Target Deconvolution of a Multikinase Inhibitor with Antimetastatic Properties Identifies TAOK3 as a Key Contributor to a Cancer Stem Cell-Like Phenotype.

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell-like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine-based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Identification of novel small molecule Beclin 1 mimetics activating autophagy.

Anti-apoptotic proteins Bcl-2 and Bcl-xL could block autophagy by binding to Beclin 1 protein, an essential inducer of autophagy. Compounds mimicking Beclin 1 might be able to disrupt Bcl-xL/2-Beclin 1 interaction, free out Beclin 1, and thus trigger autophagy. In order to identify small molecule Beclin 1 mimetics, a fluorescence polarization-based high-throughput screening of 50,316 compounds was carried out with a Z' score of 0.82 ± 0.05, and an outcome of 58 hits. After the structure analysis, three acridine analogues were unveiled and confirmed using the fluorescence polarization assay and the surface plasmon resonance assay. Moreover, a set of 17 additional acridine analogues was prepared and tested. Compound 7 showed selectivity for Bcl-xL (KD = 6.5 µM) over Bcl-2 (KD = 160 µM) protein, and potent cytotoxicity (nanomolar scale) in PC-3, PC-3a and DU145 prostate cancer cells. Furthermore, induction of autophagy was also demonstrated in PC-3 and PC-3a cells treated with some acridine compounds by LC3 conversion immunoblotting and LC3 fluorescence microscopy. These Beclin 1 mimetics will be invaluable tools for developing novel autophagy inducers, better understanding the roles of autophagy in cancer, and will contribute to cancer therapy.

Structural and Functional Evaluation of Clinically Relevant Inhibitors of Steroidogenic Cytochrome P450 17A1.

Human steroidogenic cytochrome P450 17A1 (CYP17A1) is a bifunctional enzyme that performs both hydroxylation and lyase reactions, with the latter required to generate androgens that fuel prostate cancer proliferation. The steroid abiraterone, the active form of the only CYP17A1 inhibitor approved by the Food and Drug Administration, binds the catalytic heme iron, nonselectively impeding both reactions and ultimately causing undesirable corticosteroid imbalance. Some nonsteroidal inhibitors reportedly inhibit the lyase reaction more than the preceding hydroxylase reaction, which would be clinically advantageous, but the mechanism is not understood. Thus, the nonsteroidal inhibitors seviteronel and orteronel and the steroidal inhibitors abiraterone and galeterone were compared with respect to their binding modes and hydroxylase versus lyase inhibition. Binding studies and X-ray structures of CYP17A1 with nonsteroidal inhibitors reveal coordination to the heme iron like the steroidal inhibitors. (S)-seviteronel binds similarly to both observed CYP17A1 conformations. However, (S)-orteronel and (R)-orteronel bind to distinct CYP17A1 conformations that differ in a region implicated in ligand entry/exit and the presence of a peripheral ligand. To reconcile these binding modes with enzyme function, side-by-side enzymatic analysis was undertaken and revealed that neither the nonsteroidal seviteronel nor the (S)-orteronel inhibitors demonstrated significant lyase selectivity, but the less potent (R)-orteronel was 8- to 11-fold selective for lyase inhibition. While active-site iron coordination is consistent with competitive inhibition, conformational selection for binding of some inhibitors and the differential presence of a peripheral ligand molecule suggest the possibility of CYP17A1 functional modulation by features outside the active site.

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes.

The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [(14)C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs.

Identification and validation of novel small molecule disruptors of HuR-mRNA interaction.

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR-ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ∼6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR-ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.

Cognitive sequelae of subthalamic nucleus deep brain stimulation in Parkinson's disease: a meta-analysis.

BACKGROUND: Deep brain stimulation of the subthalamic nucleus (STN DBS) is an increasingly common treatment for Parkinson's disease. Qualitative reviews have concluded that diminished verbal fluency is common after STN DBS, but that changes in global cognitive abilities, attention, executive functions, and memory are only inconsistently observed and, when present, often nominal or transient. We did a quantitative meta-analysis to improve understanding of the variability and clinical significance of cognitive dysfunction after STN DBS. METHODS: We searched MedLine, PsycLIT, and ISI Web of Science electronic databases for articles published between 1990 and 2006, and extracted information about number of patients, exclusion criteria, confirmation of target by microelectrode recording, verification of electrode placement via radiographic means, stimulation parameters, assessment time points, assessment measures, whether patients were on levodopa or dopaminomimetics, and summary statistics needed for computation of effect sizes. We used the random-effects meta-analytical model to assess continuous outcomes before and after STN DBS. FINDINGS: Of 40 neuropsychological studies identified, 28 cohort studies (including 612 patients) were eligible for inclusion in the meta-analysis. After adjusting for heterogeneity of variance in study effect sizes, the random effects meta-analysis revealed significant, albeit small, declines in executive functions and verbal learning and memory. Moderate declines were only reported in semantic (Cohen's d 0.73) and phonemic verbal fluency (0.51). Changes in verbal fluency were not related to patient age, disease duration, stimulation parameters, or change in dopaminomimetic dose after surgery. INTERPRETATION: STN DBS, in selected patients, seems relatively safe from a cognitive standpoint. However, difficulty in identification of factors underlying changes in verbal fluency draws attention to the need for uniform and detailed reporting of patient selection, demographic, disease, treatment, surgical, stimulation, and clinical outcome parameters.