Research Items (18)
- Aug 2018
Obverse Cover: The cover image, by Steven V. Molinski et al., is based on the Research Article Comprehensive mapping of cystic fibrosis mutations to CFTR protein identifies mutation clusters and molecular docking predicts corrector binding site, DOI: 10.1002/prot.25496.
- Mar 2018
Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, of which over 2000 have been reported to date. Mutations have yet to be analyzed in aggregate to assess their distribution across the tertiary structure of the CFTR protein, an approach that could provide valuable insights into the structure‐function relationship of CFTR. In addition, the binding site of Class I correctors (VX‐809, VX‐661, C18) is not well understood. In this study, exonic CFTR mutations and mutant allele frequencies described in three curated databases (ABCMdb, CFTR1 and CFTR2, comprising >130,000 data points) were mapped to two different structural models: a homology model of full‐length CFTR protein in the open‐channel state, and a cryo‐electron microscopy core‐structure of CFTR in the closed‐channel state. Accordingly, residue positions of six high‐frequency mutant CFTR alleles were found to spatially co‐localize in CFTR protein, and a significant cluster was identified at the NBD1:ICL4 interdomain interface. In addition, immunoblotting confirmed the approximate binding site of Class I correctors, demonstrating that these small molecules act via a similar mechanism in vitro, and in silico molecular docking generated binding poses for their complex with the cryo‐electron microscopy structure to suggest the putative corrector binding site is a multi‐domain pocket near residues F374‐L375. These results confirm the significance of interdomain interfaces as susceptible to disruptive mutation, and identify a putative corrector binding site. The structural pharmacogenomics approach of mapping mutation databases to protein models shows promise for facilitating drug discovery and personalized medicine for monogenetic diseases. This article is protected by copyright. All rights reserved.
- Dec 2016
The investigational compound BIA 10-2474, designed as a long-acting and reversible inhibitor of fatty acid amide hydrolase for the treatment of neuropathic pain, led to the death of one participant and hospitalization of five others due to intracranial hemorrhage in a Phase I clinical trial. Putative off-target activities of BIA 10-2474 have been suggested to be major contributing factors to the observed neurotoxicity in humans, motivating our study's proteome-wide screening approach to investigate its polypharmacology. Accordingly, we performed an in silico screen against 80,923 protein structures reported in the Protein Data Bank. The resulting list of 284 unique human interactors was further refined using target-disease association analyses to a subset of proteins previously linked to neurological, intracranial, inflammatory, hemorrhagic or clotting processes and/or diseases. Eleven proteins were identified as potential targets of BIA 10-2474, and the two highest-scoring proteins, Factor VII and thrombin, both essential blood-clotting factors, were predicted to be inhibited by BIA 10-2474 and suggest a plausible mechanism of toxicity. Once this small molecule becomes commercially available, future studies will be conducted to evaluate the predicted inhibitory effect of BIA 10-2474 on blood clot formation specifically in the brain.
- Mar 2016
We explored a selection of analogies used for introducing students to the concept of potential energy wells. Two analogy systems were developed, a spring system to explain simple harmonic motion 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 formatulation 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 carefully designing these tools.
- Jan 2014
Signal transducer and activator of transcription 3 (STAT3) protein is involved in regulatory functions in cell proliferation, differentiation and survival, and is linked to cancer phenotype and tumorigenesis. Towards developing new methodologies for screening STAT3 interactions, the electrochemical method based on the use of redox active protein was proposed. The electrochemical signal, due to the redox (ferrocene)-labeled STAT3 protein immobilized on a gold surface, was modulated due to protein dimerization with the unlabeled STAT3 molecule. The dramatic decrease in current density from 2.7 μA cm(-2) to 0.5 μA cm(-2) was observed following the STAT3-ferrocene-STAT3 dimerization. The electrochemical approach was further extended for screening the potential dimerization inhibitors. Previously published potent salicylic acid derivatives were the most promising candidates for inhibition of STAT3 dimerization in this assay. We expect that other SH2-containing proteins may be monitored by the proposed electrochemical method.
- Sep 2013
The STAT3 gene is abnormally active in glioblastoma and is a critically important mediator of tumour 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 im-portance. Herein, we report a most potent, small molecule, non-phosphorylated STAT3 inhibitor, 31 that displayed potent STAT3 binding affinity (KD = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells, effec-tively suppresses STAT3 phosphorylation and downstream transcriptional targets at low nM concentrations. In vivo studies with 31 in mice orthotopically xenografted with glioma and analyzed by immunohistochemical staining demonstrated that 31 exhibited blood brain barrier permeability, in vivo potency, on target anti-STAT3 activity and potent inhibition of tumour cell proliferation and increased apoptosis. This work demonstrates the clinical efficacy of a STAT3 inhibitor for clinical application in glioblastoma.
