ABSTRACT
Adaptor protein 2-associated kinase 1 (AAK1) is a serine/threonine kinase that was identified as a therapeutic target for the potential treatment of neuropathic pain. Inhibition of AAK1 in the central nervous system, particularly within the spinal cord, was found to be the relevant site for achieving an antinociceptive effect. We previously reported that compound 7 is a brain-penetrant, AAK1 inhibitor that showed efficacy in animal models for neuropathic pain. One approach we took to improve upon the potency of 7 involved tying the amide back into the neighboring phenyl ring to form a bicyclic heterocycle. Investigation of the structure-activity relationships (SARs) of substituents on the resultant quinazoline and quinoline ring systems led to the identification of (S)-31, a brain-penetrant, AAK1-selective inhibitor with improved enzyme and cellular potency compared to 7. The synthesis, SAR, and in vivo evaluation of a series of quinazoline and quinoline-based AAK1 inhibitors are described herein.
Subject(s)
Neuralgia , Quinolines , Amides/pharmacology , Amides/therapeutic use , Animals , Neuralgia/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Quinazolines/therapeutic use , Quinolines/pharmacology , Quinolines/therapeutic use , Structure-Activity RelationshipABSTRACT
Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3, which was confirmed by protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an enhancement in selectivity against key off-target kinases.
ABSTRACT
Affinity selection screening of macrocycle libraries derived from DNA-programmed chemistry identified XIAP BIR2 and BIR3 domain inhibitors that displace bound pro-apoptotic caspases. X-ray cocrystal structures of key compounds with XIAP BIR2 suggested potency-enhancing structural modifications. Optimization of dimeric macrocycles with similar affinity for both domains were potent pro-apoptotic agents in cancer cell lines and efficacious in shrinking tumors in a mouse xenograft model.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/therapeutic use , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Breast/drug effects , Breast/metabolism , Breast/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Caspase 3/metabolism , Cell Line, Tumor , Crystallography, X-Ray , Drug Discovery , Female , Gene Library , Humans , Macrocyclic Compounds/pharmacokinetics , Mice , Models, Molecular , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
A scheduler has been developed for an integrated laboratory robot system that operates in an always-on mode. The integrated system is designed for imaging plates containing protein crystallization experiments, and it allows crystallographers to enter plates at any time and request that they be imaged at multiple time points in the future. The scheduler must rearrange tasks within the time it takes to image one plate, trading off the quality of the schedule for the speed of the computation. For this reason, the scheduler was based on a simulated annealing algorithm with an objective function that makes use of a linear programming solver. To optimize the scheduler, extensive computational simulations were performed involving a difficult but representative scheduling problem. The simulations explore multiple configurations of the simulated annealing algorithm, including both geometric and adaptive annealing schedules, 3 neighborhood functions, and 20 neighborhood diameters. An optimal configuration was found that produced the best results in less than 60 seconds, well within the window necessary to dynamically reschedule imaging tasks as new plates are entered into the system.
Subject(s)
Automation, Laboratory/methods , Biomedical Research/organization & administration , Robotics/methods , Algorithms , Computer Simulation , Crystallization/methods , Optical Imaging/methodsABSTRACT
The crystal structure of unphosphorylated p38alpha MAP kinase complexed with a representative pyrrolotriazine-based inhibitor led to the elucidation of the high-affinity binding mode of this class of compounds at the ATP-binding site. The ligand binds in an extended conformation, with one end interacting with the adenine-pocket hinge region, including a hydrogen bond from the carboxyl O atom of Met109. The other end of the ligand interacts with the hydrophobic pocket of the binding site and with the backbone N atom of Asp168 in the DFG activation loop. Addition of an extended benzylmorpholine group forces the DFG loop to flip out of position and allows the ligand to make additional interactions with the protein.
Subject(s)
Anilides/chemistry , Benzamides/chemistry , Mitogen-Activated Protein Kinase 14/chemistry , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Pyrroles/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Models, Molecular , Protein BindingABSTRACT
A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Pyrroles/chemical synthesis , Triazines/chemical synthesis , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/drug therapy , Binding Sites , Crystallography, X-Ray , Drug Design , Female , Humans , In Vitro Techniques , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred BALB C , Microsomes, Liver/metabolism , Mitogen-Activated Protein Kinase 14/chemistry , Models, Molecular , Pyrroles/pharmacokinetics , Pyrroles/pharmacology , Rats , Rats, Inbred Lew , Structure-Activity Relationship , Triazines/pharmacokinetics , Triazines/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/bloodABSTRACT
In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.
