ABSTRACT
The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.
Subject(s)
Janus Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazines/chemistry , Pyrroles/chemistry , Drug Design , Humans , Janus Kinase 3/metabolism , Models, Molecular , Molecular Conformation , Molecular Structure , Phosphorylation , Piperidines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Structure-Activity RelationshipABSTRACT
Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.
Subject(s)
Drug Design , Janus Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Binding Sites , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Indazoles/chemistry , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 1/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Janus Kinase 3/metabolism , Molecular Docking Simulation , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Structure, Tertiary , Pyrazines/chemical synthesis , Pyrazines/chemistry , Pyrazines/metabolism , Structure-Activity RelationshipABSTRACT
The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.
ABSTRACT
Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.
Subject(s)
Apoptosis/physiology , Hydantoins/pharmacology , Models, Molecular , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Blotting, Western , Cell Cycle Proteins , Cell Line, Tumor , Crystallization , Dimerization , Fluorescence Resonance Energy Transfer , Humans , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-mdm2/chemistry , Signal Transduction/drug effects , Signal Transduction/physiology , Tetrazolium Salts , ThiazolesABSTRACT
The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K(i) = 1-3 nmol/L) and was inactive (K(i) > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC(50)s
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Clinical Trials, Phase I as Topic , Cyclin-Dependent Kinases/metabolism , Female , G1 Phase/drug effects , G2 Phase/drug effects , Genes, MDR/drug effects , Humans , Mice , Mice, Nude , Phosphorylation/drug effects , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats , Rats, Inbred F344 , Retinoblastoma/drug therapy , Retinoblastoma/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.
