Cytotoxic and Antifungal Staurosporine Derivatives from Marine-Derived Actinomycete Streptomyces sp. ZS-A121.

A novel staurosporine derivate, streptomholyrine A (1), along with 6 known compounds were identified from the rice-based solid fermentation of marine-derived Streptomyces sp. ZS-A121. The planar structure and absolute configuration of streptomholyrine A were elucidated using a combination of 1D, 2D NMR, HRESIMS data analysis, chemical transformation, ECD and NMR calculations. Screening of all these compounds revealed their cytotoxic activity against HCT-116 cell lines, with IC50 values ranging from 0.012 to 11.67 µM, except for the known 1H-indole-3-hydroxyacetyl, which showed no inhibition activity. Furthermore, streptomholyrine A, along with two known staurosporine derivatives, k252d and staurosporine, exhibited activities against Candida albicans, with MICs of 12.5, 25.0 and 50.0 µg/ml, respectively.

Matrix Thermal Shift Assay for Fast Construction of Multidimensional Ligand-Target Space.

Existing thermal shift-based mass spectrometry approaches are able to identify target proteins without chemical modification of the ligand, but they are suffering from complicated workflows with limited throughput. Herein, we present a new thermal shift-based method, termed matrix thermal shift assay (mTSA), for fast deconvolution of ligand-binding targets and binding affinities at the proteome level. In mTSA, a sample matrix, treated horizontally with five different compound concentrations and vertically with five technical replicates of each condition, was denatured at a single temperature to induce protein precipitation, and then, data-independent acquisition was employed for quick protein quantification. Compared with previous thermal shift assays, the analysis throughput of mTSA was significantly improved, but the costs as well as efforts were reduced. More importantly, the matrix experiment design allowed simultaneous computation of the statistical significance and fitting of the dose-response profiles, which can be combined to enable a more accurate identification of target proteins, as well as reporting binding affinities between the ligand and individual targets. Using a pan-specific kinase inhibitor, staurosporine, we demonstrated a 36% improvement in screening sensitivity over the traditional thermal proteome profiling (TPP) and a comparable sensitivity with a latest two-dimensional TPP. Finally, mTSA was successfully applied to delineate the target landscape of perfluorooctanesulfonic acid (PFOS), a persistent organic pollutant that is hard to perform modification on, and revealed several potential targets that might account for the toxicities of PFOS.

Inhibitor binding to mutants of protein kinase A with GGGxxG and GxGxxA glycine-rich loop motifs.

The conserved GxGxxG motif of protein kinases forms a beta turn at the tip of the flexible glycine-rich loop and creates much of the ATP pocket binding surface. Notable exceptions to this sequence include GGGxxG in ABL kinase and GxGxxA in protein kinase C isoforms. We constructed the corresponding mutants of PKA, T51G, and G55A, and tested quinazoline inhibitors that were designed to bind via glycine-rich loop interactions, testing also staurosporine for comparison. The quinazoline inhibitors have significantly reduced binding strengths in both mutants. In striking contrast to these results, the binding of the "pan-kinome" inhibitor staurosporine is strengthened in the mutants. Surface plasmon resonance (SPR) shows that the tightened binding of staurosporine arises from increased kon rates, changes not offset by more moderately increased koff rates. The SPR results fit best to a two step binding process for staurosporine in wild type PKA, but not the mutants.

Choosing Antifungals for the Midostaurin-Treated Patient: Does CYP3A4 Outweigh Recommendations? A Brief Insight from Real Life.

INTRODUCTION: Patients treated with midostaurin and chemotherapy are at risk of invasive fungal disease. Prophylactic posaconazole is recommended for these patients, but posaconazole strongly inhibits the CYP3A4 isozyme that metabolizes midostaurin. Posaconazole therefore introduces a risk of patient's overexposure to midostaurin. METHODS: Blood samples were obtained from 4 patients treated with midostaurin for newly diagnosed FLT3-mutAML. Patients had received a concomitant treatment with posaconazole, isavuconazole, or micafungin, respectively. All blood samples were drawn before daily dose administration of midostaurin. RESULTS: Posaconazole caused a ≥8-fold increase of midostaurin plasma levels at through, which was accompanied by a decreased plasma exposure to O-demethylated or hydroxylated midostaurin metabolites. We also show that hematologists react to risk perception by replacing posaco-nazole with antifungals like micafungin or isavuconazole, which lack a strong inhibition of CYP3A4 and fail to modify midostaurin pharmacokinetics but are not formally recommended in these settings. DISCUSSION: In real-life scenarios, concerns about CYP3A4 inhibition may outweigh compliance with recommendations. Large studies are needed to survey the risk:benefit of hematologist's decision to replace posaconazole with other antifungals.