A screen of a library of diverse small-molecules against a subset of phosphatases identified 7b and 7c, which potently inhibit TC-PTP, PTPσ and PTP1B with no inhibition of PTP-LAR, PRL2 A/S, MKPX or papain. In CHO-IR cells these inhibitors effectively suppress PTP1B activity restoring the insulin receptor phosphorylation levels.
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 (IC50's <5μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold's history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.
- Aug 2011
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.
- Jan 2011
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 (K(D), 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.
- Dec 2010
Aberrant activation of oncogenic signal transducer and activator of transcription 3 (STAT3) protein signaling pathways has been extensively implicated in human cancers. Given STAT3's 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.
- Nov 2010
Protection/activation strategy is applied to the preparation of the target compounds with high regioselectivity.
- Jun 2010
Guanine poses several problems to the synthetic chemist owing to its polyfunctional nature and poor solubility. Over the past few decades, synthetic guanines have found applications as anti-cancer and anti-viral agents. Coupled with the ever-growing interest in designer PNAs and G-quartets, simple and efficient synthetic routes to novel guanines would be of significant benefit. We herein report that, upon simple protection and/or activation step(s), the guanine precursor 2-amino-6-chloropurine is rendered an excellent substrate for Mitsunobu chemistry, furnishing, after subsequent hydrolytic dechlorination and appropriate deprotection step(s), the desired N9-mono-, N2-mono- or N2,N9-di-substituted guanines in excellent yields (≥80%). Importantly, we demonstrate that N9-functionalization proceeds with very good N9/N7 regioselectivity and with complete inversion of stereochemistry.
- Dec 2009
Signal transducer and activator of transcription protein 3 (STAT3) is a latent cytosolic transcription factor that is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutively activated STAT3 protein activity is routinely observed in human cancers, promoting uncontrolled cell proliferation and suppressing apoptosis. Until relatively recently, inhibition of STAT3 transcriptional activity was achieved indirectly via suppression of upstream kinase activators and extracellular cytokine and (or) growth factor stimuli. However, activated STAT3 forms transcriptionally functional STAT3-STAT3 dimers, providing a valid juncture for targeted downstream molecular inhibition. STAT3's prominent role in cancer has seen a decade of innovative and novel approaches to targeting constitutively active STAT3 protein-protein complexes. This mini-review outlines the progress made towards identifying molecular agents capable of silencing aberrant STAT3 signalling through the disruption of STAT3 complexation events.
In summary, we have developed a library of analogues of the previously reported Stat3 inhibitor S3I-201, the most potent member of which displays more than twice the original potency for Stat3 dimer disruption. Furthermore, the most active analogues SF-1-066 and SF-1-121 have shown impressive in vitro and whole-cell activities; this is possibly a result of the successful occupation of a third, hydrophobic subpocket of the Stat3 SH2 domain. In general, our studies demonstrate that appropriate scaffold extension into this subpocket, which is believed to be occupied by the R1 substituent, resulted in significant increases in potency, thereby validating our approach to enhancing the Stat3 inhibitory activity of S3I-201. Future synthetic studies are currently focused upon developing extended agents with optimized occupation of the R1 subpocket, as well as optimizing the sulfonamide portion of our inhibitors to enhance interactions in the tosylamide-binding subpocket.
The inside cover picture shows the culmination of a rational drug discovery program of Stat3 inhibitors. Computational modeling of the most potent salicylic acid-based therapeutic shows excellent occupation of the Stat3-SH2 domain, thereby inhibiting its homodimerization. For details, see the communication by P. T. Gunning et al. on p. 1959 ff.
- Jul 2009
A facile, efficient and mild synthesis of 2,6,9-tri-substituted purines is presented, starting from commercially available 2-amino-6-chloropurine, which employs sequential N9 then N2 Mitsunobu reactions as key steps. Importantly, our synthetic approach to N2-functionalization of the purine nucleus obviates the harsh conditions required by the traditional nucleophilic aromatic substitution of a 2-halo group with primary amines. Benzylic, allylic, propargylic and aliphatic alcohols all coupled in very good to excellent yields in both Mitsunobu reactions. Significantly, excellent chemoselectivity and N9-regioselectivity were observed for the first coupling, and reactions were complete within 15min at room temperature. Our novel methodology may be readily adapted to furnish N9-mono- or N2,N9-di-functionalized guanine analogues, and the utility of our protocol is further demonstrated by the efficient synthesis of the CDK inhibitor bohemine.