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1.
Cell Chem Biol ; 25(11): 1389-1402.e9, 2018 11 15.
Article in English | MEDLINE | ID: mdl-30197194

ABSTRACT

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.


Subject(s)
Amyloid/chemistry , Benzoxazoles/chemistry , Benzoxazoles/pharmacology , Protein Aggregates/drug effects , alpha-Synuclein/chemistry , Amyloid/antagonists & inhibitors , Amyloid/ultrastructure , Fluorescence Resonance Energy Transfer/methods , High-Throughput Screening Assays/methods , Humans , Protein Conformation/drug effects , Protein Multimerization/drug effects , alpha-Synuclein/antagonists & inhibitors , alpha-Synuclein/ultrastructure
2.
J Pharm Biomed Anal ; 141: 32-38, 2017 Jul 15.
Article in English | MEDLINE | ID: mdl-28419935

ABSTRACT

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody.


Subject(s)
Proteins/analysis , Animals , CHO Cells , Chromatography, Liquid , Cricetinae , Cricetulus , Enzyme-Linked Immunosorbent Assay , Humans , Tandem Mass Spectrometry
3.
J Chromatogr A ; 1481: 44-52, 2017 Jan 20.
Article in English | MEDLINE | ID: mdl-28017567

ABSTRACT

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules.


Subject(s)
Cathepsin B/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Immunoconjugates/chemistry , Immunoglobulin G/analysis , Pharmaceutical Preparations/analysis , Antineoplastic Agents/chemistry , Buffers , Humans , Lysine/chemistry , Mass Spectrometry , Pharmaceutical Preparations/chemistry , Polysorbates/chemistry , Reference Standards , Reproducibility of Results , Ultraviolet Rays
4.
J Pharm Biomed Anal ; 128: 447-454, 2016 Sep 05.
Article in English | MEDLINE | ID: mdl-27344634

ABSTRACT

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is widely used for purity analysis of monoclonal antibody therapeutics for release and stability to demonstrate product consistency and shelf life during the manufacturing and life cycle of the product. CE-SDS method development is focused on exploring the method capability to provide the information about the product purity and product related degradants (fragmentation, aggregation etc.). In order to establish the functionality of the instrumentation, software, and sample preparation; system suitability criteria need to be defined for analytical methods using a well characterized reference standard run under the same protocol and analysis as the test articles. Typically the reference standard is produced using a manufacturing process representative of the clinical material. The qualification, control, and maintenance of in-house reference standards are established through rigorous quality and regulatory guidelines. The U.S. Pharmacopeia (USP) has developed a monoclonal IgG System Suitability Reference Standard to be utilized for assessment of system suitability in CE-SDS methods. In this communication, we evaluate the system suitability acceptance criteria performance of the USP IgG standard using two methods, the recommended USP protocol provided in monograph <129> and a molecule specific Bristol-Myers Squibb (BMS) CE-SDS method. The results from USP IgG standard were compared with two in-house monoclonal antibody reference standards. The data suggest that the USP CE-SDS method may not be suitable for CE-SDS analysis for release and stability of monoclonal antibody therapeutics due to the high level of method induced partial reduction observed for all molecules tested. This high level of fragmentation observed utilizing the USP method will result in reporting lower purity levels, which will impact the overall quality assessment of the molecule. The system suitability criteria recommended by the USP method can be achieved by using the USP reference standard during the development and pre-validation stages. Furthermore, the USP reference standard does not offer significant advantages to existing SST criteria in the BMS method during release and stability testing, and therefore we propose use of the USP standard only during the optimization and pre-validation stages of method development.


Subject(s)
Antibodies, Monoclonal/analysis , Drug Liberation , Drug Stability , Electrophoresis, Capillary/standards , Reference Standards , Sodium Dodecyl Sulfate , Electrophoresis, Capillary/methods , Immunoglobulin G/analysis , Pharmacopoeias as Topic , United States
5.
Indian J Pharmacol ; 43(4): 450-4, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21845004

ABSTRACT

To investigate the antihyperglycemic and hypolipidemic effects of methanolic extract of leaves of Amaranthus viridis (MEAV) in normal and Streptozotocin (STZ) induced diabetic rats. The anti-hyperglycemic and hypolipidemic activity of methanolic extract of leaves of Amaranthus viridis was evaluated by using normal and STZ induced diabetic rats at dose of 200 mg/kg and 400 mg/kg by mouth per day for 21 days. Blood glucose levels and body weight was monitored at specific intervals, and different biochemical parameters, serum cholesterol, serum triglyceride, high density lipoprotein, low density lipoprotein, very low density lipoprotein were also assessed in the experimental animals. Histology of pancreas was performed. The statistical data indicated a significant increase in the body weight, decrease in the blood glucose, total cholesterol and serum triglycerides after treatment with MEAV. High density lipoprotein (HDL) cholesterol level was significantly increased when treated with extract. Histologically, focal necrosis was observed in the diabetic rat pancreas; however, was less obvious in treated groups. The MEAV has beneficial effects in reducing the elevated blood glucose level and body weight changes, and improves the lipid profile of STZ induced rats.

