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Results for “Enzyme Inhibitors”

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 OSTI2022

Characterization of Interactions between CTX-M-15 and Clavulanic Acid, Desfuroylceftiofur, Ceftiofur, Ampicillin, and Nitrocefin

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamases (ESBLs) are commonly associated with Gram-negative, hospital-acquired infections worldwide. Several beta-lactamase inhibitors, such as clavulanate, are used to inhibit the activity of these enzymes. To understand the mechanism of CTX-M-15 activity, we have determined the crystal structures of CTX-M-15 in complex with two specific classes of beta-lactam compounds, desfuroylceftiofur (DFC) and ampicillin, and an inhibitor, clavulanic acid. The crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of those compounds. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant structures, we determined that CTX-M-15 has a similar affinity for all beta-lactam compounds (ceftiofur, nitrocefin, DFC, and ampicillin), but with lower affinity for clavulanic acid. A catalytic mechanism for tested β-lactams and two-step inhibition mechanism of clavulanic acid were proposed. CTX-M-15 showed a higher activity toward DFC and nitrocefin, but significantly lower activity toward ampicillin and ceftiofur. The interaction between CTX-M-15 and both ampicillin and ceftiofur displayed a higher entropic but lower enthalpic effect, compared with DFC and nitrocefin. DFC, a metabolite of ceftiofur, displayed lower entropy and higher enthalpy than ceftiofur. This finding suggests that compounds containing amine moiety (e.g., ampicillin) and the furfural moiety (e.g., ceftiofur) could hinder the hydrolytic activity of CTX-M-15.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. In this work, five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

60 APPLIED LIFE SCIENCES↗
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 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 OSTI2022

Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

The Novel Carbapenem, JDB/PQ-1-219, Has Potent Broad Spectrum Activity against Multi-Drug Resistant Acinetobacter baumannii

Carbapenem resistance in Acinetobacter baumannii, driven largely by class D, along with class A and class B β- lactamases, has severely compromised the utility of these last resort antibiotics. As a result, infections caused by such pathogens are characterized by extremely high mortality rates. Here we describe the antimicrobial activity of the novel C5 methyl-substituted carbapenem JDB/PQ-1−219 against multidrug resistant A. baumannii and the mechanism of its interaction with its major carbapenemase, OXA-23. JDB/PQ-1-219 exhibits potent antimicrobial activity against A. baumannii producing various carbapenemases, with MICs that are all in the clinically susceptible range. The compound has unrestricted ingress through porins and avoids egress by efflux pumps, a unique property when compared to all commercial carbapenems. Kinetic experiments demonstrated that unlike for other carbapenems, acylation of OXA-23 by JDB/ PQ-1-219 is monophasic, and mass spectrometry studies showed that this results from the conversion of all enzyme into a reversible tetrahedral intermediate which gradually transitions into the stable acyl-enzyme complex. No deacylation of this complex is observed over a physiologically relevant time period, making JDB/PQ-1−219 an extremely potent inhibitor of OXA-23. Time-resolved crystallography revealed fine details of active site dynamics, leading to complete inhibition of the enzyme. Together, these studies identify JDB/PQ-1-219 as a uniquely effective novel carbapenem with clinically significant levels of activity against multidrug resistant A. baumannii.

Acinetobacter baumannii↗
DOE OSTI2021

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Small molecule BLVRB redox inhibitor promotes megakaryocytopoiesis and stress thrombopoiesis in vivo

Biliverdin IXβ reductase (BLVRB) is an NADPH-dependent enzyme previously implicated in a redox-regulated mechanism of thrombopoiesis distinct from the thrombopoietin (TPO)/c-MPL axis. Here, we apply computational modeling to inform molecule design, followed by de novo syntheses and screening of unique small molecules retaining the capacity for selective BLVRB inhibition as a novel platelet-enhancing strategy. Two distinct classes of molecules are identified, and NMR spectroscopy and co-crystallization studies confirm binding modes within the BLVRB active site and ring stacking between the nicotinamide moiety of the NADP+ cofactor. A diazabicyclo derivative displaying minimal off-target promiscuity and excellent bioavailability characteristics promotes megakaryocyte speciation in biphenotypic (erythro/megakaryocyte) cellular models and synergizes with TPO-dependent megakaryocyte formation in hematopoietic stem cells. Upon oral delivery into mice, this inhibitor expands platelet recovery in stress thrombopoietic models with no adverse effects. In this work, we identify and validate a cellular redox inhibitor retaining the potential to selectively promote megakaryocytopoiesis and enhance stress-associated platelet formation in vivo distinct from TPO receptor agonists.

36 MATERIALS SCIENCE↗
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 OSTI2026

Continuous Directed Evolution of a Short-Lived Plant Histidinol Dehydrogenase

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover i.e., extending the effective working life (Catalytic Cycles till Replacement, CCR) of short-lived enzymes is thus a rational strategy to conserve energy and carbon and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain lifeshortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4 Δ strain to raise cumulative HDH function and, by proxy, lifespan as functional enzymes, by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDH variants carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme working life in vivo in addition to, as expected, altering kinetic properties.

directed evolution↗
DOE OSTI2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.

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

Deciphering the structure of Arabidopsis thaliana 5-enol-pyruvyl-shikimate-3-phosphate synthase: An essential step toward the discovery of novel inhibitors to supersede glyphosate

Glyphosate interferes with plant aromatic metabolism through the inhibition of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase [EPSPS, EC 2.5.1.19]. For this reason, EPSPS has been extensively studied in a vast array of organisms. This notwithstanding, up to date, the crystal structure of the protein has been solved exclusively in a few prokaryotes, while that of the plant enzyme has been only deduced in silico by similarity. This study aimed at determining the structure of EPSPS from the plant model species Arabidopsis thaliana, which has been cloned, heterologously expressed and affinity-purified. The kinetic properties of the enzyme have been determined, as well as its susceptibility to the inhibition brought about by glyphosate. The crystal structure of the protein has been resolved at high resolution (1.4 Å), showing open conformation of the enzyme, which is the state ready for substrate/inhibitor binding. This provides a framework for the structure-based design of novel EPSPS inhibitors. Surface regions near the active-site cleft entrance or at the interdomain hinge appear promising for inhibitor selectivity, while bound chloride near the active site is a potential placeholder for anionic moieties of future herbicides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13–AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13–AcrIIA15 as unique bifunctional inhibitors of SauCas9.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Microbial β-glucuronidases drive human periodontal disease etiology

Periodontitis is a chronic inflammatory disease associated with persistent oral microbial dysbiosis. The human β-glucuronidase (GUS) degrades constituents of the periodontium and is used as a biomarker for periodontitis severity. However, the human microbiome also encodes GUS enzymes, and the role of these factors in periodontal disease is poorly understood. Here, we define the 53 unique GUSs in the human oral microbiome and examine diverse GUS orthologs from periodontitis-associated pathogens. Oral bacterial GUS enzymes are more efficient polysaccharide degraders and processers of biomarker substrates than the human enzyme, particularly at pHs associated with disease progression. Using a microbial GUS-selective inhibitor, we show that GUS activity is reduced in clinical samples obtained from individuals with untreated periodontitis and that the degree of inhibition correlates with disease severity. Together, these results establish oral GUS activity as a biomarker that captures both host and microbial contributions to periodontitis, facilitating more efficient clinical monitoring and treatment paradigms for this common inflammatory disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Toward wide-spectrum antivirals against coronaviruses: Molecular characterization of SARS-CoV-2 NSP13 helicase inhibitors

To date, effective therapeutic treatments that confer strong attenuation against coronaviruses (CoVs) remain elusive. Among potential drug targets, the helicase of CoVs is attractive due to its sequence conservation and indispensability. We rely on atomistic molecular dynamics simulations to explore the structural coordination and dynamics associated with the SARS-CoV-2 Nsp13 apo enzyme, as well as their complexes with natural ligands. A complex communication network is revealed among the five domains of Nsp13, which is differentially activated because of the presence of the ligands, as shown by shear strain analysis, principal components analysis, dynamical cross-correlation matrix analysis, and water transport analysis. The binding free energy and the corresponding mechanism of action are presented for three small molecules that were shown to be efficient inhibitors of the previous SARS-CoV Nsp13 enzyme. Together, our findings provide critical fresh insights for rational design of broad-spectrum antivirals against CoVs.

59 BASIC BIOLOGICAL SCIENCES↗