3 research outputs found
Ribonuclease-Activated Cancer Prodrug
Cancer chemotherapeutic agents often have a narrow therapeutic
index that challenges the maintenance of a safe and effective dose.
Consistent plasma concentrations of a drug can be obtained by using
a timed-release prodrug strategy. We reasoned that a ribonucleoside
3′-phosphate could serve as a pro-moiety that also increases
the hydrophilicity of a cancer chemotherapeutic agent. Herein, we
report an efficient route for the synthesis of the prodrug uridine
3′-(4-hydroxytamoxifen phosphate) (UpHT). UpHT demonstrates
timed-released activation kinetics with a half-life of approximately
4 h at the approximate plasma concentration of human pancreatic ribonuclease
(RNase 1). MCF-7 breast cancer cells treated with UpHT showed decreased
proliferation upon coincubation with RNase 1, consistent with the
release of the active drugî—¸4-hydroxytamoxifen. These data demonstrate
the utility of a human plasma enzyme as a useful activator of a prodrug
Diazo Groups Endure Metabolism and Enable Chemoselectivity in Cellulo
We
introduce a stabilized diazo group as a reporter for chemical
biology. ManDiaz, which is a diazo derivative of <i>N</i>-acetylmannosamine, is found to endure cellular metabolism and label
the surface of a mammalian cell. There its diazo group can undergo
a 1,3-dipolar cycloaddition with a strained alkyne, providing a signal
comparable to that from the azido congener, ManNAz. The chemoselectivity
of diazo and alkynyl groups enables dual labeling of cells that is
not possible with azido and alkynyl groups. Thus, the diazo group,
which is approximately half the size of an azido group, provides unique
opportunities for orthogonal labeling of cellular components
Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display
Intein-mediated expressed protein
ligation (EPL) permits the site-specific
chemical customization of proteins. While traditional techniques have
used purified, soluble proteins, we have extended these methods to
release and modify intein fusion proteins expressed on the yeast surface,
thereby eliminating the need for soluble protein expression and purification.
To this end, we sought to simultaneously release yeast surface-displayed
proteins and selectively conjugate with chemical functionalities compatible
with EPL and click chemistry. Single-chain antibodies (scFv) and green
fluorescent protein (GFP) were displayed on the yeast surface as fusions
to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released
from the yeast surface with either a sulfur nucleophile (MESNA) or
a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine
azide permitted the simultaneous release and azido functionalization
of displayed proteins, but nonspecific reactions with other yeast
proteins were detected, and cleavage efficiency was limited. In contrast,
MESNA released significantly more protein from the yeast surface while
also generating a unique thioester at the carboxy-terminus of the
released protein. These protein thioesters were subsequently reacted
with a cysteine alkyne in an EPL reaction and then employed in an
azide<i>–</i>alkyne cycloaddition to immobilize the
scFv and GFP on an azide-decorated surface with >90% site-specificity.
Importantly, the immobilized proteins retained their activity. Since
yeast surface display is also a protein engineering platform, these
approaches provide a particularly powerful tool for the rapid assessment
of engineered proteins