The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01.

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2-UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.

Intrinsic relative preference profile of pan-kinase inhibitor drug staurosporine towards the clinically occurring gatekeeper mutations in Protein Tyrosine Kinases.

Protein tyrosine kinases (PTKs) have been recognized as the attractive druggable targets of various diseases including cancer. However, many PTKs are clinically observed to establish a gatekeeper mutation in the peripheral hinge section of active site, which plays a primary role in development of acquired drug resistance to kinase inhibitors. The natural product Staurosporine, an ATP-competitive reversible pan-kinase inhibitor, has been found to exhibit wild type-sparing selectivity for some PTK gatekeeper mutants. In this study, totally 23 acquired drug-resistant gatekeeper mutations harbored on 17 PTKs involved in diverse cancers were curated, from which only five amino acid types, namely Thr, Met, Val, Leu and Ile, were observed at both wild-type and mutant residues of these clinically occurring gatekeeper sites. Here, an integrative strategy that combined molecular modeling and kinase assay was described to systematically investigate the relative preference of Staurosporine towards the five gatekeeper amino acid types in real kinase context and in a psendokinase model. A kinase-free, intrinsic relative preference profile of Staurosporine to gatekeeper amino acids was created: (dispreferred) Thr⊳Val⊳Ile⊳Leu⊳Met (preferred). It is found that kinase context has no essential effect on the profile; different kinases and even psendokinase can obtain a consistent conclusion for the preference order. Theoretically, we can use the profile to predict Staurosporine response to any gatekeeper mutation between the five amino acid types in any PTK. Structural and energetic analyses revealed that the multiple-aromatic ring system of Staurosporine can form multiple noncovalent interactions with the weakly polar side chain of Met and can pack tightly or moderately against the nonpolar side chains of Val, Ile and Leu, thus stabilizing the kinase-inhibitor system (ΔU < 0), whereas the polar side chain of Thr may cause unfavorable electronegative and solvent effects with the aromatic electrons of Staurosporine, thus destabilizing the system (ΔU > 0).

Impurity profiling and stability-indicating method development and validation for the estimation of assay and degradation impurities of midostaurin in softgel capsules using HPLC and LC-MS.

Midostaurin (MDS) is used for the treatment of acute myeloid leukemia, myelodysplastic syndrome, and advanced systemic mastocytosis. MDS softgel capsule samples were subjected to stress testing per International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines for impurity profiling study. MDS underwent extensive degradation under stress testing (acid, alkaline, oxidative, photolytic, thermolytic, and hydrolysis conditions) and formed four degradation products (DPs). MDS and its DPs were separated well from one another with good resolution using reserved-phase HPLC using an Inertsil ODS-3V column (250 × 4.6 mm, 5 µm) and a mobile phase of ammonium formate (40 mM) and acetonitrile. The stability-indicating characteristic of the newly developed method was proven for the estimation of MDS assay, and its organic impurities were free from interference. The validated method exhibited excellent linearity, accuracy, precision, specificity, detection limit, and quantitation limit within 25 min run time. Stress testing, robustness, and solution stability were performed to ensure the continuous performance of the developed method. The peak fractions of DPs formed under stress testing were isolated and characterized using LC-MS, 1 H and 13 C NMR, IR, and UV-Vis. The structure of the major DPs was predicted as DP1 based on the spectral data. The proposed method is effectively used for MDS in bulk drug and finished formulations in the pharmaceutical industry.

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.

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.

Systematic response of staurosporine scaffold-based inhibitors to drug-resistant cancer kinase mutations.

Human protein kinases have been established as promising druggable targets in cancer therapy. However, a large number of acquired drug-resistant kinase mutations are observed after first- and second-line kinase inhibitor treatments, largely limiting the application of small-molecule inhibitors in the targeted cancer therapy. Previously, the pan-kinase inhibitor staurosporine and its derivatives have been reported to selectively inhibit gatekeeper mutants over wild-type kinases, suggesting that the staurosporine scaffold is potentially helpful in developing wild-type-sparing inhibitors of drug-resistant kinase mutants. Here, a systematic response profile of 32 staurosporine scaffold-based inhibitors (SSBIs) for 61 ontology-enriched drug-resistant cancer kinase mutations is created using a combination of in silico analysis and in vitro assay, from which it is possible to identify those mutations that have the potential to cause resistance or confer sensitivity to SSBIs. The profile reveals that SSBIs exhibit distinct responses to kinase gatekeeper and nongatekeeper mutations, and SSBIs bearing p7 substituents can considerably influence their response to kinase gatekeeper mutations, particularly for the mutations of the Ile residue, which possesses a Cß methyl group that tends to cause steric clash with bound SSBIs. Nongatekeeper mutations generally have a moderate and unfavorable effect on SSBI activity, as most of them are outside the kinase active site and do not directly contact inhibitor ligands. In addition, it is found that resistance is commonly caused by mutation-induced hindrance effects, whereas sensitivity is primarily conferred by mutation-established additional interactions.

