5 research outputs found
A Carbohydrate-Derived Splice Modulator
Small-molecule splice
modulators have recently been recognized
for their clinical potential for diverse cancers. This, combined with
their use as tools to study the importance of splice-regulated events
and their association with disease, continues to fuel the discovery
of new splice modulators. One of the key challenges found in the current
class of materials arises from their instability, where rapid metabolic
degradation can lead to off-target responses. We now describe the
preparation of bench-stable splice modulators by adapting carbohydrate
motifs as a central scaffold to provide rapid access to potent splice
modulators
hBfl-1/hNOXA Interaction Studies Provide New Insights on the Role of Bflā1 in Cancer Cell Resistance and for the Design of Novel Anticancer Agents
Upregulation
of antiapoptotic Bcl-2 proteins in certain tumors confers cancer cell
resistance to chemotherapy or radiations. Members of the antiapoptotic
Bcl-2 proteins, including Bcl-2, Mcl-1, Bcl-xL, Bcl-w, and Bfl-1,
inhibit apoptosis by selectively binding to conserved Ī±-helical
regions, named BH3 domains, of pro-apoptotic proteins such as Bim,
tBid, Bad, or NOXA. Five antiapoptotic proteins have been identified
that interact with various selectivity with BH3 containing pro-apoptotic
counterparts. Cancer cells present various and variable levels of
these proteins, making the design of effective apoptosis based therapeutics
challenging. Recently, BH3 profiling was introduced as a method to
classify cancer cells based on their ability to resist apoptosis following
exposure to selected BH3 peptides. However, these studies were based
on binding affinities measured with model BH3 peptides and Bcl-2-proteins
taken from mouse sequences. While the majority of these interactions
are conserved between mice and humans, we found surprisingly that
human NOXA binds to human Bfl-1 potently and covalently <i>via</i> conserved Cys residues, with over 2 orders of magnitude increased
affinity over hMcl-1. Our data suggest that some assumptions of the
original BH3 profiling need to be revisited and that perhaps further
targeting efforts should be redirected toward Bfl-1, for which no
suitable specific inhibitors or pharmacological tools have been reported.
In this regard, we also describe the initial design and characterizations
of novel covalent BH3-based agents that potently target Bfl-1. These
molecules could provide a novel platform on which to design effective
Bfl-1 targeting therapeutics
Selectivity in Small Molecule Splicing Modulation
The dysregulation of RNA splicing
is a molecular hallmark of disease,
including different and often complex cancers. While gaining recognition
as a target for therapeutic discovery, understanding the complex mechanisms
guiding RNA splicing remains a challenge for chemical biology. The
discovery of small molecule splicing modulators has recently enabled
an evaluation of the mechanisms of aberrant splicing. We now report
on three unique features within the selectivity of splicing modulators.
First, we provide evidence that structural modifications within a
splicing modulator can alter the splicing of introns in specific genes
differently. These studies indicate that structure activity relationships
not only have an effect on splicing activity but also include specificity
for specific introns within different genes. Second, we find that
these splicing modulators also target the mRNAs encoding components
of the spliceosome itself. Remarkably, this effect includes the genes
for the SF3B complex, a target of pladienolide B and related splicing
modulators. Finally, we report on the first observation of a temporal
phenomenon associated with small molecule splicing modulation. Combined,
these three observations provide an important new perspective for
the exploration of splicing modulation in terms of both future medicinal
chemistry programs as well as understanding the key facets underlying
its timing
Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)
Tissue microarrays have become a valuable tool for high-throughput
analysis using immunohistochemical labeling. However, the large majority
of biochemical studies are carried out in cell lines to further characterize
candidate biomarkers or therapeutic targets with subsequent studies
in animals or using primary tissues. Thus, cell line-based microarrays
could be a useful screening tool in some situations. Here, we constructed
a cell microarray (CMA) containing a panel of 40 pancreatic cancer
cell lines available from American Type Culture Collection in addition
to those locally available at Johns Hopkins. As proof of principle,
we performed immunocytochemical labeling of an epithelial cell adhesion
molecule (Ep-CAM), a molecule generally expressed in the epithelium,
on this pancreatic cancer CMA. In addition, selected molecules that
have been previously shown to be differentially expressed in pancreatic
cancer in the literature were validated. For example, we observed
strong labeling of CA19-9 antigen, a prognostic and predictive marker
for pancreatic cancer. We also carried out a bioinformatics analysis
of a literature curated catalog of pancreatic cancer biomarkers developed
previously by our group and identified two candidate biomarkers, HLA
class I and transmembrane protease, serine 4 (TMPRSS4), and examined
their expression in the cell lines represented on the pancreatic cancer
CMAs. Our results demonstrate the utility of CMAs as a useful resource
for rapid screening of molecules of interest and suggest that CMAs
can become a universal standard platform in cancer research
Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)
Tissue microarrays have become a valuable tool for high-throughput
analysis using immunohistochemical labeling. However, the large majority
of biochemical studies are carried out in cell lines to further characterize
candidate biomarkers or therapeutic targets with subsequent studies
in animals or using primary tissues. Thus, cell line-based microarrays
could be a useful screening tool in some situations. Here, we constructed
a cell microarray (CMA) containing a panel of 40 pancreatic cancer
cell lines available from American Type Culture Collection in addition
to those locally available at Johns Hopkins. As proof of principle,
we performed immunocytochemical labeling of an epithelial cell adhesion
molecule (Ep-CAM), a molecule generally expressed in the epithelium,
on this pancreatic cancer CMA. In addition, selected molecules that
have been previously shown to be differentially expressed in pancreatic
cancer in the literature were validated. For example, we observed
strong labeling of CA19-9 antigen, a prognostic and predictive marker
for pancreatic cancer. We also carried out a bioinformatics analysis
of a literature curated catalog of pancreatic cancer biomarkers developed
previously by our group and identified two candidate biomarkers, HLA
class I and transmembrane protease, serine 4 (TMPRSS4), and examined
their expression in the cell lines represented on the pancreatic cancer
CMAs. Our results demonstrate the utility of CMAs as a useful resource
for rapid screening of molecules of interest and suggest that CMAs
can become a universal standard platform in cancer research