How to Separate Kinase Inhibition from Undesired Monoamine Oxidase A Inhibition—The Development of the DYRK1A Inhibitor AnnH75 from the Alkaloid Harmine

The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure–activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.     

Recent Advances in Pain Management: Relevant Protein Kinases and Their Inhibitors

The purpose of this review is to underline the protein kinases that have been established, either in fundamental approach or clinical trials, as potential biological targets in pain management. Protein kinases are presented according to their group in the human kinome: TK (Trk, RET, EGFR, JAK, VEGFR, SFK, BCR–Abl), CMGC (p38 MAPK, MEK, ERK, JNK, ASK1, CDK, CLK2, DYRK1A, GSK3, CK2), AGC (PKA, PKB, PKC, PKMζ, PKG, ROCK), CAMK, CK1 and atypical/other protein kinases (IKK, mTOR). Examples of small molecule inhibitors of these biological targets, demonstrating an analgesic effect, are described. Altogether, this review demonstrates the fundamental role that protein kinase inhibitors could play in the development of new pain treatments.     

The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site

Background  

The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1in vitro and in cultivated cells.     

DYRK2 controls a key regulatory network in chronic myeloid leukemia stem cells

Chronic myeloid leukemia is a hematological cancer driven by the oncoprotein BCR-ABL1, and lifelong treatment with tyrosine kinase inhibitors extends patient survival to nearly the life expectancy of the general population. Despite advances in the development of more potent tyrosine kinase inhibitors to induce a durable deep molecular response, more than half of patients relapse upon treatment discontinuation. This clinical finding supports the paradigm that leukemia stem cells feed the neoplasm, resist tyrosine kinase inhibition, and reactivate upon drug withdrawal depending on the fitness of the patient’s immune surveillance. This concept lends support to the idea that treatment-free remission is not achieved solely with tyrosine kinase inhibitors and that new molecular targets independent of BCR-ABL1 signaling are needed in order to develop adjuvant therapy to more efficiently eradicate the leukemia stem cell population responsible for chemoresistance and relapse. Future efforts must focus on the identification of new targets to support the discovery of potent and safe small molecules able to specifically eradicate the leukemic stem cell population. In this review, we briefly discuss molecular maintenance in leukemia stem cells in chronic myeloid leukemia and provide a more in-depth discussion of the dual-specificity kinase DYRK2, which has been identified as a novel actionable checkpoint in a critical leukemic network. DYRK2 controls the activation of p53 and proteasomal degradation of c-MYC, leading to impaired survival and self-renewal of leukemia stem cells; thus, pharmacological activation of DYRK2 as an adjuvant to standard therapy has the potential to induce treatment-free remission.Subject terms: Chronic lymphocytic leukaemia, Cancer stem cells     

A cell-based screening method for specifically detecting kinase activity

No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts.     

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.     

Targeting AMPK signalling pathway with natural medicines for atherosclerosis therapy: an integration of in silico screening and in vitro assay

An integration of virtual screening and kinase assay was reported to identify AMPK kinase inhibitors from various natural medicines.The activation of AMP-activated protein kinase (AMPK) signalling pathway plays a central role in the pathologic progression of atherosclerosis (AS). Targeting the AMPK is thus considered as a potential therapeutics to attenuate AS. Here, we report the establishment of a synthetic pipeline that integrates in silico virtual screening and in vitro kinase assay to discover new lead compounds of AMPK inhibitors. The screening is performed against a large-size pool of structurally diverse natural products, from which a number of compounds are inferred as promising candidates, and few of them are further tested in vitro by using a standard kinase assay protocol to determine their inhibitory potency against AMPK. With this scheme we successfully identify five potent AMPK inhibitors with IC₅₀ values at micromolar level. We also examine the structural basis and molecular mechanism of nonbonded interaction network across the modelled complex interface of AMPK kinase domain with a newly identified natural medicine.     

Cell-based assays and molecular simulation reveal that the anti-cancer harmine is a specific matrix metalloproteinase-3 (MMP-3) inhibitor

The biological activities of harmine have been a much clearer picture in recent years, which include anti-tumor, anti-inflammation and cytotoxic properties. Numerous in vitro and in vivo animal models have confirmed its activities, but its mode of action remains a relative unsolved issue. We therefore investigated harmine for its effects on MMP-3 and the molecular interaction was also simulated. The human glioma cancer cell line, U-87 MG cells, was subjected to different concentrations (1–10 μM) of harmine for 24 h. Methylthiazol tetrazolium (MTT) test, half maximal inhibitory concentration (IC50), western blot analysis, enzyme-linked immunosorbent assay and molecular docking through BIOVIA DiscoveryStudio™ were performed. These results showed that although harmine stimulation in vitro has very little or no effects on MMP-3 expression by U-87 MG cells, the treatment of harmine decreases MMP-3 activity in a dose dependent manner. It was further calculated that 7.9 μM is the IC50 towards MMP-3. Using a molecular dynamic simulation approach, we identified the N2, methyl of C1 and benzene ring of harmine interact with Zn²⁺ (2.4 Å), His205 (2.4 Å) and His211 (2.4 Å) as well as Val163 (2.7 Å) at the active site of MMP-3, respectively, and thus conferred a striking specific binding advantage. Taken altogether, the present study evidences that harmine acts as an MMP-3 inhibitor specially targeting the enzymatic active site and possibly efficiently ameliorates MMP-3-driven malignant and inflammatory diseases.     

Simultaneous Production of Psilocybin and a Cocktail of β-Carboline Monoamine Oxidase Inhibitors in “Magic” Mushrooms

The psychotropic effects of Psilocybe “magic” mushrooms are caused by the l -tryptophan-derived alkaloid psilocybin. Despite their significance, the secondary metabolome of these fungi is poorly understood in general. Our analysis of four Psilocybe species identified harmane, harmine, and a range of other l -tryptophan-derived β-carbolines as their natural products, which was confirmed by 1D and 2D NMR spectroscopy. Stable-isotope labeling with ¹³C₁₁-l -tryptophan verified the β-carbolines as biosynthetic products of these fungi. In addition, MALDI-MS imaging showed that β-carbolines accumulate toward the hyphal apices. As potent inhibitors of monoamine oxidases, β-carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same building block and produce dissimilar compounds, yet contribute directly or indirectly to the same pharmacological effects.     

Design of 5H-pyrrolo[2,3-b]pyrazine-2-phenyl Ether Derivatives as Potential JAK3 Inhibitor Based on Surflex Docking,3D-QSAR Modeling and Reverse Docking

Tyrosine protein kinase JAK3 has a very important significance on organ transplantation and the treatment of autoimmune diseases, which has been a potential therapeutic target. In recent years, a large number of JAK3 inhibitors have been reported. However, the poor selectivity and side effects have limited their widespread use in clinical practice. In order to solve this problem, 52 potential small-molecule inhibitors were combined with JAK1, JAK2 and JAK3 respectively to obtain the optimal conformation of small molecules. On the basis of that we established 3D quantitative structure-activity relationships(3D-QSAR) model. Comparative molecular field analysis(Co MFA) and molecular similarity analysis(Co MSIA) were used to evaluate the model. We took advantage of reverse docking to explore the underlying toxicity and side effects. Combining 3D quantitative structure-activity relationships, surflex-dock and reverse docking results, ten 5 H-pyrrolo[2,3-b]pyrazine-2-phenyl ether derivatives based on the most optimal selectivity and activity compound 39 were designed. It can be seen from Co MFA and Co MSIA predicted active values of designed molecules that the selectivity of designed small molecules was improved obviously. Among them, compounds 61 and 62 could become the potential small molecule compounds.     

Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors

The generation of synthetic compounds with exclusive target specificity is an extraordinary challenge of molecular recognition and demands novel design strategies, in particular for large and homologous protein families such as protein kinases with more than 500 members. Simple organic molecules often do not reach the necessary sophistication to fulfill this task. Here, we present six carefully tailored, stable metal-containing compounds in which unique and defined molecular geometries with natural-product-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six reported metal compounds displays high selectivity for an individual protein kinase, namely GSK3α, PAK1, PIM1, DAPK1, MLCK, and FLT4. Although being conventional ATP-competitive inhibitors, the combination of the unusual globular shape and rigidity characteristics, of these compounds facilitates the design of highly selective protein kinase inhibitors. Unique structural features of the octahedral coordination geometry allow novel interactions with the glycine-rich loop, which contribute significantly to binding potencies and selectivities. The sensitive correlation between metal coordination sphere and inhibition properties suggests that in this design, the metal is located at a "hot spot" within the ATP binding pocket, not too close to the hinge region where globular space is unavailable, and at the same time not too far out toward the solvent where the octahedral coordination sphere would not have a significant impact on potency and selectivity. This study thus demonstrates that inert (stable) octahedral metal complexes are sophisticated structural scaffolds for the design of highly selective chemical probes.     

Predicting kinase selectivity profiles using Free-Wilson QSAR analysis

Kinases are involved in a variety of diseases such as cancer, diabetes, and arthritis. In recent years, many kinase small molecule inhibitors have been developed as potential disease treatments. Despite the recent advances, selectivity remains one of the most challenging aspects in kinase inhibitor design. To interrogate kinase selectivity, a panel of 45 kinase assays has been developed in-house at Pfizer. Here we present an application of in silico quantitative structure activity relationship (QSAR) models to extract rules from this experimental screening data and make reliable selectivity profile predictions for all compounds enumerated from virtual libraries. We also propose the construction of R-group selectivity profiles by deriving their activity contribution against each kinase using QSAR models. Such selectivity profiles can be used to provide better understanding of subtle structure selectivity relationships during kinase inhibitor design.     

Highlights on Steroidal Arylidene Derivatives as a Source of Pharmacologically Active Compounds: A Review

Steroids constitute a unique class of chemical compounds, playing an important role in physiopathological processes, and have high pharmacological interest. Additionally, steroids have been associated with a relatively low toxicity and high bioavailability. Nowadays, multiple steroidal derivatives are clinically available for the treatment of numerous diseases. Moreover, different structural modifications on their skeleton have been explored, aiming to develop compounds with new and improved pharmacological properties. Thus, steroidal arylidene derivatives emerged as a relevant example of these modifications. This family of compounds has been mainly described as 17β-hydroxysteroid dehydrogenase type 1 and aromatase inhibitors, as well as neuroprotective and anticancer agents. Besides, due to their straightforward preparation and intrinsic chemical reactivity, steroidal arylidene derivatives are important synthetic intermediates for the preparation of other compounds, particularly bearing heterocyclic systems. In fact, starting from arylidenesteroids, it was possible to develop bioactive steroidal pyrazolines, pyrazoles, pyrimidines, pyridines, spiro-pyrrolidines, amongst others. Most of these products have also been studied as anti-inflammatory and anticancer agents, as well as 5α-reductase and aromatase inhibitors. This work aims to provide a comprehensive overview of steroidal arylidene derivatives described in the literature, highlighting their bioactivities and importance as synthetic intermediates for other pharmacologically active compounds.     

Targeting the PI3K/AKT/mTOR Signaling Pathway in Lung Cancer: An Update Regarding Potential Drugs and Natural Products

Lung cancer is one of the most common cancers and has a high mortality rate. Due to its high incidence, the clinical management of the disease remains a major challenge. Several reports have documented a relationship between the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway and lung cancer. The recognition of this pathway as a notable therapeutic target in lung cancer is mainly due to its central involvement in the initiation and progression of the disease. Interest in using natural and synthetic medications to target these signaling pathways has increased in recent years, with promising results in vitro, in vivo, and in clinical trials. In this review, we focus on the current understanding of PI3K/AKT/mTOR signaling in tumor development. In addition to the signaling pathway, we highlighted the therapeutic potential of recently developed PI3K/AKT/mTOR inhibitors based on preclinical and clinical trials.     

Resorcylic acid lactones: A pluripotent scaffold with therapeutic potential

The recent discoveries of potent HSP90 and MAP kinase inhibitors amongst the resorcylic acid lactones (RALs) have revived interest in this family of natural products. Both HSP90 and MAP kinase inhibition hold tremendous therapeutic potential, particularly in the treatment of cancer. Our synthetic efforts towards the RALs and, in particular, selective inhibitors of HSP90 and kinases are reviewed.     

Discovering potent inhibitors against c-Met kinase: molecular design, organic synthesis and bioassay

The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers nowadays. The discovery of small molecule inhibitors is of special interest in the blockade of the c-Met kinase pathway. Here, we initiated our study from compound 1a, a novel inhibitor against c-Met kinase. A substructure similarity search against the SPECS database and chemical synthesis methods were performed to obtain a series of pyrazolidine-3,5-dione derivatives. Through the enzyme-based assay against c-Met kinase, 4 compounds (1c, 1e, 1m and 1o) showed potential inhibitory activity, with IC(50) values mostly less than 10 μM. Based on the structure-activity relationship (SAR) and binding mode analysis, a focused combinatorial library was designed by the LD1.0 program. Taking into account ADMET properties and synthesis accessibility, seven candidate compounds (5a-g) were successfully synthesized. The activity of the most potent compounds 5b (IC(50) = 0.46 μM) was 20 fold higher than that of the lead 1a. Taken together, our findings identified the pyrazolidine-3,5-dione derivatives as potent inhibitors against c-Met kinase and demonstrated the efficiency of the strategy in the development of small molecules against c-Met kinase.