ABSTRACT
Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/genetics , Inhibitor of Apoptosis Proteins/genetics , Chromatography, Gel , Cross-Linking Reagents , Humans , Kinetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Proteolysis , Spectrometry, Mass, Electrospray Ionization , Ubiquitin-Protein Ligases , Ubiquitination , X-Ray DiffractionABSTRACT
High-throughput experimentation (HTE) has emerged as an important tool in drug discovery, providing a platform for preparing large compound libraries and enabling swift reaction screening over wide-ranging conditions. Recent advances in automated high-density, material-sparing HTE have necessitated the development of rapid analytics with sensitivity and resolution sufficient to identify products and/or assess reaction performance in a timely and data-rich manner. Combination of an ultrathroughput (UT) reader platform with Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry (ADE-OPI-MS) provides the requisite speed and sensitivity. Herein, we report the application of ADE-OPI-MS to HTE in the areas of parallel medicinal chemistry and reaction screening.
ABSTRACT
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Agammaglobulinaemia Tyrosine Kinase , Animals , Cells, Cultured , Ligands , Polyubiquitin/metabolism , Rats , ThermodynamicsABSTRACT
The drugable proteome is limited by the number of functional binding sites that can bind small molecules and respond with a therapeutic effect. Orthosteric and allosteric modulators of enzyme function or receptor signaling are well-established mechanisms of drug action. Drugs that perturb protein-protein interactions have only recently been launched. This approach is more difficult due to the extensive contact surfaces that must be perturbed antagonistically. Compounds that promote novel protein-protein interactions promise to dramatically expand opportunities for therapeutic intervention. This approach is precedented with natural products (rapamycin, FK506, sanglifehrin A), synthetic small molecules (thalidomide and IMiD derivatives) and indisulam analogues.
Subject(s)
Adhesives/pharmacology , Biological Products/pharmacology , Allosteric Regulation/drug effects , Drug Discovery , Humans , Ligands , Protein Binding , Proteolysis , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/metabolismABSTRACT
Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.
Subject(s)
Enzyme Inhibitors/chemistry , Glutathione/chemistry , Drug Design , Glutathione/metabolism , Humans , Kinetics , Mass Spectrometry , Nuclear Magnetic Resonance, Biomolecular , Pharmaceutical Preparations/chemistryABSTRACT
Herein we describe the structure-aided design and synthesis of a series of pyridone-conjugated monobactam analogues with in vitro antibacterial activity against clinically relevant Gram-negative species including Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli . Rat pharmacokinetic studies with compound 17 demonstrate low clearance and low plasma protein binding. In addition, evidence is provided for a number of analogues suggesting that the siderophore receptors PiuA and PirA play a role in drug uptake in P. aeruginosa strain PAO1.
Subject(s)
Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Monobactams/pharmacology , Pyridones/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Escherichia coli/drug effects , Inhibitory Concentration 50 , Klebsiella pneumoniae/drug effects , Male , Microbial Sensitivity Tests , Molecular Structure , Monobactams/chemistry , Monobactams/pharmacokinetics , Pseudomonas aeruginosa/drug effects , Pyridones/chemistry , Pyridones/pharmacokinetics , Rats , Rats, WistarABSTRACT
We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa . In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.
Subject(s)
Cross Infection/drug therapy , Drug Discovery , Drug Resistance, Multiple/drug effects , Gram-Negative Bacteria/drug effects , Polymyxins/chemistry , Polymyxins/pharmacology , beta-Alanine/analogs & derivatives , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/toxicity , Dogs , Female , Gram-Negative Bacteria/physiology , Humans , Male , Microbial Sensitivity Tests , Polymyxins/pharmacokinetics , Polymyxins/toxicity , Rats , beta-Alanine/chemistryABSTRACT
The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Gram-Negative Bacteria/drug effects , Hydroxamic Acids/chemistry , Amidohydrolases/metabolism , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/chemistry , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Structure-Activity Relationship , Sulfones/chemistryABSTRACT
Novel siderophore-linked monobactams with in vitro and in vivo anti-microbial activity against MDR Gram-negative pathogens are described.