Nonparametric Analysis of Thermal Proteome Profiles Reveals Novel Drug-binding Proteins.

Detecting the targets of drugs and other molecules in intact cellular contexts is a major objective in drug discovery and in biology more broadly. Thermal proteome profiling (TPP) pursues this aim at proteome-wide scale by inferring target engagement from its effects on temperature-dependent protein denaturation. However, a key challenge of TPP is the statistical analysis of the measured melting curves with controlled false discovery rates at high proteome coverage and detection power. We present nonparametric analysis of response curves (NPARC), a statistical method for TPP based on functional data analysis and nonlinear regression. We evaluate NPARC on five independent TPP data sets and observe that it is able to detect subtle changes in any region of the melting curves, reliably detects the known targets, and outperforms a melting point-centric, single-parameter fitting approach in terms of specificity and sensitivity. NPARC can be combined with established analysis of variance (ANOVA) statistics and enables flexible, factorial experimental designs and replication levels. An open source software implementation of NPARC is provided.

Microparticle-Assisted Precipitation Screening Method for Robust Drug Target Identification.

While thermal proteome profiling (TPP) shines in the field of drug target screening by analyzing the soluble fraction of the proteome samples treated at high temperature, the counterpart, the insoluble precipitate, has been overlooked for a long time. The analysis of the precipitate is hampered by the inefficient sample processing procedure. Herein, we propose a novel method, termed microparticle-assisted precipitation screening (MAPS), for drug target identification. The MAPS method exploits the principle that drug-bound proteins will be more resistant to thermal unfolding similar to the classic TPP method, but the process of protein precipitation is assisted by microparticles. Upon heating, proteins unfold and aggregate on the surface of the microparticles. The introduction of a microparticle simplifies the whole sample preparation workflow. The proteins that precipitate on the microparticles are subjected to washing, alkylation, and digestion. The whole sample preparation is processed conveniently on the surface of the microparticles without any transfer. With the assistance of microparticles, sample loss is minimized. The MAPS method is compatible with minute amounts of initial proteins. MAPS was applied to screen the targets of several well-studied drugs and the known target proteins were successfully identified with high confidence and specificity. To investigate the specificity of the method, MAPS was applied to screen the targets of the pan-kinase inhibitor, staurosporine, and 32 protein kinases (specificity of 80%) were identified using only 20 µg of initial proteins of each sample. MAPS is an unbiased robust method for drug target screening, filling the vacancy of stability-based target screening using a precipitate.

Real-Time NMR Spectroscopy for Studying Metabolism.

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in-cell NMR spectroscopy experiments. We are able to monitor real-time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer-based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.

Phosphoregulation of the intracellular termini of K+-Cl- cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K+-Cl- cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

Human neural stem cell-derived neuron/astrocyte co-cultures respond to La Crosse virus infection with proinflammatory cytokines and chemokines.

BACKGROUND: La Crosse virus (LACV) causes pediatric encephalitis in the USA. LACV induces severe inflammation in the central nervous system, but the recruitment of inflammatory cells is poorly understood. A deeper understanding of LACV-induced neural pathology is needed in order to develop treatment options. However, there is a severe limitation of relevant human neuronal cell models of LACV infection. METHODS: We utilized human neural stem cell (hNSC)-derived neuron/astrocyte co-cultures to study LACV infection in disease-relevant primary cells. hNSCs were differentiated into neurons and astrocytes and infected with LACV. To characterize susceptibility and responses to infection, we measured viral titers and levels of viral RNA, performed immunofluorescence analysis to determine the cell types infected, performed apoptosis and cytotoxicity assays, and evaluated cellular responses to infection using qRT-PCR and Bioplex assays. RESULTS: hNSC-derived neuron/astrocyte co-cultures were susceptible to LACV infection and displayed apoptotic responses as reported in previous in vitro and in vivo studies. Neurons and astrocytes are both targets of LACV infection, with neurons becoming the predominant target later in infection possibly due to astrocytic responses to IFN. Additionally, neuron/astrocyte co-cultures responded to LACV infection with strong proinflammatory cytokine, chemokine, as well as MMP-2, MMP-7, and TIMP-1 responses. CONCLUSIONS: hNSC-derived neuron/astrocyte co-cultures reproduce key aspects of LACV infection in humans and mice and are useful models to study encephalitic viruses. Specifically, we show astrocytes to be susceptible to LACV infection and that neurons and astrocytes are important drivers of the inflammatory responses seen in LACV infection through the production of proinflammatory cytokines and chemokines.

