ABSTRACT
Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.
Subject(s)
Epigenesis, Genetic , Myelin Sheath , Oligodendroglia , Remyelination , Animals , Myelin Sheath/metabolism , Humans , Mice , Remyelination/drug effects , Oligodendroglia/metabolism , Central Nervous System/metabolism , Mice, Inbred C57BL , Rejuvenation , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/drug effects , Sterol Regulatory Element Binding Protein 1/metabolism , Organoids/metabolism , Organoids/drug effects , Demyelinating Diseases/metabolism , Demyelinating Diseases/genetics , Cell Differentiation/drug effects , Small Molecule Libraries/pharmacology , Male , Regeneration/drug effects , Multiple Sclerosis/metabolism , Multiple Sclerosis/genetics , Multiple Sclerosis/drug therapy , Multiple Sclerosis/pathologyABSTRACT
Neurofibromatosis type 1, a genetic disorder caused by pathogenic germline variations in NF1, predisposes individuals to the development of tumors, including cutaneous and plexiform neurofibromas (CNs and PNs), optic gliomas, astrocytomas, juvenile myelomonocytic leukemia, high-grade gliomas and malignant peripheral nerve sheath tumors (MPNSTs), which are chemotherapy- and radiation-resistant sarcomas with poor survival. Loss of NF1 also occurs in sporadic tumors, such as glioblastoma (GBM), melanoma, breast, ovarian and lung cancers. We performed a high-throughput screen for compounds that were synthetic lethal with NF1 loss, which identified several leads, including the small molecule Y102. Treatment of cells with Y102 perturbed autophagy, mitophagy and lysosome positioning in NF1-deficient cells. A dual proteomics approach identified BLOC-one-related complex (BORC), which is required for lysosome positioning and trafficking, as a potential target of Y102. Knockdown of a BORC subunit using siRNA recapitulated the phenotypes observed with Y102 treatment. Our findings demonstrate that BORC might be a promising therapeutic target for NF1-deficient tumors.
Subject(s)
Lysosomes , Neurofibromin 1 , Humans , Lysosomes/metabolism , Neurofibromin 1/genetics , Neurofibromin 1/metabolism , Neurofibromatosis 1/metabolism , Neurofibromatosis 1/genetics , Neurofibromatosis 1/pathology , Autophagy/drug effects , Synthetic Lethal Mutations , Protein Transport/drug effectsABSTRACT
Current antiplatelet therapies have several clinical complications and are mostly irreversible in terms of suppressing platelet activity; hence, there is a need to develop improved therapeutic agents. Previous studies have implicated RhoA in platelet activation. Here, we further characterized the lead RhoA inhibitor, Rhosin/G04, in platelet function and present structure-activity relationship (SAR) analysis. A screening for Rhosin/G04 analogs in our chemical library by similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. A screening for Rhosin/G04 analogs in our chemical library using similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. SAR analysis revealed that the active compounds have a quinoline group optimally attached to the hydrazine at the 4-position and halogen substituents at the 7- or 8-position. Having indole, methylphenyl, or dichloro-phenyl substituents led to better potency. Rhosin/G04 contains a pair of enantiomers, and S-G04 is significantly more potent than R-G04 in inhibiting RhoA activation and platelet aggregation. Furthermore, the inhibitory effect is reversible, and S-G04 is capable of inhibiting diverse-agonist-stimulated platelet activation. This study identified a new generation of small-molecule RhoA inhibitors, including an enantiomer capable of broadly and reversibly modulating platelet activity.
Subject(s)
Platelet Aggregation Inhibitors , rhoA GTP-Binding Protein , Platelet Aggregation Inhibitors/pharmacology , rhoA GTP-Binding Protein/metabolism , Blood Platelets/metabolism , Organic Chemicals/pharmacology , Structure-Activity RelationshipABSTRACT
PURPOSE: Emerging evidence suggests that 5' Adenosine Monophosphate-Activated Protein Kinase (AMPK), a key regulator of cellular bioenergetics, is a novel target for the treatment of glioblastoma (GBM), a lethal brain tumor. SBI-0206965, an aminopyrimidine derivative, is a potent AMPK inhibitor being investigated for the treatment of GBM. Here we characterized the systemic and brain pharmacokinetics (PK) and hepatic metabolism of SBI-0206965. METHODS: We performed intracerebral microdialysis to determine brain partitioning of SBI-0206965 in jugular vein cannulated rats. We assessed systemic PK of SBI-0206965 in rats and C57BL/6 mice following oral administration. Employing human, mouse, and rat liver microsomes we characterized the metabolism of SBI-0206965. RESULTS: SBI-0206965 is quickly absorbed, achieving plasma and brain extracellular fluid (ECF) peak levels within 0.25 - 0.65 h. Based on the ratio of Cmax and AUC in brain ECF to plasma (corrected for protein binding), brain partitioning is ~ 0.6-0.9 in rats. However, the compound has a short elimination half-life (1-2 h) and low relative oral bioavailability (~ 0.15). The estimated in-vitro hepatic intrinsic clearance of SBI-0206965 in mouse, rat and human was 325, 76 and 68 mL/min/kg, respectively. SBI-0206965 metabolites included desmethylated products, and the metabolism was strongly inhibited by ketoconazole, a CYP3A inhibitor. CONCLUSION: SBI-0206965 has adequate brain permeability but low relative oral bioavailability which may be due to rapid hepatic metabolism, likely catalyzed by CYP3A enzymes. Our observations will facilitate further development of SBI-0206965, and/or other structurally related molecules, for the treatment of GBM and other brain tumors.
