9 research outputs found
Measuring RNA–Ligand Interactions with Microscale Thermophoresis
In
recent years, there has been dramatic growth in the study of
RNA. RNA has gone from being known as an intermediate in the central
dogma of molecular biology to a molecule with a large diversity of
structure and function that is involved in all aspects of biology.
As new functions are rapidly discovered, it has become clear that
there is a need for RNA-targeting small molecule probes to investigate
RNA biology and clarify the potential for therapeutics based on RNA–small
molecule interactions. While a host of techniques exist to measure
RNA–small molecule interactions, many of these have drawbacks
that make them intractable for routine use and are often not broadly
applicable. A newer technology called microscale thermophoresis (MST),
which measures the directed migration of a molecule and/or molecule–ligand
complex along a temperature gradient, can be used to measure binding
affinities using very small amounts of sample. The high sensitivity
of this technique enables measurement of affinity constants in the
nanomolar and micromolar range. Here, we demonstrate how MST can be
used to study a range of biologically relevant RNA interactions, including
peptide–RNA interactions, RNA–small molecule interactions,
and displacement of an RNA-bound peptide by a small molecule
Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays
The identification
of small molecules that bind to and perturb
the function of microRNAs is an attractive approach for the treatment
for microRNA-associated pathologies. However, there are only a few
small molecules known to interact directly with microRNAs. Here, we
report the use of a small molecule microarray (SMM) screening approach
to identify low molecular weight compounds that directly bind to a
pre-miR-21 hairpin. Compounds identified using this approach exhibit
good affinity for the RNA (ranging from 0.8–2.0 μM) and
are not composed of a polycationic scaffold. Several of the highest
affinity compounds inhibit Dicer-mediated processing, while in-line
probing experiments indicate that the compounds bind to the apical
loop of the hairpin, proximal to the Dicer site. This work provides
evidence that small molecules can be developed to bind directly to
and inhibit miR-21
A HaloTag-Based Small Molecule Microarray Screening Methodology with Increased Sensitivity and Multiplex Capabilities
Small Molecule Microarrays (SMMs) represent a general
platform
for screening small molecule–protein interactions independent
of functional inhibition of target proteins. In an effort to increase
the scope and utility of SMMs, we have modified the SMM screening
methodology to increase assay sensitivity and facilitate multiplex
screening. Fusing target proteins to the HaloTag protein allows us
to covalently prelabel fusion proteins with fluorophores, leading
to increased assay sensitivity and an ability to conduct multiplex
screens. We use the interaction between FKBP12 and two ligands, rapamycin
and ARIAD’s “bump” ligand, to show that the HaloTag-based
SMM screening methodology significantly increases assay sensitivity.
Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we
show that prelabeling various protein isoforms with different fluorophores
allows us to conduct multiplex screens and identify ligands to a specific
isoform. Finally, we show this multiplex screening technique is capable
of identifying ligands selective for a specific PTP1B isoform using
a 20,000 compound screening deck
Development of Small Molecules with a Noncanonical Binding Mode to HIV‑1 Trans Activation Response (TAR) RNA
Small molecules that
bind to RNA potently and specifically are
relatively rare. The study of molecules that bind to the HIV-1 transactivation
response (TAR) hairpin, a cis-acting HIV genomic element, has long
been an important model system for the chemistry of targeting RNA.
Here we report the synthesis, biochemical, and structural evaluation
of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, <b>15</b>, retained the TAR binding affinity of the initial hit and
displaced a Tat-derived peptide with an IC<sub>50</sub> of 40 ÎĽM.
NMR characterization of a soluble analogue, <b>2</b>, revealed
a noncanonical binding mode for this class of compounds. Finally,
evaluation of <b>2</b> and <b>15</b> by selective 2′-hydroxyl
acylation analyzed by primer extension (SHAPE) indicates specificity
in binding to TAR within the context of an in vitro-synthesized 365-nt
HIV-1 5′-untranslated region (UTR). Thus, these compounds exhibit
a novel and specific mode of interaction with TAR, providing important
suggestions for RNA ligand design
Macrophilones from the Marine Hydroid <i>Macrorhynchia philippina</i> Can Inhibit ERK Cascade Signaling
Six new macrophilone-type pyrroloiminoquines
were isolated and identified from an extract of the marine hydroid <i>Macrorhynchia philippina</i>. The proton-deficient and heteroatom-rich
structures of macrophilones B–G (<b>2</b>–<b>7</b>) were elucidated by spectroscopic analysis and comparison
of their data with those of the previously reported metabolite macrophilone
A (<b>1</b>). Compounds <b>1</b>–<b>7</b> are the first pyrroloiminoquines to be reported from a hydroid.