Subject(s)
Drug Resistance, Multiple, Bacterial/drug effects , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Monobactams/pharmacology , Animals , Binding Sites/drug effects , Blood Proteins/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Gram-Negative Bacterial Infections/microbiology , Humans , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Monobactams/chemical synthesis , Monobactams/chemistry , Rats , Structure-Activity RelationshipABSTRACT
The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Hydroxamic Acids/chemical synthesis , Pyridones/chemical synthesis , Sulfonic Acids/chemical synthesis , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Crystallography, X-Ray , Humans , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/pharmacology , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Protein Conformation , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Pyridones/pharmacokinetics , Pyridones/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Sulfonic Acids/pharmacokinetics , Sulfonic Acids/pharmacologyABSTRACT
In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Biphenyl Compounds/chemical synthesis , Hydroxamic Acids/chemical synthesis , Phenyl Ethers/chemical synthesis , Pseudomonas Infections/drug therapy , Sulfides/chemical synthesis , Sulfones/chemical synthesis , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Catalytic Domain , Crystallography, X-Ray , Drug Resistance, Bacterial , Hydrogen Bonding , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Mice , Models, Molecular , Molecular Conformation , Phenyl Ethers/chemistry , Phenyl Ethers/pharmacology , Pseudomonas aeruginosa , Rats , Stereoisomerism , Structure-Activity Relationship , Sulfides/chemistry , Sulfides/pharmacology , Sulfones/chemistry , Sulfones/pharmacologyABSTRACT
Linezolid (Zyvox) is the first member of an entirely new class of antibiotics to reach the market in over 35 years; it was approved for use in 2000. A member of the oxazolidinone class of antibiotics, linezolid is highly effective for the treatment of serious Gram-positive infections and has activity that compares favorably with vancomycin for most clinically relevant pathogens. Zyvox is approved for use against serious Gram-positive infections, including those caused by Streptococcus pneumoniae, and the very challenging methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium organisms. Zyvox inhibits bacterial protein synthesis by binding to 23S rRNA in the catalytic site of the 50S ribosome. It can be administered both orally and intravenously and has good tissue distribution. Recent results have demonstrated that oxazolidinone analogs related to linezolid are effective in treating pulmonary tuberculosis caused by resistant Mycobacterium tuberculosis in animal infection models and suggest additional new therapeutic applications for these antibiotics.
Subject(s)
Acetamides/therapeutic use , Anti-Bacterial Agents/therapeutic use , Gram-Positive Bacterial Infections/drug therapy , Oxazolidinones/therapeutic use , Acetamides/chemical synthesis , Acetamides/pharmacokinetics , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacokinetics , Drug Discovery/trends , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/physiology , Humans , Linezolid , Models, Biological , Models, Molecular , Oxazolidinones/chemical synthesis , Oxazolidinones/classification , Oxazolidinones/pharmacokineticsABSTRACT
To facilitate a drug discovery project, we needed to develop a robust asymmetric synthesis of (2S,5S)-5-substituted-azepane-2-carboxylate derivatives. Two key requirements for the synthesis were flexibility for elaboration at C5 and suitability for large scale preparation. To this end we have successfully developed a scalable asymmetric synthesis of these derivatives that starts with known hydroxy-ketone 8. The key step features an oxidative cleavage of aza-bicyclo[3.2.2]nonene 14, which simultaneously generates the C2 and C5 substituents in a stereoselective manner.
Subject(s)
Carboxylic Acids/chemistry , Carboxylic Acids/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Drug Discovery , Ketones/chemistryABSTRACT
A novel series of monocarbam compounds exhibiting promising antibacterial activity against multidrug resistant Gram-negative microorganisms is reported, along with the synthesis of one such molecule MC-1 (1). Also reported are structure-activity relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in addition to the hydrolytic stability of such compounds and possible implications thereof.
ABSTRACT
Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.