Subject(s)
Brain Neoplasms , Glioblastoma , AMP-Activated Protein Kinases/metabolism , Animals , Benzamides , Brain/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Drugs, Investigational , Glioblastoma/drug therapy , Glioblastoma/metabolism , Humans , Mice , Mice, Inbred C57BL , Pyrimidines , RatsABSTRACT
Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa (RP). Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening (HTS) of 68, 979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type (WT) rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection (IVI) of 25 pmol MTX increased electroretinogram (ERG) response and rhodopsin level in the retinae of RhoP23H/+ knock-in mice at 1 month of age. Additionally, four weekly IVIs increased the photoreceptor cell number in the retinae of RhoP23H/+ mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin-associated RP.
Subject(s)
Retinitis Pigmentosa/metabolism , Rhodopsin/metabolism , Animals , Cell Line , Electroretinography/methods , Female , HEK293 Cells , Humans , Male , Mice , Mutant Proteins/metabolism , Mutation/genetics , NIH 3T3 Cells , Photoreceptor Cells/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Folding , Retina/metabolism , Retinitis Pigmentosa/genetics , Rhodopsin/geneticsABSTRACT
The vacuolar (Hâº)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I-IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a-e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 µM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.
Subject(s)
Antineoplastic Agents/chemistry , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemistry , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Bisbenzimidazole/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor/methods , Female , Humans , Ovarian Neoplasms/drug therapy , Triple Negative Breast Neoplasms/drug therapyABSTRACT
The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.
Subject(s)
Enzyme Inhibitors , ras GTPase-Activating Proteins/antagonists & inhibitors , ras Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Dose-Response Relationship, Drug , Drug Delivery Systems/methods , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , ras GTPase-Activating Proteins/genetics , ras GTPase-Activating Proteins/metabolism , ras Guanine Nucleotide Exchange Factors/genetics , ras Guanine Nucleotide Exchange Factors/metabolismABSTRACT
The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a â¼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein-coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG-RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme-substrate pair of GEF-RhoA at their binding interface that leads to enhanced efficacy and specificity.
Subject(s)
Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Amino Acid Sequence , Cell Division/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Humans , Neoplasm Invasiveness/prevention & control , Sequence Homology, Amino AcidABSTRACT
All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.
