38 research outputs found
Continuing evolution of Burkholderia mallei through genome reduction and large-scale rearrangements
Burkholderia mallei (Bm), the causative agent of the predominately equine disease glanders, is a genetically uniform species that is very closely related to the much more diverse species Burkholderia pseudomallei (Bp), an opportunistic human pathogen and the primary cause of melioidosis. To gain insight into the relative lack of genetic diversity within Bm, we performed whole-genome comparative analysis of seven Bm strains and contrasted these with eight Bp strains. The Bm core genome (shared by all seven strains) is smaller in size than that of Bp, but the inverse is true for the variable gene sets that are distributed across strains. Interestingly, the biological roles of the Bm variable gene sets are much more homogeneous than those of Bp. The Bm variable genes are found mostly in contiguous regions flanked by insertion sequence (IS) elements, which appear to mediate excision and subsequent elimination of groups of genes that are under reduced selection in the mammalian host. The analysis suggests that the Bm genome continues to evolve through random IS-mediated recombination events, and differences in gene content may contribute to differences in virulence observed among Bm strains. The results are consistent with the view that Bm recently evolved from a single strain of Bp upon introduction into an animal host followed by expansion of IS elements, prophage elimination, and genome rearrangements and reduction mediated by homologous recombination across IS elements
AI is a viable alternative to high throughput screening: a 318-target study
: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery
Direct Detection of Products from <i>S</i>‑Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay
<i>S</i>-Adenosylmethionine (AdoMet)-dependent methyltransferases
(MTases) are an essential superfamily of enzymes that catalyze the
transfer of a methyl group to several biomolecules. Alterations in
the methylation of cellular components crucially impact vital biological
processes, making MTases attractive drug targets for treating infectious
diseases and diseases caused by overactive human-encoded MTases. Several
methods have been developed for monitoring the activity of MTases,
but most MTase assays have inherent limitations or are not amenable
for high-throughput screening. We describe a universal, competitive
fluorescence polarization (FP) assay that directly measures the production
of <i>S</i>-adenosylhomocysteine (AdoHcy) from MTases. Our
developed assay monitors the generation of AdoHcy by displacing a
fluorescently labeled AdoHcy molecule complexed to a catalytically
inert 5′-methylthioadenosine nucleosidase (MTAN-D198N) variant
performed in a mix-and-read format. Producing the fluorescently labeled
molecule involves a one-pot synthesis by combining AdoHcy with an
amine-reactive rhodamine derivative, which possesses a <i>K</i><sub>d</sub> value of 11.3 ± 0.7 nM to MTAN-D198N. The developed
competitive FP assay expresses a limit of detection for AdoHcy of
6 nM and exhibits a 34-fold preference to AdoHcy in comparison to
AdoMet. We demonstrate the utility of the developed assay by performing
a pilot screen with the NIH Clinical Collection as well as determining
the kinetic parameters of l-histidine methylation for EgtD
from Mycobacterium tuberculosis. Additionally,
the developed assay is applicable to other AdoMet-dependent and ATP-dependent
enzymes by detecting various adenosine-containing molecules including
5′-methylthioadenosine, AMP, and ADP
Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states
MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs
Exploring Covalent Allosteric Inhibition of Antigen 85C from <i>Mycobacterium tuberculosis</i> by Ebselen Derivatives
Previous
studies identified ebselen as a potent <i>in vitro</i> and <i>in vivo</i> inhibitor of the <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) antigen 85 (Ag85) complex, comprising three
homologous enzymes required for the biosynthesis of the mycobacterial
cell wall. In this study, the <i>Mtb</i> Ag85C enzyme was
cocrystallized with azido and adamantyl ebselen derivatives, resulting
in two crystallographic structures of 2.01 and 1.30 Å resolution,
respectively. Both structures displayed the anticipated covalent modification
of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide
bond. Continuous difference density for both thiol modifiers allowed
for the assessment of interactions that influence ebselen binding
and inhibitor orientation that were unobserved in previous Ag85C ebselen
structures. The <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> values for ebselen, adamantyl ebselen, and azido ebselen
support the importance of observed constructive chemical interactions
with Arg239 for increased <i>in vitro</i> efficacy toward
Ag85C. To better understand the <i>in vitro</i> kinetic
properties of these ebselen derivatives, the energetics of specific
protein–inhibitor interactions and relative reaction free energies
were calculated for ebselen and both derivatives using density functional
theory. These studies further support the different <i>in vitro</i> properties of ebselen and two select ebselen derivatives from our
previously published ebselen library with respect to kinetics and
protein–inhibitor interactions. In both structures, the α9
helix was displaced farther from the enzyme active site than the previous
Ag85C ebselen structure, resulting in the restructuring of a connecting
loop and imparting a conformational change to residues believed to
play a role in substrate binding specific to Ag85C. These notable
structural changes directly affect protein stability, reducing the
overall melting temperature by up to 14.5 °C, resulting in the
unfolding of protein at physiological temperatures. Additionally,
this structural rearrangement due to covalent allosteric modification
creates a sizable solvent network that encompasses the active site
and extends to the modified Cys209 residue. In all, this study outlines
factors that influence enzyme inhibition by ebselen and its derivatives
while further highlighting the effects of the covalent modification
of Cys209 by said inhibitors on the structure and stability of Ag85C.
Furthermore, the results suggest a strategy for developing new classes
of Ag85 inhibitors with increased specificity and potency
Exploring Covalent Allosteric Inhibition of Antigen 85C from <i>Mycobacterium tuberculosis</i> by Ebselen Derivatives
Previous
studies identified ebselen as a potent <i>in vitro</i> and <i>in vivo</i> inhibitor of the <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) antigen 85 (Ag85) complex, comprising three
homologous enzymes required for the biosynthesis of the mycobacterial
cell wall. In this study, the <i>Mtb</i> Ag85C enzyme was
cocrystallized with azido and adamantyl ebselen derivatives, resulting
in two crystallographic structures of 2.01 and 1.30 Å resolution,
respectively. Both structures displayed the anticipated covalent modification
of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide
bond. Continuous difference density for both thiol modifiers allowed
for the assessment of interactions that influence ebselen binding
and inhibitor orientation that were unobserved in previous Ag85C ebselen
structures. The <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> values for ebselen, adamantyl ebselen, and azido ebselen
support the importance of observed constructive chemical interactions
with Arg239 for increased <i>in vitro</i> efficacy toward
Ag85C. To better understand the <i>in vitro</i> kinetic
properties of these ebselen derivatives, the energetics of specific
protein–inhibitor interactions and relative reaction free energies
were calculated for ebselen and both derivatives using density functional
theory. These studies further support the different <i>in vitro</i> properties of ebselen and two select ebselen derivatives from our
previously published ebselen library with respect to kinetics and
protein–inhibitor interactions. In both structures, the α9
helix was displaced farther from the enzyme active site than the previous
Ag85C ebselen structure, resulting in the restructuring of a connecting
loop and imparting a conformational change to residues believed to
play a role in substrate binding specific to Ag85C. These notable
structural changes directly affect protein stability, reducing the
overall melting temperature by up to 14.5 °C, resulting in the
unfolding of protein at physiological temperatures. Additionally,
this structural rearrangement due to covalent allosteric modification
creates a sizable solvent network that encompasses the active site
and extends to the modified Cys209 residue. In all, this study outlines
factors that influence enzyme inhibition by ebselen and its derivatives
while further highlighting the effects of the covalent modification
of Cys209 by said inhibitors on the structure and stability of Ag85C.
Furthermore, the results suggest a strategy for developing new classes
of Ag85 inhibitors with increased specificity and potency