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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 973 records · Page 54
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DOE OSTI2017

Azomethines, isoxazole, N-substituted pyrazoles and pyrimidine containing curcumin derivatives: Urease inhibition and molecular modeling studies

Curcumin has shown large number of pharmacological properties against different phenotypes of various disease models. Different synthetic routes have been employed to develop its various derivatives for diverse biological functions. In this study, curcumin derived azomethine, isoxazole, pyrimidines and N-substituted pyrazoles were synthesized to investigate their urease enzyme inhibition. The structures of newly synthesized compounds were described by IR, MS, {sup 1}H NMR and {sup 13}C NMR spectral data. Urease enzyme inhibition was evaluated through in vitro assays in which compound 8b was found to be the most potent (IC{sub 50} = 2.44 ± 0.07 μM) among the tested compounds. The compounds with diazine ring system except the 4d showed better urease inhibition (IC{sub 50} = 11.43 ± 0.21–19.63 ± 0.28 μM) than the standard urease inhibitor thiourea (IC{sub 50} = 22.61 ± 0.23 μM). Similarly enzyme kinetics data revealed that compounds 3c-3e and 8b were competitive inhibitors with Ki values of 20.0, 19.87, 20.23 and 19.11 μM respectively while the compounds 4b, 4c and 4e were mixed type of inhibitors with Ki values 6.72, 19.69 and 6.72 μM respectively. Molecular docking studies were also performed to identify the plausible binding modes of the most active compounds. - Highlights: • Azomethines, Isoxazole, N-substituted pyrazoles and pyrimidine with curcumin scaffold were synthesized. • Anti-urease studies of target compounds were performed. • Molecular modeling of most active compounds at active site of urease. • Thiourea conjugated with curcumin showed excellent urease inhibition with IC{sub 50} = 2.44 ± 0.07 μM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3

Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of β-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-β1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α. - Highlights: • Ran-binding protein 3 (RanBP3) is an accessory factor in the Ran GTPase system. • The N-terminal region of RanBP3 contains a nuclear localization signal (NLS). • Crystal structure of importin-α3 bound to the NLS of RanBP3 is solved. • Phosphorylation of RanBP3 at Ser58 may modulate binding affinity to importin-α.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Integration of in silico approaches to determination of endocrine-disrupting perfluorinated chemicals binding potency with steroidogenic acute regulatory protein

A myriad of perfluorinated compounds (PFCs) have the ability to interfere with steroidogenic acute regulatory (StAR) protein. Consequently, PFCs breaches cholesterol biotransformation in mitochondria and cause fatal consequences in steroidogenesis, however, these were poorly characterized. In the present study, we have evaluated toxic potencies, nuclear mediated probabilities and interaction profiles with StAR of PFCs using computational system biology tools. Toxicity endpoints revealed that PFCs contain high carcinogenicity, developmental toxicity, skin sensitization effects with low mutagenic activity. Consensus qualitative nuclear receptor agonist models show higher probability rates towards ER and PPAR-γ receptor than AR and AhR models were observed. To poise the subtle fluctuations of actual predictions, balanced accuracy and MCC were computed, and they signify perfect correlation ranges in all models. Screening studies resulting protein-ligand interaction profiles showed that residues Asn148, Asn150, Glu169, Ala171, Arg182, Phe184, Arg188, Trp241, Thr263 and Phe267 were identified as novel hotspots, participated in halogen bonds, H-bonds, atomic π-stacking, π-cation interactions and salt-bridges formation. Thus, the additional bonds contribute conformer stability that holds the protein structure at flexible state, so that PFCs acts as a barrier to cholesterol binding. From docking outcomes, representation space was created, that specifies high and medium StAR binders were occupied in toxic endpoints space with active concern. PFCs restrain molecular features and mitochondrial membrane disruption functions were revealed by efficient toxicogenomics studies. These data indicate toxicity and StAR protein binding levels of PFCs, sorted pinpoints could be useful in a promising way to know the other environmental pollutants and health risks. - Highlights: • In this study, QSAR models were revealed that PFCs have greater adverse toxicity along with nuclear receptors interaction potencies. • Binding potencies of PFCs against the StAR protein revealed only potential novel residue hotspots showed various bond patterns, and they sustain stability and complexity of StAR. • Through that, PFCs acts as a barrier in cholesterol binding and leads fatal consequences in steroidogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structural insights and binding of a natural ligand, succinic acid with serine and cysteine proteases

