28 research outputs found
The Many Faces of Histidine Triad Nucleotide Binding Protein 1 (HINT1)
The histidine triad nucleotide binding protein 1 (HINT1)
is a nucleoside
phosphoramidase that has garnered interest due to its widespread expression
and participation in a broad range of biological processes. Herein,
we discuss the role of HINT1 as a regulator of several CNS functions,
tumor suppressor, and mast cell activator via its interactions with
multiple G-protein-coupled receptors and transcription factors. Importantly,
altered HINT1 expression and mutation are connected to the progression
of multiple disease states, including several neuropsychiatric disorders,
peripheral neuropathy, and tumorigenesis. Additionally, due to its
involvement in the activation of several clinically used phosphoramidate
prodrugs, tremendous efforts have been made to better understand the
interactions behind nucleoside binding and phosphoramidate hydrolysis
by HINT1. We detail the substrate specificity and catalytic mechanism
of HINT1 hydrolysis, while highlighting the structural biology behind
these efforts. The aim of this review is to summarize the multitude
of biological and pharmacological functions in which HINT1 participates
while addressing the areas of need for future research
Chemically Self-Assembled Antibody Nanostructures as Potential Drug Carriers
Chemically self-assembled antibody nanorings (CSANs)
displaying
multiple copies of single-chain variable fragments can be prepared
from dihydrofolate reductase (DHFR) fusion proteins and bis-methotrexate
(bisMTX). We have designed and synthesized a bisMTX chemical dimerizer
(<b>bisMTX-NH<sub>2</sub></b>) that contains a third linker
arm that can be conjugated to fluorophores, radiolabels, and drugs.
Monovalent, divalent, and higher-order AntiCD3 CSANs were assembled
with a fluorescein isothiocyanate (FITC)-labeled bis-methotrexate
ligand (<b>bisMTX-FITC</b>) and found to undergo rapid internalization
and trafficking by HPB-MLT, a CD3+ T-leukemia cell line, to the early
and late endosome and lysosome. Because the fluorescence of <b>bisMTX-FITC</b> when incorporated into CSANs was found to be significantly
greater than that of the free ligand, the stability of the endocytosed
AntiCD3 CSANs could be monitored. The internalized CSANs were found
to be stable for several hours, while treatment with the nontoxic
DHFR inhibitor trimethoprim resulted in a rapid loss (>80%) of
cellular
fluorescence within minutes, consistent with efficient intracellular
disassembly of the nanorings. Over longer time periods (24 h), cellular
fluorescence decreased by 75–90%, regardless of whether cells
had been treated with DMSO or trimethoprim. Although bisMTX is a potent
inhibitor of DHFR, it was found to be nontoxic (GI<sub>50</sub> >
20 μM) to HPB-MLT cells. In contrast, AntiCD3 CSANs prepared
with bisMTX were found to be at least 13-fold more cytotoxic (GI<sub>50</sub> = 0.5–1.5 μM) than bisMTX at 72 h. Consistent
with our findings from CSAN stability studies, no increase in cytotoxicity
was observed upon treatment with trimethoprim. Taken together, our
results suggest that cell receptor targeting CSANs prepared with trifunctional
bisMTX could be used as potential tissue selective drug carriers
Prosthetic Antigen Receptors
Chimeric antigen
receptors (CARs) have shown great promise for
the immunological treatment of cancer. Nevertheless, the need to genetically
engineer a patient’s T-cells has presented significant production
and safety challenges. To address these issues, we have demonstrated
that chemically self-assembled nanorings (CSANs) displaying single
chain antibodies can bind to both the CD3 ε subunit of the T-cell-receptor/CD3
complex and the CD22 antigen on malignant B cells such as B-leukemias
or lymphomas. We demonstrate that the multivalent and bispecific format
allows the antiCD3/antiCD22 CSANs to stably bind to T-cell surfaces
for greater than 4 days, while being easily disassembled on the cell
membrane by treatment with the nontoxic FDA approved drug, trimethoprim.
In the presence of CD22+ Raji cells, T-cells modified with antiCD3/antiCD22
CSANs were shown to selectively up-regulate the production of interleukin-2
(IL-2) and interferon-γ (IFN-γ) and to initiate cytotoxicity.