Subject(s)
Bacteria/enzymology , Protease Inhibitors/pharmacology , Aminopeptidases/chemistry , Aminopeptidases/isolation & purification , Bacteria/drug effects , Bacterial Proteins/pharmacology , Crystallization , Crystallography, X-Ray , Kinetics , Magnetic Resonance Spectroscopy , Methionyl Aminopeptidases , Models, Molecular , Protease Inhibitors/chemistry , Protein Conformation , Quantum TheoryABSTRACT
Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.
Subject(s)
Catalytic Domain , Drug Design , Protein Engineering , Protein Tyrosine Phosphatases/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Utilizing modeling information from a recently resolved structure of human HIF-1alpha prolyl hydroxylase (EGLN1) and structure-based design, a novel series of imidazo[1,2-a]pyridine derivatives was prepared. The activity of these compounds was determined in a human EGLN1 assay and a limited SAR was developed.
Subject(s)
Procollagen-Proline Dioxygenase/antagonists & inhibitors , Pyridines/pharmacology , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases , Structure-Activity RelationshipABSTRACT
Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.
Subject(s)
Drug Design , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Cell Line , Humans , Hypoxia-Inducible Factor-Proline DioxygenasesABSTRACT
Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series with consistent activity against EGLN-1.
Subject(s)
Drug Design , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Pyridines/chemical synthesis , Pyridines/pharmacology , Humans , Hypoxia-Inducible Factor-Proline DioxygenasesABSTRACT
A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.
Subject(s)
Drug Design , Oxyquinoline/analogs & derivatives , Oxyquinoline/pharmacology , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Hypoxia-Inducible Factor-Proline Dioxygenases , Oxyquinoline/chemical synthesis , Oxyquinoline/chemistry , Procollagen-Proline Dioxygenase/chemistry , Structure-Activity RelationshipABSTRACT
The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.
Subject(s)
Amides/pharmacology , Drug Design , Indoles/pharmacology , Osteoporosis/drug therapy , PPAR gamma/metabolism , 3T3 Cells , Amides/chemical synthesis , Animals , Indoles/chemical synthesis , MiceABSTRACT
Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.
Subject(s)
Arthropods/chemistry , Copepoda/chemistry , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Protein Biosynthesis , Animals , Arthropods/metabolism , Copepoda/metabolism , Crystallography, X-Ray/methods , Embryo, Nonmammalian , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Models, Molecular , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Structural Homology, Protein , Xenopus laevis/embryologyABSTRACT
The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly selective and potent inhibitors of HPTPbeta. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadiazoles without a significant effect on HPTPbeta potency.
Subject(s)
Chemistry, Pharmaceutical/methods , Nerve Tissue Proteins/antagonists & inhibitors , Protein Tyrosine Phosphatases/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Hydrogen Bonding , Hydrolysis , Models, Chemical , Models, Molecular , Molecular Structure , Receptor-Like Protein Tyrosine Phosphatases, Class 5 , Structure-Activity RelationshipABSTRACT
High-throughput screening of the P&GP corporate repository against several protein tyrosine phosphatases identified the sulfamic acid moiety as potential phosphotyrosine mimetic. Incorporation of the sulfamic acid onto a 1,2,3,4-tetrahydroisoquinoline scaffold provided a promising starting point for PTP1B inhibitor design.
Subject(s)
Enzyme Inhibitors/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Sulfonic Acids/pharmacology , Tetrahydroisoquinolines/chemistry , Binding Sites , Binding, Competitive , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Ligands , Models, Molecular , Molecular Mimicry , Phosphotyrosine/metabolism , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistry , src Homology DomainsABSTRACT
Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS. FliS adopts a novel fold that is clearly distinct from those of the type III secretion chaperones, indicating that they do not share a common evolutionary origin. However, the structure of FliS in complex with a fragment of FliC (flagellin) reveals that, like the type III secretion chaperones, flagellar export chaperones bind their target proteins in extended conformation and suggests that this mode of recognition may be widely used in bacteria.
Subject(s)
Flagella/metabolism , Molecular Chaperones/metabolism , Proteins/metabolism , Bacteria/chemistry , Bacteria/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Flagellin/chemistry , Flagellin/metabolism , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO(4)) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds.