ABSTRACT
Modifications at C6 and C7 positions of 3-cyanoquinolines 6 and 7 led to potent inhibitors of the ErbB family of kinases particularly against EGFRWT and Her4 enzymes in the radioisotope filter binding assay. The lead (4, SAB402) displayed potent dual biochemical activities with EGFRWT/Her4 IC50 ratio of 80 due to its potent inhibition of Her4 activity (IC50 0.03 nM), however, the selectivity towards activating mutations (EGFRL858R, EGFRex19del) was decreased. Inhibitor 4 also exhibited excellent growth inhibition in seven different cancer types and reduced cell viability in female NMRI nude mice in the intraperitoneally implanted hollow fibers which have been loaded with MOLT-4 (leukemia) and NCI-H460 (NSCLC) cells in a statistically significant manner.
Subject(s)
Acetamides/pharmacology , Protein Kinase Inhibitors/pharmacology , Receptor, ErbB-4/antagonists & inhibitors , Sulfhydryl Compounds/pharmacology , Acetamides/chemical synthesis , Acetamides/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Receptor, ErbB-4/metabolism , Structure-Activity Relationship , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.
Subject(s)
Drug Design , Matrix Metalloproteinase 12/metabolism , Matrix Metalloproteinase Inhibitors , Pulmonary Disease, Chronic Obstructive/enzymology , Animals , Asthma/drug therapy , Asthma/enzymology , Chemistry, Pharmaceutical/methods , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Humans , Inflammation , Inhibitory Concentration 50 , Mice , Models, Chemical , Models, Molecular , Molecular Conformation , Pulmonary Disease, Chronic Obstructive/drug therapy , Structure-Activity Relationship , Sulfonamides/chemistry , X-RaysABSTRACT
Cortisol and the glucocorticoid receptor (GR) signaling pathway has been linked to the development of diabetes and metabolic syndrome. In vivo, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) catalyzes the conversion of inactive cortisone to its active form, cortisol. Existing clinical data have supported 11ß-HSD1 as a valid therapeutic target for type 2 diabetes. In our research program, (R)-1,1,1-trifluoro-2-(3-((R)-4-(4-fluoro-2-(trifluoromethyl)phenyl)-2-methylpiperazin-1-ylsulfonyl)phenyl)propan-2-ol (HSD-016) was discovered to be a potent, selective, and efficacious 11ß-HSD1 inhibitor and advanced as a clinical candidate. Herein, a reliable and scalable synthesis of HSD-016 is described. Key transformations include an asymmetric synthesis of a chiral tertiary alcohol via Sharpless dihydroxylation, epoxide formation, and subsequent mild reduction. This route ensured multikilogram quantities of HSD-016 necessary for clinical studies.
Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/chemical synthesis , Piperazines/chemical synthesis , Propanols/chemical synthesis , 11-beta-Hydroxysteroid Dehydrogenase Type 1/antagonists & inhibitors , 11-beta-Hydroxysteroid Dehydrogenase Type 1/chemistry , Administration, Oral , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Molecular Structure , Piperazines/chemistry , Piperazines/pharmacology , Propanols/chemistry , Propanols/pharmacologyABSTRACT
The synthesis and stereochemical determination of 1-(4-(4-((1R,5R,6R)-6-hydroxy-3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-yl)phenyl)-3-(pyridin-4-yl)urea (2), an active metabolite of the potent PI3 kinase inhibitor PKI-179 (1), is described. Stereospecific hydroboration of the double bond of 2,5-dihydro-1H-pyrrole 8 gave the 2,3-trans alcohol 9 exclusively. The configuration of the 3-hydroxyl group in 9 was inverted by an oxidation and stereoselective reduction sequence to give the corresponding 2,3-cis isomer 23. Both exo (21) and endo (27) isomers of the metabolite 2 were prepared via a practical synthetic route from 9 and 23, respectively, and the stereochemistry of 2 was determined to be endo. The endo isomer (27) was separated into two enantiomers 28 and 29 by chiral HPLC. Compound 2 was found to be enantiomerically pure and identical to the enantiomer 28. The absolute stereochemistry of the enantiomer 28 was determined by Mosher's method, thus establishing the stereochemistry of the active metabolite 2.
