14 research outputs found
Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)
Human
histidine triad nucleotide binding protein 1 (hHint1) is
classified as an efficient nucleoside phosphoramidase and acyl-adenosine
monophosphate hydrolase. Human Hint1 has been shown to be essential
for the metabolic activation of nucleotide antiviral pronucleotides
(i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir.
The active site of hHint1 comprises an ensemble of strictly conserved
histidines, including nucleophilic His112. To structurally investigate
the mechanism of hHint1 catalysis, we have designed and prepared nucleoside
thiophosphoramidate substrates that are able to capture the transiently
formed nucleotidylated-His112 intermediate (<b>E*</b>) using
time-dependent crystallography. Utilizing a catalytically inactive
hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme–substrate
(<b>ES</b><sup><b>1</b></sup>) and product (<b>EP</b><sup><b>2</b></sup>) complexes were also cocrystallized, respectively,
thus providing a structural map of the reaction trajectory. On the
basis of these observations and the mechanistic necessity of proton
transfers, proton inventory studies were carried out. Although we
cannot completely exclude the possibility of more than one proton
in flight, the results of these studies were consistent with the transfer
of a single proton during the formation of the intermediate. Interestingly,
structural analysis revealed that the critical proton transfers required
for intermediate formation and hydrolysis may be mediated by a conserved
active site water channel. Taken together, our results provide mechanistic
insights underpinning histidine nucleophilic catalysis in general
and hHint1 catalysis, in particular, thus aiding the design of future
proTides and the elucidation of the natural function of the Hint family
of enzymes
Design, Synthesis, and Characterization of Sulfamide and Sulfamate Nucleotidomimetic Inhibitors of hHint1
Hint1 has recently
emerged to be an important target of interest
due to its involvement in the regulation of a broad range of CNS functions
including opioid signaling, tolerance, neuropathic pain, and nicotine
dependence. A series of inhibitors were rationally designed, synthesized,
and tested for their inhibitory activity against hHint1 using isothermal
titration calorimetry (ITC). The studies resulted in the development
of the first small-molecule inhibitors of hHint1 with submicromolar
binding affinities. A combination of thermodynamic and high-resolution
X-ray crystallographic studies provides an insight into the biomolecular
recognition of ligands by hHint1. These novel inhibitors have potential
utility as molecular probes to better understand the role and function
of hHint1 in the CNS
A Highly Efficient Catalyst for Oxime Ligation and Hydrazone–Oxime Exchange Suitable for Bioconjugation
Imine-based
reactions are useful for a wide range of bioconjugation
applications. Although aniline is known to catalyze the oxime ligation
reaction under physiological conditions, it suffers from slow reaction
kinetics, specifically when a ketone is being used or when hydrazone–oxime
exchange is performed. Here, we report on the discovery of a new catalyst
that is up to 15 times more efficient than aniline. That catalyst, <i>m</i>-phenylenediamine (mPDA), was initially used to analyze
the kinetics of oxime ligation on aldehyde- and ketone-containing
small molecules. While mPDA is only modestly more effective than aniline
when used in equal concentrations (∼2-fold), its much greater
aqueous solubility relative to aniline allows it to be used at higher
concentrations, resulting in significantly more efficient catalysis.
In the context of protein labeling, it was first used to site-specifically
label an aldehyde-functionalized protein through oxime ligation, and
its kinetics were compared to reaction with aniline. Next, a protein
was labeled with an aldehyde-containing substrate in crude cell lysate,
captured with hydrazide-functionalized beads and then the kinetics
of immobilized protein release via hydrazone-oxime exchange were analyzed.
Our results show that mPDA can release and label 15 times more protein
than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic
factor, a protein with therapeutic potential, was successfully labeled
with a fluorophore in only 5 min. Finally, a protein containing the
unnatural amino acid, <i>p</i>-acetyl phenylalanine, a ketone-containing
residue, was prepared and PEGylated efficiently via oxime ligation
using mPDA. This new catalyst should have a significant impact on
the field of bioconjugation, where oxime ligation and hydrazone–oxime
exchange are commonly employed
A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides
Nucleotide
analogues that incorporate a metabolically labile nucleoside
phosphoramidate (a ProTide) have found utility as prodrugs. In humans,
ProTides can be cleaved by human histidine triad nucleotide binding
protein 1 (hHint1) to expose the nucleotide monophosphate. Activation
by this route circumvents highly selective nucleoside kinases that
limit the use of nucleosides as prodrugs. To better understand the
diversity of potential substrates of hHint1, we created and studied
a series of phosphoramidate nucleosides. Using a combination of enzyme
kinetics, X-ray crystallography, and isothermal titration calorimetry
with both wild-type and inactive mutant enzymes, we have been able
to explore the energetics of substrate binding and establish a structural
basis for catalytic efficiency. Diverse nucleobases are well tolerated,
but portions of the ribose are needed to position substrates for catalysis.
Beneficial characteristics of the amine leaving group are also revealed.
Structural principles revealed by these results may be exploited to
tune the rate of substrate hydrolysis to strategically alter the intracellular
release of the product nucleoside monophosphate from the ProTide