4 research outputs found
Unlocking the Binding and Reaction Mechanism of Hydroxyurea Substrates as Biological Nitric Oxide Donors
Hydroxyurea is the only FDA approved treatment of sickle
cell disease. It is believed that the primary mechanism of action
is associated with the pharmacological elevation of nitric oxide in
the blood; however, the exact details of this are still unclear. In
the current work, we investigate the atomic level details of this
process using a combination of flexible-ligand/flexible-receptor virtual
screening coupled with energetic analysis that decomposes interaction
energies. Utilizing these methods, we were able to elucidate the previously
unknown substrate binding modes of a series of hydroxyurea analogs
to hemoglobin and the concomitant structural changes of the enzyme.
We identify a backbone carbonyl that forms a hydrogen bond with bound
substrates. Our results are consistent with kinetic and electron paramagnetic resonance (EPR) measurements of hydroxyurea–hemoglobin reactions, and a full
mechanism is proposed that offers new insights into possibly improving
substrate binding and/or reactivity
How Does Catalase Release Nitric Oxide? A Computational Structure–Activity Relationship Study
Hydroxyurea (HU) is the only FDA
approved medication for treating sickle cell disease in adults. The
primary mechanism of action is pharmacological elevation of nitric
oxide (NO) levels which induces propagation of fetal hemoglobin. HU
is known to undergo redox reactions with heme based enzymes like hemoglobin
and catalase to produce NO. However, specific details about the HU
based NO release remain unknown. Experimental studies indicate that
interaction of HU with human catalase compound I produces NO. Presently,
we combine flexible receptor–flexible substrate induced fit
docking (IFD) with energy decomposition analyses to examine the atomic
level details of a possible key step in the clinical conversion of
HU to NO. Substrate binding modes of nine HU analogs with catalase
compound I were investigated to determine the essential properties
necessary for effective NO release. Three major binding orientations
were found that provide insight into the possible reaction mechanisms
for producing NO. Further results show that anion/radical intermediates
produced as part of these mechanisms would be stabilized by hydrogen
bonding interactions from distal residues His75, Asn148, Gln168, and
oxoferryl-heme. These details will ideally contribute to both a clearer
mechanistic picture and provide insights for future structure based
drug design efforts
Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β‑Lactamases
Bacterial
resistance to standard (i.e., β-lactam-based) antibiotics
has become a global pandemic. Simultaneously, research into the underlying
causes of resistance has slowed substantially, although its importance
is universally recognized. Key to unraveling critical details is characterization
of the noncovalent interactions that govern binding and specificity
(DD-peptidases, antibiotic targets, versus β-lactamases, the
evolutionarily
derived enzymes that play a major role in resistance) and ultimately
resistance as a whole. Herein, we describe a detailed investigation
that elicits new chemical insights into these underlying intermolecular
interactions. Benzylpenicillin and a novel β-lactam peptidomimetic
complexed to the Stremptomyces R61
peptidase are examined using an arsenal of computational techniques:
MD simulations, QM/MM calculations, charge perturbation analysis,
QM/MM orbital analysis, bioinformatics, flexible receptor/flexible
ligand docking, and computational ADME predictions. Several key molecular
level interactions are identified that not only shed light onto fundamental
resistance mechanisms, but also offer explanations for observed specificity.
Specifically, an extended π–π network is elucidated
that suggests antibacterial resistance has evolved, in part, due to
stabilizing aromatic interactions. Additionally, interactions between
the protein and peptidomimetic substrate are identified and characterized.
Of particular interest is a water-mediated salt bridge between Asp217
and the positively charged N-terminus of the peptidomimetic, revealing
an interaction that may significantly contribute to β-lactam
specificity. Finally, interaction information is used to suggest modifications
to current β-lactam compounds that should both improve binding
and specificity in DD-peptidases and their physiochemical properties
Disaggregation is a Mechanism for Emission Turn-On of <i>ortho</i>-Aminomethylphenylboronic Acid-Based Saccharide Sensors
<i>ortho</i>-Aminomethylphenylboronic acid-based receptors
with appended fluorophores are commonly used as molecular sensors
for saccharides in aqueous media. The mechanism for fluorescence modulation
in these sensors has been attributed to some form of photoinduced
electron transfer (PET) quenching, which is diminished in the presence
of saccharides. Using a well-known boronic acid-based saccharide sensor
(<b>3</b>), this work reveals a new mechanism for fluorescence
turn-on in these types of sensors. Compound <b>3</b> exhibits
an excimer, and the associated ground-state aggregation is responsible
for fluorescence modulation under certain conditions. When fructose
was titrated into a solution of <b>3</b> in 2:1 water/methanol
with NaCl, the fluorescence intensity increased. Yet, when the same
titration was repeated in pure methanol, a solvent in which the sensor
does not aggregate, no fluorescence response to fructose was observed.
This reveals that the fluorescence increase is not fully associated
with fructose binding, but instead disaggregation of the sensor in
the presence of fructose. Further, an analogue of the sensor that
does not contain a boronic acid (<b>4</b>) responded nearly
identically to <b>3</b> in the presence of fructose, despite
having no functional group with which to bind the saccharide. This
further supports the claim that fluorescence modulation is not primarily
a result of binding, but of disaggregation. Using an indicator displacement
assay and isothermal titration calorimetry, it was confirmed that
fructose does indeed bind to the sensor. Thus, our evidence reveals
that while binding occurs with fructose in the aqueous solvent system
used, it is not related to the majority of the fluorescence modulation.
Instead, disaggregation dominates the signal turn-on, and is thus
a mechanism that should be investigated in other <i>ortho</i>-aminomethylphenylboronic acid-based sensors