29 research outputs found
DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors
Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an ICâ
â value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the ICâ
â (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (ICâ
â: 1.25 ÎŒM) inhibits growth of PD002 at 0.0024â0.16 ÎŒM in combination with 0.10â2.0 ÎŒM CPPB (ICâ
â: 35 ÎŒM)
DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors
Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an ICâ
â value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the ICâ
â (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (ICâ
â: 1.25 ÎŒM) inhibits growth of PD002 at 0.0024â0.16 ÎŒM in combination with 0.10â2.0 ÎŒM CPPB (ICâ
â: 35 ÎŒM)
A statistical framework to evaluate virtual screening
<p>Abstract</p> <p>Background</p> <p>Receiver operating characteristic (ROC) curve is widely used to evaluate virtual screening (VS) studies. However, the method fails to address the "early recognition" problem specific to VS. Although many other metrics, such as RIE, BEDROC, and pROC that emphasize "early recognition" have been proposed, there are no rigorous statistical guidelines for determining the thresholds and performing significance tests. Also no comparisons have been made between these metrics under a statistical framework to better understand their performances.</p> <p>Results</p> <p>We have proposed a statistical framework to evaluate VS studies by which the threshold to determine whether a ranking method is better than random ranking can be derived by bootstrap simulations and 2 ranking methods can be compared by permutation test. We found that different metrics emphasize "early recognition" differently. BEDROC and RIE are 2 statistically equivalent metrics. Our newly proposed metric SLR is superior to pROC. Through extensive simulations, we observed a "seesaw effect" â overemphasizing early recognition reduces the statistical power of a metric to detect true early recognitions.</p> <p>Conclusion</p> <p>The statistical framework developed and tested by us is applicable to any other metric as well, even if their exact distribution is unknown. Under this framework, a threshold can be easily selected according to a pre-specified type I error rate and statistical comparisons between 2 ranking methods becomes possible. The theoretical null distribution of SLR metric is available so that the threshold of SLR can be exactly determined without resorting to bootstrap simulations, which makes it easy to use in practical virtual screening studies.</p
The Vacuolar Ca2+ ATPase Pump Pmc1p Is Required for Candida albicans Pathogenesis
Maintenance of Ca2+ homeostasis is important for fungal cells to respond to a multitude of stresses, as well as antifungal treatment, and for virulence in animal models. Here, we demonstrate that a P-type ATPase, Pmc1p, is required for Candida albicans to respond to a variety of stresses, affects azole susceptibility, and is required to sustain tissue invasive hyphal growth and to cause disease in a mouse model of disseminated infection. Defining the mechanisms responsible for maintaining proper Ca2+ homeostasis in this important human pathogen can ultimately provide opportunities to devise new chemotherapeutic interventions that dysregulate intracellular signaling and induce Ca2+ toxicity.Calcium is a critically important secondary messenger of intracellular signal transduction in eukaryotes but must be maintained at low levels in the cytoplasm of resting cells to avoid toxicity. This is achieved by several pumps that actively transport excess cytoplasmic Ca2+ out of the cell across the plasma membrane and into other intracellular compartments. In fungi, the vacuole serves as the major storage site for excess Ca2+, with two systems actively transporting cytoplasmic calcium ions into the vacuole. The H+/Ca2+ exchanger, Vcx1p, harnesses the proton-motive force across the vacuolar membrane (generated by the V-ATPase) to drive Ca2+ transport, while the P-type ATPase Pmc1p uses ATP hydrolysis to translocate Ca2+ into the vacuole. Ca2+-dependent signaling is required for the prevalent human fungal pathogen Candida albicans to endure exposure to the azole antifungals and to cause disease within the mammalian host. The purpose of this study was to determine if the Pmc1p or Vcx1p Ca2+ pumps are required for C. albicans pathogenicity and if these pumps impact antifungal resistance. Our results indicate that Pmc1p is required by C. albicans to transition from yeast to hyphal growth, to form biofilms in vitro, and to cause disease in a mouse model of disseminated infection. Moreover, loss of Pmc1p function appears to enhance C. albicans azole tolerance in a temperature-dependent manner
Expression, Purification and Characterization of EnoylâACP Reductase II, FabK, from Porphyromonas gingivalis
The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FASâII, are distinct from their mammalian counterparts, FASâI, in terms of both structure and enzymatic mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. One such enzyme, enoylâacyl carrier protein (ACP) reductase II, FabK, is a key, rateâlimiting enzyme in the FASâII pathway. The bacterial organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the U.S. population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FASâII cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a PingâPong BiâBi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction has been optimized for highâthroughput, 384âwell format. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract xârays to high resolution
Fragment-Based Drug Discovery Using a Multidomain, Parallel MD-MM/PBSA Screening Protocol
We
have developed a rigorous computational screening protocol to identify
novel fragment-like inhibitors of N<sup>5</sup>-CAIR mutase (PurE),
a key enzyme involved in de novo purine synthesis that represents
a novel target for the design of antibacterial agents. This computational
screening protocol utilizes molecular docking, graphics processing
unit (GPU)-accelerated molecular dynamics, and Molecular Mechanics/PoissonâBoltzmann
Surface Area (MM/PBSA) free energy estimations to investigate the
binding modes and energies of fragments in the active sites of PurE.
