5 research outputs found
Diiron Oxidation State Control of Substrate Access to the Active Site of Soluble Methane Monooxygenase Mediated by the Regulatory Component
The regulatory component (MMOB) of
soluble methane monooxygenase
(sMMO) has a unique N-terminal tail not found in regulatory proteins
of other bacterial multicomponent monooxygenases. This N-terminal
tail is indispensable for proper function, yet its solution structure
and role in catalysis remain elusive. Here, by using double electron–electron
resonance (DEER) spectroscopy, we show that the oxidation state of
the hydroxylase component, MMOH, modulates the conformation of the
N-terminal tail in the MMOH–2MMOB complex, which in turn facilitates
catalysis. The results reveal that the N-terminal tail switches from
a relaxed, flexible conformational state to an ordered state upon
MMOH reduction from the diironÂ(III) to the diironÂ(II) state. This
observation suggests that some of the crystallographically observed
allosteric effects that result in the connection of substrate ingress
cavities in the MMOH–2MMOB complex may not occur in solution
in the diironÂ(III) state. Thus, O<sub>2</sub> may not have easy access
to the active site until after reduction of the diiron center. The
observed conformational change is also consistent with a higher binding
affinity of MMOB to MMOH in the diironÂ(II) state, which may allow
MMOB to displace more readily the reductase component (MMOR) from
MMOH following reduction
Discovery of Inactive Conformation-Selective Kinase Inhibitors by Utilizing Cascade Assays
Achieving
selectivity across the human kinome is a major hurdle
in kinase inhibitor drug discovery. Targeting inactive (vs active)
kinase conformations offers advantages in achieving selectivity because
of their more diversified structures. Discovery of inactive conformation-selective
inhibitors, however, has been hampered partly by the lack of general
assay methods. Herein, we show that such inhibitors can be discovered
by utilizing kinase cascade assays. This type of assay is initiated
with the target kinase in its unphosphorylated, inactive conformation,
which is activated during the assay. Inactive conformation-selective
inhibitors stabilize the inactive kinase, block activation, and yield
reduced kinase activity. We investigate the properties of the assay
by mathematical modeling, as well as by proof-of-concept experiments
using the BRAF-MEK1 cascade. This study demonstrates effective identification
of inactive conformation-selective inhibitors by cascade assays, reveals
key factors that impact results, and provides guidelines for successful
cascade assay development
Are Free Radicals Involved in IspH Catalysis? An EPR and Crystallographic Investigation
The [4Fe–4S] protein IspH in the methylerythritol
phosphate
isoprenoid biosynthesis pathway is an important anti-infective drug
target, but its mechanism of action is still the subject of debate.
Here, by using electron paramagnetic resonance (EPR) spectroscopy
and <sup>2</sup>H, <sup>17</sup>O, and <sup>57</sup>Fe isotopic labeling,
we have characterized and assigned two key reaction intermediates
in IspH catalysis. The results are consistent with the bioorganometallic
mechanism proposed earlier, and the mechanism is proposed to have
similarities to that of ferredoxin, thioredoxin reductase, in that
one electron is transferred to the [4Fe–4S]<sup>2+</sup> cluster,
which then performs a formal two-electron reduction of its substrate,
generating an oxidized high potential iron–sulfur protein (HiPIP)-like
intermediate. The two paramagnetic reaction intermediates observed
correspond to the two intermediates proposed in the bioorganometallic
mechanism: the early π-complex in which the substrate’s
3-CH<sub>2</sub>OH group has rotated away from the reduced iron–sulfur
cluster, and the next, η<sup>3</sup>-allyl complex formed after
dehydroxylation. No free radical intermediates are observed, and the
two paramagnetic intermediates observed do not fit in a Birch reduction-like
or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy
and X-ray crystallography that two substrate analogues (<b>4</b> and <b>5</b>) follow the same reaction mechanism
Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors
Dihydroorotate dehydrogenase (DHODH) is a mitochondrial
enzyme
that affects many aspects essential to cell proliferation and survival.
Recently, DHODH has been identified as a potential target for acute
myeloid leukemia therapy. Herein, we describe the identification of
potent DHODH inhibitors through a scaffold hopping approach emanating
from a fragment screen followed by structure-based drug design to
further improve the overall profile and reveal an unexpected novel
binding mode. Additionally, these compounds had low P-gp efflux ratios,
allowing for applications where exposure to the brain would be required