ABSTRACT
The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.
Subject(s)
Antineoplastic Agents , Colonic Neoplasms , Lung Neoplasms , Humans , Animals , Mice , Proto-Oncogene Proteins p21(ras)/genetics , Precision Medicine , Antineoplastic Agents/pharmacology , Oncogenes , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , MAP Kinase Kinase 4ABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infected over 688 million people worldwide, causing public health concern and approximately 6.8 million deaths due to COVID-19. COVID-19, especially severe cases, is characterized by exacerbated lung inflammation with an increase of pro-inflammatory cytokines. In addition to antiviral drugs, there is a need for anti-inflammatory therapies to treat all phases of COVID-19. One of the most attractive drug targets for COVID-19 is the SARS-CoV-2 main protease (MPro), an enzyme responsible for cleaving polyproteins formed after the translation of viral RNA, which is essential for viral replication. MPro inhibitors, therefore, have the potential to stop viral replication and act as antiviral drugs. Considering that several kinase inhibitors are known for their action in inflammatory pathways, this could also be investigated toward a potential anti-inflammatory treatment for COVID-19. Therefore, the use of kinase inhibitors against SARS-CoV-2 MPro may be a promising strategy to find molecules with dual activityâantiviral and anti-inflammatory. Considering this, the potential of six kinase inhibitors against SARS-CoV-2 MPro were evaluated in silico and in vitro, including Baricitinib, Tofacitinib, Ruxolitinib, BIRB-796, Skepinone-L, and Sorafenib. To assess the inhibitory potential of the kinase inhibitors, a continuous fluorescent-based enzyme activity assay was optimized with SARS-CoV-2 MPro and MCA-AVLQSGFR-K(Dnp)-K-NH2 (substrate). BIRB-796 and Baricitinib were identified as inhibitors of SARS-CoV-2 MPro, presenting IC50 values of 7.99 and 25.31 µM, respectively. As they are also known for their anti-inflammatory action, both are prototype compounds with the potential to present antiviral and anti-inflammatory activity against SARS-CoV-2 infection.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Antiviral Agents/pharmacology , Protease Inhibitors/pharmacology , Anti-Inflammatory Agents/pharmacology , Molecular Docking SimulationABSTRACT
Glycogen synthase kinase-3ß (GSK-3ß) is a potential target in the field of Alzheimer's disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3ß inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure-activity relationships against GSK-3ß supported by 1 µs molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.
Subject(s)
Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , CHO Cells , Cell Line , Cricetulus , Drug Discovery , Drug Evaluation, Preclinical/methods , Drug Stability , Female , Glycogen Synthase Kinase 3 beta/chemistry , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Indoles/chemistry , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Glycogen synthase kinase-3ß (GSK-3ß) represents a relevant drug target for the treatment of neurodegenerative pathologies including Alzheimer's disease. We herein report on the optimization of a novel class of GSK-3ß inhibitors based on the tofacitinib-derived screen hit 3-((3R,4R)-3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile (1). We synthesized a series of 19 novel 7-chloro-9H-pyrimido[4,5-b]indole-based derivatives and studied their structure-activity relationships with focus on the cyanoacetyl piperidine moiety. We unveiled the crucial role of the nitrile group and its importance for the activity of this compound series. A successful rigidization approach afforded 3-(3aRS,7aSR)-(1-(7-chloro-9H-pyrimido[4,5-b]indol-4-yl)octahydro-6H-pyrrolo[2,3-c]pyridin-6-yl)-propanenitrile (24), which displayed an IC50 value of 130 nM on GSK-3ß and was further characterized by its metabolic stability. Finally, we disclosed the putative binding modes of the most potent inhibitors within the ATP binding site of GSK-3ß by 1 µs molecular dynamics simulations.
Subject(s)
Chemistry Techniques, Synthetic , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Binding Sites , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
An alternative strategy for the synthesis of 1-aryl- and 1-alkyl-2-methylsulfanyl-4-(4-fluorophenyl)-5-(pyridin-4-yl)imidazoles as potential p38α mitogen-activated protein kinase inhibitors is reported. The regioselective N-substitution of the imidazole ring was achieved by treatment of α-aminoketones with different aryl or alkyl isothiocyanates. In contrast to previously published synthesis routes starting from 2-amino-4-methylpyridine, the presented route is characterized by a higher flexibility and a lower number of steps. This strategy was also applied to access 1-alkyl-2-methylsulfanyl-5-(4-fluorophenyl)-4-(pyridin-4-yl)imidazoles in six steps starting from 2-chloro-4-methylpyridine.