Subject(s)
Diterpenes/metabolism , Photic Stimulation/adverse effects , Retinal Degeneration/metabolism , Retinal Degeneration/prevention & control , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/radiation effects , Retinaldehyde/metabolism , Animals , Cattle , Diterpenes/pharmacology , Diterpenes/therapeutic use , Female , Male , Mice , Mice, Knockout , Retinal Pigment Epithelium/drug effectsABSTRACT
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential therapeutic agent that induces apoptosis selectively in tumor cells. However, numerous solid tumor types are resistant to TRAIL. Sensitization to TRAIL has been an area of great research interest, but has met significant challenges because of poor bioavailability, half-life, and solubility of sensitizing compounds such as curcumin. Soluble, TRAIL-sensitizing compounds were screened on the basis of similarity to the redox-active substructure of curcumin and sensitization to TRAIL-induced apoptosis. We determined the effect of the lead compound, C25, in combination with TRAIL in human cancer cell lines using MTS proliferation assays, apoptosis assays, and western blotting. Short hairpin RNA knockdown of death receptor 5 (DR5) was performed to determine whether DR5 upregulation was required for TRAIL-mediated apoptosis. In-vivo efficacy was determined using human lung tumor xenograft models. C25 helped overcome TRAIL resistance by upregulating the expression of the TRAIL receptor DR5 and apoptosis in several tumor cell lines. Blockade of DR5 expression abrogated C25 sensitization to TRAIL, demonstrating the requirement for DR5 upregulation for C25-mediated potentiation of TRAIL-mediated apoptosis. The combination of C25 and TRAIL effectively inhibited tumorigenesis in vivo. This study demonstrates the synergistic efficacy of C25 in sensitization to TRAIL-induced apoptosis in multiple tumor cell types, including highly resistant lung and ovarian tumor cell lines. Furthermore, C25 was efficacious against tumor growth in vivo. Thus, C25 may be a potential therapeutic for cancer in combination with TRAIL or DR5 agonist therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Hydrazones/pharmacology , Pyrazoles/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Caspase 3/metabolism , Cell Line, Tumor , Databases, Chemical , Drug Screening Assays, Antitumor , Drug Synergism , Female , Heterografts , Humans , Hydrazones/chemistry , Hydrazones/therapeutic use , Mice, Nude , Mitochondria/metabolism , Neoplasm Transplantation , Pyrazoles/chemistry , Pyrazoles/therapeutic use , Signal Transduction , Solubility , TNF-Related Apoptosis-Inducing Ligand/therapeutic use , Tumor Burden/drug effects , Up-RegulationABSTRACT
Aspartyl aminopeptidase (DNPEP) has been implicated in the control of angiotensin signaling and endosome trafficking, but its precise biologic roles remain incompletely defined. We performed a high-throughput screen of â¼25,000 small molecules to identify inhibitors of DNPEP for use as tools to study its biologic functions. Twenty-three confirmed hits inhibited DNPEP-catalyzed hydrolysis of angiotensin II with micromolar potency. A counter screen against glutamyl aminopeptidase (ENPEP), an enzyme with substrate specificity similar to that of DNPEP, identified eight DNPEP-selective inhibitors. Structure-activity relationships and modeling studies revealed structural features common to the identified inhibitors, including a metal-chelating group and a charged or polar moiety that could interact with portions of the enzyme active site. The compounds identified in this study should be valuable tools for elucidating DNPEP physiology.
Subject(s)
Enzyme Inhibitors/pharmacology , Glutamyl Aminopeptidase/antagonists & inhibitors , Glutamyl Aminopeptidase/metabolism , Angiotensin II/metabolism , Animals , Catalytic Domain/drug effects , Humans , Hydrolysis/drug effects , Mammals , Sf9 Cells , Spodoptera , Structure-Activity RelationshipABSTRACT
BACKGROUND: Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. METHODS: We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. RESULTS: High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. CONCLUSIONS: Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.
Subject(s)
Brain Stem Neoplasms , Diffuse Intrinsic Pontine Glioma , Imidazoles , Pyridines , Pyrimidines , Child , Humans , Epigenesis, Genetic , Histones/genetics , Diffuse Intrinsic Pontine Glioma/genetics , Brain Stem Neoplasms/drug therapy , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/pathology , Methyltransferases/genetics , Methyltransferases/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , PiperazinesABSTRACT
We present an in silico approach for drug discovery, dubbed connectivity enhanced structure activity relationship (ceSAR). Building on the landmark LINCS library of transcriptional signatures of drug-like molecules and gene knockdowns, ceSAR combines cheminformatic techniques with signature concordance analysis to connect small molecules and their targets and further assess their biophysical compatibility using molecular docking. Candidate compounds are first ranked in a target structure-independent manner, using chemical similarity to LINCS analogs that exhibit transcriptomic concordance with a target gene knockdown. Top candidates are subsequently rescored using docking simulations and machine learning-based consensus of the two approaches. Using extensive benchmarking, we show that ceSAR greatly reduces false-positive rates, while cutting run times by multiple orders of magnitude and further democratizing drug discovery pipelines. We further demonstrate the utility of ceSAR by identifying and experimentally validating inhibitors of BCL2A1, an important antiapoptotic target in melanoma and preterm birth-associated inflammation.
Subject(s)
Drug Discovery , Drug Repositioning , Molecular Docking Simulation , Drug Discovery/methods , Drug Repositioning/methods , Humans , Transcriptome , Machine Learning , Structure-Activity RelationshipABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0071093.].
ABSTRACT
Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and â¼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.