Simultaneous Physiologically Based Pharmacokinetic (PBPK) Modeling of Parent and Active Metabolites to Investigate Complex CYP3A4 Drug-Drug Interaction Potential: A Case Example of Midostaurin.

Midostaurin (PKC412) is being investigated for the treatment of acute myeloid leukemia (AML) and advanced systemic mastocytosis (advSM). It is extensively metabolized by CYP3A4 to form two major active metabolites, CGP52421 and CGP62221. In vitro and clinical drug-drug interaction (DDI) studies indicated that midostaurin and its metabolites are substrates, reversible and time-dependent inhibitors, and inducers of CYP3A4. A simultaneous pharmacokinetic model of parent and active metabolites was initially developed by incorporating data from in vitro, preclinical, and clinical pharmacokinetic studies in healthy volunteers and in patients with AML or advSM. The model reasonably predicted changes in midostaurin exposure after single-dose administration with ketoconazole (a 5.8-fold predicted versus 6.1-fold observed increase) and rifampicin (90% predicted versus 94% observed reduction) as well as changes in midazolam exposure (1.0 predicted versus 1.2 observed ratio) after daily dosing of midostaurin for 4 days. The qualified model was then applied to predict the DDI effect with other CYP3A4 inhibitors or inducers and the DDI potential with midazolam under steady-state conditions. The simulated midazolam area under the curve ratio of 0.54 and an accompanying observed 1.9-fold increase in the CYP3A4 activity of biomarker 4ß-hydroxycholesterol indicated a weak-to-moderate CYP3A4 induction by midostaurin and its metabolites at steady state in patients with advSM. In conclusion, a simultaneous parent-and-active-metabolite modeling approach allowed predictions under steady-state conditions that were not possible to achieve in healthy subjects. Furthermore, endogenous biomarker data enabled evaluation of the net effect of midostaurin and its metabolites on CYP3A4 activity at steady state and increased confidence in DDI predictions.

Integrated Exploitation of the Structural Diversity Space of Chemotherapy Drugs to Selectively Inhibit HER2 T798M Mutant in Lung Cancer.

An acquired T798M gatekeeper mutation in human epidermal growth factor receptor 2 (HER2) kinase can cause drug resistance to anti-HER2 chemotherapy drugs in lung cancer. Previously, the reversible pan-kinase inhibitor staurosporine has been found to selectively inhibit the HER2 T798M mutant over wild-type kinase, suggesting that the staurosporine scaffold is potentially to develop mutant-selective inhibitors. Here, we systematically evaluated the chemical space of staurosporine scaffold-based compounds in response to HER2 T798M mutation at structural, energetic and molecular levels by using an integrated analysis strategy. With this strategy, we were able to identify several novel wild-type sparing inhibitors with high or moderate selectivity, which are comparable to or even better than that of the parent compound staurosporine. Molecular modeling and structural analysis revealed that noncovalent contacts can form between the side chain of mutated residue Met798 and selective inhibitor ligands, which may improve the favorable interaction energy between the kinase and inhibitor and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding.

Activation of HIV-1 expression in latently infected CD4+ T cells by the small molecule PKC412.

BACKGROUND: HIV-1 latency is a major obstacle for HIV-1 eradication. Extensive efforts are being directed toward the reactivation of latent HIV reservoirs with the aim of eliminating latently infected cells via the host immune system and/or virus-mediated cell lysis. RESULTS: We screened over 1,500 small molecules and kinase inhibitors and found that a small molecule, PKC412 (midostaurin, a broad-spectrum kinase inhibitor), can stimulate viral transcription and expression from the HIV-1 latently infected ACH2 cell line and primary resting CD4+ T cells. PKC412 reactivated HIV-1 expression in ACH2 cells in a dose- and time-dependent manner. Our results also suggest that the nuclear factor κB (NF-κB) signaling could be one of cellular pathways activated during PKC412-mediated activation of latent HIV-1 expression. Additionally, combining PKC412 with the HDAC inhibitor vorinostat (VOR) had an additive effect on HIV-1 reactivation in both ACH2 cells and infected resting CD4+ T cells. CONCLUSIONS: These studies provide evidence that PKC412 is a new compound with the potential for optimization as a latency-reactivator to eradicate HIV-1 infection.