Systematic profiling of staralog response to acquired drug resistant kinase gatekeeper mutations in targeted cancer therapy.

Kinase-targeted therapy has been widely used as a lifesaving strategy for cancer patients. However, many patients treated with targeted cancer drugs are clinically observed to rapidly develop acquired resistance. Kinase gatekeeper mutation is one of the most chief factors contributing to the resistance, which modulates the accessibility of kinase's ATP-binding pocket. Previously, the pan-kinase inhibitor Staurosporine and its analogs (termed as Staralogs) have been reported to exhibit wild-type sparing selectivity for some kinase gatekeeper mutants, such as EGFR T790M, Her2 T798M and cSrc T338M. Here, we describe an integrative approach to systematically profile the molecular response of 15 representative Staralogs to 17 kinase gatekeeper mutations in targeted cancer therapy. With the profile we are able to divide gatekeeper mutations into three classes (i.e. classes I, II and III) and to divide Staralogs into two groups (i.e. groups 1 and 2) using heuristic clustering. The class I and II mutations confer consistent sensitivity and resistance for all Staralogs, respectively, while the class III mutations address divergent effects on different Staralogs. The mutations to Ile residue can generally reduce Staralog affinity by inducing unfavorable steric hindrance, whereas the mutations to Met and Leu residues would improve Staralog affinity by establishing favorable S···π interaction, van der Waals packing and/or hydrophobic contact. The group 1 and 2 Staralogs are primarily determined by carbonyl or hydroxyl substitution state at the position 7 of Staralog core, where points to kinase gatekeeper residue and can thus be directly influenced by gatekeeper mutation.

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.

Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale.

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.

Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application.

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.

ATP-Competitive Inhibitors Midostaurin and Avapritinib Have Distinct Resistance Profiles in Exon 17-Mutant KIT.

KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17-mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations. SIGNIFICANCE: This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.

Semisynthetic Derivatives of Fradcarbazole A and Their Cytotoxicity against Acute Myeloid Leukemia Cell Lines.

Fourteen derivatives of the marine-derived fradcarbazole A were synthesized from staurosporine. Their structures were identified by NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS). The derivatives were screened in vitro for antiproliferative activity against three human leukemic cell lines (MV4-11, HL-60, K562). All of the derivatives displayed cytotoxicity against the human FLT-3 internal tandem duplication (ITD) mutant acute myeloid leukemia (AML) cell line MV4-11 with IC50 values of 0.32-0.96 µM. The mechanism of action studies indicated that the most effective 3-chloro-5‴-fluorofradcarbazole A (6) induced apoptosis of the MV4-11 cells and arrested the cell cycle at the G0/G1 phase. Furthermore, compound 6 can reduce the expression of FLT-3, CDK2, and c-kit. The results suggest that 3-chloro-5‴-fluorofradcarbazole A (6) is a potential candidate for developing novel anti-AML agents in the future.

An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

Minicapsules encapsulating nanoparticles for targeting, apoptosis induction and treatment of colon cancer.

Cancer therapies are aimed at eliminating the rapidly growing tumor cells by surgery and radiotherapy. The present therapies are only fruitful in early identified cases. The present study involves the preparation and characterization of eudragit S100-coated mini-capsules filled with chitosan nanoparticles-unconjugated and folic acid (FA)-conjugated encapsulating caspase 3 activator (7-hydroxystaurosporine). The formulated nanoparticles were compared for the cancer targeting and curing ability of the same by pre-investigation through drug release in organ-imitated fluids and ex vivo studies (cell viability, DNA fragmentation, caspase 3 activity), and then its confirmation through in vivo studies (tumor regression and distribution). The prepared nanoparticles were nearly spherical in shape, having positive zeta potential. From the cell line studies, it can be concluded that both the conjugated formulations showed better uptake, apoptosis, caspase 3 activation and DNA fragmentation. Stability study was performed according to ICH guidelines and formulation stored at 5° ± 3 °C was found to be most stable. The in vivo studies also supported the findings and showed better comprehensive residence time (23.61 ± 1.75 h), tumor distribution profile than UCN 01 alone. The results of in vitro, ex vivo and in vivo studies lead to the conclusion that the coated minicapsules specifically deliver the drug in the colon showing high therapeutic value and low side effects.

The FLT3 inhibitor midostaurin selectively resensitizes ABCB1-overexpressing multidrug-resistant cancer cells to conventional chemotherapeutic agents.