Highlights: • Identification of a natural ligand through structural and functional studies, which can inhibit serine and cystein proteases. • Crystallographic and ITC binding studies support the inhibition of serine protease. • This is supported by both radial diffusion assay and modelling studies with serine and cystein protease. • The ligand activity can be accounted for the anti-viral nature of plant extracts where dicarboxilic fatty acids are abundant. In the age of growing infectious diseases, there is a great demand for new inhibitors which can exhibit minimum side effects. Owing to the importance of proteases in life cycle and invasion, they have been projected as attractive targets for structure based drug designing against microbes including viruses. Here we report the inhibitory activity of a well known natural compound succinic acid against both serine and cysteine proteases. The ligand is found co-crystallized with Bovine pancreatic trypsin in one of our crystallization trials and the diffraction data up to1.9 Å reveal its interactions with the catalytic triad residues Histidine 57 and Serine 195. Binding of the ligand with these proteases have been validated using caseinolysis inhibition. With trypsin, ITC analysis showed tight binding of the ligand, resulting in change in Gibb's free energy (ΔG) by −20.31 kJ/mol. To understand the existence of succinic acid at the active site, molecular docking was performed and it revealed binding of it with trypsin and papain at corresponding active sites. This dual inhibitory activity of natural ligand, succinic acid can be accounted for the recent reports on anti-viral property of plant extracts where dicarboxilic fatty acids are normally abundant.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Rational design of small molecules that modulate the transcriptional function of the response regulator PhoP

Highlights: • Development of a successful computational strategy to identify both inhibitors as well as enhancers of the PhoP dimerization. • Identification of the first 3 different classes compounds that have experimentally validated activity. • These compounds are the first druglike compounds targeting the PhoP dimerization. • The most potent molecule has an EC50 value 2.0 μM. The response regulator PhoP, which is part of the PhoP/PhoQ two-component system, regulates the expression of multiple genes involved in controlling virulence in Salmonella enterica serovar Typhimurium and other species of Gram-negative bacteria. Modulating the phosphorylation-mediated dimerization in the receiver domain may interfere with the transcriptional function of PhoP. In this study, we analyzed the therapeutic potential of the PhoP receiver domain by exploring it as a potential target for drug design. The structural information was then applied to identify the first hit compounds from commercial chemical libraries by combining pharmacophore modelling and docking methods with a GFP (Green Fluorescent Protein)-based promoter-fusion bioassay. In total, one hundred and forty compounds were selected, purchased, and tested for biological activity. Several novel scaffolds showed acceptable potency to modulate the transcriptional function of PhoP, either by enhancing or inhibiting the expression of PhoP-dependent genes. These compounds may be used as the starting point for developing modulators that target the protein-protein interface of the PhoP protein as an alternative strategy against antibiotic resistance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Monoterpenoid perillyl alcohol impairs metabolic flexibility of Candida albicans by inhibiting glyoxylate cycle

Highlights: • PA is efficient inhibitor of glyoxylate cycle in Candida albicans which is absent in humans. • PA has negligible hemolytic activity making it viable drug candidate. • PA showed enhanced survival of C. albicans in nematode model, C. elegans demonstrating its in-vivo efficacy. The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans. We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Suppression of tau propagation using an inhibitor that targets the DK-switch of nSMase2

Highlights: • Cambinol inhibits spread of AD synaptosome derived tau seeds, from donor to recipient cells. • Cambinol inhibits nSMase2 through targeting the DK-switch in its active site. • Cambinol inhibits EV-mediated tau propagation. • Cambinol has low brain permeability and inhibition of nSMase activity in vivo. Targeting of molecular pathways involved in the cell-to-cell propagation of pathological tau species is a novel approach for development of disease-modifying therapies that could block tau pathology and attenuate cognitive decline in patients with Alzheimer's disease and other tauopathies. We discovered cambinol through a screening effort and show that it is an inhibitor of cell-to-cell tau propagation. Our in vitro data demonstrate that cambinol inhibits neutral sphingomyelinase 2 (nSMase2) enzyme activity in dose response fashion, and suppresses extracellular vesicle (EV) production while reducing tau seed propagation. Our in vivo testing with cambinol shows that it can reduce the nSMase2 activity in the brain after oral administration. Our molecular docking and simulation analysis reveals that cambinol can target the DK-switch in the nSMase2 active site.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation

Highlights: • Acylation of CyaA-Hly by CyaC-acyltransferase requires AR-acylation and HP-hydrophobic regions, hence hemolysis activation. • ∼100-kDa AR-containing CyaA-RTX lacking HP cannot be acylated by functional CyaC, thus hemolytically inactive. • ∼70-kDa hemolytically inactive-CyaA-HP/BI containing both AR and HP could interact with co-expressed CyaC. • ∼20-kDa CyaA-HP comprising α1-α5 potentially interacted with CyaC via multiple H-bonding and ionic interactions. • HP is required for not only membrane-pore formation but also CyaC-association, hence toxin acylation. Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met{sup 482}-Arg{sup 1706}) from Bordetella pertussis was palmitoylated at Lys{sup 983} when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met{sup 482}-Leu{sup 750}) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala{sup 751}-Arg{sup 1706}) containing the Lys{sup 983}-acylation region (AR, Ala{sup 751}-Gln{sup 1000}), but lacking HP, was co-produced with CyaC in E. coli. Hemolysis assay indicated that CyaA-RTX showed no hemolytic activity. Additionally, MALDI-TOF/MS and LC-MS/MS analyses confirmed that CyaA-RTX was non-acylated, although the co-expressed CyaC-acyltransferase was able to hydrolyze its chromogenic substrate−p-nitrophenyl palmitate and acylate CyaA-Hly to become hemolytically active. Unlike CyaA-RTX, the ∼70-kDa His-tagged CyaA-HP/BI fragment which is hemolytically inactive and contains both HP and AR was constantly co-eluted with CyaC during IMAC-purification as the presence of CyaC was verified by Western blotting. Such potential interactions between the two proteins were also revealed by semi-native PAGE. Moreover, structural analysis via electrostatic potential calculations and molecular docking suggested that CyaA-HP comprising α1-α5 (Leu{sup 500}-Val{sup 698}) can interact with CyaC through several hydrogen and ionic bonds formed between their opposite electrostatic surfaces. Overall, our results demonstrated that the HP region of CyaA-Hly is conceivably required for not only membrane-pore formation but also functional association with CyaC-acyltransferase, and hence effective palmitoylation at Lys{sup 983}.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Designing an effective drug combination for ER stress loading in cancer therapy using a real-time monitoring system

Excess stress caused by accumulation of misfolded proteins inside the endoplasmic reticulum (ER) lumen can cause cells to undergo apoptosis. Misfolded proteins exported from ER to cytoplasm are ubiquitinated and mostly degraded by the proteasome, but can also be destroyed by autophagy mediated by the docking proteins p62 and NBR1. When misfolded proteins accumulate beyond the capacity of these clearance systems, they are transported to the microtubule organization center to form aggresomes, which are also degraded by autophagy. Together, these phenomena suggest the existence of a coordinated intracellular network for coping with the accumulation of misfolded proteins. Thus, rational inhibition of this network system might enhance killing of cancer cells subjected to pronounced ER stress loading. Based on this rationale, we sought to establish a quantitative assay for monitoring ER stress loading. MDA-MB231 cells stably transfected with the ERAI-Venus vector exhibited a strong XBP1 splicing signal in response to ER stress. Using the IncuCyte cell imaging system, we monitored the fluorescence intensity of XBP1-Venus, normalized against cell density, as an ER stress indicator. This parameter correlated closely with other reporters of unfolded protein responses. Assessment of the XBP1-Venus signal during exposure to various drug combinations revealed that simultaneous inhibition of the proteasome, autophagy, and aggresome formation led to more effective ER stress loading and higher cytotoxicity than inhibition of only two components. Our data suggest that this monitoring system is a useful tool for designing effective drug combinations for ER stress loading in cancer therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Enhanced PKMT-substrate recognition through non active-site interactions

