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1.
Cell ; 168(1-2): 86-100.e15, 2017 Jan 12.
Article in English | MEDLINE | ID: mdl-27916275

ABSTRACT

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.


Subject(s)
Artemisinins/pharmacology , Diabetes Mellitus, Type 1/drug therapy , Disease Models, Animal , Receptors, GABA-A/metabolism , Signal Transduction , Animals , Artemether , Artemisinins/administration & dosage , Carrier Proteins/metabolism , Cell Transdifferentiation/drug effects , Cells, Cultured , Diabetes Mellitus/drug therapy , Diabetes Mellitus, Type 1/pathology , Gene Expression Profiling , Homeodomain Proteins/metabolism , Humans , Insulin/genetics , Insulin/metabolism , Islets of Langerhans/drug effects , Membrane Proteins/metabolism , Mice , Protein Stability/drug effects , Rats , Single-Cell Analysis , Transcription Factors/metabolism , Zebrafish , gamma-Aminobutyric Acid/metabolism
2.
Nat Immunol ; 17(12): 1361-1372, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27798618

ABSTRACT

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.


Subject(s)
Gram-Negative Bacterial Infections/immunology , Guanine Nucleotide Exchange Factors/metabolism , Heme/metabolism , Hemolysis/immunology , Macrophages/immunology , Phagocytosis , Sepsis/immunology , Animals , Anti-Bacterial Agents/therapeutic use , Cytoskeleton/metabolism , Female , Gram-Negative Bacterial Infections/drug therapy , Guanine Nucleotide Exchange Factors/genetics , Heme Oxygenase-1/genetics , Hemolysis/drug effects , Humans , Immune Evasion , Macrophages/drug effects , Macrophages/microbiology , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Phagocytosis/drug effects , Quinine/therapeutic use , RAW 264.7 Cells , Sepsis/drug therapy , cdc42 GTP-Binding Protein/metabolism
3.
Bioorg Med Chem Lett ; 98: 129546, 2024 Jan 15.
Article in English | MEDLINE | ID: mdl-37944866

ABSTRACT

Epigenetic proteins containing YEATS domains (YD) are an emerging target class in drug discovery. Described herein are the discovery and characterization efforts associated with PFI-6, a new chemical probe for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). For hit identification, fragment-like mimetics of endogenous YD ligands (crotonylated histone-containing proteins), were synthesized via parallel medicinal chemistry (PMC) and screened for MLLT1 binding. Subsequent SAR studies led to iterative MLLT1/3 binding and selectivity improvements, culminating in the discovery of PFI-6. PFI-6 demonstrates good affinity and selectivity for MLLT1/3 vs. other human YD proteins (YEATS2/4) and engages MLLT3 in cells. Small-molecule X-ray co-crystal structures of two molecules, including PFI-6, bound to the YD of MLLT1/3 are also described. PFI-6 may be a useful tool molecule to better understand the biological effects associated with modulation of MLLT1/3.


Subject(s)
Histones , Nuclear Proteins , Humans , Nuclear Proteins/metabolism , Histones/metabolism , Protein Domains , Drug Discovery , Neoplasm Proteins/metabolism , Transcription Factors/metabolism
4.
EMBO J ; 37(17)2018 09 03.
Article in English | MEDLINE | ID: mdl-30026309

ABSTRACT

RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between ß2 and ß3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.


Subject(s)
Nod2 Signaling Adaptor Protein/metabolism , Protein Kinase Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Cell Line, Tumor , Female , Humans , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Mice , Nod2 Signaling Adaptor Protein/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Signal Transduction/genetics , X-Linked Inhibitor of Apoptosis Protein/genetics , X-Linked Inhibitor of Apoptosis Protein/metabolism
5.
Biol Chem ; 403(4): 391-402, 2022 03 28.
Article in English | MEDLINE | ID: mdl-35191283

ABSTRACT

Ubiquitination is a key regulatory mechanism vital for maintenance of cellular homeostasis. Protein degradation is induced by E3 ligases via attachment of ubiquitin chains to substrates. Pharmacological exploitation of this phenomenon via targeted protein degradation (TPD) can be achieved with molecular glues or bifunctional molecules facilitating the formation of ternary complexes between an E3 ligase and a given protein of interest (POI), resulting in ubiquitination of the substrate and subsequent proteolysis by the proteasome. Recently, the development of novel covalent fragment screening approaches has enabled the identification of first-in-class ligands for E3 ligases and deubiquitinases revealing so far unexplored binding sites which highlights the potential of these methods to uncover and expand druggable space for new target classes.


Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin , Ligands , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
6.
Br J Cancer ; 124(4): 817-830, 2021 02.
Article in English | MEDLINE | ID: mdl-33214684

ABSTRACT

BACKGROUND: Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. METHODS: In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. RESULTS: Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. CONCLUSIONS: Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.


Subject(s)
Colorectal Neoplasms/enzymology , Colorectal Neoplasms/immunology , Cytokines/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Ubiquitin Thiolesterase/metabolism , Ubiquitins/metabolism , Antigenic Variation , Cell Line, Tumor , Colorectal Neoplasms/radiotherapy , Gene Knockout Techniques , HCT116 Cells , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/radiotherapy , Radiation Tolerance/genetics , Radiation Tolerance/immunology , Ubiquitin Thiolesterase/deficiency , Ubiquitin Thiolesterase/genetics
7.
Chembiochem ; 22(12): 2116-2123, 2021 06 15.
Article in English | MEDLINE | ID: mdl-33887086

ABSTRACT

Selinexor, a covalent XPO1 inhibitor, is approved in the USA in combination with dexamethasone for penta-refractory multiple myeloma. Additional XPO1 covalent inhibitors are currently in clinical trials for multiple diseases including hematologic malignancies, solid tumor malignancies, glioblastoma multiforme (GBM), and amyotrophic lateral sclerosis (ALS). It is important to measure the target engagement and selectivity of covalent inhibitors to understand the degree of engagement needed for efficacy, while avoiding both mechanism-based and off-target toxicity. Herein, we report clickable probes based on the XPO1 inhibitors selinexor and eltanexor for the labeling of XPO1 in live cells to assess target engagement and selectivity. We used mass spectrometry-based chemoproteomic workflows to profile the proteome-wide selectivity of selinexor and eltanexor and show that they are highly selective for XPO1. Thermal profiling analysis of selinexor further offers an orthogonal approach to measure XPO1 engagement in live cells. We believe these probes and assays will serve as useful tools to further interrogate the biology of XPO1 and its inhibition in cellular and in vivo systems.


Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Antineoplastic Agents/pharmacology , Glioblastoma/drug therapy , Hydrazines/pharmacology , Karyopherins/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Triazoles/pharmacology , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Hydrazines/chemistry , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Triazoles/chemistry , Exportin 1 Protein
8.
Nat Chem Biol ; 15(8): 822-829, 2019 08.
Article in English | MEDLINE | ID: mdl-31285596

ABSTRACT

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.


Subject(s)
Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , CRISPR-Cas Systems , Cell Line , Cell Proliferation/drug effects , Cell Survival , Gene Expression Regulation/drug effects , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Domains , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism
9.
Nature ; 519(7544): 477-81, 2015 Mar 26.
Article in English | MEDLINE | ID: mdl-25561175

ABSTRACT

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.


Subject(s)
Amino Acid Transport Systems/metabolism , Amino Acids/metabolism , Lysosomes/metabolism , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cell Line , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice , Monomeric GTP-Binding Proteins/metabolism , Nucleotides/metabolism
10.
Nature ; 508(7495): 222-7, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24695225

ABSTRACT

Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.


Subject(s)
Antineoplastic Agents/pharmacology , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Aminoquinolines/pharmacology , Animals , Antineoplastic Agents/chemistry , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Crizotinib , Crystallization , DNA Breaks, Single-Stranded/drug effects , DNA Repair , DNA Repair Enzymes/biosynthesis , DNA Repair Enzymes/chemistry , Disease Models, Animal , Female , Homeostasis/drug effects , Humans , Mice , Mice, SCID , Models, Molecular , Nucleotides/metabolism , Phosphoric Monoester Hydrolases/biosynthesis , Phosphoric Monoester Hydrolases/chemistry , Protein Conformation , Protein Kinase Inhibitors/chemistry , Proteomics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Pyrazoles/chemistry , Pyridines/chemistry , Substrate Specificity , Xenograft Model Antitumor Assays , ras Proteins/genetics
11.
J Am Chem Soc ; 141(22): 8951-8968, 2019 06 05.
Article in English | MEDLINE | ID: mdl-31060360

ABSTRACT

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.


