Carpatamides A–C, Cytotoxic Arylamine Derivatives from a Marine-Derived <i>Streptomyces</i> sp.

Three new acylated arylamine derivatives (<b>1</b>–<b>3</b>), carpatamides A–C, were isolated from a marine-derived <i>Streptomyces</i> sp. based on activity screening against non-small-cell lung cancer (NSCLC). The structures of <b>1</b>–<b>3</b> were established on the basis of comprehensive spectroscopic analyses and chemical methods. Compounds <b>1</b> and <b>3</b> showed moderate cytotoxicity against NSCLC cell lines HCC366, A549, and HCC44 with IC₅₀ values ranging from 2.2 to 8.4 μM

Confirmation screen and dose-response studies.

<p><b>A</b>) Flowchart of high-throughput screening used to identify and validate the hits from the 8000-compound screen. The 42 preliminary hits were chosen based on their RZ score and lack of intrinsic fluorescence. These were further narrowed down to 4 compounds that were chosen based on their ability to inhibit the GTPase activity of Dyn1 in a dose-dependent manner and their commercial availability <b>B)</b> Chemical structures of the 4 compounds that were tested in dose-response studies. <b>C)</b> 11-point dose response curve of the 4 validated hits from the primary screen of 8000 compounds measured using 50 nM Dyn1 and 10 μM GTP (n = 1, measured in triplicates). Data are presented as mean ± SD.</p

Optimization of assay sensitivity to measure GDP production.

<p><b>A)</b> Cartoon depicting the Transcreener GDP fluorescence polarization reaction <b>B)</b> The GDP antibody was titrated to determine its optimal concentrations for 10, 100 and 500 μM GTP. Optimal antibody concentrations are represented by the highlighted points (n = 1, measured in triplicates). <b>C)</b> Standard curve representing the conversion of 0 to 100% GDP from 10 μM GTP. This curve was used to convert the fluorescence polarization data to GDP released (n = 1, measured in triplicates). Data are presented as mean ± SD.</p

Assay validation and application for high-throughput screening of 8000 compounds.

<p><b>A)</b> The assay was validated in a preliminary mock screen that compared reactions containing no enzyme (positive control for inhibition) to uninhibited reactions containing DMSO (negative control for inhibition). <b>B)</b> A quantile-quantile (Q-Q) plot of the pilot screen of 8000 compounds. The compounds are ranked from 0 to 8000 according to their two-point robust Z score. Primary hits were chosen based on a RZ score of greater than 3.</p

Detection of basal GTP hydrolysis by Dyn1.

<p><b>A)</b> Dyn1 was titrated from 0.3 nM to 5000 nM in the presence of 10 μM, 100 μM or 500 μM GTP and GTP hydrolysis was measured as ΔmP after 60 min incubation at room temperature (n = 1, measured in triplicates). <b>B)</b> The GTP hydrolysis by Dyn1 was linear during 60-minute incubations at room temperature (n = 4, each measured in duplicates). <b>C)</b> The GTP binding mutant, Dyn1^S45N, shows no activity at any concentration of GTP (n = 1, measured in triplicates). <b>D)</b> The basal GTPase activity is proportional to Dyn1 concentration from 0 to 1000 nM, indicating no cooperativity under these concentrations (n = 3, each measured in triplicates). Data are presented as mean ± SD.</p

Comparison of commercially available inhibitors with compound 24.

<p><b>A)</b> Dynasore and Dyngo-4a were tested alongside compound 24 in the Transcreener assay (n = 1, measured in triplicates). <b>B)</b> Dose response curves for the inhibitory effects of Dynasore, Dyngo-4a and compound 24 on the lipid nanotube-stimulated GTPase activity of 100 nM Dyn1 assayed in the presence of 300 μM lipid nanotubes and 25 μM GTP and measured using the malachite green assay (n = 3, each measured in triplicates). Data are presented as mean ± SD.</p

Table_2_Small molecule glucagon release inhibitors with activity in human islets.pdf

PurposeType 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet β-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. ResultsHere, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 β-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. ConclusionGiven the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.</p

Table_1_Small molecule glucagon release inhibitors with activity in human islets.pdf

PurposeType 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet β-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. ResultsHere, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 β-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. ConclusionGiven the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.</p

Discovery of Cytochrome P450 4F11 Activated Inhibitors of Stearoyl Coenzyme A Desaturase

Stearoyl-CoA desaturase (SCD) catalyzes the first step in the conversion of saturated fatty acids to unsaturated fatty acids. Unsaturated fatty acids are required for membrane integrity and for cell proliferation. For these reasons, inhibitors of SCD represent potential treatments for cancer. However, systemically active SCD inhibitors result in skin toxicity, which presents an obstacle to their development. We recently described a series of oxalic acid diamides that are converted into active SCD inhibitors within a subset of cancers by CYP4F11-mediated metabolism. Herein, we describe the optimization of the oxalic acid diamides and related <i>N</i>-acyl ureas and an analysis of the structure–activity relationships related to metabolic activation and SCD inhibition

Discovery of Cytochrome P450 4F11 Activated Inhibitors of Stearoyl Coenzyme A Desaturase

Stearoyl-CoA desaturase (SCD) catalyzes the first step in the conversion of saturated fatty acids to unsaturated fatty acids. Unsaturated fatty acids are required for membrane integrity and for cell proliferation. For these reasons, inhibitors of SCD represent potential treatments for cancer. However, systemically active SCD inhibitors result in skin toxicity, which presents an obstacle to their development. We recently described a series of oxalic acid diamides that are converted into active SCD inhibitors within a subset of cancers by CYP4F11-mediated metabolism. Herein, we describe the optimization of the oxalic acid diamides and related <i>N</i>-acyl ureas and an analysis of the structure–activity relationships related to metabolic activation and SCD inhibition