Vijay M. Shahani

Disruption of Transcriptionally Active Stat3 Dimers with Non‐phosphorylated, Salicylic Acid‐Based Small Molecules: Potent in vitro and Tumor Cell Activities

Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that relays signals from receptors in the plasma membrane directly to the nucleus, and is routinely hyperactivated in many human cancers and diseases.[1] Regarded as an oncogene, Stat3 is well-recognized as a master regulator of cellular events that lead to the cancer phenotype, making this protein viable target for molecular therapeutic design.[2] Stat3 inhibitors have included peptides,[3–4] peptidomimetics,[5–9] small molecules[10–14] and metal complexes.[15] Despite significant advances in Stat3 inhibition,[1] truly potent (in vivo), isoform-selective, small molecule Stat3 agents have not been readily forthcoming; this is likely due in part to the challenge of disrupting protein–protein interactions.[16]

Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma

The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood-brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.

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 (KD, 0.8−12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro 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.

Design, synthesis, and in vitro characterization of novel hybrid peptidomimetic inhibitors of STAT3 protein.

Aberrant activation of oncogenic signal transducer and activator of transcription 3 (STAT3) protein signaling pathways has been extensively implicated in human cancers. Given STAT3s prominent dysregulatory role in malignant transformation and tumorigenesis, there has been a significant effort to discover STAT3-specific inhibitors as chemical probes for defining the aberrant STAT3-mediated molecular events that support the malignant phenotype. To identify novel, STAT3-selective inhibitors suitable for interrogating STAT3 signaling in tumor cells, we explored the design of hybrid molecules by conjugating a known STAT3 inhibitory peptidomimetic, ISS610 to the high-affinity STAT3-binding peptide motif derived from the ILR/gp-130. Several hybrid molecules were examined in in vitro biophysical and biochemical studies for inhibitory potency against STAT3. Lead inhibitor 14aa was shown to strongly bind to STAT3 (K(D)=900 nM), disrupt STAT3:phosphopeptide complexes (K(i)=5 μM) and suppress STAT3 activity in in vitro DNA binding activity/electrophoretic mobility shift assay (EMSA). Moreover, lead STAT3 inhibitor 14aa induced a time-dependent inhibition of constitutive STAT3 activation in v-Src transformed mouse fibroblasts (NIH3T3/v-Src), with 80% suppression of constitutively-active STAT3 at 6h following treatment of NIH3T3/v-Src. However, STAT3 activity recovered at 24h after treatment of cells, suggesting potential degradation of the compound. Results further showed a suppression of aberrant STAT3 activity in NIH3T3/v-Src by the treatment with compound 14aa-OH, which is the non-pTyr version of compound 14aa. The effect of compounds 14aa and 14aa-OH are accompanied by a moderate loss of cell viability.

Identification of NAE Inhibitors Exhibiting Potent Activity in Leukemia Cells: Exploring the Structural Determinants of NAE Specificity

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.

A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC50s <5μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffolds history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.

The efficacy of interactive analogical models in the instruction of bond energy curves in undergraduate chemistry

We explored analogies used for introducing students to the concept of potential energy wells. Two analogy systems were developed, a spring system and a novel system consisting of electrostatic spheres. These two, distinct analogies were housed within an interactive tool that allowed students to manipulate the analogous systems and witness changes to potential energy curves in real time. A pre-test/post-test evaluation provided insight into the impact the formulation of an analogy system can have on understanding. Students modified written descriptions to include new details in accordance to the structure-mapping theory of analogies. However, students failed to correct visual descriptions of energy wells. The failure of participants to apply key concepts after using the interactive and animated analogy systems highlights the importance of designing for education.