Subject(s)
Bridged-Ring Compounds/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Morpholines/chemical synthesis , Phosphoinositide-3 Kinase Inhibitors , Urea/analogs & derivatives , Binding Sites , Bridged-Ring Compounds/chemistry , Enzyme Inhibitors/pharmacology , Molecular Structure , Morpholines/chemistry , Morpholines/pharmacology , Oxidation-Reduction , Phosphatidylinositol 3-Kinases/chemistry , Stereoisomerism , Urea/chemical synthesis , Urea/chemistry , Urea/pharmacologyABSTRACT
A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitumor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure-activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was comparable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher metabolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against MCF-7 and MDA-MB-361 lines and was selected for further evaluation.
Subject(s)
Aneuploidy , Antineoplastic Agents/pharmacology , Benzothiazoles/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Benzothiazoles/chemical synthesis , Benzothiazoles/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Neoplasm Proteins/deficiency , Neoplasm Proteins/metabolism , Securin , Stereoisomerism , Structure-Activity RelationshipABSTRACT
We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3Kalpha. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3Kgamma revealed the key hydrogen bonding interactions.
Subject(s)
Benzofurans/chemistry , Benzofurans/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Adenosine Triphosphate/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Crystallography, X-Ray , Humans , Inhibitory Concentration 50 , Mice , Mice, Nude , Microsomes/metabolism , Models, Molecular , Neoplasms/drug therapy , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Structure-Activity Relationship , TOR Serine-Threonine KinasesABSTRACT
A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed.
Subject(s)
Morpholines/chemistry , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , TOR Serine-Threonine Kinases/antagonists & inhibitors , Triazines/chemistry , Urea/analogs & derivatives , Administration, Oral , Animals , Cell Line, Tumor , Humans , Mice , Mice, Nude , Morpholines/chemical synthesis , Morpholines/pharmacokinetics , Phosphatidylinositol 3-Kinase/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity Relationship , TOR Serine-Threonine Kinases/metabolism , Triazines/chemical synthesis , Triazines/pharmacokinetics , Tropanes/chemistry , Urea/chemical synthesis , Urea/chemistry , Urea/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
This article describes the syntheses and SAR of a series of imidazolopyrimidine derivatives, which are evaluated as inhibitors of PI3-Kinase (PI3K) and mTOR. These compounds were found to be ATP competitive with good tumor cell growth inhibition, and suppression of pathway specific biomakers such as phosphorylation of Akt at T308.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Binding Sites , Binding, Competitive , Cell Line, Tumor , Computer Simulation , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , TOR Serine-Threonine KinasesABSTRACT
The palladium-catalyzed oxidative coupling of pyrido- and benzotriazol-1-yloxyquinazolines and -thienopyrimidines with aryl boronic acids in the presence of Pd(PPh(3))(4) and Cs(2)CO(3) under oxygen in DME containing 0.4-0.8% water for the preparation of heteroaryl ethers is described. These transformations of triazol-1-yloxy reagents demonstrate excellent O-chemoselective control under mild conditions and good yields. Mechanistic studies based on (18)O labeling indicate that phenols as intermediates in S(N)Ar reactions with ethers are formed in oxidative and nonoxidative pathways.