Taken together, our results demonstrate that antiCD3/antiCD22 bispecific
CSANs offer a potential alternative to CARs, as prosthetic antigen
receptors
Tunable Supramolecular Assemblies from Amphiphilic Nucleoside Phosphoramidate Nanofibers by Enzyme Activation
Enzymes
possess unique qualities that make them ideal regulators
of supramolecular assembly. They are uniquely sensitive to biomolecules
and biological compartments, catalytic in effecting chemical reactions,
and present a biocompatible and degradable platform for assembly regulation.
We demonstrate the novel utility of Histidine Triad Nucleotide Binding
Protein 1 (HINT1) in regulating supramolecular hydrogel formation.
We synthesized nucleoside-phosphoramidate-functionalized self-assembling
peptides that we observed to form nanofibers. We found HINT1’s
catalytic hydrolysis of the nucleoside phosphoramidate moieties within
the nanofiber structures to induce nanofiber organization and higher
ordered assembly. The role of HINT1 in effecting this structural change
was confirmed with experiments utilizing a high-affinity HINT1 inhibitor
and catalytically dead HINT1 mutant. In addition, the kinetics and
morphology of hydrogel formation were found to be dependent on the
structure of the released nucleoside monophosphate. This work highlights
the self-assembly of phosphoramidate nanofibers and their higher organization
triggered by HINT1 enzymatic activity
Programmable Self-Assembly of Antibody–Oligonucleotide Conjugates as Small Molecule and Protein Carriers
Dihydrofolate reductase single-chain variable fragment
(scFv) fusion
proteins can be used for the targeted cellular delivery of oligonucleotides,
conjugated small molecules, and proteins via labeling of oligonucleotides
by bis-methotrexate
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
Titratable Avidity Reduction Enhances Affinity Discrimination in Mammalian Cellular Selections of Yeast-Displayed Ligands
Yeast
surface display selections against mammalian cell monolayers
have proven effective in isolating proteins with novel binding activity.
Recent advances in this technique allow for the recovery of clones
with even micromolar binding affinities. However, no efficient method
has been shown for affinity-based selection in this context. This
study demonstrates the effectiveness of titratable avidity reduction
using dithiothreitol to achieve this goal. A series of epidermal growth
factor receptor binding fibronectin domains with a range of affinities
are used to quantitatively identify the number of ligands per yeast
cell that yield the strongest selectivity between strong, moderate,
and weak affinities. Notably, reduction of ligand display to 3,000–6,000
ligands per yeast cell of a 2 nM binder yields 16-fold better selectivity
than that to a 17 nM binder. These lessons are applied to affinity
maturation of an EpCAM-binding fibronectin population, yielding an
enriched pool of ligands with significantly stronger affinity than
that of an analogous pool sorted by standard cellular selection methods.
Collectively, this study offers a facile approach for affinity selection
of yeast-displayed ligands against full-length cellular targets and
demonstrates the effectiveness of this method by generating EpCAM-binding
ligands that are promising for further applications
Targeted Delivery of Antisense Oligonucleotides by Chemically Self-Assembled Nanostructures
Synthetic nucleic acids have shown
great potential in the treatment
of various diseases. Nevertheless, the selective delivery to a target
tissue has proved challenging. The coupling of nucleic acids to targeting
peptides, proteins, and antibodies has been explored as an approach
for their selective tissue delivery. Nevertheless, the preparation
of covalently coupled peptides and proteins that can also undergo
intracellular release as well as deliver more than one copy of the
nucleic acid has proved challenging. Recently, we have developed a
novel method for the rapid noncovalent conjugation of nucleic acids
to targeting single chain antibodies (scFv) using chemically self-assembled
nanostructures (CSANs). CSANs have been prepared by the self-assembly
of two dihydrofolate reductase molecules (DHFR<sup>2</sup>) and a
targeting scFv in the presence of bis-methotrexate (bis-MTX). The
valency of the nanorings can be tuned from one to eight subunits,
depending on the length and composition of the linker between the
dihydrofolate reductase molecules. To explore their potential for
the therapeutic delivery of nucleic acids as well as the ability to
expand the capabilities of CSANs by incorporating smaller cyclic targeting
peptides, we prepared DHFR<sup>2</sup> proteins fused through a flexible
peptide linker to cyclic-RGD, which targets αvβ3 integrins,
and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide
(bis-MTX-ASO) that has been shown to silence expression of eukaryotic
translation initiation factor 4E (eIF4E). Monomeric and multimeric
cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo
endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses
αvβ3. The bis-MTX-ASO was shown to undergo endosomal escape
resulting in the knock down of eIF4E with at least the same efficiency
as ASO delivered by oligofectamine. The modularity, flexibility, and
common method of conjugation may prove to be a useful general approach
for the targeted delivery of ASOs, as well as other nucleic acids
to cells
<i>In Vivo</i> Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures
Chemically
self-assembled nanorings (CSANs) are made of dihydrofolate
reductase (DHFR) fusion proteins and have been successfully used <i>in vitro</i> for cellular cargo delivery and cell surface engineering
applications. However, CSANs have yet to be evaluated for their <i>in vivo</i> stability, circulation, and tissue distribution.