The structure of a dual-specificity tyrosine phosphorylation-regulated kinase 1A-PKC412 complex reveals disulfide-bridge formation with the anomalous catalytic loop HRD(HCD) cysteine.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase associated with neuronal development and brain physiology. The DYRK kinases are very unusual with respect to the sequence of the catalytic loop, in which the otherwise highly conserved arginine of the HRD motif is replaced by a cysteine. This replacement, along with the proximity of a potential disulfide-bridge partner from the activation segment, implies a potential for redox control of DYRK family activities. Here, the crystal structure of DYRK1A bound to PKC412 is reported, showing the formation of the disulfide bridge and associated conformational changes of the activation loop. The DYRK kinases represent emerging drug targets for several neurological diseases as well as cancer. The observation of distinct activation states may impact strategies for drug targeting. In addition, the characterization of PKC412 binding offers new insights for DYRK inhibitor discovery.

Proteome-wide discovery of unknown ATP-binding proteins and kinase inhibitor target proteins using an ATP probe.

ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich other ATP-binding proteins. However, a robust method to identify ATP-binding proteins with systematic elimination of nonspecific binding proteins has yet to be established. Here, we describe an ATP competition assay that permitted establishment of a rigorous ATP-binding protein list with virtual elimination of nonspecific proteins. A total of 539 ATP-binding protein candidates were identified, including 178 novel candidates. In informatics analysis, ribosomal proteins were overrepresented in the list of novel candidates. We also found multiple ATP-competitive sites for several kinases, including epidermal growth factor receptor, serine/threonine-protein kinase PRP4 homologue, cyclin-dependent kinase 12, eukaryotic elongation factor 2 kinase, ribosomal protein S6 kinase alpha-1, and SRSF protein kinase 1. Using our cataloged ATP-binding protein list, a selectivity profiling method that covers the kinome and ATPome was established to identify off-target binding sites of ATP-competitive kinase inhibitors, staurosporine and crizotinib.

Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors.

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Applying conformational selection theory to improve crossdocking efficiency in 3-phosphoinositide dependent protein kinase-1.

The emerging picture of biomolecular recognition is that of conformational selection followed by induced-fit. Conformational selection theory states that binding partners exist in various conformations in solution, with binding involving a "selection" between complementary conformers. In this study, we devise a docking protocol that mimics conformational selection in protein-ligand binding and demonstrate that it significantly enhances crossdocking accuracy over Glide's flexible docking protocol, which is widely used in the pharmaceutical industry. Our protocol uses a pregenerated conformational ensemble to simulate ligand flexibility. The ensemble was generated by thorough conformational sampling coupled with conformer minimization. The generated conformers were then rigidly docked in the active site of the protein along with a postdocking minimization step that allows limited induced fit effects to be modeled for the ligand. We illustrate the improved performance of our protocol through crossdocking of 31 ligands to cocomplexed proteins of the kinase 3-phosphoinositide dependent protein kinase-1 extracted from the crystal structures 1H1W (ATP bound), 1OKY (staurosporine bound) and 3QD0 (bound to a potent inhibitor). Consistent with conformational selection theory, the performance of our protocol was the best for crossdocking to the cognate protein bound to the natural ligand, ATP.

Thermostability-assisted limited proteolysis-coupled mass spectrometry for capturing drug target proteins and sites.