Subject(s)
Boronic Acids/chemistry , Chemistry, Organic/methods , Ethers/chemistry , Palladium/chemistry , Quinazolines/chemistry , Catalysis , Models, Chemical , Molecular Structure , Nitrogen/chemistry , Oxygen/chemistry , Phenol/chemistry , Pyrimidines/chemistry , Sulfur/chemistry , Time FactorsABSTRACT
The novel bicyclic penem inhibitor BLI-489 has demonstrated activity as an inhibitor of class A, C, and D beta-lactamases. To determine the combination of piperacillin and BLI-489 to be used in susceptibility testing that would most accurately identify susceptible and resistant isolates, a predictor panel of beta-lactamase-producing bacteria was utilized to determine the reliability of the combination of piperacillin-BLI-489 at a constant inhibitor concentration of 2 or 4 microg/ml and at ratios of 1:1, 2:1, 4:1, and 8:1. There were a number of strains that would be falsely reported as susceptible or intermediate if tested with the ratios of 1:1 and 2:1, whereas the constant concentration of 2 microg/ml of BLI-489 and the ratio of 8:1 had a tendency to overpredict resistance. Similar MICs were obtained with piperacillin-BLI-489 in a 4:1 ratio and when BLI-489 was held constant at 4 microg/ml. Based on these results, an in vitro testing methodology employing a constant concentration of 4 microg/ml BLI-489 was used to evaluate the combination of piperacillin-BLI-489 against a larger panel of recently identified clinical isolates. Approximately 55% of all of the enteric bacilli tested were nonsusceptible to piperacillin alone (MIC > or = 32 microg/ml). However, 92% of these piperacillin nonsusceptible strains were inhibited by < or =16 microg/ml piperacillin-BLI-489; in contrast, only 66% were inhibited by < or =16 microg/ml piperacillin-tazobactam. The combination of piperacillin-BLI-489 also demonstrated improved activity compared to that of piperacillin-tazobactam against the problematic extended-spectrum beta-lactamase- and AmpC-expressing strains.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Microbial Sensitivity Tests/methods , Piperacillin/pharmacology , beta-Lactamase Inhibitors , Bacteria/drug effects , Bacterial Infections/microbiology , Drug Combinations , Humans , Lactams/pharmacologyABSTRACT
Src kinase signaling has been implicated in multiple mechanisms of ischemic injury, including vascular endothelial growth factor (VEGF)-mediated vascular permeability that leads to vasogenic edema, a major clinical complication in stroke and brain trauma. Here we report the effects of two novel Src kinase inhibitors, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile (SKI-606) and 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient [transient middle cerebral artery occlusion (tMCAO)] and permanent [permanent middle cerebral artery occlusion (pMCAO)] focal ischemia models in the rat were used with drug treatments initiated up to 6 h after onset of stroke to mimic the clinical scenario. Brain penetration of Src inhibitors, their effect on blood-brain barrier integrity and VEGF signaling in human endothelial cells were also evaluated. Our results demonstrate that both agents potently block VEGF-mediated signaling in human endothelial cells, penetrate rat brain upon systemic administration, and inhibit postischemic Src activation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3-30 mg/kg i.v.) resulted in a dose-dependent reduction in infarct volume and robust protection from neurological impairments even when the therapy was initiated up to 4- to 6-h after tMCAO. Src blockade after pMCAO resulted in accelerated improvement in recovery from motor, sensory, and reflex deficits during a long-term (3 weeks) testing period poststroke. These data demonstrate that the novel Src kinase inhibitors provide effective treatment against ischemic conditions within a clinically relevant therapeutic window and may constitute a viable therapy for acute stroke.
Subject(s)
Aniline Compounds/therapeutic use , Brain Ischemia/drug therapy , Neuroprotective Agents/therapeutic use , Nitriles/therapeutic use , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Quinolines/therapeutic use , src-Family Kinases/antagonists & inhibitors , Aniline Compounds/administration & dosage , Aniline Compounds/chemistry , Aniline Compounds/pharmacokinetics , Animals , Blood Platelets/drug effects , Blood Platelets/enzymology , Brain/drug effects , Brain/enzymology , Brain/metabolism , Brain Ischemia/enzymology , Capillary Permeability , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Flow Cytometry , Humans , Injections, Intravenous , Male , Molecular Structure , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacokinetics , Neuropsychological Tests , Nitriles/administration & dosage , Nitriles/chemistry , Nitriles/pharmacokinetics , Piperazines/administration & dosage , Piperazines/chemistry , Piperazines/pharmacokinetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Quinolines/administration & dosage , Quinolines/chemistry , Quinolines/pharmacokinetics , Rats , Rats, Wistar , Time FactorsABSTRACT
The synthesis and SAR of a series of 2,4-diamino-quinazoline derivatives as beta-catenin/Tcf-4 inhibitors are described. This series was developed by modifying the initial lead 1, which was identified by screening of our compound library and found to inhibit the beta-catenin/Tcf-4 pathway. Replacement of the biphenyl moiety in compound 1 with the N-phenylpiperidine-4-carboxamide chain as in 2, resulted in a number of new analogues, which are potent inhibitors of the beta-catenin/Tcf-4 pathway. Compound such as 16k exhibited good cellular potency, solubility, metabolic stability and oral bioavailability.
