Discovery of novel bacterial topoisomerase I inhibitors by use of in silico docking and in vitro assays

Topoisomerases are important targets for antibacterial and anticancer therapies. Bacterial topoisomerase I remains to be exploited for antibiotics that can be used in the clinic. Inhibitors of bacterial topoisomerase I may provide leads for novel antibacterial drugs against pathogens resistant to current antibiotics. TB is the leading infectious cause of death worldwide, and new TB drugs against an alternative target are urgently needed to overcome multi-drug resistance. Mycobacterium tuberculosis topoisomerase I (MtbTopI) has been validated genetically and chemically as a TB drug target. Here we conducted in silico screening targeting an active site pocket of MtbTopI. The top hits were assayed for inhibition of MtbTopI activity. The shared structural motif found in the active hits was utilized in a second round of in silico screening and in vitro assays, yielding selective inhibitors of MtbTopI with ICs as low as 2 µM. Growth inhibition of Mycobacterium smegmatis by these compounds in combination with an efflux pump inhibitor was diminished by the overexpression of recombinant MtbTopI. This work demonstrates that in silico screening can be utilized to discover new bacterial topoisomerase I inhibitors, and identifies a novel structural motif which could be explored further for finding selective bacterial topoisomerase I inhibitors

Structural insights into the repair mechanism of AGT for methyl-induced DNA damage

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- A nd post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex

Airglow observations and modeling of F region depletion zonal velocities over Christmas Island

We report image measurements of plasma depletions in the equatorial F region ionosphere over Christmas Island (2.1°N, 157.4°W; dip latitude 2.8°N) in the central Pacific Ocean. The observations were made during the equinox period, September‐October 1995, using a Utah State University CCD imaging system filtered to observe thermospheric O I (630.0 nm) airglow emissions centered at ∼280 km altitude. Well‐defined magnetic field‐aligned depletions were observed on 18 nights during the campaign, including strong postmidnight fossilized structures, enabling detailed measurements of their morphology and dynamics. The number of depletions was influenced by their initial onset times and their persistence. The separations between adjacent depletions ranged from ∼150 to ∼250 km in good agreement with prior observations from other sites. However, measurements of their eastward zonal drift speeds indicated normal behavior peaking around 90–100 m/s prior to local midnight, with exceptionally high velocities, ∼80 m/s during the postmidnight period that persisted until dawn. These results differ markedly from optical measurements at similar equatorial latitudes but at different longitude sectors, suggesting that the zonal drift velocities can have a significant longitudinal dependence. Model drift velocities calculated using a simple electric field model with winds defined by the horizontal wind model (HWM‐07) produced an eastward drift throughout the night, but their postmidnight magnitudes were much smaller than observed. Using a modified HWM‐07 wind field, a basic nighttime trend similar to the Christmas Island trend was successfully obtained

Quantifying the regional water footprint of biofuel production by incorporating hydrologic modeling

A spatially explicit life cycle water analysis framework is proposed, in which a standardized water footprint methodology is coupled with hydrologic modeling to assess blue water, green water (rainfall), and agricultural grey water discharge in the production of biofuel feedstock at county-level resolution. Grey water is simulated via SWAT, a watershed model. Evapotranspiration (ET) estimates generated with the Penman-Monteith equation and crop parameters were verified by using remote sensing results, a satellite-imagery-derived data set, and other field measurements. Crop irrigation survey data are used to corroborate the estimate of irrigation ET. An application of the concept is presented in a case study for corn-stover-based ethanol grown in Iowa (United States) within the Upper Mississippi River basin. Results show vast spatial variations in the water footprint of stover ethanol from county to county. Producing 1 L of ethanol from corn stover growing in the Iowa counties studied requires from 4.6 to 13.1 L of blue water (with an average of 5.4 L), a majority (86%) of which is consumed in the biorefinery. The county-level green water (rainfall) footprint ranges from 760 to 1000 L L-1. The grey water footprint varies considerably, ranging from 44 to 1579 L, a 35-fold difference, with a county average of 518 L. This framework can be a useful tool for watershed-or county-level biofuel sustainability metric analysis to address the heterogeneity of the water footprint for biofuels

Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer

Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane

Simultaneous observations of equatorial F-region plasma depletions over Brazil during the Spread-F Experiment (SpreadFEx)

From September to November 2005, the NASA Living with a Star program supported the Spread-F Experiment campaign (SpreadFEx) in Brazil to study the effects of convectively generated gravity waves on the ionosphere and their role in seeding Rayleigh-Taylor instabilities, and associated equatorial plasma bubbles. Several US and Brazilian institutes deployed a broad range of instruments (all-sky imagers, digisondes, photometers, meteor/VHF radars, GPS receivers) covering a large area of Brazil. The campaign was divided in two observational phases centered on the September and October new moon periods. During these periods, an Utah State University (USU) all-sky CCD imager operated at São João d'Aliança (14.8° S, 47.6° W), near Brasilia, and a Brazilian all-sky CCD imager located at Cariri (7.4° S, 36° W), observed simultaneously the evolution of the ionospheric bubbles in the OI (630 nm) emission and the mesospheric gravity wave field. The two sites had approximately the same magnetic latitude (9–10° S) but were separated in longitude by ~1500 km. <br><br> Plasma bubbles were observed on every clear night (17 from Brasilia and 19 from Cariri, with 8 coincident nights). These joint datasets provided important information for characterizing the ionospheric depletions during the campaign and to perform a novel longitudinal investigation of their variability. Measurements of the drift velocities at both sites are in good agreement with previous studies, however, the overlapping fields of view revealed significant differences in the occurrence and structure of the plasma bubbles, providing new evidence for localized generation. This paper summarizes the observed bubble characteristics important for related investigations of their seeding mechanisms associated with gravity wave activity

Covalent Complex of DNA and Bacterial Topoisomerase: Implications in Antibacterial Drug Development

A topoisomerase-DNA transient covalent complex can be a druggable target for novel topoisomerase poison inhibitors that represent a new class of antibacterial or anticancer drugs. Herein, we have investigated molecular features of the functionally important Escherichia coli topoisomerase I (EctopoI)-DNA covalent complex (EctopoIcc) for molecular simulations, which is very useful in the development of new antibacterial drugs. To demonstrate the usefulness of our approach, we used a model small molecule (SM), NSC76027, obtained from virtual screening. We examined the direct binding of NSC76027 to EctopoI as well as inhibition of EctopoI relaxation activity of this SM via experimental techniques. We then performed molecular dynamics (MD) simulations to investigate the dynamics and stability of EctopoIcc and EctopoI-NSC76027-DNA ternary complex. Our simulation results show that NSC76027 forms a stable ternary complex with EctopoIcc. EctopoI investigated here also serves as a model system for investigating a complex of topoisomerase and DNA in which DNA is covalently attached to the protein

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers