Discovery of benzimidazole-indazole derivatives as potent FLT3-tyrosine kinase domain mutant kinase inhibitors for acute myeloid leukemia.

The FMS-like tyrosine kinase 3 (FLT3) gene encodes a class III receptor tyrosine kinase that is expressed in hematopoietic stem cells. The mutations of FLT3 gene found in 30% of acute myeloid leukemia (AML), leads to an abnormal constitutive activation of FLT3 kinase of the receptor and results in immature myeloblast cell proliferation. Although small molecule drugs targeting the FLT3 kinase have been approved, new FLT3 inhibitors are needed owing to the side effects and drug resistances arising from kinase domain mutations, such as D835Y and F691L. In this study, we have developed benzimidazole-indazole based novel inhibitors targeting mutant FLT3 kinases through the optimization of diverse chemical moieties substituted around the core skeleton. The most optimized compound 22f exhibited potent inhibitory activities against FLT3 and FLT3/D835Y, with IC50 values of 0.941 and 0.199 nM, respectively. Furthermore, 22f exhibited strong antiproliferative activity against an AML cell line, MV4-11 cells with a GI50 of 0.26 nM. More importantly, 22f showed single-digit nanomolar GI50 values in the mutant FLT kinase expressed Ba/F3 cell lines including FLT-D835Y (GI50 = 0.29 nM) and FLT3-F691L (GI50 = 2.87 nM). Molecular docking studies indicated that the compound exhibits a well-fitted binding mode as a type 1 inhibitor in the homology model of active conformation of FLT3 kinase.

Methotrexate recognition by the human reduced folate carrier SLC19A1.

Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4-6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4-8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.

Novel Thioxothiazolo[3,4-a]quinazolin-5(4H)-one Derivatives as BKCa Channel Activators for Urinary Incontinence.

Overactive bladder (OAB) is a syndrome causing a sudden and unstoppable need to urinate with significant global prevalence. Several drugs are used to treat OAB; however, they have various side effects. Therefore, new treatment options for OAB are required. A series of novel 5-oxo-N-phenyl-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide derivatives were synthesized and evaluated for their large-conductance voltage- and Ca2+-activated K+ channel activation through a cell-based fluorescence assay and electrophysiological recordings. Several compounds, including a 7-bromo substituent on the heterocyclic system, showed increased channel currents. Among the derivatives, compound 12h exhibited potent in vitro activity with a half-maximal effective concentration (EC50) of 2.89 µM, good oral pharmacokinetic properties (area under the curve and half-life), and in vivo efficacy in a spontaneously hypertensive rat model.

Discovery of indirubin-3'-aminooxy-acetamide derivatives as potent and selective FLT3/D835Y mutant kinase inhibitors for acute myeloid leukemia.

Mutations in Fms-like tyrosine kinase 3 (FLT3) have been implicated in the pathogenesis of acute myeloid leukemia (AML) by affecting the proliferation and differentiation of hematopoietic stem and progenitor cells. Although several FLT3 inhibitors have been developed, the occurrence of secondary TKD mutations of FLT3 such FLT3/D835Y and FLT3/F691L lead to drug resistance and has become a key area of unmet medical needs. To overcome the obstacle of secondary TKD mutations, a new series of indirubin-3'-aminooxy-acetamide derivatives was discovered as potent and selective FLT3 and FLT3/D835Y inhibitors that were predicted to bind at the DFG-in active conformation of FLT3 in molecular docking studies. Through structure-activity relationship studies, the most optimized compound 13a was developed as a potent inhibitor at FLT3 and FLT3/D835Y with IC50 values of 0.26 nM and 0.18 nM, respectively, which also displayed remarkably strong in vitro anticancer activities, with single-digit nanomolar GI50 values for several AML (MV4-11 and MOLM14) and Ba/F3 cell lines expressed with secondary TKD mutated FLT3 kinases as well as FLT3-ITD. The selectivity profiles of compound 13a in the oncology kinase panel and various human cancer cell lines were prominent, demonstrating that its inhibitory activities were mainly focused on a few members of the receptor tyrosine kinase family and AML versus solid tumor cell lines. Furthermore, significant in vivo anticancer efficacy of compound 13a was confirmed in a xenograft animal model implanted with FLT3-ITD/D835Y-expressing MOLM-14 cells related to secondary TKD mutation.

Synthesis and structure-activity relationship studies of 1,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 1 (JAK1) inhibitors.

