ABSTRACT
Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.
Subject(s)
Anti-HIV Agents/pharmacology , Anti-HIV Agents/therapeutic use , Capsid Proteins/antagonists & inhibitors , HIV-1/drug effects , Adolescent , Adult , Anti-HIV Agents/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line , Cells, Cultured , Drug Resistance, Viral/genetics , Female , HIV-1/growth & development , Humans , Male , Middle Aged , Models, Molecular , Virus Replication/drug effects , Young AdultABSTRACT
Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.
Subject(s)
B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/chemistry , Antibodies, Monoclonal/chemistry , B7-H1 Antigen/metabolism , Crystallography, X-Ray/methods , Humans , Immunotherapy , Ligands , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Mass Spectrometry , Models, Molecular , Oxidation-Reduction , Peptides/chemistry , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , Protein Footprinting/methods , Small Molecule Libraries/pharmacologyABSTRACT
A tremendous research and development effort was exerted toward combating chronic hepatitis C, ultimately leading to curative oral treatments, all of which are targeting viral proteins. Despite the advantage of numerous targets allowing for broad hepatitis C virus (HCV) genotype coverage, the only host target inhibitors that advanced into clinical development were Cyclosporin A based cyclophilin inhibitors. While cyclosporin-based molecules typically require a fermentation process, Gilead successfully pursued a fully synthetic, oral program based on Sanglifehrin A. The drug discovery process, though greatly helped by facile crystallography, was still hampered by the limitations in the accuracy of predictive computational methods for prioritizing compound ideas. Recent advances in accuracy and speed of free energy perturbation (FEP) methods, however, are attractive for prioritizing and derisking synthetically challenging molecules and potentially could have had a significant impact on the speed of the development of this program. Here in our simulated prospective study, the binding free energies of 26 macrocyclic cyclophilin inhibitors were blindly predicted using FEP+ to test this hypothesis. The predictions had a low mean unsigned error (MUE) (1.1 kcal/mol) and accurately reproduced many design decisions from the program, suggesting that FEP+ has the potential to drive synthetic chemistry efforts by more accurately ranking compounds with nonintuitive structure-activity relationships (SARs).
Subject(s)
Drug Discovery , Entropy , Prospective Studies , Structure-Activity Relationship , ThermodynamicsABSTRACT
Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity. Towards this goal, distinct interactions with the conserved catalytic triad of the NS3/4A protease were identified that improved genotype 3 antiviral activity. We further discovered that protein adduct formation strongly correlated with clinical ALT elevation for this therapeutic class. Improving metabolic stability and decreasing protein adduct formation through structural modifications ultimately resulted in voxilaprevir. Voxilaprevir, in combination with sofosbuvir and velpatasvir, has demonstrated pan-genotypic antiviral clinical activity. Furthermore, hepatotoxicity was not observed in Phase 3 clinical trials with voxilaprevir, consistent with our design strategy. Vosevi® (sofosbuvir, velpatasvir, and voxilaprevir) is now an approved pan-genotypic treatment option for the most difficult-to-cure individuals who have previously failed direct acting antiviral therapy.
Subject(s)
Antiviral Agents/pharmacology , Carbamates/chemistry , Drug Discovery , Hepacivirus/drug effects , Heterocyclic Compounds, 4 or More Rings/chemistry , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Protease Inhibitors/pharmacology , Sofosbuvir/chemistry , Sulfonamides/chemistry , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Aminoisobutyric Acids , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cyclopropanes , Dose-Response Relationship, Drug , Drug Combinations , Hepacivirus/genetics , Humans , Lactams, Macrocyclic , Leucine/analogs & derivatives , Macrocyclic Compounds/chemical synthesis , Microbial Sensitivity Tests , Molecular Structure , Proline/analogs & derivatives , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Quinoxalines , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Colitis, Ulcerative/drug therapy , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase Inhibitors/chemistry , Stomach Neoplasms/drug therapy , Allosteric Site , Antibodies/chemistry , Catalytic Domain , Crystallography, X-Ray , Drug Design , Drug Evaluation, Preclinical , Gelatin/chemistry , Gene Deletion , HEK293 Cells , Humans , Inhibitory Concentration 50 , Protein Binding , Recombinant Proteins/chemistry , Surface Plasmon ResonanceABSTRACT
We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/ß). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat Fâ¯=â¯73%) and IV pharmacological studies.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Drug Discovery , Protein Kinase Inhibitors/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.