The macrophilones were shown to inhibit the enzymatic conjugation
of SUMO to peptide substrates, and macrophilones A (<b>1</b>) and C (<b>3</b>) exhibit potent and selective cytotoxic properties
in the NCI-60 anticancer screen. Bioinformatic analysis revealed a
close association of the cytotoxicity profiles of <b>1</b> and <b>3</b> with two known B-Raf kinase inhibitory drugs. While compounds <b>1</b> and <b>3</b> showed no kinase inhibitory activity,
they resulted in a dramatic decrease in cellular protein levels of
selected components of the ERK signal cascade. As such, the chemical
scaffold of the macrophilones could provide small-molecule therapeutic
leads that target the ERK signal transduction pathway
Small Molecule Microarrays Enable the Identification of a Selective, Quadruplex-Binding Inhibitor of MYC Expression
The
transcription factor MYC plays a pivotal role in cancer initiation,
progression, and maintenance. However, it has proven difficult to
develop small molecule inhibitors of MYC. One attractive route to
pharmacological inhibition of MYC has been the prevention of its expression
through small molecule-mediated stabilization of the G-quadruplex
(G4) present in its promoter. Although molecules that bind globally
to quadruplex DNA and influence gene expression are well-known, the
identification of new chemical scaffolds that selectively modulate
G4-driven genes remains a challenge. Here, we report an approach for
the identification of G4-binding small molecules using small molecule
microarrays (SMMs). We use the SMM screening platform to identify
a novel G4-binding small molecule that inhibits MYC expression in
cell models, with minimal impact on the expression of other G4-associated
genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated
that this molecule binds reversibly to the MYC G4 with single digit
micromolar affinity, and with weaker or no measurable binding to other
G4s. Biochemical and cell-based assays demonstrated that the compound
effectively silenced MYC transcription and translation via a G4-dependent
mechanism of action. The compound induced G1 arrest and was selectively
toxic to MYC-driven cancer cell lines containing the G4 in the promoter
but had minimal effects in peripheral blood mononucleocytes or a cell
line lacking the G4 in its MYC promoter. As a measure of selectivity,
gene expression analysis and qPCR experiments demonstrated that MYC
and several MYC target genes were downregulated upon treatment with
this compound, while the expression of several other G4-driven genes
was not affected. In addition to providing a novel chemical scaffold
that modulates MYC expression through G4 binding, this work suggests
that the SMM screening approach may be broadly useful as an approach
for the identification of new G4-binding small molecules
Macrophilone A: Structure Elucidation, Total Synthesis, and Functional Evaluation of a Biologically Active Iminoquinone from the Marine Hydroid <i>Macrorhynchia philippina</i>
A previously
uncharacterized pyrroloiminoquinone natural product,
macrophilone A, was isolated from the stinging hydroid <i>Macrorhynchia
philippina</i>. The structure was assigned utilizing long-range
NMR couplings and DFT calculations and proved by a concise, five-step
total synthesis. Macrophilone A and a synthetic analogue displayed
potent biological activity, including increased intracellular reactive
oxygen species levels and submicromolar cytotoxicity toward lung adenocarcinoma
cells
Proximity-Induced Nucleic Acid Degrader (PINAD) Approach to Targeted RNA Degradation Using Small Molecules
Nature has evolved
intricate machinery to target and
degrade RNA,
and some of these molecular mechanisms can be adapted for therapeutic
use. Small interfering RNAs and RNase H-inducing oligonucleotides
have yielded therapeutic agents against diseases that cannot be tackled
using protein-centered approaches. Because these therapeutic agents
are nucleic acid-based, they have several inherent drawbacks which
include poor cellular uptake and stability. Here we report a new approach
to target and degrade RNA using small molecules, proximity-induced
nucleic acid degrader (PINAD). We have utilized this strategy to design
two families of RNA degraders which target two different RNA structures
within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral
pseudoknot. We demonstrate that these novel molecules degrade their
targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy
allows any RNA binding small molecule to be converted into a degrader,
empowering RNA binders that are not potent enough to exert a phenotypic
effect on their own. PINAD raises the possibility of targeting and
destroying any disease-related RNA species, which can greatly expand
the space of druggable targets and diseases
Proximity-Induced Nucleic Acid Degrader (PINAD) Approach to Targeted RNA Degradation Using Small Molecules
Nature has evolved
intricate machinery to target and
degrade RNA,
and some of these molecular mechanisms can be adapted for therapeutic
use. Small interfering RNAs and RNase H-inducing oligonucleotides
have yielded therapeutic agents against diseases that cannot be tackled
using protein-centered approaches. Because these therapeutic agents
are nucleic acid-based, they have several inherent drawbacks which
include poor cellular uptake and stability. Here we report a new approach
to target and degrade RNA using small molecules, proximity-induced
nucleic acid degrader (PINAD). We have utilized this strategy to design
two families of RNA degraders which target two different RNA structures
within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral
pseudoknot. We demonstrate that these novel molecules degrade their
targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy
allows any RNA binding small molecule to be converted into a degrader,
empowering RNA binders that are not potent enough to exert a phenotypic
effect on their own. PINAD raises the possibility of targeting and
destroying any disease-related RNA species, which can greatly expand
the space of druggable targets and diseases