BACKGROUND: Identifying drug-binding targets and their corresponding sites is crucial for drug discovery and mechanism studies. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a sophisticated method used for the detection of compound and protein interactions. However, in some cases, LiP-MS cannot identify the target proteins due to the small structure changes or the lack of enrichment of low-abundant protein. To overcome this drawback, we developed a thermostability-assisted limited proteolysis-coupled mass spectrometry (TALiP-MS) approach for efficient drug target discovery. RESULTS: We proved that the novel strategy, TALiP-MS, could efficiently identify target proteins of various ligands, including cyclosporin A (a calcineurin inhibitor), geldanamycin (an HSP90 inhibitor), and staurosporine (a kinase inhibitor), with accurately recognizing drug-binding domains. The TALiP protocol increased the number of target peptides detected in LiP-MS experiments by 2- to 8-fold. Meanwhile, the TALiP-MS approach can not only identify both ligand-binding stability and destabilization proteins but also shows high complementarity with the thermal proteome profiling (TPP) and machine learning-based limited proteolysis (LiP-Quant) methods. The developed TALiP-MS approach was applied to identify the target proteins of celastrol (CEL), a natural product known for its strong antioxidant and anti-cancer angiogenesis effect. Among them, four proteins, MTHFD1, UBA1, ACLY, and SND1 were further validated for their strong affinity to CEL by using cellular thermal shift assay. Additionally, the destabilized proteins induced by CEL such as TAGLN2 and CFL1 were also validated. SIGNIFICANCE: Collectively, these findings underscore the efficacy of the TALiP-MS method for identifying drug targets, elucidating binding sites, and even detecting drug-induced conformational changes in target proteins in complex proteomes.

Effect of Mir-4270 Inhibitor and Mimic on Viability and Stemness in Gastric Cancer Stem-Like Cells Derived from MKN-45 Cell Line.

Background: MicroRNAs (miRNAs) are significant regulatory factors in stem cell proliferation, and change in miRNA expression influences the cancer stem cell viability and gene expression. Herein, we evaluated the effect of the hsa-miR-4270 inhibitor and its mimic on the expression of stem cell markers in gastric cancer (GC) stem-like cells. Methods: GC stem-like cells were isolated from the MKN-45 cell line by a non-adherent surface system. The cells were confirmed by differentiation assays using dexamethasone and insulin as adipogenesis-inducing agents and also Staurosporine as a neural-inducing agent. Isolated GC stem-like cells were treated with different concentrations (0, 15, 20, 25, 30, 40, 50, and 60 nM) of hsa-miR-4270 inhibitor and its mimic. The quantity of cell viability was determined by trypan blue method. Transcription of the stem cell marker genes, including CD44, OCT3/4, SOX2, Nanog, and KLF4, was evaluated by real-time RT-PCR. Results: The results showed that GC stem-like cells were differentiated into both adipose cells using dexamethasone and insulin and neural cells by Staurosporine. Treatment of GC stem-like cells with hsa-miR-4270 inhibitor decreased cell viability and downregulated OCT3/4, CD44, and Nanog to 86%, 79%, and 91% respectively. Also, SOX2 and KLF4 were overexpressed to 8.1- and 1.94-folds, respectively. However, hsa-miR-4270 mimic had opposite effects on the cell viability and gene expression of the stem cell markers. Conclusion: The effect of hsa-miR-4270 inhibitor and its mimic on the expression of the stem cell markers in GCSCs indicated that hsa-miR-4270 stimulates the stemness property of GCSCs, likely through stimulating the development of gastric stem cells.

A Novel Role of Secretory Cytosolic Tryparedoxin Peroxidase in Delaying Apoptosis of Leishmania-Infected Macrophages.

The cytosolic tryparedoxin peroxidase (cTXNPx) of Leishmania donovani is a defensive enzyme. Apart from the nonsecretory form, the cTXNPx is released in the spent media of Leishmania cultures and also in the host cell cytosol. The secretory form of the enzyme from the parasite interacts with multiple proteins in the host cell cytosol, the apoptosis-inducing factor (AIF) being one of them. Immunoprecipitation with anti-cTXNPx and anti-AIF antibodies suggests a strong interaction between AIF and cTXNPx. Consequent to parasite invasion, the migration of AIF to the nucleus to precipitate apoptosis is inhibited in the presence of recombinant cTXNPx expressed in the host cell. This inhibition of AIF movement results in lesser host cell death, giving an advantage to the parasite for continued survival. Staurosporine-induced AIF migration to the nucleus was also inhibited in the presence of recombinant cTXNPx in the host cell. Therefore, this study demonstrates the ability of a Leishmania parasite enzyme, cTXNPx, to interfere with the migration of the host AIF protein, providing a survival advantage to the Leishmania parasite.