Set of 94 differentially up- or down-regulated genes identified via RNA-sequencing with opposite fold changes in MDA-MB-231 and T47D cells chronically treated with SH-4-54 relative to respective wild-type cells.

<p>Genes containing putative STAT3 or STAT5 binding elements in their promoter region are highlighted <b>in bold</b>. As <i>MYC</i> is a known STAT3/5 target gene, differentially expressed genes regulated by MYC are also indicated in bold. Genes validated by qPCR are further highlighted in grey.</p

Primers used for relative qPCR to validate RNA-sequencing results.

<p>Melt peaks listed were obtained on a BioRad CFX Connect Real-Time System and may vary by -/+ 1°C depending on the thermocycler.</p

A visual summary of differential gene expression patterns derived from RNA-sequencing data.

<p>Scatter plots illustrate overall gene expression similarities and differences between (A) wild-type (WT) MDA-MB-231 (MDA) cells and MDA clone #2, and (B) WT T47D cells and T47D clone #1. Volcano plots highlight genes that were differentially expressed between (C) WT MDA cells and MDA clone #2, and (D) WT T47D cells and T47D clone #1. Density plots illustrate expression level distribution, with non-overlapping segments representing differential gene expression between (E) WT MDA cells and MDA clone #2, and (F) WT T47D cells and T47D clone #1. Heatmaps illustrate the level of gene expression [in log10(FPKM+1)] for genes that were differentially expressed between (G) WT MDA cells and MDA clone #2, and (H) WT T47D cells and T47D clone #1. FPKM: fragments per kilobase of transcript per million mapped reads. (I) Linear regression analysis of qPCR results compared with RNA-sequencing results. All pairwise comparisons and a wide range of fold-changes are represented in the analysis. Regression revealed high concordance between the two methods.</p

11 genes selected for graphical representation of relative qPCR fold-changes indicate significant differences, which confirmed the status of 18 total DEGs identified by RNA-sequencing (refer to Table 3).

<p>For each group, data represents the mean of 3 independent biological replicates, each analyzed in duplicate, with error bars indicating the SEM calculated using the 2^-[Δ][Δ]Ct method. Data represent the mean of three independent experiments (±SEM) calculated relative to appropriate controls. A star (*) denotes statistically significant differences determined using a t-test (p<0.05).</p

Subset of select genes reported by others to be regulated by STAT3 or STAT5 [16,20,48–50].

<p>These genes were identified by RNA-sequencing to be differentially expressed in MDA-MB-231 and T47D SH-4-54-resistant clones relative to wild-type (untreated) cells.</p

Phosphopeptide Selective Coordination Complexes as Promising Src Homology 2 Domain Mimetics

Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein–protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein–protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (<i>K</i>_a ∼ 10⁷ M^–1) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines

Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies

To facilitate the discovery of clinically useful Stat3 inhibitors, computational analysis of the binding to Stat3 of the existing Stat3 dimerization disruptors and quantitative structure−activity relationships (QSAR) were pursued, by which a pharmacophore model was derived for predicting optimized Stat3 dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good affinities (<i>K</i>_D, 0.8−12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity <i>in vitro</i> and intracellular phosphorylation at 20−60 μM. Furthermore, agents selectively suppressed viability of human prostate, breast and pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor aberrant Stat3 activity. Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors

Small Molecule STAT5-SH2 Domain Inhibitors Exhibit Potent Antileukemia Activity

A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an <i>in vitro</i> screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (<i>K</i>_i < 5 μM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, <b>BP-1-107</b>, <b>BP-1-108</b>, <b>SF-1-087</b>, and <b>SF-1-088</b>. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC₅₀’s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes <i>cyclin D1</i>, <i>cyclin D2</i>, <i>C-MYC</i>, and <i>MCL-1</i>. Moreover, lead agent <b>BP-1-108</b> showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date

Identification of Bidentate Salicylic Acid Inhibitors of PTP1B

PTP1B is a master regulator in the insulin and leptin metabolic pathways. Hyper-activated PTP1B results in insulin resistance and is viewed as a key factor in the onset of type II diabetes and obesity. Moreover, inhibition of PTP1B expression in cancer cells dramatically inhibits cell growth <i>in vitro</i> and <i>in vivo</i>. Herein, we report the computationally guided optimization of a salicylic acid-based PTP1B inhibitor <b>6</b>, identifying new and more potent bidentate PTP1B inhibitors, such as <b>20h</b>, which exhibited a > 4-fold improvement in activity. In CHO-IR cells, <b>20f</b>, <b>20h</b>, and <b>20j</b> suppressed PTP1B activity and restored insulin receptor phosphorylation levels. Notably, <b>20f</b>, which displayed a 5-fold selectivity for PTP1B over the closely related PTPσ protein, showed no inhibition of PTP-LAR, PRL2 A/S, MKPX, or papain. Finally, <b>20i</b> and <b>20j</b> displayed nanomolar inhibition of PTPσ, representing interesting lead compounds for further investigation

Nanomolar-Potency Small Molecule Inhibitor of STAT5 Protein

We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound <b>13a</b>, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5–SH2 domain complexation events <i>in vitro</i>, silences activated STAT5 in leukemic cells, as well as STAT5′s downstream transcriptional targets, including <i>MYC</i> and <i>MCL1</i>, and, as a result, leads to apoptosis. We believe <b>13a</b> represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials