Neoprzewaquinone A Inhibits Breast Cancer Cell Migration and Promotes Smooth Muscle Relaxation by Targeting PIM1 to Block ROCK2/STAT3 Pathway.

Salvia miltiorrhiza Bunge (Danshen) has been widely used to treat cancer and cardiovascular diseases in Chinese traditional medicine. Here, we found that Neoprzewaquinone A (NEO), an active component of S. miltiorrhiza, selectively inhibits PIM1. We showed that NEO potently inhibits PIM1 kinase at nanomolar concentrations and significantly suppresses the growth, migration, and Epithelial-Mesenchymal Transition (EMT) in the triple-negative breast cancer cell line, MDA-MB-231 in vitro. Molecular docking simulations revealed that NEO enters the PIM1 pocket, thereby triggering multiple interaction effects. Western blot analysis revealed that both NEO and SGI-1776 (a specific PIM1 inhibitor), inhibited ROCK2/STAT3 signaling in MDA-MB-231 cells, indicating that PIM1 kinase modulates cell migration and EMT via ROCK2 signaling. Recent studies indicated that ROCK2 plays a key role in smooth muscle contraction, and that ROCK2 inhibitors effectively control the symptoms of high intraocular pressure (IOP) in glaucoma patients. Here, we showed that NEO and SGI-1776 significantly reduce IOP in normal rabbits and relax pre-restrained thoracic aortic rings in rats. Taken together, our findings indicated that NEO inhibits TNBC cell migration and relaxes smooth muscles mainly by targeting PIM1 and inhibiting ROCK2/STAT3 signaling, and that PIM1 may be an effective target for IOP and other circulatory diseases.

In-silico and in-vitro investigation of STAT3-PIM1 heterodimeric complex: Its mechanism and inhibition by curcumin for cancer therapeutics.

The functional activity among STAT3 and PIM1, are key signaling events for cancer cell function. Curcumin, a diarylheptanoid isolated from turmeric, effectively inhibits STAT3 signaling. Selectively, we attempted to address interactions of STAT3, PIM1 and Curcumin for therapeutic intervention using in silico and in vitro experimental approaches. Firstly, protein-protein interactions (PPI) between STAT3-PIM1 by molecular docking studies reflected salt bridges among Arg279 (STAT3)-Glu140 (PIM1) and Arg282 (STAT3)-Asp100 (PIM1), with a binding affinity of -38.6 kcal/mol. Secondly, molecular dynamics simulations of heterodimeric STAT3-PIM1 complex with curcumin revealed binding of curcumin on PIM-1 interface of the complex through hydrogen bonds (Asp155) and hydrophobic interactions (Leu13, Phe18, Val21, etc.) with a binding energy of -7.3 kcal/mol. These PPIs were confirmed in vitro by immunoprecipitation assays in MDA-MB-231 cells. Corroborating our results, expression levels of STAT3 and PIM1 decreased after curcumin treatment. We observed that PIM1 interacts with STAT3 and these functional interactions are disrupted by curcumin. The calculated band energy gap of heterodimeric STAT3-PIM1-Curcumin complex was of 9.621 kcal/mol. The present study revealed the role of curcumin in STAT3/PIM1 signaling and its binding affinity to the complex for design of advanced cancer therapeutics.

Phytoconstituents and Medicinal Plants for Anticancer Drug Discovery: Computational Identification of Potent Inhibitors of PIM1 Kinase.

Natural products, medicinal plants, and phytoconstituents serve as important sources and accelerators for anticancer drug discovery, especially when they are combined with virtual screening and molecular simulations against molecular drug targets. Proto-oncogene serine/threonine-protein kinase Pim1 (PIM1) is involved in cell survival and proliferation, with great relevance for oncogenesis. PIM1 plays a major role in the progression of various common complex human cancers, including prostate cancer, acute myeloid leukemia, and other hematopoietic malignancies. The overexpression of PIM1 leads to cancer progression, and thus it is considered as a potential target for drug design and development purposes. Here, we report original in silico findings by employing structure-based virtual screening to discover potential phytoconstituents from the medicinal plants-based compounds, which could inhibit the PIM1 activity, using the IMPPAT (a curated database of Indian Medicinal Plants, Phytochemistry And Therapeutics) database. The initial hits were selected based on their binding affinity toward PIM1 calculated through the molecular docking approach. Subsequently, interaction analyses and molecular dynamics (MD) simulation for 100 ns was carried out to study the conformational dynamics and complex stability of PIM1 with the identified compounds. Importantly, we found that PIM1 forms stable protein-ligand complexes with the phytoconstituents Dehydrotectol and Nordracorubin in particular. Our findings suggest that identified phytoconstituents Dehydrotectol and Nordracorubin bind to PIM1 in ATP-competitive binding mode. These findings and the compounds reported herein warrant further exploration as promising scaffolds for anticancer drug design, discovery, and development.

Discovery of novel pyrazolo[3,4-b]pyridine scaffold-based derivatives as potential PIM-1 kinase inhibitors in breast cancer MCF-7 cells.

Pim-1 kinase targeted recently has proved an essential goal of breast cancer therapy. We report the design, synthesis with full characterization analysis of pyrazolo[3,4-b]pyridine scaffold-based derivatives targeting Pim-1 kinase as anti-breast cancer agents. All the newly synthesized compounds were screened for their in vitro cytotoxic activity against two breast cancer cell lines MCF-7 and MDA-MB-231, and non-cancerous MCF-10A cells. Four derivatives notably, 17 and 19 exhibited a remarkable cytotoxic activity with IC50 values 5.98 and 5.61 µM against MCF-7 (ERα-dependent) cells in a selective way, as they weren't active against MDA-MB-231 (non-ERα-dependent) and safe against MCF-10A. The most active compounds through in vitro screening were subjected to PIM-1 kinase to elucidate the Pim-1 kinase inhibitory activity as the mechanistic mode of action. Among the tested derivatives, Compounds 17 and 19 showed the highest inhibitory activity with IC50 values 43 and 26 nM, respectively, compared to the 5-FU with IC50 value 17 nM. Moreover, apoptotic investigation through flow cytometry and gene expression analysis of the apoptosis-related genes for the most active compound 19 against MCF-7. It was found that compound 19 induced apoptotic MCF-7 cell death by cell cycle arrest at G2/M phase and by elevation the expression of pro-apoptotic genes and inhibition of anti-apoptotic genes expression. Finally, the PIM-1 inhibition activities for compounds 17 and 19 were in accordance with the molecular docking study that revealed good interaction with the Pim-1 kinase active site.

Discovery of novel (+)-Usnic acid derivatives as potential anti-leukemia agents with pan-Pim kinases inhibitory activity.

Usnic acid (UA) is the main secondary metabolite isolated from lichens, with moderate anticancer activity. A small group of (+)-UA derivatives characterized with flavanone moiety was designed and synthesized, and their anticancer activities were evaluated in leukemia cells. It was demonstrated that (+)-UA derivatives 6a-6g inhibited the proliferation of leukemia cells HL-60 and K562 with low micromolar IC50 values. Mechanisms of action were investigated to find that 6g induced apoptosis in HL-60 and K562 cell lines, and affected the expression of MNK/eIF4E axis-related proteins, such as Mcl-1, p-eIF4E, p-4E-BP1. Finally, kinase inhibition assay suggested 6g was a potential inhibitor of pan-Pim kinases. Meanwhile, the blocking of phosphorylation of BAD and 4E-BP1 by 6g, together with the proposed binding mode of 6g with Pim-1 further confirmed its Pim inhibition effects. Our finding provides the sight towards the potential mechanism of (+)-UA derivatives 6g as anti-leukemia agents.

Quercetin Therapy for Selected Patients with PIM1 Kinase-Positive Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Pilot Study.

We reported that PIM1 kinase is expressed in the lymphocytes of patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Quercetin, a naturally occurring flavonoid, is a dietary supplement and inhibits many kinases, including PIM1, in vitro. Under an Institutional Review Board-approved protocol, we performed an open-label, single-arm pilot study to evaluate the antitumor activity of quercetin in patients with CLL/SLL. Q-ForceTM chews were administered orally, 500 mg twice daily, for 3 months. Eligible patients had failed prior therapies, had had no other standard treatment, or refused other therapies. Response was assessed based on objective change in disease parameters. Patients were included if their lymphocyte counts were rising and ≥10,000/µL but not > 100,000/µL. Three patients received quercetin treatment. There was no toxicity. Two responded with stabilization of rising lymphocyte counts (p < 0.001 for each), which remained stable during their follow-up (5 and 11 months after cessation of treatment, respectively). The CLL cells in the nonresponder harbored a TP53 mutation. Although our data from this pilot translational study are based on a small sample, further studies of quercetin as a potential therapeutic agent in selected patients with CLL/SLL appear warranted.

Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors - Part I.

Novel N-substituted azaindoles have been discovered as PIM1 inhibitors. X-ray structures have played a significant role in orienting the chemistry effort in the initial phase of hit confirmation. Disclosure of an unconventional binding mode for 1 and 2, as demonstrated by X-ray crystallography, is presented and was an important factor in selecting and advancing a lead series.

Discovery of N-substituted 7-azaindoles as Pan-PIM kinase inhibitors - Lead series identification - Part II.

N-Substituted azaindoles have been discovered as pan-PIM kinase inhibitors. Initial SAR, early ADME and PK/PD data of a series of compounds is described and led to the identification of promising pan-PIM inhibitors which validated our interest in the 7-azaindole scaffold and led us to pursue the identification of a clinical candidate.

Neuroprotective effects of Danggui-Jakyak-San on rat stroke model through antioxidant/antiapoptotic pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dangui-Jakyak-San (DJ) is a traditional Korean medicinal polyherb, prescribed typically in patients with insufficient blood supply in Eastern Asia. The DJ also has been reported to have neuroprotective effects in vitro and in vivo studies. AIM OF STUDY: The therapeutic potential of DJ was examined in stroke rat model, in comparison with donepezil, a reversible acetylcholinesterase inhibitor. MATERIALS AND METHODS: Ischemic stroke rat model was induced by surgery of permanent occlusion of middle cerebral artery (pMCAO). The model was orally administered with distilled water (pMCAO control), donepezil at 10mg/kg (Donepezil) and DJ at 200, 100 and 50mg/kg (DJ 200, DJ 100 and DJ 50, respectively). Sham had the same surgery excepting for the pMCAO, and it was administered with distilled water (sham control). RESULTS: After the administration for 28 days, the groups of DJ exhibited dose-dependent reduction in infarct/defect volumes with improvement in sensorimotor and cognitive motor function, comparing to pMCAO control. The DJ treatments seemed to enhance antiapoptotic and antioxidant effects; increases in antiapoptotic expressions (STAT3 and Pim-1) and decreases in lipid peroxidation (MDA) together with increases in contents of endogenous antioxidant (GSH) and activities of antioxidant enzymes (catalase and SOD). The histopathological analyses revealed significant reduction in neuronal apoptosis (caspase-3 and PARP) and neuronal degradation with atrophy and degeneration, in the DJ treatments. Furthermore, the oxidative stresses (nitrotyrosine as an iNOS factor and 4-HNE as a marker of lipid peroxidation) were observed mild. Although the similar neuroprotective effects were observed, the body weight loss was scarcely alleviated in Donepezil comparing to pMCAO control. CONCLUSION: These suggest that DJ ameliorate the neurological dysfunction of cerebral ischemia through augmentation of antioxidant defense system and up-regulation of STAT3 and Pim-1.

Inhibitors of the Cyclin-Dependent Kinase and PIM Kinase Pathways in the Treatment of Myeloma.

Multiple myeloma (MM) cells are characterized by genomic alternations that lead to increased cell proliferation and resistance to therapeutic interventions. Up-regulation of cyclins is a characteristic finding in a significant proportion of myeloma patients, mediated through a variety of mechanisms including chromosomal translocations. Cyclins and the cyclin-dependent kinases (CDKs) play a critical role in the cell proliferation seen in MM, especially in the high-risk disease. Given this, CDK inhibitors have been evaluated in this disease, and studies so far have led to a mixed picture. Recent studies with targeted CDK inhibitors have shown early promise, and trials of these drugs in combination with other myeloma drugs are ongoing. The malignant plasma cells in MM are highly dependent on the microenvironment for their growth and survival. Multiple signaling pathways have been found to mediate the interactions between the microenvironment and the plasma cells, whether mediated through cytokines or adhesion molecules. The PIM kinase pathway appears to play a major role in the myeloma cell survival, and PIM kinase inhibitors have shown efficacy in the laboratory, and a recent clinical trial also demonstrates important clinical activity.

Targeting Pim kinases for cancer treatment: opportunities and challenges.

Pim oncogenes are highly expressed in many types of hematological and solid cancers. Pim kinases regulate the network of signaling pathways that are critical for tumorigenesis and development, making Pim kinases the attractive drug targets. Currently, two approaches have been employed in designing Pim kinase inhibitors: ATP-mimetics and non-ATP mimetics; but all target the ATP-binding pocket and are ATP-competitive. In this review, we summarize the current progress in understanding the Pim-related structure and biology, and provide insights into the binding modes of some prototypical Pim-1 inhibitors. The challenges as well as opportunities are highlighted for development of Pim kinase inhibitors as potential anticancer agents.

Discovery and identification of PIM-1 kinase inhibitors through a hybrid screening approach.

PIM-1 kinase is an important therapeutic target in the treatment of cancer. Discovery and identification of PIM-1 Inhibitors with novel scaffolds are an effective way for developing potent therapeutic agents for the treatment of cancers. Here we proposed a hybrid screening approach which combines an optimal structure-based drug design strategy and a simple pharmacophore model to discover PIM-1 kinase inhibitors. With the proposed hybrid screening approach, the SPECS database containing 204,580 molecules was screened. In total, 89 hits were obtained. Forty three of them were purchased and tested in bioassays. Finally, 5 lead compounds with novel scaffolds were identified to exhibit promising antitumor activities against human leukemia cell line MV4-11, K-562 and human prostate cancer cell line PC-3 and DU145. Their IC(50) values range from 4.40 to 37.96 µM. Three hits with 3 different scaffolds were selected from these five hits for binding mode analysis. It was demonstrated that the subtle differences in the interactions of the representatives with PIM-1 kinase contribute to the different inhibitory activities. It was also demonstrated that the suggested hybrid screening approach is an effective method to discover PIM-1 inhibitors possessing different scaffolds. These leads have a strong likelihood to act as further starting points for us in the optimization and development of potent PIM-1 inhibitors.

Discovery of novel pyrazolo[1,5-a]pyrimidines as potent pan-Pim inhibitors by structure- and property-based drug design.

Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.

A combination strategy to inhibit Pim-1: synergism between noncompetitive and ATP-competitive inhibitors.

Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance.

Boosting the pentose phosphate pathway restores cardiac progenitor cell availability in diabetes.

AIMS: Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway. METHODS AND RESULTS: Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt. CONCLUSION: We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.

A novel Pim-1 kinase inhibitor targeting residues that bind the substrate peptide.

A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC(50) value of 150 nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G(1)/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors.

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

Discovery of novel Pim-1 kinase inhibitors by a hierarchical multistage virtual screening approach based on SVM model, pharmacophore, and molecular docking.

In this investigation, we describe the discovery of novel potent Pim-1 inhibitors by employing a proposed hierarchical multistage virtual screening (VS) approach, which is based on support vector machine-based (SVM-based VS or SB-VS), pharmacophore-based VS (PB-VS), and docking-based VS (DB-VS) methods. In this approach, the three VS methods are applied in an increasing order of complexity so that the first filter (SB-VS) is fast and simple, while successive ones (PB-VS and DB-VS) are more time-consuming but are applied only to a small subset of the entire database. Evaluation of this approach indicates that it can be used to screen a large chemical library rapidly with a high hit rate and a high enrichment factor. This approach was then applied to screen several large chemical libraries, including PubChem, Specs, and Enamine as well as an in-house database. From the final hits, 47 compounds were selected for further in vitro Pim-1 inhibitory assay, and 15 compounds show nanomolar level or low micromolar inhibition potency against Pim-1. In particular, four of them were found to have new scaffolds which have potential for the chemical development of Pim-1 inhibitors.

Vitamin B1 analog benfotiamine prevents diabetes-induced diastolic dysfunction and heart failure through Akt/Pim-1-mediated survival pathway.

BACKGROUND: The increasing incidence of diabetes mellitus will result in a new epidemic of heart failure unless novel treatments able to halt diabetic cardiomyopathy early in its course are introduced. This study aimed to determine whether the activity of the Akt/Pim-1 signaling pathway is altered at critical stages of diabetic cardiomyopathy and whether supplementation with vitamin B1 analog benfotiamine (BFT) helps to sustain the above prosurvival mechanism, thereby preserving cardiomyocyte viability and function. METHODS AND RESULTS: Untreated streptozotocin-induced type 1 or leptin-receptor mutant type 2 diabetic mice showed diastolic dysfunction evolving to contractile impairment and cardiac dilatation and failure. BFT (70 mg/kg(-1)/d(-1)) improved diastolic and systolic function and prevented left ventricular end-diastolic pressure increase and chamber dilatation in both diabetic models. Moreover, BFT improved cardiac perfusion and reduced cardiomyocyte apoptosis and interstitial fibrosis. In hearts of untreated diabetic mice, the expression and activity of Akt/Pim-1 signaling declined along with O-N-acetylglucosamine modification of Akt, inhibition of pentose phosphate pathway, activation of oxidative stress, and accumulation of glycation end products. Furthermore, diabetes reduced pSTAT3 independently of Akt. BFT inhibited these effects of diabetes mellitus, thereby conferring cardiomyocytes with improved resistance to high glucose-induced damage. The phosphoinositide-3-kinase inhibitor LY294002 and dominant-negative Akt inhibited antiapoptotic action of BFT-induced and Pim-1 upregulation in high glucose-challenged cardiomyocytes. CONCLUSIONS: These results show that BFT protects from diabetes mellitus-induced cardiac dysfunction through pleiotropic mechanisms, culminating in the activation of prosurvival signaling pathway. Thus, BFT merits attention for application in clinical practice.

Constitutive Fms-like tyrosine kinase 3 activation results in specific changes in gene expression in myeloid leukaemic cells.

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukaemogenesis. We have examined, by cDNA microarray analysis, the changes in gene expression induced by FLT3/ITD or constitutively activated wild type FLT3 signalling. A limited set of genes was consistently affected by FLT3 inhibition. In confirmation of their FLT3 dependence, these genes returned toward pretreatment levels of expression after reversal of FLT3 inhibition. Several of the most significantly affected genes are involved in the RAS/mitogen-activated protein kinase, Janus kinase/signal transducer and activator of transcription and phosphatidylinositol 3 kinase (PI3K)/AKT pathways. These data suggest that constitutively activated FLT3 works through multiple signal transduction pathways. PIM1, MYC and CCND3 were chosen from this gene set to explore their biological roles. Knock-down of these genes by small interfering RNA showed that these genes play important roles in constitutively activated FLT3 expressing cells. The alterations of the gene expression profiles in these cells help to further elucidate the mechanisms of FLT3-mediated leukaemogenesis.