ABSTRACT
Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Cysteine/chemistry , Immunoconjugates/pharmacology , Lung Neoplasms/drug therapy , Trastuzumab/pharmacology , Animals , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Blotting, Western , Cell Proliferation/drug effects , Cross-Linking Reagents , Female , Flow Cytometry , Fluorescent Antibody Technique , Fusion Regulatory Protein-1/immunology , Humans , Immunoconjugates/chemistry , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Rats , Rats, Sprague-Dawley , Receptor, ErbB-2/antagonists & inhibitors , Trastuzumab/chemistry , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. ß-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aß, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (ka) measured at 25 °C ranged from a low of 2.42×10(4) M(-1) s(-1) to the highest value of 8.3×10(5) M(-1) s(-1). Rate constants of dissociation (kd) ranged from 1.09×10(-4) s(-1) (corresponding to a residence time of close to three hours), to the fastest of 0.028 s(-1). Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.
Subject(s)
Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Ethanolamines , Protease Inhibitors/pharmacology , Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Proteases/antagonists & inhibitors , Aspartic Acid Proteases/chemistry , Enzymes, Immobilized/antagonists & inhibitors , Enzymes, Immobilized/chemistry , Humans , Kinetics , Protease Inhibitors/chemistry , Protein Conformation , ThermodynamicsABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of [1,2,4]triazolo[4,3-b]pyridazines that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays and show an unprecedented selectivity towards the G2019S mutant. A structural rational for the observed selectivity is proposed.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridazines/pharmacology , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/metabolism , Pyridazines/chemical synthesis , Pyridazines/chemistry , Structure-Activity RelationshipABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial Parkinson's disease (PD). The kinase activity of this complex protein is increased by pathogenic mutations. Inhibition of LRRK2 kinase activity has therefore emerged as a promising approach for the treatment of PD. Herein we report our findings on a series of 4-alkylamino-7-aryl-3-cyanoquinolines that exhibit kinase inhibitory activity against both wild type and G2019S mutant LRRK2. Activity was determined in both biochemical and cellular assays. Compound 14 was further evaluated in an in vivo pharmacodynamic study and found to significantly inhibit Ser935 phosphorylation after oral dosing.
Subject(s)
Drug Discovery , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinolines/pharmacology , Animals , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Mice, Knockout , Mice, Transgenic , Models, Molecular , Molecular Structure , Mutation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Quinolines/chemical synthesis , Quinolines/chemistry , Structure-Activity RelationshipABSTRACT
The structure-activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid/chemistry , Drug Design , Isoquinolines/pharmacology , Protease Inhibitors/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Catalysis , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration.
Subject(s)
Heterocyclic Compounds, 2-Ring/chemistry , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Amides/chemical synthesis , Amides/chemistry , Amides/metabolism , Animals , Binding Sites , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Molecular Docking Simulation , Mutation , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , TransfectionABSTRACT
The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Chromans/chemistry , Drug Design , Enzyme Inhibitors/chemical synthesis , Binding Sites , Cells, Cultured , Ethylamines/chemical synthesis , Ethylamines/chemistry , Ethylamines/pharmacology , Inhibitory Concentration 50 , Models, Molecular , Structure-Activity RelationshipABSTRACT
Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Pyrazoles/pharmacology , Sulfonamides/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Animals , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Mice , Pyrazoles/chemistry , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
Extensive knowledge has been gained on the transcription network controlled by ERα, however, the mechanism underlying ESR1 (encoding ERα) expression is less understood. We recently discovered that the Hippo pathway is required for the proper expression of ESR1. YAP/TAZ are transcription coactivators that are phosphorylated and inhibited by the Hippo pathway kinase LATS. Here we delineated the molecular mechanisms underlying ESR1 transcription repression by the Hippo pathway. Mechanistically, YAP binds to TEAD to increase local chromatin accessibility to stimulate transcription of nearby genes. Among the YAP target genes, Vestigial-Like Protein 3 (VGLL3) competes with YAP/TAZ for binding to TEAD transcription factor and recruits the NCOR2/SMRT repressor to the super-enhancer of ESR1 gene, leading to epigenetic alteration and transcriptional silencing. We developed a potent LATS inhibitor VT02956. Targeting the Hippo pathway by VT02956 represses ESR1 expression and inhibits the growth of ER+ breast cancer cells as well as patient-derived tumour organoids. Moreover, histone deacetylase inhibitors, such as Entinostat, induce VGLL3 expression to inhibit ER+ breast cancer cells. Our study suggests LATS as unexpected cancer therapeutic targets, especially for endocrine-resistant breast cancers.
Subject(s)
Breast Neoplasms , Estrogen Receptor alpha , Hippo Signaling Pathway , Female , Humans , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , YAP-Signaling Proteins/metabolismABSTRACT
The SAR of a series of tri-substituted thiophene JNK3 inhibitors is described. By optimizing both the N-aryl acetamide region of the inhibitor and the 4-position of the thiophene we obtained single digit nanomolar compounds, such as 47, which demonstrated an in vivo effect on JNK activity when dosed orally in our kainic acid mouse model as measured by phospho-c-jun reduction.