Subject(s)
Drug Evaluation, Preclinical/methods , Electrons , HEK293 Cells , Humans , Ligands , Models, Molecular , Molecular Weight , Protein Conformation , Time Factors
12.
Int J Med Microbiol ; 309(2): 130-142, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30665874

ABSTRACT

Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.


Subject(s)
Antiprotozoal Agents/pharmacology , Giardia lamblia/drug effects , Histone Deacetylase Inhibitors/pharmacology , Trophozoites/drug effects , Caco-2 Cells , Cytokinesis/drug effects , Giardia lamblia/cytology , Giardia lamblia/growth & development , Humans , Inhibitory Concentration 50 , Microscopy , Microscopy, Electron , Parasitic Sensitivity Tests , Trophozoites/cytology , Trophozoites/growth & development
13.
Nat Chem Biol ; 13(6): 681-690, 2017 06.
Article in English | MEDLINE | ID: mdl-28437395

ABSTRACT

Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.


Subject(s)
Immunomodulation/drug effects , Small Molecule Libraries/pharmacology , Cardiac Myosins/metabolism , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Crizotinib , Humans , Myosin Heavy Chains/metabolism , Pyrazoles/pharmacology , Pyridines/pharmacology
14.
Angew Chem Int Ed Engl ; 58(2): 515-519, 2019 01 08.
Article in English | MEDLINE | ID: mdl-30431220

ABSTRACT

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

15.
Nat Methods ; 12(11): 1055-7, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26389571

ABSTRACT

Thermal stabilization of proteins after ligand binding provides an efficient means to assess the binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry, the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. We profiled the targets of the drugs methotrexate and (S)-crizotinib and the metabolite 2'3'-cGAMP in intact cells and identified the 2'3'-cGAMP cognate transmembrane receptor STING, involved in immune signaling.


Subject(s)
Proteome/metabolism , Pyrazoles/chemistry , Pyridines/chemistry , Animals , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Computational Biology , Crizotinib , Drug Design , Humans , Immune System , K562 Cells , Ligands , Mass Spectrometry , Methotrexate/chemistry , Mice , Protein Binding , Protein Kinase Inhibitors/chemistry , Proteomics , Signal Transduction , Systems Biology , Temperature
16.
Nat Chem Biol ; 12(7): 504-10, 2016 07.
Article in English | MEDLINE | ID: mdl-27159579

ABSTRACT

Bromodomain-containing proteins of the BET family recognize histone lysine acetylation and mediate transcriptional activation of target genes such as the MYC oncogene. Pharmacological inhibitors of BET domains promise therapeutic benefits in a variety of cancers. We performed a high-diversity chemical compound screen for agents capable of modulating BRD4-dependent heterochromatization of a generic reporter in human cells. In addition to known and new compounds targeting BRD4, we identified small molecules that mimic BRD4 inhibition without direct engagement. One such compound was a potent inhibitor of the second bromodomain of TAF1. Using this inhibitor, we discovered that TAF1 synergizes with BRD4 to control proliferation of cancer cells, making TAF1 an attractive epigenetic target in cancers driven by MYC.


Subject(s)
Chromatin/chemistry , Histone Acetyltransferases/antagonists & inhibitors , Histone Acetyltransferases/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Small Molecule Libraries/pharmacology , TATA-Binding Protein Associated Factors/antagonists & inhibitors , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/antagonists & inhibitors , Transcription Factor TFIID/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Cell Cycle Proteins , Cell Line , Cell Proliferation/drug effects , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Histone Acetyltransferases/chemistry , Humans , Molecular Structure , Nuclear Proteins/chemistry , Protein Domains/drug effects , Small Molecule Libraries/chemistry , TATA-Binding Protein Associated Factors/chemistry , Transcription Factor TFIID/chemistry , Transcription Factors/chemistry
17.
Angew Chem Int Ed Engl ; 57(50): 16302-16307, 2018 12 10.
Article in English | MEDLINE | ID: mdl-30288907

ABSTRACT

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine-binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small-molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3-histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small-molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first-in-class probe molecule can be used to understand MLLT1/3-associated biology and the therapeutic potential of small-molecule YD inhibitors.