Subject(s)
Imidazoles/chemical synthesis , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Isothiocyanates/chemistry , Ketones/chemistry , Picolines/chemistry , StereoisomerismABSTRACT
Drugs that function through covalent bond formation represent a considerable fraction of our repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clinical applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Herein, we review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.
Subject(s)
Cysteine/antagonists & inhibitors , Drug Design , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Cysteine/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistryABSTRACT
Inflammasomes activate caspase-1, initiating a lytic form of programmed cell death termed pyroptosis, which is an important innate immune defense mechanism against intracellular infections. We recently demonstrated in a mouse infection model of pyroptosis that instead of releasing bacteria into the extracellular space, bacteria remain trapped within the pyroptotic cell corpse, termed the pore-induced intracellular trap (PIT). This trapping mediates efferocytosis of the PIT and associated bacteria by neutrophils; bacteria are subsequently killed via neutrophil ROS. Using this pyroptosis model, we now show that the pro-inflammatory cytokines IL-1ß and IL-18 and inflammatory lipid mediators termed eicosanoids are required for effective clearance of bacteria downstream of pyroptosis. We further show that IL-1ß, IL-18, and eicosanoids affect this in part by mediating neutrophil recruitment to the PIT. This is in addition to our prior findings that complement is also important to attract neutrophils. Thus, the PIT initiates a robust and coordinated innate immune response involving multiple mediators that attract neutrophils to efferocytose the PIT and its entrapped bacteria.
Subject(s)
Gram-Positive Bacterial Infections/immunology , Neutrophils/immunology , Salmonella enterica/immunology , Animals , Caspase 1/metabolism , Cell Movement , Cells, Cultured , Eicosanoids/metabolism , Extracellular Traps/immunology , Immunity, Innate , Inflammasomes/metabolism , Interleukin-18/metabolism , Interleukin-1beta/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Porins/metabolism , Pyroptosis , Reactive Oxygen Species/metabolismABSTRACT
Janus kinases (JAKs) are a family of four cytosolic protein kinases with a high degree of structural similarity. Due to its very restricted role in immune regulation, JAK3 was promoted as an excellent target for immunosuppression for more than a decade, but clinical validation of this concept is still elusive. During the last years, speculation arose that kinase activity of JAK1, which cooperates with JAK3 in cytokine receptor signaling, may have a dominant role over the one of JAK3. Until recently, however, this issue could not be appropriately addressed due to a lack of highly isoform-selective tool compounds. With the recent resurgence of covalent drugs, targeting of a specific cysteine that distinguishes JAK3 from other JAK family members became an attractive design option. By applying this strategy, a set of JAK3 inhibitors with excellent selectivity against other JAK isoforms and the kinome was developed during the last three years and used to decipher JAK3-dependent signaling. The data obtained with these tool compounds demonstrates that selective JAK3 inhibition is sufficient to block downstream signaling. Since one of these inhibitors is currently under evaluation in phase II clinical studies against several inflammatory disorders, it will soon become apparent whether selective JAK3 inhibition translates into clinical efficacy.
Subject(s)
Cysteine/antagonists & inhibitors , Janus Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Cysteine/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Janus Kinase 3/metabolism , Protein Kinase Inhibitors/chemistryABSTRACT
A safe BET: Apart from target engagement, subcellular location and tissue distribution are critical parameters in drug discovery. A novel, generalizable approach has been reported that involves exploiting click chemistry to elucidate these parameters. By using clickable analogues of known Bromodomain and extraterminal domain (BET) inhibitors, various labels were introduced in situ and utilized to determine the biological fate of these probes.
Subject(s)
Azepines/pharmacology , Benzodiazepines/pharmacology , Drug Discovery , Proteins/antagonists & inhibitors , Triazoles/pharmacology , Cell Biology , Click Chemistry , Humans , Molecular StructureABSTRACT
BACKGROUND/AIMS: The diterpene alcohol Sclareol has been proposed for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, p38 kinase and casein kinase 1α. The present study explored, whether Sclareol induces eryptosis and, if so, shed light on the mechanisms involved. METHODS: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was estimated from haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Sclareol (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly modifying the average forward scatter, DCF-fluorescence or ceramide abundance. Sclareol (≥ 50 µM) further triggered hemolysis. Sclareol (100 µM) significantly increased Fluo3-fluorescence, but the effect of Sclareol on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+. Instead, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone (2 µM) and in the presence of casein kinase 1α inhibitor D4476 (10 µM). CONCLUSIONS: Sclareol triggers phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to activation of p38 kinase and casein kinase 1α.
Subject(s)
Calcium/metabolism , Diterpenes/pharmacology , Eryptosis/drug effects , Erythrocytes/drug effects , Phosphatidylserines/metabolism , Benzamides/pharmacology , Casein Kinase I/antagonists & inhibitors , Casein Kinase I/metabolism , Ceramides/metabolism , Dibenzocycloheptenes/pharmacology , Dose-Response Relationship, Drug , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/metabolism , Erythrocytes/metabolism , Flow Cytometry , Hemolysis/drug effects , Humans , Imidazoles/pharmacology , Reactive Oxygen Species/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Endoplasmic reticulum (ER) homeostasis is regulated by a network of signaling pathways to which stearoyl-CoA desaturase (SCD)-1, p38 mitogen-activated protein kinase (MAPK) and the unfolded protein response (UPR) belong. Because all these pathways are located at the interface of cell cycle control and cell stress, we hypothesized a cross-regulation. Interference with SCD-1, either by small interfering (si)RNA or the specific SCD-1 inhibitor CAY10566 (EC50 1 µM; ≥ 24 h), specifically induced phosphorylation and thus activation of p38 MAPK in NIH-3T3 mouse fibroblasts (1.5- to 2-fold; 48 hours). During lipotoxic and cell cycle stress, prolonged activation of p38 MAPK due to SCD-1 inhibition induced ER stress, the UPR, and ER/Golgi remodeling as shown by Western blot and immunofluorescence microscopy (1.2- to 3.5-fold). Specific inhibition of p38 MAPK by Skepinone-L [half maximal inhibitory concentration (IC50) 25-50 nM] reversed these effects (at 1 µM; 48 hours). The specificity by which SCD-1 modulates the phospholipid composition and inhibits p38 MAPK signaling (among survival/stress pathways), thereby preventing ER stress (but not other SCD-1-dependent responses), suggests selective protein-lipid interactions. Palmitoleoyl/oleoyl-phosphatidylinositol (PI) was accordingly identified as potential lipid mediator using chromatography-coupled ESI tandem mass spectrometry. We conclude that the negative regulation of p38 MAPK mediates the protective effects of SCD-1 on ER homeostasis under distinct stress conditions.
Subject(s)
Endoplasmic Reticulum/metabolism , Homeostasis , Stearoyl-CoA Desaturase/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , 3T3-L1 Cells , Animals , Blotting, Western , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Chromatography, Reverse-Phase , Dibenzocycloheptenes/pharmacology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Enzyme Inhibitors/pharmacology , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Mice , Microscopy, Fluorescence , NIH 3T3 Cells , Palmitates/pharmacology , Phospholipids/metabolism , Phosphorylation/drug effects , RNA Interference , Signal Transduction/drug effects , Spectrometry, Mass, Electrospray Ionization , Stearoyl-CoA Desaturase/antagonists & inhibitors , Stearoyl-CoA Desaturase/genetics , Tandem Mass Spectrometry , Unfolded Protein Response/drug effects , Unfolded Protein Response/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Multiple mutations in the EGFR gene are a major cause for the failure of Erlotinib and Gefitinib in the treatment of patients harboring non-small-cell lung cancer (NSCLC) who initially responded to this therapy. The development of these tyrosine kinase inhibitors (TKIs) is going back to the early 90s, where cancer was widely considered and fully treated as a disease of an organ. Fundamental gain of knowledge in cell biology in general and cancer genetics in particular led us to where we currently stand: cancer is a disease that originates in the genome. Fast and affordable gene sequencing paved the way and opened our eyes for the genetic instability of many cancers, particularly EGFR driven NSCLC. This might allow highly rational and personal therapies by aiming at a very particular wild type and mutant kinase pattern. However, the paradigm "one disease - one target - one drug" is currently challenged. Both activating and deactivating EGFR mutations are known to render the development of novel targeted drugs difficult. Among all lung adenocarcinomas, only 20% are driven by EGFR and only a subpopulation has an activating mutation (e.g. L858R), making them sensitive to first generation EGFR inhibitors. Unfortunately, most of them acquire second deactivating mutations (e.g. T790M) during treatment, leading to a complete loss of response. Are specific inhibitors of the double EGFR mutant L858R/T790M the magic bullet? Much scientific evidence but also high expectations justify this approach. Structural biology of EGFR mutants constitutes the basis for highly rational approaches. Second generation pan EGFR inhibitors inhibiting wild type (WT) and mutant EGFR like Afatinib suffer from dose-limiting adverse effects. Inhibition of WT EGFR is considered to be the culprit. Third generation EGFR inhibitors follow two strategies. Mutant selectivity and improved target residential time. These inhibitors display high mutant selectivity and irreversible binding patterns while sparing WT EGFR activity, hence enhancing tumor selectivity while minimizing adverse effects. Third generation EGFR inhibitors are still undergoing preclinical and clinical evaluation. The most advanced are Rociletinib and AZD9291 which displayed encouraging preliminary clinical phase II data regarding response and adverse effects. In the current review we show both a medicinal chemists' approach toward the design of third generation EGFR inhibitors as well as a detailed overview of the development of EGFR inhibitors over the last decade. High interdisciplinary approaches, such as structural biology and time-resolved tumor genetics pave the way toward the development of drugs that target EGFR mutants. This might lead to highly effective targeted and personalized therapies with enhanced response rates for a minor cohort of patients which have to undergo continuous gene sequencing, hence enabling therapies with tailor-made TKIs.
Subject(s)
Drug Resistance, Neoplasm/genetics , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Molecular Targeted Therapy/methods , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma of Lung , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , MutationABSTRACT
The activity of protein kinase B (Akt)--a major kinase promoting cell proliferation and survival--oscillates during the cell cycle. To investigate whether membrane phospholipids may regulate Akt phosphorylation and thus activity, we monitored the lipid profile of nocodazole-synchronized mouse NIH 3T3 fibroblasts during the cell cycle by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The proportion of sn-2-arachidonoyl-phosphatidylcholine (20:4-PC) inversely correlated with Akt activity. Increasing the cellular ratio of 20:4-PC by supplementation of 20:4-PC to the cell culture medium diminished Akt [serine (Ser)473] phosphorylation. Saturated and monounsaturated phosphatidylcholines, used as control had no effect; 20:4-PC reduced cell proliferation relative to controls, interfered with S-phase transition, and suppressed Akt downstream signaling and cyclin expression like LY294002, which is a specific inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. Additive effects of 20:4-PC and LY294002 were not observed, underlining the critical role of Akt for 20:4-PC signaling; 20:4-PC suppressed Akt membrane translocation as shown by immunofluorescence microscopy but left the concentration of the anchor lipid phosphatidylinositol-3,4,5-trisphosphate unchanged. An in vitro binding assay suggests that 20:4-PC attenuates the interaction of Akt with its membrane binding site. We conclude that 20:4-PC oscillates during the cell cycle and delays cell cycle progression by inhibiting Akt membrane binding.
Subject(s)
Cell Cycle/physiology , Cell Proliferation/drug effects , Lysophosphatidylcholines/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Chromatography, Liquid , Chromones/metabolism , Chromones/pharmacology , Lysophosphatidylcholines/pharmacology , Mice , Microscopy, Fluorescence , Morpholines/metabolism , Morpholines/pharmacology , NIH 3T3 Cells , Nocodazole , Phosphorylation/drug effects , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
We target the gatekeeper MET146 of c-Jun N-terminal kinase 3 (JNK3) to exemplify the applicability of X···S halogen bonds in molecular design using computational, synthetic, structural and biophysical techniques. In a designed series of aminopyrimidine-based inhibitors, we unexpectedly encounter a plateau of affinity. Compared to their QM-calculated interaction energies, particularly bromine and iodine fail to reach the full potential according to the size of their σ-hole. Instead, mutation of the gatekeeper residue into leucine, alanine, or threonine reveals that the heavier halides can significantly influence selectivity in the human kinome. Thus, we demonstrate that, although the choice of halogen may not always increase affinity, it can still be relevant for inducing selectivity. Determining the crystal structure of the iodine derivative in complex with JNK3 (4X21) reveals an unusual bivalent halogen/chalcogen bond donated by the ligand and the back-pocket residue MET115. Incipient repulsion from the too short halogen bond increases the flexibility of Cε of MET146, whereas the rest of the residue fails to adapt being fixed by the chalcogen bond. This effect can be useful to induce selectivity, as the necessary combination of methionine residues only occurs in 9.3% of human kinases, while methionine is the predominant gatekeeper (39%).