In an effort to evaluate CSANs <i>in vivo</i>, we engineered
a site-specifically PEGylated epidermal growth factor receptor (EGFR)
targeting DHFR molecules, characterized their self-assembly into CSANs
with bivalent methotrexates (bis-MTX), visualized their <i>in
vivo</i> tissue localization by microPET/CT imaging, and determined
their <i>ex vivo</i> organ biodistribution by tissue-based
gamma counting. A dimeric DHFR (DHFR<sup>2</sup>) molecule fused with
a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate
a site-specific ketone functionality using unnatural amino acid mutagenesis.
Aminooxy-PEG, of differing chain lengths, was successfully conjugated
to the protein using oxime chemistry. These proteins were self-assembled
into CSANs with bis-MTX DHFR dimerizers and characterized by size
exclusion chromatography and dynamic light scattering. <i>In
vitro</i> binding studies were performed with fluorescent CSANs
assembled using bis-MTX-FITC, while <i>in vivo</i> microPET/CT
imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTAÂ[<sup>64</sup>Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by
mouse macrophages (RAW 264.7) up to 40% without altering the CSAN’s
binding affinity toward U-87 MG glioblastoma cells <i>in vitro</i>. A significant time dependent tumor accumulation of <sup>64</sup>Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and
biodistribution studies in mice bearing U-87 MG xenografts. PEGylated
CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen
resulting in high contrast tumor imaging within an hour of intravenous
injection (9.6% ID/g), and continued to increase up to 24 h (11.7%
ID/g) while the background signal diminished. CSANs displayed an <i>in vivo</i> profile between those of rapidly clearing small
molecules and slow clearing antibodies. Thus, CSANs offer a modular,
programmable, and stable protein based platform that can be used for <i>in vivo</i> drug delivery and imaging applications
Anchimerically Activated ProTides as Inhibitors of Cap-Dependent Translation and Inducers of Chemosensitization in Mantle Cell Lymphoma
The cellular delivery of nucleotides
through various pronucleotide
strategies has expanded the utility of nucleosides as a therapeutic
class. Although highly successful, the highly popular ProTide system
relies on a four-step enzymatic and chemical process to liberate the
corresponding monophosphate. To broaden the scope and reduce the number
of steps required for monophosphate release, we have developed a strategy
that depends on initial chemical activation by a sulfur atom of a
methylthioalkyl protecting group, followed by enzymatic hydrolysis
of the resulting phosphoramidate monoester. We have employed this
ProTide strategy for intracellular delivery of a nucleotide antagonist
of eIF4E in mantle cell lymphoma (MCL) cells. Furthermore, we demonstrated
that chemical inhibition of cap-dependent translation results in suppression
of c-Myc expression, increased p27 expression, and enhanced chemosensitization
to doxorubicin, dexamethasone, and ibrutinib. In addition, the new
ProTide strategy was shown to enhance oral bioavailability of the
corresponding monoester phosphoramidate