Subject(s)
Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Small Molecule Libraries/chemistry , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Crystallography, X-Ray , Histones/metabolism , Humans , Molecular Docking Simulation , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Protein Domains , Protein Interaction Maps/drug effects , Small Molecule Libraries/pharmacology , Transcription Factors/metabolism
18.
Apoptosis ; 22(9): 1169-1188, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28685254

ABSTRACT

Treatment of leishmaniasis involves the use of antimonials, miltefosine, amphotericin B or pentamidine. However, the side effects of these drugs and the reports of drug-resistant parasites demonstrate the need for new treatments that are safer and more efficacious. Histone deacetylase inhibitors are a new class of compounds with potential to treat leishmaniasis. Herein, we evaluated the effects of KH-TFMDI, a novel histone deacetylase inhibitor, on Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values of this compound for promastigotes and intracellular amastigotes were 1.976 and 1.148 µM, respectively, after 72 h of treatment. Microscopic analyses revealed that promastigotes became elongated and thinner in response to KH-TFMDI, indicating changes in cytoskeleton organization. Immunofluorescence microscopy, western blotting and flow cytometry using an anti-acetylated tubulin antibody revealed an increase in the expression of acetylated tubulin. Furthermore, transmission electron microscopy revealed several ultrastructural changes, such as (a) mitochondrial swelling, followed by the formation of many vesicles inside the matrix; (b) presence of lipid bodies randomly distributed through the cytoplasm; (c) abnormal chromatin condensation; and (d) formation of blebs on the plasma membrane. Physiological studies for mitochondrial function, flow cytometry with propidium iodide and TUNEL assay confirmed the alterations in the mitochondrial metabolism, cell cycle, and DNA fragmentation, respectively, which could result to cell death by mechanisms related to apoptosis-like. All these together indicate that histone deacetylases are promising targets for the development of new drugs to treat Leishmania, and KH-TFMDI is a promising drug candidate that should be tested in vivo.


Subject(s)
Benzylidene Compounds/pharmacology , Cell Death/drug effects , Cytoskeleton/drug effects , Histone Deacetylase Inhibitors/pharmacology , Indoles/pharmacology , Leishmania/drug effects , Mitochondria/drug effects , Sirtuins/antagonists & inhibitors , Animals , Antiparasitic Agents/pharmacology , Antiparasitic Agents/toxicity , Apoptosis/drug effects , Benzylidene Compounds/toxicity , Cell Cycle Checkpoints/drug effects , Cell Survival/drug effects , Cytoskeleton/metabolism , Histone Deacetylase Inhibitors/toxicity , Indoles/toxicity , Inhibitory Concentration 50 , Leishmania/cytology , Leishmania/growth & development , Leishmania/ultrastructure , Leishmaniasis/drug therapy , Leishmaniasis/parasitology , Lipid Droplets/drug effects , Lipid Droplets/metabolism , Microtubules/drug effects , Microtubules/metabolism , Oxidative Stress/drug effects
19.
Nat Chem Biol ; 11(8): 571-578, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26167872

ABSTRACT

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.


Subject(s)
Antineoplastic Agents/pharmacology , Biphenyl Compounds/pharmacology , Dihydropyridines/pharmacology , Gene Expression Regulation, Neoplastic , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Leukemia, Myeloid, Acute/metabolism , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Animals , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Molecular Docking Simulation , Molecular Sequence Data , Molecular Targeted Therapy , Mutation , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Signal Transduction , Tumor Cells, Cultured
20.
Bioorg Med Chem ; 25(9): 2657-2665, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28341403

ABSTRACT

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75µM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Rhodanine/analogs & derivatives , Sulfonamides/pharmacology , Thiazolidines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , K562 Cells , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Molecular Docking Simulation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Rhodanine/chemical synthesis , Rhodanine/pharmacokinetics , Rhodanine/pharmacology , Solubility , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Thiazolidines/chemical synthesis , Thiazolidines/pharmacokinetics
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