Subject(s)
Phosphatidylinositol 3-Kinases/chemistry , Purines/chemistry , Quinazolinones/chemistry , Adenosine Triphosphate/chemistry , Androstadienes/chemistry , Animals , Binding, Competitive , Catalytic Domain , Class I Phosphatidylinositol 3-Kinases , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Crystallography, X-Ray , Humans , Hydrogen Bonding , Kinetics , Mice , Models, Molecular , Phosphoinositide-3 Kinase Inhibitors , Protein Binding , WortmanninABSTRACT
Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.
Subject(s)
Crystallization , Crystallography, X-Ray/methods , Acoustics , Animals , Chickens , Egg White/chemistry , Glycols/chemistry , HIV Reverse Transcriptase/chemistry , Hepacivirus/metabolism , Humans , Muramidase/chemistry , Nanoparticles , Nanotechnology/methods , Protein-Tyrosine Kinases/chemistry , Proteins/chemistry , Serum Albumin/chemistry , Viral Proteins/chemistry , ViscosityABSTRACT
A crystal seeding technique is introduced that uses acoustic waves to deliver nanolitre volumes of seed suspension into protein drops. The reduction in delivery volume enables enhanced crystal growth in matrix-seeding experiments without concern for bias from chemical components in the seed-carrying buffer suspension. Using this technique, it was found that while buffer components alone without seed can marginally promote crystal growth in some cases, crystal seeding is far more effective in boosting the number of sparse-matrix conditions that yield protein crystals.
Subject(s)
Crystallization/methods , Proteins/chemistry , HumansABSTRACT
JNK2 and p38alpha are closely related mitogen-activated protein kinases that regulate various cellular activities and are considered drug targets for inflammatory diseases. We have determined the X-ray crystal structure of the clinical phase II p38alpha inhibitor BIRB796 bound to its off-target JNK2. This shows for the first time a JNK subfamily member in the DFG-out conformation. The fully resolved activation loop reveals that BIRB796 inhibits JNK2 activation by stabilizing the loop in a position that does not allow its phosphorylation by upstream kinases. The structure suggests that substituents at the BIRB796 morpholino group and modifications of the t-butyl moiety should further increase the p38alpha to JNK2 potency ratio. For the design of selective DFG-out binding JNK2 inhibitors, the binding pocket of the BIRB796 tolyl group may have the best potential.
Subject(s)
Mitogen-Activated Protein Kinase 9/chemistry , Mitogen-Activated Protein Kinases/chemistry , Naphthalenes/chemistry , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Models, Molecular , Molecular StructureABSTRACT
An analysis of the binding motifs of known HIV-1 non-nucleoside reverse transcriptase inhibitors has led to discovery of novel piperidine-linked aminopyrimidine derivatives with broad activity against wild-type as well as drug-resistant mutant viruses. Notably, the series retains potency against the K103N/Y181C and Y188L mutants, among others. Thus, the N-benzyl compound 5k has a particularly attractive profile. Synthesis and SAR are presented and discussed, as well as crystal structures relating to the binding motifs.
Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , Mutation , Pyrimidines/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Drug Discovery , Drug Resistance, Viral/genetics , HIV-1/genetics , Models, Molecular , Pyrimidines/chemistry , Structure-Activity RelationshipABSTRACT
Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.