JAK inhibitors have been considered as useful targets for the treatment of related diseases. However, first-generation JAK inhibitors have side effects such as anemia, thrombocytopenia, neutropenia and headaches which have been suggested to result from high JAK2 inhibition. Second-generation JAK inhibitors with more specific JAK isozyme inhibition have been studied to eliminate these adverse effects. In this study, novel 4-(1,5- or 2,5-triazole)-pyrrolopyrimidine derivatives with aromatic moieties were synthesized as JAK1 inhibitors, and an in vitro enzyme assay was used to evaluate the JAK inhibitory effects. Among these JAK1 inhibitors, the compound 23a showed an IC50 level of 72 nM, as well as being selective against other JAKs by 12 times or more: the results of molecular docking studies suggested that the high JAK1 selectivity resulted from a key interaction between the iodine atom of compound 23a and His-885 of hJAK1.

Quantification of Active Site Density and Turnover Frequency: From Single-Atom Metal to Nanoparticle Electrocatalysts.

Single-atom catalysts (SACs) featuring atomically dispersed metal cations covalently embedded in a carbon matrix show significant potential to achieve high catalytic performance in various electrocatalytic reactions. Although considerable advances have been achieved in their syntheses and electrochemical applications, further development and fundamental understanding are limited by a lack of strategies that can allow the quantitative analyses of their intrinsic catalytic characteristics, that is, active site density (SD) and turnover frequency (TOF). Here we show an in situ SD quantification method using a cyanide anion as a probe molecule. The decrease in cyanide concentration triggered by irreversible adsorption on metal-based active sites of a model Fe-N-C catalyst is precisely measured by spectrophotometry, and it is correlated to the relative decrease in electrocatalytic activity in the model reaction of oxygen reduction reaction. The linear correlation verifies the surface-sensitive and metal-specific adsorption of cyanide on Fe-N x sites, based on which the values of SD and TOF can be determined. Notably, this analytical strategy shows versatile applicability to a series of transition/noble metal SACs and Pt nanoparticles in a broad pH range (1-13). The SD and TOF quantification can afford an improved understanding of the structure-activity relationship for a broad range of electrocatalysts, in particular, the SACs, for which no general electrochemical method to determine the intrinsic catalytic characteristics is available.

Discovery of Novel Pyrimidine-Based Capsid Assembly Modulators as Potent Anti-HBV Agents.

Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.

Synthesis and evaluation of cyclopentane-based muraymycin analogs targeting MraY.

Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for the development of new antibiotics. In this regard, phospho-MurNAc-pentapeptide translocase (MraY), an essential enzyme in the early stages of peptidoglycan biosynthesis, has emerged as a promising new antibiotic target. We recently reported the crystal structures of MraY in complex with representative members of naturally occurring nucleoside antibiotics, including muraymycin D2. However, these nucleoside antibiotics are synthetically challenging targets, which limits the scope of medicinal chemistry efforts on this class of compounds. To gain access to active muraymycin analogs with reduced structural complexity and improved synthetic tractability, we prepared and evaluated cyclopentane-based muraymycin analogs for targeting MraY. For the installation of the 1,2-syn-amino alcohol group of analogs, the diastereoselective isocyanoacetate aldol reaction was explored. The structure-activity relationship analysis of the synthesized analogs suggested that a lipophilic side chain is essential for MraY inhibition. Importantly, the analog 20 (JH-MR-23) showed antibacterial efficacy against Staphylococcus aureus. These findings provide insights into designing new muraymycin-based MraY inhibitors with improved chemical tractability.

Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer.

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

Discovery of orally active indirubin-3'-oxime derivatives as potent type 1 FLT3 inhibitors for acute myeloid leukemia.

FMS-like receptor tyrosine kinase-3 (FLT3) is expressed on acute leukemia cells and is implicated in the survival, proliferation and differentiation of hematopoietic cells in most acute myeloid leukemia (AML) patients. Despite recent achievements in the development of FLT3-targeted small-molecule drugs, there are still unmet medical needs related to kinase selectivity and the progression of some mutant forms of FLT3. Herein, we describe the discovery of novel orally available type 1 FLT3 inhibitors from structure-activity relationship (SAR) studies for the optimization of indirubin derivatives with biological and pharmacokinetic profiles as potential therapeutic agents for AML. The SAR exploration provided important structural insights into the key substituents for potent inhibitory activities of FLT3 and in MV4-11 cells. The profile of the most optimized inhibitor (36) showed IC50 values of 0.87 and 0.32 nM against FLT3 and FLT3/D835Y, respectively, along with potent inhibition against MV4-11 and FLT3/D835Y expressed MOLM14 cells with a GI50 value of 1.0 and 1.87 nM, respectively. With the high oral bioavailability of 42.6%, compound 36 displayed significant in vivo antitumor activity by oral administration of 20 mg/kg once daily dosing schedule for 21 days in a mouse xenograft model. The molecular docking study of 36 in the homology model of the DFG-in conformation of FLT3 resulted in a reasonable binding mode in type 1 kinases similar to the reported type 1 FLT3 inhibitors Crenolanib and Gilteritinib.