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on many different cellular proteins. Despite extensive biochemical and structural studies, focusing on PKMT active site-peptide interactions, little is known regarding how PKMTs recognize globular substrates. To examine whether these enzymes recognize protein substrates through interactions that take place outside of the active site, we have measured SETD6 and SETD7 activity with both protein and peptide RelA substrate. We have utilized the MTase-Glo™ methyltransferase assay to measure the activity of SETD6 and SETD7 with the different RelA substrates and calculated the Michaelis-Menten (MM) parameters. We found an up to ∼12-fold increase in K{sub M} of the PKMTs activity with RelA peptide relative to the respective full-length protein, emphasizing the significantly higher PKMT-protein interaction affinity. Examination of SETD6 and SETD7 activity toward the same RelA substrates highlight the similarity in substrate recognition for both PKMTs. Our results show that the interaction affinity of SETD6 and SETD7 with RelA is enhanced through interactions that occur outside of the active site leading to higher catalytic efficiency and specificity. These interactions can significantly vary depending on the PKMT and the specific methylation site on RelA. Overall, our results underline that PKMTs can recognize their substrates through docking interactions that occur out of the active site-peptide region for enhancing their activity and specificity in the cellular environment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Erybraedin A is a potential Src inhibitor that blocks the adhesion and viability of non-small-cell lung cancer cells

Highlights: • Erybraedin A inhibited the adhesion of lung cancer cells to fibronectin. • Erybraedin A suppressed the activation of Src and its downstream targets. • Erybraedin A inhibited the interaction between integrin β1 or integrin β3 and Src. • Erybraedin A interacted with the ATP-binding pocket and the regulatory site of Src. • Erybraedin A inhibited the viability of lung cancer cells by inducing apoptosis. The adhesion of cancer cells to the extracellular matrix (ECM) is crucial for cell proliferation, survival, and metastasis. Thus, it is necessary to inhibit cell-ECM adhesion by blocking the activation of the associated signaling to control cancer. Here, we identify erybraedin A (EBA) as a potential Src inhibitor that blocks cell adhesion and viability in non-small-cell lung cancer (NSCLC). EBA significantly inhibited the adhesion of NSCLC cells to fibronectin. EBA also markedly inhibited the activation of Src and its downstream targets, including FAK and Akt. The interaction between integrin β1 or integrin β3 and Src was inhibited by EBA treatment. A docking study revealed the bindings of EBA to the ATP-binding pocket and the allosteric regulatory site of the Src kinase. Additionally, EBA markedly inhibited the viability and the colony formation of NSCLC cells and induced apoptotic cell death. These results describe novel biological properties of EBA, which can block the Src-mediated adhesion and survival of NSCLC cells, suggesting the potential of EBA as an anticancer Src inhibitor that warrants further development in advanced preclinical and clinical settings.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Sphingomyelin phosphodiesterase 1 (SMPD1) mediates the attenuation of myocardial infarction-induced cardiac fibrosis by astaxanthin

Highlights: • Astaxanthin (ASX) attenuated cardiac fibrosis following myocardial infarction (MI). • MI-induced overaccumulation of myocardial ceramides was normalized by ASX. • Sphingomyelin phosphodiesterase 1 (SMPD1) mediates the anti-fibrotic effect of ASX. • SMPD1 knockout abrogated the cardioprotective functions of ASX. Uncontrolled cardiac fibrosis following myocardial infarction (MI) is a critical pathological change leading to heart failure. Current pharmacotherapies are limited by unsatisfactory efficacy and undesired systemic side effects. Astaxanthin (ASX) is a natural carotenoid with strong antioxidant and anti-inflammatory activities. The effects of ASX on MI-induced cardiac fibrosis and the underlying mechanisms remain largely unknown. In this study, after the establishment of MI model, mice were administrated with ASX (200 mg/kg⋅d) for 4 weeks. We found that ASX treatment attenuated cardiac fibrosis and improved heart function following MI, as evidenced by reduced collagen I/III ratio, hydroxyproline content and left ventricular end diastolic pressure (LVEDP). Lipidomic analysis revealed the overaccumulation of myocardial ceramides in mice with cardiac fibrosis, which was normalized by ASX treatment. Molecular docking analysis showed that ASX produced a tight fit in the pocket of sphingomyelin phosphodiesterase 1 (SMPD1), a key enzyme in the production of ceramides. Western blot analysis confirmed the significant inhibition of SMPD1 expression by ASX. Furthermore, MI-induced overexpression of transforming growth factor β1 (TGF-β1) and phosphorylated SMAD2/3 were attenuated by ASX administration. SMPD1 knockout (KO) abrogated the beneficial effect of ASX. Taken together, our results suggest that the cardioprotective effects of ASX are mediated by SMPD1 through the indirection inhibition of TGF- β1/SMAD signaling cascade.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structural insights into inhibitor binding to a fungal ortholog of aspartate semialdehyde dehydrogenase

Highlights: • Structure of an inhibitor bound to an essential fungal enzyme. • Effects of cofactor binding on oligomeric assembly. • Structure guided docking of a potent fungal enzyme inhibitor. • Structural comparison to related microbial enzymes. The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The C-terminus of ubiquitin plays a critical role in deamidase Lpg2148 recognition

Highlights: • Docking of Lpg2148/ub∆c model provides a clue that the C-terminus of ubiquitin plays a vital role in interaction. • The Leu71 in C-terminus of ubiquitin is necessary for interaction and His68 is important in deamidation. • The Val33 of Lpg2148 plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin. By bearing a papain-like core structure and a cysteine-based catalytic triad, deamidase can convert glutamine to glutamic acid or asparagine to aspartic acid to modify the functions of host target proteins resulting in the blocking of eukaryotic host cell function. Legionella pneumophila effector Lpg2148 (MvcA) is a deamidase, a structural homolog of cycle inhibiting factor (Cif) effectors. Lpg2148 and Cif effectors are functionally diverse, with Lpg2148 only catalyzing ubiquitin but not NEDD8. However, a detailed understanding of substrate specificity is still missing. Here, we resolved the crystal structure of Lpg2148 at 2.5 Å resolution and obtained rigid-body modeling of Lpg2148 with C-terminus deleted ubiquitin (1–68) (ubΔc) complex using HADDOCK, which shows that the C-terminus of ubiquitin is flexible in recognition. We also conducted the truncated analysis to demonstrate that Leu71 of ubiquitin is necessary for its interaction with Lpg2148. Moreover, Val33 of Lpg2148 at the edge of a channel plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin, which may help to explain the selectivity of ubiquitin over NEDD8. In summary, these results enrich our knowledge of substrate recognition of deamidase.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

E3317 promotes cholesterol efflux in macrophage cells via enhancing ABCA1 expression

Highlights: • E3317 was first found as an ABCA1 upregulator in an ABCA1 transcriptional activity assay. • E3317 increased ABCA1 mRNA and protein expression in both L02 and RAW264.7 cells. • E3317 promoted cholesterol efflux and reduced cellular lipid accumulation in RAW264.7 macrophages dependent on ABCA1. • E3317 upregulated ABCA1 expression through activating PPARγ. • Y327 and F363 are the key PPARγ binding epitopes of E3317. Reverse cholesterol transport (RCT) plays an important role in cholesterol and lipid metabolism. Regulating the activities of key transporters and receptors in RCT, such as ATP-binding cassette transporter A1 (ABCA1), helps to prevent atherosclerotic cardiovascular disease. In this study, we used an ABCA1 promoter luciferase reporter assay to screen 20,000 compounds for ABCA1 upregulators. Compound E3317 (N-(6-butylbenzo[d]thiazol-2(3H)-ylidene)-3-(N-(2-cyanoethyl)sulfamoyl)benzamide)) was identified as a positive hit with an EC{sub 50} value of 0.2 μM in ABCA1p-LUC HepG2 cells. Thus, we hypothesized that E3317 might have cholesterol- and lipid metabolism-regulating effects through ABCA1 upregulation. E3317 significantly increased ABCA1 mRNA and protein expression in hepatic L02 cells and RAW264.7 macrophages. E3317 promoted cholesterol efflux to apolipoprotein A-I in RAW264.7 macrophages and significantly decreased lipid accumulation in oxidized low-density lipoprotein-induced murine RAW264.7 macrophages. Further studies using ABCA1 siRNA showed that the promotion of cholesterol efflux and decrease of lipid accumulation by E3317 depended on ABCA1 expression. Mechanistic studies indicated that E3317 regulated ABCA1 expression via activating nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays an important role in the regulation of glucose homeostasis and lipid metabolism. The structure of E3317 was docked in the ligand-binding domain of PPARγ (PBD code: 4EMA) to find the key binding amino acids. Site mutation assays confirmed that Y327 and F363 were the key PPARγ binding epitopes of E3317. Our results revealed that E3317 upregulates ABCA1 expression and thereby promotes cholesterol efflux. E3317 may regulate ABCA1 expression through PPARγ. Our findings provide a new compound, E3317, which may have beneficial cardiovascular effects.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of p-nitrophenol 4-monooxygenase PnpA from Pseudomonas putida DLL-E4: The key enzyme involved in p-nitrophenol degradation