Subject(s)
Azetidines/chemistry , Drug Resistance, Multiple/drug effects , Ketolides/chemistry , Ketolides/pharmacology , Respiratory Tract Infections/drug therapy , Animals , Bacteria/drug effects , Community-Acquired Infections/drug therapy , Disease Susceptibility , Drug Discovery , Drug-Related Side Effects and Adverse Reactions , Humans , Ketolides/adverse effects , Ketolides/chemical synthesis , Ketolides/therapeutic use , Mice , Microbial Sensitivity TestsABSTRACT
The asymmetric synthesis of (2R,3R)-3-methyl-3-hydroxypipecolic acid, a key intermediate in the synthesis of dual MMP-13/aggrecanase inhibitors, is described. The title compound is prepared in seven steps with an overall yield of 41% starting from geraniol. Key steps in the synthesis include Sharpless asymmetric epoxidation, which establishes the chiral centers, and a one-pot oxidative olefin cleavage/reductive amination sequence that closes the piperidine ring.
Subject(s)
Pipecolic Acids/chemical synthesis , Terpenes/chemistry , Acyclic Monoterpenes , Hydrolysis , Molecular Structure , Ozone/chemistry , Pipecolic Acids/chemistry , StereoisomerismABSTRACT
Explorations in the pyrimidinetrione series of MMP-13 inhibitors led to the discovery of a series of spiro-fused compounds that are potent and selective inhibitors of MMP-13. While other spiro-fused motifs are hydrolytically unstable, presumably due to electronic destabilization of the pyrimidinetrione ring, the spiropyrrolidine series does not share this liability. Greater than 100-fold selectivity versus other MMP family members was achieved by incorporation of an extended aryl-heteroaryl P1'group. When dosed as the sodium salt, these compounds displayed excellent oral absorption and pharmacokinetic properties. Despite the selectivity, a representative of this series produced fibroplasia in a 14 day rat study.
Subject(s)
Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemistry , Pyrimidines/chemistry , Pyrrolidines/chemistry , Spiro Compounds/chemistry , Animals , Enzyme Stability/drug effects , Matrix Metalloproteinase 13/metabolism , Protease Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrrolidines/pharmacology , Rats , Spiro Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
The design, synthesis, and biological evaluation of potent inhibitors of the TrkA kinase is presented. A homology model is created to aid in the enhancement of potency and selectivity of isothiazole inhibitors found during a high-throughput screen. Three different syntheses are utilized to make diverse analogs within this series. Aminoheterocycles are found to be good urea surrogates, whereas bicyclic substituents on the C3 thio group were found to be extremely potent TrkA inhibitors in kinase and cell assays.
Subject(s)
Computer Simulation , Enzyme Inhibitors , Receptor, trkA/antagonists & inhibitors , Thiazoles , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Molecular Structure , Stereoisomerism , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
A series of 3-hydroxy-3-methylpipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was designed based on the observation of increased aggrecanase activity with substitution at the 3-position of the piperidine ring. Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group that binds in the S1' pocket. These compounds also possess markedly improved bioavailability and lower metabolic clearance compared to analogous 3,3-dimethyl-5-hydroxypipecolic hydroxamates. These improvements are attributed to lowered lipophilicity proximal to the metabolically labile hydroxamic acid. Synthesis, structure activity relationships, and in vivo efficacy data are described.
Subject(s)
Endopeptidases/drug effects , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacokinetics , Matrix Metalloproteinase Inhibitors , Pipecolic Acids/chemical synthesis , Pipecolic Acids/pharmacokinetics , Administration, Oral , Animals , Collagenases/metabolism , Drug Design , Endopeptidases/metabolism , Humans , Hydroxamic Acids/chemistry , Matrix Metalloproteinase 13 , Molecular Structure , Pipecolic Acids/chemistry , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
A series of pipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was discovered based on screening known inhibitors of TNF-alpha converting enzyme (TACE). Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group. Incorporation of geminal alkyl substitution at the 3-position of the piperidine ring improved metabolic stability, presumably by increasing steric hindrance around the metabolically labile hydroxamic acid. This modification also resulted in dramatic improvement of aggrecanase activity with a slight reduction in selectivity versus MMP-1. Synthesis, structure activity relationships, and strategies to reduce metabolic clearance are described.