Chemical characterization and biological activity data for a novel indirubin derivative, LDD-1819.

This article contains chemical characterization and biological activity data for a novel indirubin derivative, termed LDD-1819. The detailed synthesis procedure and associated NMR data are presented. The concentration-dependent inhibition data of two biological targets, glycogen synthase kinase-3 ß and aurora kinase A are described. The following biological data are also contained in this article: 1) the cellularization of skeletal muscle myotubes by LDD-1819 or two small molecule inhibitors of glycogen synthase kinase-3 ß and aurora kinase A (BIO and reversine) and gene expression data for the myoblast markers Pax-7 and Myf5, 2) Cell viability of hTERT human immortalized fibroblasts, colon cancer cells and breast cancer cells, and 3) Western blotting analysis of full length and cleaved caspse-7, and cleaved poly (ADP-ribose) polymerase (PARP) in hTERT fibroblasts treated with LDD-1819. A schematic diagram of the biological activities of LDD-1819 is also presented. Further interpretation and discussion of these data are provided in the associated research article 'A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity' (Kim et al., 2019).

Discovery of Novel Biased Opioid Receptor Ligands through Structure-Based Pharmacophore Virtual Screening and Experiment.

Gi -protein-biased agonists with minimal ß-arrestin recruitment represent opportunities to overcome the serious adverse effects of human mu opioid receptor (µ-OR) agonists and developing alternative and safe treatments for pain. In order to discover novel non-morphinan opioid receptor agonists, we applied hierarchical virtual screening of our in-house database against a pharmacophore based on modeling the active conformations of opioid receptors. We discovered an initial hit compound, a novel µ-OR agonist with a pyrazoloisoquinoline scaffold. We applied computational R-group screening to this compound and synthesized 14 derivatives predicted to be the best. Of these, a new Gi -protein-biased compound, 1-{5-(3-chlorophenyl)-7,8-dimethoxy-3-[4-(methylsulfonyl)benzyl]-3H-pyrazolo[3,4-c]isoquinolin-1-yl}-N,N-dimethylmethanamine, showed an EC50 value of 179 nm against the µ-OR. This resulted in significant pain relief for mice in the phase II period of formalin response tests. This study provides a new strategy to identify diverse sets of promising compounds that might prove useful for the development of drugs that target other G-protein-coupled receptors.

A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity.

Indirubin-based compounds affect diverse biological processes, such as inflammation and angiogenesis. In this study, we tested a novel indirubin derivative, LDD-1819 (2-((((2Z,3E)-5-hydroxy-5'-nitro-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethan-1-aminium chloride) for two major biological activities: cell plasticity and anti-cancer activity. Biological assays indicated that LDD-1819 induced somatic cell plasticity. LDD-1819 potentiated myoblast reprogramming into osteogenic cells and fibroblast reprogramming into adipogenic cells. Interestingly, in an assay of skeletal muscle dedifferentiation, LDD-1819 induced human muscle cellularization and blocked residual proliferative activity to produce a population of mononuclear refractory cells, which is also observed in the early stages of limb regeneration in urodele amphibians. In cancer cell lines, LDD-1819 treatment inhibited cell invasion and selectively induced apoptosis compared to normal cells. In an animal tumor xenograft model, LDD-1819 reduced human cancer cell metastasis in vivo at doses that did not produce toxicity. Biochemical assays showed that LDD-1819 possessed inhibitory activity against glycogen synthase kinase-3ß, which is linked to cell plasticity, and aurora kinase, which regulates carcinogenesis. These results indicate that novel indirubin derivative LDD-1819 is a dual inhibitor of glycogen synthase kinase-3ß and aurora A kinase, and has potential for development as an anti-cancer drug or as a reprogramming agent for cell-therapy based approaches to treat degenerative diseases.

Correction: Discovery of a FLT3 inhibitor LDD1937 as an anti-leukemic agent for acute myeloid leukemia.

