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1.
Nat Nanotechnol ; 18(11): 1351-1363, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37443252

ABSTRACT

Intravenously administered cyclic dinucleotides and other STING agonists are hampered by low cellular uptake and poor circulatory half-life. Here we report the covalent conjugation of cyclic dinucleotides to poly(ß-amino ester) nanoparticles through a cathepsin-sensitive linker. This is shown to increase stability and loading, thereby expanding the therapeutic window in multiple syngeneic tumour models, enabling the study of how the long-term fate of the nanoparticles affects the immune response. In a melanoma mouse model, primary tumour clearance depends on the STING signalling by host cells-rather than cancer cells-and immune memory depends on the spleen. The cancer cells act as a depot for the nanoparticles, releasing them over time to activate nearby immune cells to control tumour growth. Collectively, this work highlights the importance of nanoparticle structure and nano-biointeractions in controlling immunotherapy efficacy.


Subject(s)
Melanoma , Nanoparticles , Neoplasms , Animals , Mice , Polymers/pharmacology , Neoplasms/drug therapy , Signal Transduction , Nanoparticles/therapeutic use , Nanoparticles/chemistry
2.
Science ; 376(6597): 1074-1079, 2022 06 03.
Article in English | MEDLINE | ID: mdl-35653481

ABSTRACT

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.


Subject(s)
Antimalarials , Malaria, Falciparum , Molecular Targeted Therapy , Plasmodium falciparum , Protein Biosynthesis , Protozoan Proteins , Tyrosine-tRNA Ligase , Adenosine/analogs & derivatives , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Antimalarials/therapeutic use , Crystallography, X-Ray , Humans , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Mice , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Protein Biosynthesis/drug effects , Protein Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Sulfonic Acids/chemistry , Tyrosine-tRNA Ligase/chemistry , Tyrosine-tRNA Ligase/metabolism
3.
J Med Chem ; 64(10): 6902-6923, 2021 05 27.
Article in English | MEDLINE | ID: mdl-34000802

ABSTRACT

Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.


Subject(s)
Membrane Proteins/agonists , Nucleotides, Cyclic/chemistry , Pyrimidines/chemistry , Administration, Intravenous , Animals , Binding Sites , Cell Line, Tumor , Half-Life , Humans , Immunotherapy , Membrane Proteins/metabolism , Mice , Molecular Docking Simulation , Neoplasms/pathology , Neoplasms/therapy , Nucleotides, Cyclic/metabolism , Nucleotides, Cyclic/therapeutic use , Phosphates/chemistry , Rats , Structure-Activity Relationship , Transplantation, Heterologous
4.
J Med Chem ; 64(5): 2501-2520, 2021 03 11.
Article in English | MEDLINE | ID: mdl-33631934

ABSTRACT

SUMOylation is a reversible post-translational modification that regulates protein function through covalent attachment of small ubiquitin-like modifier (SUMO) proteins. The process of SUMOylating proteins involves an enzymatic cascade, the first step of which entails the activation of a SUMO protein through an ATP-dependent process catalyzed by SUMO-activating enzyme (SAE). Here, we describe the identification of TAK-981, a mechanism-based inhibitor of SAE which forms a SUMO-TAK-981 adduct as the inhibitory species within the enzyme catalytic site. Optimization of selectivity against related enzymes as well as enhancement of mean residence time of the adduct were critical to the identification of compounds with potent cellular pathway inhibition and ultimately a prolonged pharmacodynamic effect and efficacy in preclinical tumor models, culminating in the identification of the clinical molecule TAK-981.


Subject(s)
Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Neoplasms/drug therapy , Sulfonic Acids/therapeutic use , Sumoylation/drug effects , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Mice , Molecular Structure , Protein Binding , Protein Processing, Post-Translational/drug effects , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/metabolism , Ubiquitin-Activating Enzymes/metabolism , Xenograft Model Antitumor Assays
5.
Bioorg Med Chem Lett ; 26(4): 1156-60, 2016 Feb 15.
Article in English | MEDLINE | ID: mdl-26804230

ABSTRACT

Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.


Subject(s)
Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Tetrahydronaphthalenes/chemistry , Animals , Cell Line, Tumor , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Inhibitory Concentration 50 , Male , Mice , Mutagenesis , Neoplasms/drug therapy , Neoplasms/pathology , Protein Binding , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Tetrahydronaphthalenes/pharmacokinetics , Tetrahydronaphthalenes/therapeutic use , Transplantation, Heterologous
6.
J Med Chem ; 54(6): 1836-46, 2011 Mar 24.
Article in English | MEDLINE | ID: mdl-21341678

ABSTRACT

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.


Subject(s)
Antineoplastic Agents/chemical synthesis , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Tetrahydronaphthalenes/chemical synthesis , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Availability , Crystallography, X-Ray , Drug Design , Melanoma, Experimental/drug therapy , Melanoma, Experimental/enzymology , Melanoma, Experimental/pathology , Mice , Mice, Nude , Models, Molecular , Mutation , Proto-Oncogene Proteins B-raf/genetics , Stereoisomerism , Structure-Activity Relationship , Tetrahydronaphthalenes/chemistry , Tetrahydronaphthalenes/pharmacology , Xenograft Model Antitumor Assays
7.
Bioorg Med Chem Lett ; 20(16): 4800-4, 2010 Aug 15.
Article in English | MEDLINE | ID: mdl-20634068

ABSTRACT

The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.


Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazoles/chemistry , Amino Acid Substitution , Binding Sites , Computer Simulation , Drug Evaluation, Preclinical , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship
8.
Blood ; 116(9): 1515-23, 2010 Sep 02.
Article in English | MEDLINE | ID: mdl-20525923

ABSTRACT

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.


Subject(s)
Cyclopentanes/pharmacology , Germinal Center/drug effects , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/pathology , NF-kappa B/metabolism , Pyrimidines/pharmacology , Ubiquitins/antagonists & inhibitors , Animals , Apoptosis/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , DNA Replication/drug effects , Female , Flow Cytometry , Germinal Center/metabolism , Germinal Center/pathology , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , NEDD8 Protein , NF-kappa B/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitins/metabolism , Xenograft Model Antitumor Assays
9.
Mol Cell ; 37(1): 102-11, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-20129059

ABSTRACT

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.


Subject(s)
Adenosine Monophosphate/metabolism , Cyclopentanes/metabolism , Enzyme Inhibitors/metabolism , Pyrimidines/metabolism , Ubiquitins/metabolism , Adenosine Monophosphate/chemistry , Binding Sites , Binding, Competitive , Cell Line, Tumor , Crystallography, X-Ray , Cyclopentanes/chemistry , Cyclopentanes/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , NEDD8 Protein , Protein Structure, Tertiary , Pyrimidines/chemistry , Pyrimidines/pharmacology , Ubiquitins/chemistry
10.
Nature ; 458(7239): 732-6, 2009 Apr 09.
Article in English | MEDLINE | ID: mdl-19360080

ABSTRACT

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Cyclopentanes/pharmacology , Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Pyrimidines/pharmacology , Ubiquitin-Activating Enzymes/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Cullin Proteins/metabolism , Female , Humans , Mice , NEDD8 Protein , Proteasome Inhibitors , Transplantation, Heterologous , Ubiquitins/metabolism
11.
Med Res Rev ; 25(3): 310-30, 2005 May.
Article in English | MEDLINE | ID: mdl-15593285

ABSTRACT

This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an 'in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection.


Subject(s)
Drug Design , Protein Kinase Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Isoquinolines/chemical synthesis , Isoquinolines/pharmacology , Naphthalenes/chemical synthesis , Naphthalenes/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Purines/chemical synthesis , Purines/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology
12.
J Biol Chem ; 279(14): 13976-83, 2004 Apr 02.
Article in English | MEDLINE | ID: mdl-14722102

ABSTRACT

Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic beta-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K(m) and V(max) values determined using glucose-6-phosphate as the substrate were 0.45 mm and 32 nmol/mg/min by malachite green assay, and 0.29 mm and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.


Subject(s)
Diabetes Mellitus, Type 2/metabolism , Islets of Langerhans/enzymology , Proteins/genetics , Proteins/metabolism , Animals , Baculoviridae/genetics , Buffers , COS Cells , Coloring Agents , Dimethyl Sulfoxide/pharmacology , Enzyme Activation/drug effects , Free Radical Scavengers/pharmacology , Gene Expression , Glucose-6-Phosphatase/antagonists & inhibitors , Glucose-6-Phosphatase/metabolism , Humans , Hydrogen-Ion Concentration , Hyperglycemia/metabolism , Hyperglycemia/physiopathology , Insecta , Liver/enzymology , Male , Metals/pharmacology , Mice , Mice, Inbred C57BL , Mice, Obese , RNA, Messenger/analysis , Rosaniline Dyes
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