursor polyproteins. Upon expression, a model precursor of MPro WT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MPro M ) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MPro WT . MPro M and the catalytic domain (MPro 1-199 ) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MPro M and MPro 1-199 , respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir

We investigate the effects of two naturally selected substitution and deletion (Δ) mutations, constituting part of the substrate binding subsites S2 and S4, on the structure, function, and inhibition of SARS CoV-2 main protease. Comparable to wild-type, MPro D48Y/ΔP168 undergoes N-terminal autoprocessing essential for stable dimer formation and mature-like catalytic activity. The structures are similar, but for an open active site conformation in MPro D48Y/ΔP168 and increased dynamics of the S2 helix, S5 loop, and the helical domain. Some dimer interface contacts exhibit shorter H bond distances corroborating the ~40-fold enhanced dimerization of the mutant although its thermal sensitivity to unfolding is 8 °C lower, relative to wild-type. ITC reveals a 3- and 5-fold decrease in binding affinity for nirmatrelvir and ensitrelvir, respectively, and similar GC373 affinity, to MPro D48Y/ΔP168 relative to wild-type. Structural differences in four inhibitor complexes of MPro D48Y/ΔP168 compared to wild-type are described. Consistent with enhanced dynamics, the S2 helix and S5 loop adopting a more open conformation appears to be a unique feature of MPro D48Y/ΔP168 both in the inhibitor-free and bound states. Our results suggest that mutational effects are compensated by changes in the conformational dynamics and thereby modulate N-terminal autoprocessing, K dimer , catalytic efficiency, and inhibitor binding.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

DNA-encoded chemical libraries yield non-covalent and non-peptidic SARS-CoV-2 main protease inhibitors

The development of SARS-CoV-2 main protease (M pro ) inhibitors for the treatment of COVID-19 has mostly benefitted from X-ray structures and preexisting knowledge of inhibitors; however, an efficient method to generate M pro inhibitors, which circumvents such information would be advantageous. As an alternative approach, we show here that DNA-encoded chemistry technology (DEC-Tec) can be used to discover inhibitors of M pro . An affinity selection of a 4-billion-membered DNA-encoded chemical library (DECL) using M pro as bait produces novel non-covalent and non-peptide-based small molecule inhibitors of M pro with low nanomolar K i values. Furthermore, these compounds demonstrate efficacy against mutant forms of M pro that have shown resistance to the standard-of-care drug nirmatrelvir. Overall, this work demonstrates that DEC-Tec can efficiently generate novel and potent inhibitors without preliminary chemical or structural information.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 μM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1–2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Exact reaction coordinates for flap opening in HIV-1 protease

The primary goal of protein science is to understand how proteins function, which requires understanding the functional dynamics responsible for transitions between different functional structures of a protein. A central concept is the exact reaction coordinates that can determine the value of committor for any protein configuration, which provide the optimal description of functional dynamics. Despite intensive efforts, identifying the exact reaction coordinates (RCs) in complex molecules remains a formidable challenge. Using the recently developed generalized work functional, we report the discovery of the exact RCs for an important functional process—the flap opening of HIV-1 protease. Our results show that this process has six RCs, each one is a linear combination of ~240 backbone dihedrals, providing the precise definition of collectivity and cooperativity in the functional dynamics of a protein. Applying bias potentials along each RC can accelerate flap opening by 10 3 to 10 4 folds. The success in identifying the RCs of a protein with 198 residues represents a significant progress beyond that of the alanine dipeptide, currently the only other complex molecule for which the exact RCs for its conformational changes are known. Our results suggest that the generalized work functional (GWF) might be the fundamental operator of mechanics that controls protein dynamics.

Science & Technology - Other Topics↗
DOE OSTI2025

Engineering a protease-stable, oral single-domain antibody to inhibit IL-23 signaling

Interleukin (IL)-23 is a validated therapeutic target in inflammatory bowel disease. While antibodies targeting IL23 demonstrate clinical efficacy, they face challenges such as high costs, safety risks, and the necessity of parenteral administration. Here, we present a workflow to simultaneously enhance the affinity and protease stability of an inhibitory anti-IL23R VHH for oral use. Cocrystal structure analysis reveals that the anti-IL23R VHH employs both CDR and framework residues to achieve picomolar affinity for IL23R. The engineered VHH remains stable for over 8 h in intestinal fluid and 24 h in fecal samples. Oral administration of this VHH achieves deep pathway inhibition in a murine colitis model. Furthermore, a single pill provides sustained IL23R inhibition in nonhuman primate blood for over 24 h. With high potency, gut stability, high production yield, and favorable drug-like properties, oral VHHs offer a promising approach for inflammatory bowel diseases.