PurE is a functional octamer comprised of identical subunits. The
octameric structure, with its eight active sites, provided a distinct
advantage in these studies because, for a given simulation length,
we were able to place eight separate fragment compounds in the active
sites to increase the throughput of the MM/PBSA analysis. To validate
this protocol, we have screened an in-house fragment library consisting
of 352 compounds. The theoretical results were then compared with
the results of two experimental fragment screens, Nuclear Magnetic
Resonance (NMR) and Surface Plasmon Resonance (SPR) binding analyses.
In these validation studies, the protocol was able to effectively
identify the competitive binders that had been independently identified
by experimental testing, suggesting the potential utility of this
method for the identification of novel fragments for future development
as PurE inhibitors
Structural characterization of Porphyromonas gingivalis enoyl-ACP reductase II (FabK)
Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9â
Ă
resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection
Discovery of a Novel and Potent Class of F. tularensis Enoyl-Reductase (FabI) Inhibitors by Molecular Shape and Electrostatic Matching
Enoyl-acyl carrier protein (ACP) reductase, FabI, is
a key enzyme
in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is
an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates
in a final step of the pathway. The absence of this enzyme in humans
makes it an attractive target for the development of new antibacterial
agents. FabI is known to be unresponsive to structure-based design
efforts due to a high degree of induced fit and a mobile flexible
loop encompassing the active site. Here we discuss the development,
validation, and careful application of a ligand-based virtual screen
used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study,
four known classes of FabI inhibitors were used as templates for virtual
screens that involved molecular shape and electrostatic matching.
The program ROCS was used to search a high-throughput screening library
for compounds that matched any of the four molecular shape queries.
Matching compounds were further refined using the program EON, which
compares and scores compounds by matching electrostatic properties.
Using these techniques, 50 compounds were selected, ordered, and tested.
The tested compounds possessed novel chemical scaffolds when compared
to the input query compounds. Several hits with low micromolar activity
were identified and follow-up scaffold-based searches resulted in
the identification of a lead series with submicromolar enzyme inhibition,
high ligand efficiency, and a novel scaffold. Additionally, one of
the most active compounds showed promising whole-cell antibacterial
activity against several Gram-positive and Gram-negative species,
including the target pathogen. The results of a preliminary structureâactivity
relationship analysis are presented
Hit Identification and Optimization in Virtual Screening: Practical Recommendations Based on a Critical Literature Analysis
A critical
analysis of virtual screening results published between
2007 and 2011 was performed. The activity of reported hit compounds
from over 400 studies was compared to their hit identification criteria.
Hit rates and ligand efficiencies were calculated to assist in these
analyses, and the results were compared with factors such as the size
of the virtual library and the number of compounds tested. A series
of promiscuity, druglike, and ADMET filters were applied to the reported
hits to assess the quality of compounds reported, and a careful analysis
of a subset of the studies that presented hit optimization was performed.
These data allowed us to make several practical recommendations with
respect to selection of compounds for experimental testing, definition
of hit identification criteria, and general virtual screening hit
criteria to allow for realistic hit optimization. A key recommendation
is the use of size-targeted ligand efficiency values as hit identification
criteria