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1.
J Mol Biol ; 433(24): 167309, 2021 12 03.
Article in English | MEDLINE | ID: mdl-34687713

ABSTRACT

The NLRP3 inflammasome assembles in response to a variety of pathogenic and sterile danger signals, resulting in the production of interleukin-1ß and interleukin-18. NLRP3 is a key component of the innate immune system and has been implicated as a driver of a number of acute and chronic diseases. We report the 2.8 Å crystal structure of the NLRP3 NACHT domain in complex with an inhibitor. The structure defines a binding pocket formed by the four subdomains of the NACHT domain, and shows the inhibitor acts as an intramolecular glue, which locks the protein in an inactive conformation. It provides further molecular insight into our understanding of NLRP3 activation, helps to detail the residues involved in subdomain coordination within the NLRP3 NACHT domain, and gives molecular insights into how gain-of-function mutations de-stabilize the inactive conformation of NLRP3. Finally, it suggests stabilizing the auto-inhibited form of the NACHT domain is an effective way to inhibit NLRP3, and will aid the structure-based development of NLRP3 inhibitors for a range of inflammatory diseases.


Subject(s)
Inflammasomes/antagonists & inhibitors , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , NLR Family, Pyrin Domain-Containing 3 Protein/chemistry , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Furans/chemistry , Furans/pharmacology , Humans , Indenes/chemistry , Indenes/pharmacology , Inflammasomes/metabolism , Protein Domains , Sulfonamides/chemistry , Sulfonamides/pharmacology
2.
Biochem Biophys Res Commun ; 545: 177-182, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33561652

ABSTRACT

The NLRP3 inflammasome is a critical component of the innate immune response to sterile inflammation. Its regulation involves a priming step, required for up-regulation of inflammasome protagonists and an activation step leading to NLRP3 inflammasome complex assembly, which triggers caspase-1 activity. The IκKß kinase regulates canonical NF-κB, a key pathway involved in transcriptional priming. We found that IκKß also regulates the activation and function of the NLRP3 inflammasome beyond the priming step. Two unrelated IκKß inhibitors, AFN700 and TPCA-1, when applied after priming, fully blocked IL-1ß secretion triggered by nigericin in THP-1 cells. Both inhibitors prevented neither inflammasome assembly, as monitored by measuring the formation of ASC specks, nor the generation of caspase-1 p20, a hallmark of caspase-1 activity, but they impaired the initial cleavage and activation of procaspase-1. These data thus indicate that IκKß activity is required for efficient activation of NLRP3, suggesting that IκKß may fulfill a dual role in coupling priming and activation of the NLRP3 inflammasome.


Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Amides/pharmacology , Caspase 1/metabolism , Humans , Immunity, Innate/drug effects , Inflammasomes/drug effects , Inflammasomes/immunology , Interleukin-1beta/biosynthesis , NF-kappa B/metabolism , Nigericin/pharmacology , Protein Kinase Inhibitors/pharmacology , THP-1 Cells , Thiophenes/pharmacology
3.
J Med Chem ; 63(23): 14594-14608, 2020 12 10.
Article in English | MEDLINE | ID: mdl-33216547

ABSTRACT

The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.


Subject(s)
Blood/metabolism , Caspase Inhibitors/therapeutic use , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/antagonists & inhibitors , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Urea/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Caspase Inhibitors/chemical synthesis , Caspase Inhibitors/metabolism , Caspase Inhibitors/pharmacokinetics , Cell Line, Tumor , Female , Half-Life , Humans , Mice, Inbred BALB C , Mice, SCID , Microsomes, Liver/metabolism , Neoplasms/drug therapy , Pyrazoles/chemical synthesis , Pyrazoles/metabolism , Pyrazoles/pharmacokinetics , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Pyrimidines/pharmacokinetics , Rats, Sprague-Dawley , Sheep , Urea/chemical synthesis , Urea/metabolism , Urea/pharmacokinetics , Xenograft Model Antitumor Assays
4.
J Med Chem ; 63(23): 14576-14593, 2020 12 10.
Article in English | MEDLINE | ID: mdl-33252239

ABSTRACT

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.