The occurrence of multidrug resistance (MDR) associated with the overexpression of the ATP-binding cassette (ABC) protein ABCB1 in cancer cells remains a significant obstacle to successful cancer chemotherapy. Therefore, discovering modulators that are capable of inhibiting the drug efflux function or expression of ABCB1 and re-sensitizing multidrug-resistant cancer cells to anticancer agents is of great clinical importance. Regrettably, due to potential adverse events associated with drug-drug interactions and toxicity in patients, researchers have struggled to develop a synthetic inhibitor of ABCB1 that is clinically applicable to improve the effectiveness of chemotherapy. Alternatively, through drug repositioning of approved drugs, we discovered that the FMS-like tyrosine kinase-3 (FLT3) inhibitor midostaurin blocks the drug transport function of ABCB1 and re-sensitizes ABCB1-overexpressing multidrug-resistant cancer cells to conventional chemotherapeutic drugs. Our findings were further supported by results demonstrating that midostaurin potentiates drug-induced apoptosis in ABCB1-overexpressing cancer cells and inhibits the ATPase activity of ABCB1. Considering that midostaurin is a clinically approved anticancer agent, our findings revealed an additional action of midostaurin and that patients with multidrug-resistant tumors may benefit from a combination therapy of midostaurin with standard chemotherapy, which should be further investigated.

Design of a Novel and Selective IRAK4 Inhibitor Using Topological Water Network Analysis and Molecular Modeling Approaches.

Protein kinases are deeply involved in immune-related diseases and various cancers. They are a potential target for structure-based drug discovery, since the general structure and characteristics of kinase domains are relatively well-known. However, the ATP binding sites in protein kinases, which serve as target sites, are highly conserved, and thus it is difficult to develop selective kinase inhibitors. To resolve this problem, we performed molecular dynamics simulations on 26 kinases in the aqueous solution, and analyzed topological water networks (TWNs) in their ATP binding sites. Repositioning of a known kinase inhibitor in the ATP binding sites of kinases that exhibited a TWN similar to interleukin-1 receptor-associated kinase 4 (IRAK4) allowed us to identify a hit molecule. Another hit molecule was obtained from a commercial chemical library using pharmacophore-based virtual screening and molecular docking approaches. Pharmacophoric features of the hit molecules were hybridized to design a novel compound that inhibited IRAK4 at low nanomolar levels in the in vitro assay.

FoxO3 an important player in fibrogenesis and therapeutic target for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal parenchymal lung disease with limited therapeutic options, with fibroblast-to-myofibroblast transdifferentiation and hyperproliferation playing a major role. Investigating ex vivo-cultured (myo)fibroblasts from human IPF lungs as well as fibroblasts isolated from bleomycin-challenged mice, Forkhead box O3 (FoxO3) transcription factor was found to be less expressed, hyperphosphorylated, and nuclear-excluded relative to non-diseased controls. Downregulation and/or hyperphosphorylation of FoxO3 was reproduced by exposure of normal human lung fibroblasts to various pro-fibrotic growth factors and cytokines (FCS, PDGF, IGF1, TGF-ß1). Moreover, selective knockdown of FoxO3 in the normal human lung fibroblasts reproduced the transdifferentiation and hyperproliferation phenotype. Importantly, mice with global- (Foxo3-/-) or fibroblast-specific (Foxo3f.b-/-) FoxO3 knockout displayed enhanced susceptibility to bleomycin challenge, with augmented fibrosis, loss of lung function, and increased mortality. Activation of FoxO3 with UCN-01, a staurosporine derivative currently investigated in clinical cancer trials, reverted the IPF myofibroblast phenotype in vitro and blocked the bleomycin-induced lung fibrosis in vivo These studies implicate FoxO3 as a critical integrator of pro-fibrotic signaling in lung fibrosis and pharmacological reconstitution of FoxO3 as a novel treatment strategy.

First Approved Kinase Inhibitor for AML.

Activating mutations of FLT3 occur in about 30% of acute myeloid leukemia (AML) cases and are associated with relapse and poor prognosis. Midostaurin is the first drug approved for AML since 2000, and the first multi-kinase inhibitor approved for the FLT3-mutant subtype. To view this Bench to Bedside, open or download the PDF.

Synthesis of piperazine-based thiazolidinones as VEGFR2 tyrosine kinase inhibitors inducing apoptosis.

AIM: VEGFR2 tyrosine kinase is a main target in suppressing cancer growth and metastasis. Materials & methods: Piperazine-based thiazolidinones were synthesized and screened for their anticancer and VEGFR2 tyrosine kinase inhibitory activity. Results: Compounds 11, 13 and 16 displayed potent anticancer activity against HepG-2 with IC50 values 0.03-0.06 µM. They were safe on normal human fibroblasts with selectivity indices 8.09, 11.40 and 4.37, respectively. Also, these compounds showed VEGFR2 tyrosine kinase inhibitory activities more than the reference staurosporine with IC50 values <0.3 µM. Lineweaver-Burk plot revealed that these compounds behaved as uncompetitive VEGFR2 tyrosine kinase inhibitors. They also induced caspase-dependent apoptosis in HepG-2. In addition, these compounds revealed good binding within VEGFR2 tyrosine kinase enzyme in comparison with sorafenib reference. CONCLUSION: Compounds 11, 13 and 16 comprise a new promising scaffold of selective VEGFR2 tyrosine kinase inhibitors with caspase-dependent apoptotic activities.