Structure–Activity 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, n = 11) CpB/C neurons respond to a 1 s pulse of 1-octen-3-ol with increased activity, while preparations including VU0183254 (red squares)(n = 11) do not respond as strongly (*, p<0.05; **, p<0.005).</p

VU0183254 reduces OR-mediated currents.

<p><b>A</b>–<b>B</b>. Whole-cell patch clamp recordings of odorant- and VUAA1-induced currents in AgOr-expressing cells (n = 3). <b>A.</b> VU0183254 (100 µM) decreased responses to delta-undecalactone (1 µM) and VUAA1 (100 µM) in HEK cells expressing AgOrco+AgOr48. <b>B.</b> Responses to eugenol (1 µM) and VUAA1 (100 µM) in AgOrco+AgOr65 cells were reduced by VU0183254 (100 µM). <b>C</b>–<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 µM) currents in the presence of 100 µM VU0183254. <b>E.</b> Capsaicin (10 µM) currents in HEK cells expressing rat TRPVI were not reduced with by 100 µM VU0183254. Holding potential for all figures is −60 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+/−SEM, n = 4.</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 (n = 8). 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 CO₂ 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 (n = 8). All spikes were distinguished and quantified using AutoSpike software (Syntech).</p

Discovery of Protein–Protein 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 protein–protein 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 protein–protein 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 structure–activity 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 Protein–Protein 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 protein–protein 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 protein–protein 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 RPA70N–ligand 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 RPA’s 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 RPA–protein 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 protein–protein 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 structure–activity 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 RAS–GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase−extracellular regulated kinase (MAPK–ERK) pathway, resulting in a decrease in pERK1/2^T202/Y204 protein levels at higher compound concentrations