Subject(s)
Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , HIV-1/enzymology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Anti-HIV Agents/chemical synthesis , Drug Resistance, Viral , HIV Infections/drug therapy , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , Humans , Models, Molecular , Mutation , Piperidines/chemical synthesis , Piperidines/chemistry , Piperidines/pharmacology , Pyrimidines/chemical synthesis , Structure-Activity RelationshipABSTRACT
Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 µM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B, Chronic/drug therapy , Hexanols/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacology , Toll-Like Receptor 8/agonists , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Crystallography, X-Ray , Dogs , Drug Discovery , Hepatitis B virus/drug effects , Hexanols/administration & dosage , Hexanols/chemical synthesis , Hexanols/metabolism , Humans , Macaca fascicularis , Molecular Structure , Protein Domains , Pyridines/administration & dosage , Pyridines/chemical synthesis , Pyridines/metabolism , Pyrimidines/administration & dosage , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Rats , Structure-Activity Relationship , Toll-Like Receptor 8/metabolismABSTRACT
Atropisomerism is a type of axial chirality in which enantiomers or diastereoisomers arise due to hindered rotation around a bond axis. In this manuscript, we report a case in which torsional scan studies guided the thoughtful creation of a restricted axis of rotation between two heteroaromatic systems of a phosphoinositide 3-kinase (PI3K) ß inhibitor, generating a pair of atropisomeric compounds with significantly different pharmacological and pharmacokinetic profiles. Emblematic of these differences, the metabolism of inactive ( M)-28 is primarily due to the cytosolic enzyme aldehyde oxidase, while active ( P)-28 has lower affinity for aldehyde oxidase, resulting in substantially better metabolic stability. Additionally, we report torsional scan and experimental studies used to determine the barriers of rotation of this novel PI3Kß inhibitor.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Adenosine Triphosphate/metabolism , Animals , Enzyme Inhibitors/metabolism , Inhibitory Concentration 50 , Mice , Molecular Docking Simulation , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Conformation , Quinazolines/chemistry , Quinazolines/metabolism , Quinazolines/pharmacology , Stereoisomerism , Substrate SpecificityABSTRACT
Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.
ABSTRACT
Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.
Subject(s)
Arthritis, Experimental/drug therapy , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinazolinones/pharmacology , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/enzymology , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/enzymology , Cells, Cultured , Collagen/toxicity , Crystallography, X-Ray , Disease Models, Animal , Female , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Models, Molecular , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Quinazolinones/chemistry , Quinazolinones/pharmacokinetics , Rats , Rats, Inbred Lew , Tissue DistributionABSTRACT
In the past few years, there have been many advances in the efforts to cure patients with hepatitis C virus (HCV). The ultimate goal of these efforts is to develop a combination therapy consisting of only direct-antiviral agents (DAAs). In this paper, we discuss our efforts that led to the identification of a bicyclic template with potent activity against the NS5B polymerase, a critical enzyme on the life cycle of HCV. In continuation of our exploration to improve the stilbene series, the 3,5,6,8-tetrasubstituted quinoline core was identified as replacement of the stilbene moiety. 6-Methoxy-2(1H)-pyridone was identified among several heterocyclic headgroups to have the best potency. Solubility of the template was improved by replacing a planar aryl linker with a saturated pyrrolidine. Profiling of the most promising compounds led to the identification of quinoline 41 (RG7109), which was selected for advancement to clinical development.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Quinolines/pharmacology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Dogs , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Hepacivirus/enzymology , Humans , Models, Molecular , Quinolines/chemistry , Quinolines/pharmacokinetics , Rats , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazines/chemical synthesis , Pyrroles/chemical synthesis , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/enzymology , Blood Proteins/metabolism , Crystallography, X-Ray , Humans , Mice , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Pyrazines/pharmacology , Pyrazines/toxicity , Pyrroles/pharmacology , Pyrroles/toxicity , Structure-Activity Relationship , Syk KinaseABSTRACT
A novel approach to design selective spleen tyrosine kinase (Syk) inhibitors is described. Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of autoimmune diseases such as asthma, rheumatoid arthritis, and SLE. Fostamatinib, a Syk inhibitor that successfully completed phase II clinical trials, also exhibits some undesirable side effects. More selective Syk inhibitors could offer safer, alternative treatments. Through a systematic evaluation of the kinome, we identified Pro455 and Asn457 in the Syk ATP binding site as a rare combination among sequence aligned kinases and hypothesized that optimizing the interaction between them and a Syk inhibitor molecule would impart high selectivity for Syk over other kinases. We report the structure-guided identification of three series of selective spleen tyrosine kinase inhibitors that support our hypothesis and offer useful guidance to other researchers in the field.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Spleen/enzymology , Drug Design , Humans , Models, Molecular , Protein Kinase Inhibitors/chemistryABSTRACT
Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10)- and α-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.