Subject(s)
Anilides/chemistry , Antineoplastic Agents/chemistry , Colorectal Neoplasms/drug therapy , Quinazolines/chemistry , TCF Transcription Factors/antagonists & inhibitors , beta Catenin/antagonists & inhibitors , Anilides/chemical synthesis , Anilides/pharmacokinetics , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , Quinazolines/chemical synthesis , Quinazolines/pharmacokinetics , Structure-Activity Relationship , TCF Transcription Factors/metabolism , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/metabolism , Xenograft Model Antitumor Assays , beta Catenin/metabolismABSTRACT
The major drawback of melanoma therapy with BRAF and MAPK inhibitors is the innate and acquired drug resistance. We therefore explored alternative targets and developed a new compound, SAB298, that is a SRC-family kinase (SFK) inhibitor. The drug is cytotoxic to patient-derived melanoma cells regardless of oncogene expression and inhibits tumor growth in vivo. As expected, it inhibited SRC and PI3K activity, and had the additional property of ERBB2 inhibition, that lead to inactivation of the two ERK phosphatases PP2A and SHP2. In 57% of the melanoma cell lines tested, the consequent increase in ERK activity lead to proteolytic degradation of its substrate, the lineage specific transcription factor MITF, likely contributing to growth arrest. Treatment with a combination of SAB298 and AZD6244 (selumetinib), induced a synergistic growth inhibition, suggesting that the new compound could be used in the clinic as a substitute for, or in combination with MAPK inhibitors.
ABSTRACT
The partnering of a beta-lactam with a beta-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to beta-lactam antibiotics mediated by serine beta-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D beta-lactamase that is resistant to inactivation by tazobactam. The K(i) of each penem inhibitor for OXA-1 was in the nM range (K(i) of penem 1, 45 +/- 8 nM; K(i) of penem 2, 12 +/- 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 beta-lactamase were 0.13 +/- 0.01 s(-1) and 0.11 +/- 0.01 s(-1), respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in Subject(s)
Bridged Bicyclo Compounds/pharmacology
, Enzyme Inhibitors/pharmacology
, Escherichia coli/drug effects
, Escherichia coli/enzymology
, Heterocyclic Compounds/pharmacology
, beta-Lactamase Inhibitors
, Anti-Bacterial Agents/pharmacology
, Bridged Bicyclo Compounds/chemistry
, Enzyme Inhibitors/chemistry
, Escherichia coli/genetics
, Heterocyclic Compounds/chemistry
, Kinetics
, Microbial Sensitivity Tests
, Penicillanic Acid/analogs & derivatives
, Penicillanic Acid/pharmacology
, Piperacillin/pharmacology
, Structure-Activity Relationship
, Tazobactam
, beta-Lactamases/chemistry
ABSTRACT
Beta-lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against beta-lactam antibiotics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital settings, have reduced activity against newly emerging beta-lactamases. Bacterial production of diverse beta-lactamases including class-A, class-C, and ESBLs has motivated several research groups to search for inhibitors with a broader spectrum of activity. Previously, several novel 6-methylidene penems bearing, [5,5] [5,6] and [5,5,5] heterocycles have been synthesized in our laboratory and were shown to be potent and broad-spectrum beta-lactamase inhibitors. As a continuation of our previous work and in order to extend the structure-activity relationships, in this paper, we describe herein the synthesis and in vitro, in vivo activities of several novel 5,5,6-fused tricyclic heterocycles attached to the 6-methylidene penem core. The compounds presented in the current paper are potent and broad-spectrum inhibitors of the TEM-1 and AmpC beta-lactamases. In combination with piperacillin, their in vitro activities showed enhanced susceptibility to class A- and C-resistant strains studied in various bacteria. Some of the newly synthesized compounds such as 12a-c were shown to have in vivo activity in the acute lethal infection model against TEM-1 producing organisms. The 5,5,6-fused heterocyclic ring cores such as 21, 25, and 35 reported here are hitherto unknown in the literature.
Subject(s)
Anti-Bacterial Agents/chemistry , Heterocyclic Compounds, 3-Ring/pharmacology , Lactams/pharmacology , beta-Lactamase Inhibitors , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Heterocyclic Compounds, 3-Ring/chemistry , Imidazoles/chemistry , Imidazoles/pharmacology , Lactams/chemistry , Microbial Sensitivity Tests , Pyrazoles/chemistry , Pyrazoles/pharmacology , Structure-Activity Relationship , beta-LactamasesABSTRACT
The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC(50), 0.149 +/- 0.045 micromol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors.
Subject(s)
Cysteine/metabolism , Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Allosteric Regulation/drug effects , Animals , Catalysis/drug effects , Cell Line, Tumor , Down-Regulation/drug effects , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Kinetics , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Phosphorylation/drug effects , Protein Biosynthesis/drug effects , Protein Kinases/metabolism , RNA Caps/metabolism , Rats , Structure-Activity Relationship , Substrate Specificity/drug effects , TOR Serine-Threonine Kinases , Time FactorsABSTRACT
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin receptor pathways and thus an attractive therapeutic target for diabetes and obesity. Starting with a high micromolar lead compound, structure-based optimization of novel PTP1B inhibitors by extension of the molecule from the enzyme active site into the second phosphotyrosine binding site is described. Medicinal chemistry, guided by X-ray complex structure and molecular modeling, has yielded low nanomolar PTP1B inhibitors in an efficient manner. Compounds from this chemical series were found to be actively transported into hepatocytes. This active uptake into target tissues could be one of the possible avenues to overcome the poor membrane permeability of PTP1B inhibitors.
Subject(s)
Models, Molecular , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatases/antagonists & inhibitors , Thiophenes/chemical synthesis , Animals , Binding Sites , Caco-2 Cells , Catalytic Domain , Cell Membrane Permeability , Crystallography, X-Ray , Half-Life , Hepatocytes , Humans , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Molecular Structure , Phosphotyrosine/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/chemistry , Rats , Rats, Sprague-Dawley , Solubility , Structure-Activity Relationship , Thiophenes/pharmacokinetics , Thiophenes/pharmacology , Tissue DistributionABSTRACT
A series of potent anthranilic acid-based inhibitors of the hepatitis C NS5B polymerase has been identified. The inhibitors bind to a site on NS5B between the thumb and palm regions adjacent to the active site as determined by X-ray crystallography of the enzyme-inhibitor complex. Guided by both molecular modeling and traditional SAR, the enzyme activity of the initial hit was improved by approximately 100-fold, yielding a series of potent and selective NS5B inhibitors with IC50 values as low as 10 nM. These compounds were also inhibitors of the HCV replicon in cultured HUH7 cells.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , ortho-Aminobenzoates/chemical synthesis , Allosteric Regulation , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Cell Line , Crystallography, X-Ray , Hepacivirus/enzymology , Hepacivirus/genetics , Humans , Models, Molecular , Replicon , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacologyABSTRACT
The design and synthesis of a series of 6-methylidene penems containing [6,5]-fused bicycles (thiophene, imidazole, or pyrazle-fused system) as novel class A, B, and C beta-lactamase inhibitors is described. These penems proved to be potent inhibitors of the TEM-1 (class A) and AmpC (class C) beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. Their in vitro and in vivo activities in combination with piperacillin are discussed. On the basis of the crystallographic structures of a serine-bound reaction intermediate of 2 with SHV-1 (class A) and GC1 (class C) enzymes, compounds 14a-l were designed and synthesized. Penems are proposed to form a seven-membered 1,4 thiazepine ring in both class A and C beta-lactamases. The interaction energy calculation for the enzyme-bound intermediates favor the formation of the C7 R enantiomer over the S enantiomer of the 1,4-thiazepine in both beta-lactamases, which is consistent with those obtained from the crystal structure of 2 with SHV-1 and GC1.