Highlights: • This is the first structure of PnpA determined by X-ray crystallography. • Structure of PnpA is similar to Class A flavoprotein monooxygenases. • The substrate of PnpA is coordinated by cysteine and arginine, which is different from other PHBH family enzymes. p-Nitrophenol 4-monooxygenase PnpA, the key enzyme in the hydroquinone pathway of p-nitrophenol (PNP) degradation, catalyzes the monooxygenase reaction of PNP to p-benzoquinone in the presence of FAD and NADH. Here, we determined the first crystal structure of PnpA from Pseudomonas putida DLL-E4 in its apo and FAD-complex forms to a resolution of 2.04 Å and 2.48 Å, respectively. The PnpA structure shares a common fold with hydroxybenzoate hydroxylases, despite a low amino sequence identity of 14–18%, confirming it to be a member of the Class A flavoprotein monooxygenases. However, substrate docking studies of PnpA indicated that the residues stabilizing the substrate in an orientation suitable for catalysis are not observed in other homologous hydroxybenzoate hydroxylases, suggesting PnpA employs a unique catalytic mechanism. This work expands our understanding on the reaction mode for this enzyme class.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Elucidation of the crystal structure of FabD from the multidrug-resistant bacterium Acinetobacter baumannii

Highlights: • We solved the crystal structure of A. baumannii FabD. • Highly conserved active site residues were present among bacterial homologs. • The critical conserved residues Lys195 and Lys200 were involved in AbACP binding. • There was a unique inhibitor binding pocket near the substrate binding site. • This study will be useful for structure-based design of AbFabD inhibitors. Bacterial fatty acid synthesis (FAS) has been extensively studied as a potential target of antimicrobials. In FAS, FabD mediates transacylation of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP). The mounting threat of nosocomial infection by multidrug-resistant Acinetobacter baumannii warrants a deeper understanding of its essential cellular mechanisms, which could lead to effective control of this highly competent pathogen. The molecular mechanisms involved in A. baumannii FAS are poorly understood, and recent research has suggested that Pseudomonas aeruginosa, a closely related nosocomial pathogen of A. baumannii, utilizes FAS to produce virulence factors. In this study, we solved the crystal structure of A. baumannii FabD (AbFabD) to provide a platform for the development of new antibacterial agents. Analysis of the structure of AbFabD confirmed the presence of highly conserved active site residues among bacterial homologs. Binding constants between AbFabD variants and A. baumannii ACP (AbACP) revealed critical conserved residues Lys195 and Lys200 involved in AbACP binding. Computational docking of a potential inhibitor, trifluoperazine, revealed a unique inhibitor-binding pocket near the substrate-binding site. The structural study presented herein will be useful for the structure-based design of potent AbFabD inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Closed- and open-state models of human skeletal muscle sodium channel

Highlights: • Closed- and open-state models of hNav1.4 are shown. • The incomplete outward movement of S4 of domain III holds the channel closed. • The presence of intermediate state in S4 outward movement is revealed. • Movement of ∼6.5 elementary charges is required to activate the channel from hyperpolarized state. • Beta-scorpion toxin Ts1 binds to the closed-state model. Voltage-gated sodium channels play important roles in human physiology. However, their complexity hinders the understanding of their physiology and pathology at atomic level. We took advantage of the structural reports of similar channels obtained by cryo-EM (EeNav1.4, and NavPaS), and constructed models of human Nav1.4 channels at closed and open states. The open-state model is very similar to the recently published cryo-EM structure of hNav1.4. The comparison of both models shows shifts of the voltage sensors (VS) of DIII and DIV. The activated position of VS-DII in the closed model was demonstrated by Ts1 docking, thereby confirming the requirement that VS-DI, VS-DII and VS-DIII must be activated for the channel to open. The interactions observed with VS-DIII suggest a stepwise, yet fast, transition from resting to activated state. These models provide structural insights on the closed-open transition of the channel.

60 APPLIED LIFE SCIENCES↗