[This corrects the article DOI: 10.18632/oncotarget.23221.].

Discovery of a FLT3 inhibitor LDD1937 as an anti-leukemic agent for acute myeloid leukemia.

FMS-like receptor tyrosine kinase-3 (FLT3) belongs to the family of receptor tyrosine kinase (RTK), and the FLT3 mutation is observed in 1/3 of all acute myeloid leukemia (AML) patients. Potential FLT3 inhibitors have been investigated as potential therapeutic agents of AML. In this study, we identified a potent FLT3 inhibitor LDD1937 containing an indirubin skeleton. The potent inhibitory activity of LDD1937 against FLT3 was shown with an in vitro kinase assay (IC50 = 3 nM). The LDD1937 compound selectively inhibited the growth of MV-4-11 cells (GI50 = 1 nM) and induced apoptotic cell death. LDD1937 caused cell cycle arrest at the G2/M phase and increased the cell population at the sub-G1 phase. Phosphorylation of STAT5, which is the downstream signaling of FLT3, was significantly reduced by LDD1937 in a dose-dependent manner. The pharmacokinetic properties of LDD1937 were investigated in mice. Then, the in vivo anti-tumor effect was investigated using a MV-4-11 xenograft. With the intravenous administration of 5 and 10 mg/kg in nu/nu mice, the tumor volume and weight were significantly reduced compared to the control. LDD1937 is a promising therapeutic candidate to treat AML patients because of its ability to suppress tumor cell growth in vitro and in vivo.

Benserazide, the first allosteric inhibitor of Coxsackievirus B3 3C protease.

Coxsackievirus B3 is the main cause of human viral myocarditis and cardiomyopathy. Virally encoded Coxsackievirus 3C protease (3C(pro)) plays an essential role in viral proliferation. Here, benserazide was discovered as a novel inhibitor from a drug library screen targeting Coxsackievirus 3C(pro) using a FRET-based enzyme assay. Benserazide, whose chemical structure has no electrophilic functional groups, was characterized as a non-competitive inhibitor by enzyme kinetic studies. A molecular docking study with benserazide and its analogs indicated that a novel putative allosteric binding site was involved. Specifically, a 2,3,4-trihydroxybenzyl moiety was determined to be a key pharmacophore for the enzyme's inhibitory activity. We suggest that the putative allosteric binding site may be a novel target for future therapeutic strategies.

Discovery and structure-activity relationships of pyrazolodiazepine derivatives as the first small molecule agonists of the Drosophila sex peptide receptor.

In behavioral research, the sex peptide receptor in Drosophila melanogaster (DrmSPR) is the most interesting G protein-coupled receptor (GPCR) and is involved in post-mating responses such as increased egg-laying and decreased receptivity of the female; during these responses, the receptors are activated by a specific natural peptide agonist (sex peptide, SP). To discover small molecule agonists for DrmSPR, a compound library based on a pyrazolodiazepine scaffold, which was previously reported as a potential privileged structure, was screened. Structure-activity relationship (SAR) studies of the hit compounds, which exhibited weak agonistic effects (69-72% activation at 100µM), were explored through the synthesis of various analogs with substituents at the R1, R2, R3 and R4 positions of the pyrazolodiazepine skeleton. As a result, compounds 21 and 31 of the 6-benzyl pyrazolodiazepine derivative series were found to be small molecule agonists for DrmSPR with EC50 values of 3-4µM.

Pheophorbide-a conjugates with cancer-targeting moieties for targeted photodynamic cancer therapy.

Pheophorbide-a, a non-selective photosensitizer, was conjugated with cancer-targeting moieties, such as folic acid, the CRGDLASLC peptide, the cRGDfK peptide and leuprorelin, for the purpose of targeted photodynamic cancer therapy. The cellular uptake of pheophorbide-a conjugates in cancer cells overexpressing the corresponding receptors of the targeting moieties was largely enhanced compared with that in the receptor-negative cells. In the study of in vitro photodynamic activity and selectivity of pheophorbide-a conjugates in the receptor-positive and receptor-negative cells, a pheophorbide-a conjugate, (14) with an αvß6 ligand (CRGDLASLC) exhibited the highest selectivity in the positive FaDu cells. Targeted PDT with 14 induced cell death through apoptosis and morphological apoptosis-like characteristics. These results suggest that pheophorbide-a conjugate 14 could be utilized in selective photodynamic therapy for oral cancers primarily expressing the αvß6 receptor.