Subject(s)
Brain/metabolism , MAP Kinase Kinase 4/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Administration, Oral , Drug Design , Hydrogen Bonding , Models, Molecular , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity RelationshipABSTRACT
The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Protein Precursor/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 3-Ring/chemical synthesis , Heterocyclic Compounds, 3-Ring/pharmacology , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Amyloid beta-Peptides/metabolism , Animals , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Heterocyclic Compounds, 3-Ring/chemistry , Inhibitory Concentration 50 , Mice , Mice, Inbred Strains , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
In this Letter, we describe the discovery of selective JNK2 and JNK3 inhibitors, such as 10, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs, p38α and ERK2. Substitution of the naphthalene ring affords an isoform selective JNK3 inhibitor, 30, with approximately 10-fold selectivity over both JNK1 and JNK2. A naphthalene ring penetrates deep into the selectivity pocket accounting for the differentiation amongst the kinases. Interestingly, the gatekeeper Met146 sulfide interacts with the naphthalene ring in a sulfur-π stacking interaction. Compound 38 ameliorates neurotoxicity induced by amyloid-ß in human cortical neurons. Lastly, we demonstrate how to install propitious in vitro CNS-like properties into these selective inhibitors.
Subject(s)
Aminopyridines/chemistry , Mitogen-Activated Protein Kinase 10/antagonists & inhibitors , Mitogen-Activated Protein Kinase 9/antagonists & inhibitors , Neurodegenerative Diseases/drug therapy , Neuroprotective Agents/chemistry , Protein Kinase Inhibitors/chemistry , Triazines/chemistry , Aminopyridines/pharmacokinetics , Aminopyridines/therapeutic use , Animals , Binding Sites , Central Nervous System/metabolism , Computer Simulation , Humans , Mice , Microsomes, Liver/metabolism , Mitogen-Activated Protein Kinase 10/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Neuroprotective Agents/pharmacokinetics , Neuroprotective Agents/therapeutic use , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Structure-Activity Relationship , Triazines/pharmacokinetics , Triazines/therapeutic useABSTRACT
The SAR of a series of brain penetrant, trisubstituted thiophene based JNK inhibitors with improved pharmacokinetic properties is described. These compounds were designed based on information derived from metabolite identification studies which led to compounds such as 42 with lower clearance, greater brain exposure and longer half life compared to earlier analogs.
Subject(s)
Brain/metabolism , Drug Design , Nerve Degeneration/prevention & control , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Thiophenes/pharmacology , Thiophenes/pharmacokinetics , Animals , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Dose-Response Relationship, Drug , Half-Life , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Stereoisomerism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistryABSTRACT
From high throughput screening, we discovered compound 1, the prototype for a series of disubstituted thiophene inhibitors of JNK which is selective towards closely related MAP kinases p38 and Erk2. Herein we describe the evolution of these compounds to a novel class of thiophene and thiazole JNK inhibitors that retain favorable solubility, permeability, and P-gp properties for development as CNS agents for treatment of neurodegeneration. Compound 61 demonstrated JNK3 IC(50)=77 nM and retained the excellent broad kinase selectivity observed for the series.
Subject(s)
JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Quinolines/chemical synthesis , Thiazoles/chemistry , Thiophenes/chemistry , Animals , Drug Design , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Microsomes, Liver/metabolism , Mitogen-Activated Protein Kinase 10/antagonists & inhibitors , Mitogen-Activated Protein Kinase 10/metabolism , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Mitogen-Activated Protein Kinase 8/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Thiophenes/chemical synthesis , Thiophenes/pharmacologyABSTRACT
The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Ethylamines/chemistry , Ethylamines/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/chemistry , Crystallography, X-Ray , Ethylamines/chemical synthesis , Humans , Models, Molecular , Protease Inhibitors/chemical synthesis , Protein Binding , Structure-Activity RelationshipABSTRACT
In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16 which lowers Αß by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30mpk BID for 2.5days.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Ethylamines/chemical synthesis , Ethylamines/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Alkylation , Alzheimer Disease , Animals , Brain/metabolism , Cell Line , Dogs , Drug Design , Guinea Pigs , Humans , Indicators and Reagents , Protease Inhibitors/pharmacokinetics , Protein Binding , Structure-Activity RelationshipABSTRACT
Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2' position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.
Subject(s)
Acetamides/chemistry , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Butanols/chemistry , Cyclohexylamines/chemistry , Protease Inhibitors/chemistry , Acetamides/chemical synthesis , Acetamides/pharmacokinetics , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Butanols/chemical synthesis , Butanols/pharmacokinetics , Cell Membrane Permeability/drug effects , Crystallography, X-Ray , Cyclohexylamines/chemical synthesis , Cyclohexylamines/pharmacokinetics , Humans , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The structural modification of a series of [3.3.1] bicyclic sulfonamide based gamma-secretase inhibitors is described. Appropriate substitution on the bicyclic scaffold provides a significant increase in the metabolic stability of the compounds resulting in an improved in vivo metabolic profile.
Subject(s)
Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Bridged Bicyclo Compounds/chemistry , Protease Inhibitors/chemistry , Sulfonamides/chemistry , Alzheimer Disease/drug therapy , Bridged Bicyclo Compounds/pharmacology , Bridged Bicyclo Compounds/therapeutic use , Humans , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Structure-Activity Relationship , Sulfonamides/pharmacology , Sulfonamides/therapeutic useABSTRACT
In this Letter, we report our strategy to design potent and metabolically stable gamma-secretase inhibitors that are efficacious in reducing the cortical Abetax-40 levels in FVB mice via a single PO dose.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Pyrazoles/chemistry , Quinolines/chemistry , Sulfonamides/chemistry , Administration, Oral , Amyloid Precursor Protein Secretases/metabolism , Animals , Drug Design , Mice , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Structure-Activity RelationshipABSTRACT
Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Abeta cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S1' residues critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity but poor permeability and high P-gp efflux.