Subject(s)
Chalcogens/chemistry , Halogens/chemistry , Methionine/metabolism , Mitogen-Activated Protein Kinase 10/chemistry , Crystallography, X-Ray , Fluorescence PolarizationABSTRACT
An optimized strategy for the synthesis of the potent p38α mitogen-activated protein kinase inhibitors 2-(2-hydroxyethylsulfanyl)-4-(4-fluorophenyl)-5-(2-aminopyridin-4-yl)imidazole (3) and 2-(2,3-dihydroxypropylsulfanyl)-4-(4-fluorophenyl)-5-(2-aminopyridin-4-yl)imidazole (4) starting from 2-fluoro-4-methylpyridine is reported. In contrast to a previously published synthesis starting from 2-bromo-4-methylpyridine, the overall yield could be increased from 3.6% to 29.4%. Moreover, this strategy avoids the use of palladium as a catalyst and is more diverse and versatile. Using this optimized protocol, both enantiomers of potent inhibitor 3 were synthesized. Biological data demonstrated that the (S)-enantiomer is the two times more potent eutomer.
Subject(s)
Aminopyridines/chemistry , Aminopyridines/pharmacology , Imidazoles/chemistry , Imidazoles/pharmacology , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Aminopyridines/chemical synthesis , Humans , Imidazoles/chemical synthesis , Mitogen-Activated Protein Kinase 14/metabolism , Protein Kinase Inhibitors/chemical synthesis , StereoisomerismABSTRACT
In the title compound, C18H24N6O·H2O, the piperidine ring adopts a chair conformation with an N-C-C-C torsion angle of 39.5â (5)° between the cis-related substituents. The pyrrole N-H group forms a water-mediated inter-molecular hydrogen bond to one of the N atoms of the annelated pyrimidine ring. The water mol-ecule connects two organic mol-ecules and is disorderd over two positions (occupancies of 0.48 and 0.52). The crystal packing shows zigzag chains of alternating organic and water mol-ecules running parallel to the a axis.
ABSTRACT
Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.
Subject(s)
Enzyme Inhibitors , ErbB Receptors , Reproducibility of Results , Enzyme Inhibitors/pharmacology , Drug Design , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistryABSTRACT
ACKR3, an atypical chemokine receptor, has been associated with prothrombotic events and the development of cardiovascular events. We designed, synthesized, and evaluated a series of novel small molecule ACKR3 agonists. Extensive structure-activity relationship studies resulted in several promising agonists with potencies ranging from the low micromolar to nanomolar range, for example, 23 (EC50 = 111 nM, Emax = 95%) and 27 (EC50 = 69 nM, Emax = 82%) in the ß-arrestin-recruitment assay. These compounds are selective for ACKR3 versus ACKR2, CXCR3, and CXCR4. Several agonists were subjected to investigations of their P-selectin expression reduction in the flow cytometry experiments. In particular, compounds 23 and 27 showed the highest potency for platelet aggregation inhibition, up to 80% and 97%, respectively. The most promising compounds, especially 27, exhibited good solubility, metabolic stability, and no cytotoxicity, suggesting a potential tool compound for the treatment of platelet-mediated thrombosis.
Subject(s)
Drug Design , Platelet Aggregation Inhibitors , Platelet Aggregation , Receptors, CXCR , Humans , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation Inhibitors/chemical synthesis , Platelet Aggregation Inhibitors/chemistry , Structure-Activity Relationship , Platelet Aggregation/drug effects , Receptors, CXCR/agonists , Receptors, CXCR/metabolism , Animals , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/chemical synthesis , P-Selectin/metabolism , Blood Platelets/drug effects , Blood Platelets/metabolismABSTRACT
Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent "warheads" based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.
Subject(s)
Protein Kinase Inhibitors , Receptor, Fibroblast Growth Factor, Type 4 , Receptor, Fibroblast Growth Factor, Type 4/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Humans , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity Relationship , Microsomes, Liver/metabolismABSTRACT
Fibroblast growth factor receptor (FGFR)-2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.