14 research outputs found
StructureâActivity Relationship of a Broad-Spectrum Insect Odorant Receptor Agonist
Agonism of insect odorant receptor (OR) cation channels
may represent
a new strategy for the manipulation of destructive insect olfactory-driven
behaviors. We have explored the chemical space around VUAA1, the first
in class agonist of the obligate OR co-receptor ion channel (Orco),
and describe novel compound analogues with increased potency across
insect taxa. Functional analyses reveal several of these VUAA1 structural
analogues display significantly greater potency as compared to the
activity of the previously described active compounds in mobility-based
behavioral assays on mosquito larvae
VU0183254 antagonizes odor-evoked ORN activity <i>in vivo</i>.
<p><b>A.</b> Representative traces of Cp neuron activity in response to a 1 s pulse of 1-octen-3-ol (dark bar above traces). Vertical lines under each raw trace indicates spike counter for CpB and CpC spikes only. Vehicle-only trace (top) responds strongly to 1-octen-3-ol, while preparations exposed to VU0183254 (bottom) do not respond. <b>B.</b> Quantification of traces as in (A). Spike frequencies were binned every second for 5 seconds prior to 1-octen-3-ol pulse and for 2 seconds afterwards. In the presence of vehicle alone (blue circles, nâ=â11) CpB/C neurons respond to a 1 s pulse of 1-octen-3-ol with increased activity, while preparations including VU0183254 (red squares)(nâ=â11) do not respond as strongly (*, p<0.05; **, p<0.005).</p
VU0183254 reduces OR-mediated currents.
<p><b>A</b>â<b>B</b>. Whole-cell patch clamp recordings of odorant- and VUAA1-induced currents in AgOr-expressing cells (nâ=â3). <b>A.</b> VU0183254 (100 ”M) decreased responses to delta-undecalactone (1 ”M) and VUAA1 (100 ”M) in HEK cells expressing AgOrco+AgOr48. <b>B.</b> Responses to eugenol (1 ”M) and VUAA1 (100 ”M) in AgOrco+AgOr65 cells were reduced by VU0183254 (100 ”M). <b>C</b>â<b>D</b>. Cells expressing Orco from either <i>An. gambiae</i> (<b>C</b>) or <i>Harpegnathos saltator</i> (<b>D</b>) had reduced VUAA1 (100 ”M) currents in the presence of 100 ”M VU0183254. <b>E.</b> Capsaicin (10 ”M) currents in HEK cells expressing rat TRPVI were not reduced with by 100 ”M VU0183254. Holding potential for all figures is â60 mV.</p
VU0183254 is an allosteric antagonist.
<p><b>A.</b> Concentration response curves of AgOrco+AgOr65 expressing HEK cells in the presence of a series of steady concentrations (expressed as logM) of pre-loaded VU0183254 (different colored lines, see inset) followed by increasing concentrations of eugenol as measured using Fluo-4AM and an FDSS6000. <b>B.</b> As in A with VUAA1. <b>C.</b> As in A, with AgOrco+AgOr48 and delta-undecalactone. <b>D.</b> As in C with VUAA1. In all cases results are shown as means+/âSEM, nâ=â4.</p
VU0183254 reduces Orco-mediated activity <i>in vivo.</i>
<p><b>A.</b> Representative traces of Cp neuron activity in response to vehicle (DMSO) or VU0183254 as measured by single-sensillum electrophysiology. Activity was allowed to stabilize for 10 seconds and then recorded for 60 seconds. CpA spikes can be distinguished by their larger amplitude in expansions (right panel). CpB/C spikes (counts below traces) are reduced in the presence of VU0183254. <b>B.</b> Spontaneous CpA spike rates are unaffected by the presence of VU0183254 (nâ=â8). Spikes were counted for each of the first 10 seconds and then averaged across the remaining 10-second intervals. Normal CpA activity is confirmed by CO<sub>2</sub> pulse delivered at the end of the recording period. <b>C.</b> Spontaneous firing rates of CpB/C neurons are reduced by VU0183254 in a dose-dependent manner (nâ=â8). All spikes were distinguished and quantified using AutoSpike software (Syntech).</p
Discovery of ProteinâProtein Interaction Inhibitors of Replication Protein A
Replication
protein A (RPA) is a ssDNA binding protein that is
essential for DNA replication and repair. The initiation of the DNA
damage response by RPA is mediated by proteinâprotein interactions
involving the N-terminal domain of the 70 kDa subunit with partner
proteins. Inhibition of these interactions increases sensitivity toward
DNA damage and replication stress and may therefore be a potential
strategy for cancer drug discovery. Toward this end, we have discovered
two lead series of compounds, derived from hits obtained from a fragment-based
screen, that bind to RPA70N with low micromolar affinity and inhibit
the binding of an ATRIP-derived peptide to RPA. These compounds may
offer a promising starting point for the discovery of clinically useful
RPA inhibitors
Discovery of a Potent Stapled Helix Peptide That Binds to the 70N Domain of Replication Protein A
Stapled helix peptides can serve
as useful tools for inhibiting
proteinâprotein interactions but can be difficult to optimize
for affinity. Here we describe the discovery and optimization of a
stapled helix peptide that binds to the N-terminal domain of the 70
kDa subunit of replication protein A (RPA70N). In addition to applying
traditional optimization strategies, we employed a novel approach
for efficiently designing peptides containing unnatural amino acids.
We discovered hot spots in the target protein using a fragment-based
screen, identified the amino acid that binds to the hot spot, and
selected an unnatural amino acid to incorporate, based on the structureâactivity
relationships of small molecules that bind to this site. The resulting
stapled helix peptide potently and selectively binds to RPA70N, does
not disrupt ssDNA binding, and penetrates cells. This peptide may
serve as a probe to explore the therapeutic potential of RPA70N inhibition
in cancer
Discovery of a Potent Inhibitor of Replication Protein A ProteinâProtein Interactions Using a Fragment-Linking Approach
Replication protein A (RPA), the
major eukaryotic single-stranded
DNA (ssDNA)-binding protein, is involved in nearly all cellular DNA
transactions. The RPA N-terminal domain (RPA70N) is a recruitment
site for proteins involved in DNA-damage response and repair. Selective
inhibition of these proteinâprotein interactions has the potential
to inhibit the DNA-damage response and to sensitize cancer cells to
DNA-damaging agents without affecting other functions of RPA. To discover
a potent, selective inhibitor of the RPA70N proteinâprotein
interactions to test this hypothesis, we used NMR spectroscopy to
identify fragment hits that bind to two adjacent sites in the basic
cleft of RPA70N. High-resolution X-ray crystal structures of RPA70Nâligand
complexes revealed how these fragments bind to RPA and guided the
design of linked compounds that simultaneously occupy both sites.
We have synthesized linked molecules that bind to RPA70N with submicromolar
affinity and minimal disruption of RPAâs interaction with ssDNA
Diphenylpyrazoles as Replication Protein A Inhibitors
Replication Protein A is the primary
eukaryotic ssDNA binding protein
that has a central role in initiating the cellular response to DNA
damage. RPA recruits multiple proteins to sites of DNA damage via
the N-terminal domain of the 70 kDa subunit (RPA70N). Here we describe
the optimization of a diphenylpyrazole carboxylic acid series of inhibitors
of these RPAâprotein interactions. We evaluated substituents
on the aromatic rings as well as the type and geometry of the linkers
used to combine fragments, ultimately leading to submicromolar inhibitors
of RPA70N proteinâprotein interactions
Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling
Deregulated RAS activity,
often the result of mutation, is implicated
in approximately 30% of all human cancers. Despite this statistic,
no clinically successful treatment for RAS-driven tumors has yet been
developed. One approach for modulating RAS activity is to target and
affect the activity of proteins that interact with RAS, such as the
guanine nucleotide exchange factor (GEF) son of sevenless homologue
1 (SOS1). Here, we report on structureâactivity relationships
(SAR) in an indole series of compounds. Using structure-based design,
we systematically explored substitution patterns on the indole nucleus,
the pendant amino acid moiety, and the linker unit that connects these
two fragments. Best-in-class compounds activate the nucleotide exchange
process at submicromolar concentrations in vitro, increase levels
of active RASâGTP in HeLa cells, and elicit signaling changes
in the mitogen-activated protein kinaseâextracellular regulated
kinase (MAPKâERK) pathway, resulting in a decrease in pERK1/2<sup>T202/Y204</sup> protein levels at higher compound concentrations