Subject(s)
Caspase Inhibitors/therapeutic use , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/antagonists & inhibitors , Neoplasms/drug therapy , Urea/analogs & derivatives , Urea/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Caspase Inhibitors/chemical synthesis , Caspase Inhibitors/pharmacology , Drug Discovery , Female , Humans , Immunity, Humoral/drug effects , Male , Mice, Inbred BALB C , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Rats, Sprague-Dawley , Structure-Activity Relationship , T-Lymphocytes/drug effects , Urea/pharmacology , Xenograft Model Antitumor Assays
5.
J Med Chem ; 63(11): 5697-5722, 2020 06 11.
Article in English | MEDLINE | ID: mdl-32073845

ABSTRACT

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.


Subject(s)
Benzoic Acid/chemistry , Complement Factor B/antagonists & inhibitors , Indoles/chemistry , Atypical Hemolytic Uremic Syndrome/metabolism , Atypical Hemolytic Uremic Syndrome/pathology , Benzoic Acid/metabolism , Benzoic Acid/pharmacokinetics , Binding Sites , Catalytic Domain , Complement Factor B/metabolism , Crystallography, X-Ray , Drug Evaluation, Preclinical , Half-Life , Humans , Indoles/metabolism , Indoles/pharmacokinetics , Inhibitory Concentration 50 , Macular Degeneration/metabolism , Macular Degeneration/pathology , Molecular Dynamics Simulation , Structure-Activity Relationship
6.
Proc Natl Acad Sci U S A ; 116(16): 7926-7931, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30926668

ABSTRACT

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.


Subject(s)
Complement Factor B/antagonists & inhibitors , Complement Pathway, Alternative/drug effects , Drug Discovery/methods , Immunologic Factors/pharmacology , Animals , Disease Models, Animal , Glomerulonephritis, Membranous/physiopathology , Humans , Male , Mice , Mice, Inbred C57BL , Rats, Sprague-Dawley
7.
Bioorg Med Chem Lett ; 28(12): 2153-2158, 2018 07 01.
Article in English | MEDLINE | ID: mdl-29759726

ABSTRACT

Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.


Subject(s)
Antineoplastic Agents/pharmacology , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/antagonists & inhibitors , Piperidines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Hepatocytes/drug effects , Humans , Jurkat Cells , Microsomes/drug effects , Molecular Structure , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , Piperidines/chemical synthesis , Piperidines/chemistry , Proteolysis/drug effects , Rats , Structure-Activity Relationship
8.
J Mol Biol ; 419(1-2): 4-21, 2012 May 25.
Article in English | MEDLINE | ID: mdl-22366302

ABSTRACT

The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.


Subject(s)
Caspases/chemistry , Caspases/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , B-Cell CLL-Lymphoma 10 Protein , Catalytic Domain , Cells, Cultured , Dimerization , Enzyme Activation , HEK293 Cells , Humans , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Structure, Tertiary , Receptors, Antigen/chemistry , Receptors, Antigen/genetics , Receptors, Antigen/metabolism , Signal Transduction , Structure-Activity Relationship
9.
J Biomol Screen ; 15(9): 1029-41, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20855559

ABSTRACT

Fragment-based screening (FBS) has gained acceptance in the pharmaceutical industry as an attractive approach for the identification of new chemical starting points for drug discovery programs in addition to classical strategies such as high-throughput screening. There is the concern that screening of fragments at high µM concentrations in biochemical assays results in increased false-positive and false-negative rates. Here the authors systematically compare the data quality of FBS obtained by enzyme activity-based fluorescence intensity, fluorescence lifetime, and mobility shift assays with the data quality from surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) methods. The serine protease trypsin and the matrix metalloprotease MMP12 were selected as model systems. For both studies, 352 fragments were selected each. From the data generated, all 3 biochemical protease assay methods can be used for screening of fragments with low false-negative and low false-positive rates, comparable to those achieved with the SPR-based assays. It can also be concluded that only fragments with a solubility higher than the screening concentration determined by means of NMR should be used for FBS purposes. Extrapolated to 10,000 fragments, the biochemical assays speed up the primary FBS process by approximately a factor of 10 and reduce the protease consumption by approximately 10,000-fold compared to NMR protein observation experiments.


Subject(s)
Biological Assay/methods , Drug Evaluation, Preclinical/methods , Matrix Metalloproteinase 12/metabolism , Peptide Fragments/analysis , Trypsin/metabolism , Animals , Cattle , Chromatography, Liquid , False Negative Reactions , False Positive Reactions , Feasibility Studies , Fluorescence , Humans , Kinetics , Light , Magnetic Resonance Spectroscopy , Mass Spectrometry , Peptide Fragments/chemistry , Scattering, Radiation , Solubility , Surface Plasmon Resonance
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