7 research outputs found
Modified Bleomycin Disaccharides Exhibiting Improved Tumor Cell Targeting
The
bleomycins (BLMs) are a family of antitumor antibiotics used
clinically for anticancer chemotherapy. Their antitumor selectivity
derives at least in part from their ability to target tumor cells,
a property that resides in the carbohydrate moiety of the antitumor
agent. In earlier studies, we have demonstrated that the tumor cell
selectivity resides in the mannose carbamoyl moiety of the BLM saccharide
and that both the BLM disaccharide and monosaccharide containing the
carbamoyl moiety were capable of the delivery/uptake of a conjugated
cyanine dye into cultured cancer cell lines. Presently, the nature
of the participation of the carbamoyl moiety has been explored further
to provide compounds of utility for defining the nature of the mechanism
of tumor cell recognition and uptake by BLM saccharides and in the
hope that more efficient compounds could be identified. A library
of seven disaccharide–Cy5** dye conjugates was prepared that
are structural analogues of the BLM disaccharide. These differed from
the natural BLM disaccharide in the position, orientation, and substitution
of the carbamoyl group. Studies of these compounds in four matched
sets of tumor and normal cell lines revealed a few that were both
tumor cell selective and internalized 2–4-fold more efficiently
than the natural BLM disaccharide
Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro
Plasmids containing 23S rRNA randomized
at positions 2057–2063
and 2502–2507 were introduced into <i>Escherichia coli</i>, affording a library of clones which produced modified ribosomes
in addition to the pre-existing wild-type ribosomes. These clones
were screened with a derivative of puromycin, a natural product
which acts as an analogue of the 3′-end of aminoacyl-tRNA and
terminates protein synthesis by accepting the growing polypeptide
chain, thereby killing bacterial cells. The puromycin
derivative in this study contained the dipeptide <i>p</i>-methoxyphenylalanylglycine, implying the ability
of the modified ribosomes in clones sensitive to this puromycin
analogue to recognize dipeptides. Several clones inhibited by the
puromycin derivative were used to make S-30 preparations, and
some of these were shown to support the incorporation of dipeptides
into proteins. The four incorporated species included two dipeptides
(Gly-Phe (<b>2</b>) and Phe-Gly (<b>3</b>)), as well as
a thiolated dipeptide analogue (<b>4</b>) and a fluorescent
oxazole (<b>5</b>) having amine and carboxyl groups approximately
the same distance apart as in a normal dipeptide. A protein containing
both thiolated dipeptide <b>4</b> and a 7-methoxycoumarin
fluorophore was found to undergo fluorescence quenching. Introduction
of the oxazole fluorophore <b>5</b> into dihydrofolate reductase or green fluorescent
protein resulted in quite strong enhancement of its fluorescence emission,
and the basis for this enhancement was studied. The aggregate results
demonstrate the feasibility of incorporating dipeptides as a single
ribosomal event, and illustrate the lack of recognition of the central
peptide bond in the dipeptide, potentially enabling the incorporation
of a broad variety of structural analogues
The Dynamic Character of the <i>BCL2</i> Promoter i‑Motif Provides a Mechanism for Modulation of Gene Expression by Compounds That Bind Selectively to the Alternative DNA Hairpin Structure
It
is generally accepted that DNA predominantly exists in duplex
form in cells. However, under torsional stress imposed by active transcription,
DNA can assume nonduplex structures. The <i>BCL2</i> promoter
region forms two different secondary DNA structures on opposite strands
called the G-quadruplex and the i-motif. The i-motif is a highly dynamic
structure that exists in equilibrium with a flexible hairpin species.
Here we identify a pregnanol derivative and a class of piperidine
derivatives that differentially modulate gene expression by stabilizing
either the i-motif or the flexible hairpin species. Stabilization
of the i-motif structure results in significant upregulation of the <i>BCL2</i> gene and associated protein expression; in contrast,
stabilization of the flexible hairpin species lowers <i>BCL2</i> levels. The <i>BCL2</i> levels reduced by the hairpin-binding
compound led to chemosensitization to etoposide in both in vitro and
in vivo models. Furthermore, we show antagonism between the two classes
of compounds in solution and in cells. For the first time, our results
demonstrate the principle of small molecule targeting of i-motif structures
in vitro and in vivo to modulate gene expression
Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA–Protein Interaction
Described herein are the syntheses
and photophysical characterization
of three novel cyanotryptophans, and their efficient incorporation
into proteins as fluorescent probes. Photophysical characteristics
indicated that each was significantly brighter and red-shifted in
fluorescence emission relative to tryptophan. Each analogue was used
to activate a suppressor tRNA transcript and was incorporated with
good efficiency into two different positions (Trp22 and Trp74) of <i>Escherichia coli</i> dihydrofolate reductase (<i>ec</i>DHFR). The Trp analogues could be monitored selectively in the presence
of multiple native Trp residues in DHFR. 6-CNTrp (<b>A</b>)
formed an efficient Förster resonance energy transfer (FRET)
pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, <b>D</b>) at position 17. Further, 6-CNTrp (<b>A</b>) was incorporated
into two DNA binding proteins, including the Klenow fragment of DNA
polymerase I and an RNA recognition motif (RRM2) of heterogeneous
nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins,
we demonstrated the use of FRET involving <b>A</b> as a fluorescence
donor and benzo[<i>g</i>]quinazoline-2,4-(1<i>H</i>,3<i>H</i>)-dione 2′-deoxyriboside (T<sub>f</sub>) or 4-aminobenzo[<i>g</i>]quinazoline-2-one 2′-deoxyriboside
(C<sub>f</sub>) as fluorescent acceptors to study the binding interaction
of the Klenow fragment with duplex DNA oligomers (labeled with T<sub>f</sub>), or the domain-specific association between hnRNP LL and
the <i>BCL2</i> i-motif DNA (labeled with C<sub>f</sub>).
Thus, the non-natural amino acid could be used as a FRET partner for
studying protein–nucleic acid interactions. Together, these
findings demonstrate the potential utility of 6-CNTrp (<b>A</b>) as a fluorescence donor for the study of protein conformational
events