Science & Technology - Other Topics↗
DOE OSTI2020

Molecular characterization of ebselen binding activity to SARS-CoV-2 main protease

There is an urgent need to repurpose drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent computational-experimental screenings have identified several existing drugs that could serve as effective inhibitors of the virus’ main protease, M pro , which is involved in gene expression and replication. Among these, ebselen (2-phenyl-1,2-benzoselenazol-3-one) appears to be particularly promising. Here, we examine, at a molecular level, the potential of ebselen to decrease Mpro activity. We find that it exhibits a distinct affinity for the catalytic region. Our results reveal a higher-affinity, previously unknown binding site localized between the II and III domains of the protein. A detailed strain analysis indicates that, on such a site, ebselen exerts a pronounced allosteric effect that regulates catalytic site access through surface-loop interactions, thereby inducing a reconfiguration of water hotspots. Together, these findings highlight the promise of ebselen as a repurposed drug against SARS-CoV-2.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti–COVID-19 drug design

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structure of coxsackievirus cloverleaf RNA and 3C pro dimer establishes the RNA-binding mechanism of enterovirus protease 3C pro

In positive-strand RNA viruses, the genome serves as a template for both protein translation and negative-strand RNA synthesis. Enteroviruses use the cloverleaf RNA structure at the 5′ end of the genome to balance these two processes. Cloverleaf acts as a promoter for RNA synthesis and forms a complex with viral 3CD protein, the precursor to 3C pro protease, and 3D pol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3C pro domain, yet how 3C pro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3C pro complex, wherein two 3C pro molecules interact with cloverleaf stem-loop D. 3C pro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3C pro is a previously undiscovered type of RNA binding protein. We show that 3CD protein also dimerizes on cloverleaf RNA and binds the RNA with higher affinity than 3C pro . The structure provides insight into the RNA-binding mechanism of 3C pro or 3CD with other cis-acting replication elements.

Science & Technology - Other Topics↗
DOE OSTI2020

3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV–infected mice

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in the early stage of infection. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro). The test system consists of a peptide that fluoresces when cleaved by SARS PLpro that is active in crude, unprocessed lysates from human tongue scrapes and saliva. Test results are obtained in 30 minutes or less using widely available fluorescence plate readers, or a battery-operated portable instrument for on-site testing. Proof-of-concept was obtained in a study on clinical specimens collected from patients with COVID-19 like symptoms who tested positive (n = 10) or negative (n = 10) with LIAT RT-PCR using nasal mid turbinate swabs. When saliva from these patients was tested with in-house endpoint RT-PCR, 17 were positive and only 5 specimens were negative, of which 2 became positive when tested 5 days later. PLpro activity correlated in 17 of these cases (3 out of 3 negatives and 14 out of 16 positives, with one invalid specimen). Despite the small number of samples, the agreement was significant (p value = 0.01). Two false negatives were detected, one from a sample with a late Ct value of 35 in diagnostic RT-PCR, indicating that an active infection was no longer present. The PLpro assay is easily scalable and expected to detect all viable SARS-CoV-2 variants, making it attractive as a screening and surveillance tool. Additionally, we show feasibility of the platform as a new homogeneous phenotypic assay for rapid screening of SARS-CoV-2 antiviral drugs and neutralizing antibodies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Chloropyridinyl Esters of Nonsteroidal Anti-Inflammatory Agents and Related Derivatives as Potent SARS-CoV-2 3CL Protease Inhibitors

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using 1 H-NMR, 13 C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE 6 cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC 50 value of 160 nM. Compound 13b exhibited an enzyme IC 50 value of 4.9 µM. However, it exhibited a potent antiviral EC 50 value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC 50 value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site.

3CLpro inhibitors↗
DOE OSTI2021

A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes

Abstract Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography–coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1’) and reveals a surprising role for C1r B loop–interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway–specific inhibition by a human pathogen.

Garrigues, Ryan J. (ORCID:0000000259233250)↗