Subject(s)
Antineoplastic Agents/chemistry , Benzamides/chemistry , Benzeneacetamides/chemistry , Hydrazines/chemistry , Indoles/chemistry , Phosphodiesterase Inhibitors/chemistry , Phosphoric Diester Hydrolases/chemistry , Sulfonamides/chemistry , Tetrazoles/chemistry , Animals , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Benzeneacetamides/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , High-Throughput Screening Assays , Humans , Hydrazines/pharmacology , Hydrophobic and Hydrophilic Interactions , Indoles/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Melanoma, Experimental/drug therapy , Melanoma, Experimental/secondary , Mice , Mice, Inbred C57BL , Molecular Docking Simulation , Mutation , Neoplasm Invasiveness , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Structure-Activity Relationship , Sulfonamides/pharmacology , Tetrazoles/pharmacologyABSTRACT
Despite significant advancements in developing selective FMS-like tyrosine kinase 3 (FLT3) inhibitors, resistance to treatment is common even on continued therapy. Acquisition of on-target mutations or adaptation to MAPK, JAK2, and ABL signaling pathways drive treatment failure and disease relapse. Although combinatorial targeting of all escape routes in preclinical models demonstrated its efficacy, the clinical application is challenging owing to drug-drug interaction and differing pharmacokinetics of the inhibitors. We reasoned that selective polypharmacological targeting could lead to a durable response with reduced toxicity. A cell-based screening was carried out to identify inhibitors targeting FLT3, RAS-MAPK, BCR-ABL, and JAK2 to target the adaptive resistance observed with FLT3 inhibitors. Here, we show that pluripotin is an equipotent inhibitor of FLT3, BCR-ABL, and JAK2 in addition to inhibiting Ras-GAP and extracellular signal-regulated kinase 1 (ERK1). Structural modeling studies revealed that pluripotin is a type II kinase inhibitor that selectively binds with inactive conformations of FLT3, ABL, and JAK2. Pluripotin showed potent inhibitory activity on both mouse and human cells expressing FLT3ITD, including clinically challenging resistant mutations of the gatekeeper residue, F691L. Likewise, pluripotin suppressed the adaptive resistance conferred by the activation of RAS-MAPK pathways, BCR-ABL, and JAK2 signaling. Treatment with pluripotin curbed the progression of acute myeloid leukemia (AML) in multiple in vivo models including patient-derived primary AML cells in mouse xenotransplants. As a proof of concept, we demonstrate that targeted polypharmacological inhibition of key signaling nodes driving adaptive resistance can provide a durable response.
Subject(s)
Leukemia, Myeloid, Acute , fms-Like Tyrosine Kinase 3 , Humans , Animals , Mice , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/therapeutic use , Mitogen-Activated Protein Kinase 3 , Leukemia, Myeloid, Acute/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction , Janus Kinase 2/geneticsABSTRACT
Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 µM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 µM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 µM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.
Subject(s)
Cell Division , Chromatin/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Animals , Cell Cycle , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Inhibitory Concentration 50 , Male , Mice , Proliferating Cell Nuclear Antigen/drug effectsABSTRACT
As a continuation of our efforts to discover and develop small molecules as anticancer agents, we identified GRI-394837 as an initial hit from similarity search on RGD and its analogs. Based on GRI-394837, we designed and synthesized a focused set of novel chromenes (4a-e) in a single step using microwave method. All five compounds showed activity in the nanomolar range (IC(50): 7.4-640 nM) in two melanoma, three prostate and four glioma cancer cell lines. The chromene 4e is active against all the cell lines and particularly against the A172 human glioma cell line (IC(50): 7.4 nM). Interestingly, in vitro tubulin polymerization assay shows 4e to be a weak tubulin polymerization inhibitor but it shows very strong cytotoxicity in cellular assays, therefore there must be additional unknown mechanism(s) for the anticancer activity. Additionally, the strong antiproliferative activity was verified by one of the selected chromene (4a) by the NCI 60 cell line screen. These results strongly suggest that the novel chromenes could be further developed as a potential therapeutic agent for a variety of aggressive cancers.
Subject(s)
Antineoplastic Agents/chemistry , Benzopyrans/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , Astrocytes/drug effects , Benzopyrans/chemical synthesis , Benzopyrans/toxicity , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Computer Simulation , Drug Screening Assays, Antitumor , Humans , Oligopeptides/chemistry , Protein Structure, Tertiary , Tubulin/chemistry , Tubulin/metabolism , Tubulin Modulators/chemical synthesis , Tubulin Modulators/chemistry , Tubulin Modulators/toxicityABSTRACT
Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-vav/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Antineoplastic Agents/therapeutic use , Female , Humans , Male , Mice, Inbred C57BL , Mice, SCID , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-vav/metabolism , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic use , Tumor Cells, CulturedABSTRACT
Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.