6.
Bioorg Med Chem Lett ; 20(1): 398-402, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19939680

ABSTRACT

Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.


Subject(s)
Anti-HIV Agents/chemistry , Enzyme Inhibitors/chemistry , Ribonuclease H, Human Immunodeficiency Virus/antagonists & inhibitors , Thiocarbamates/chemistry , Triazoles/chemistry , Amino Acid Sequence , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Cell Line , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Humans , Molecular Sequence Data , Ribonuclease H, Human Immunodeficiency Virus/metabolism , Structure-Activity Relationship , Thiocarbamates/chemical synthesis , Thiocarbamates/pharmacology , Triazoles/chemical synthesis , Triazoles/pharmacology , Virus Replication/drug effects
7.
Bioorg Med Chem ; 18(1): 190-201, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19932972

ABSTRACT

Piperidinyl diphenylsulfonyl sulfonamides are a novel class of molecules that have inhibitory binding affinity for sFRP-1. As a secreted protein sFRP-1 inhibits the function of the secreted Wnt glycoprotein. Therefore, as inhibitors of sFRP-1 these small molecules facilitate the Wnt/beta-catenin canonical signaling pathway. Details of the structure-activity relationships and biological activity of this structural class of compounds will be discussed.


Subject(s)
Glycoproteins/antagonists & inhibitors , Signal Transduction/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacology , Wnt Proteins/metabolism , Animals , Cell Line, Tumor , Glycoproteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Mice , Microsomes, Liver/metabolism , Organ Culture Techniques , Osteogenesis/drug effects , Rats , Skull/cytology , Skull/drug effects , Structure-Activity Relationship , beta Catenin/metabolism
8.
Bioorg Med Chem Lett ; 20(1): 366-70, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19897365

ABSTRACT

A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone.


Subject(s)
Imidazoles/chemistry , Pyrimidines/chemistry , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Bone Development/drug effects , Cell Line, Tumor , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred C57BL , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Skull/metabolism , Wnt Proteins/agonists , Wnt Proteins/genetics , Wnt3 Protein , Wnt3A Protein , beta Catenin/agonists
9.
Bioorg Med Chem Lett ; 19(22): 6337-9, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19819694

ABSTRACT

Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.


Subject(s)
Osteogenesis/physiology , Proteins/isolation & purification , Calcification, Physiologic , Cell Differentiation/physiology , Cells, Cultured , Frizzled Receptors/antagonists & inhibitors , Frizzled Receptors/isolation & purification , Intracellular Signaling Peptides and Proteins , Molecular Structure , Proteins/antagonists & inhibitors , RANK Ligand
10.
Bone ; 44(6): 1063-8, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19254787

ABSTRACT

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K(D) of 0.35 microM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC(50) of 3.9 microM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K(D) of 0.08 microM and inhibited it with an EC(50) of 0.65 microM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 microM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.


Subject(s)
Osteogenesis/drug effects , Proteins/antagonists & inhibitors , Sulfonamides/pharmacology , Wnt Proteins/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Humans , Intracellular Signaling Peptides and Proteins , Mice , Organ Culture Techniques , Osteoblasts/cytology , Osteoblasts/drug effects , Osteocytes/cytology , Osteocytes/drug effects , Proteins/genetics , Proteins/metabolism , Skull/cytology , Skull/drug effects , Spectrometry, Fluorescence , Sulfonamides/chemistry
11.
J Med Chem ; 52(8): 2181-4, 2009 Apr 23.
Article in English | MEDLINE | ID: mdl-19309081

ABSTRACT

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.


Subject(s)
Antineoplastic Agents/chemical synthesis , Cysteine/metabolism , Lactones/chemical synthesis , Oncogene Protein v-akt/antagonists & inhibitors , Pyrans/chemical synthesis , Alkylation , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biomarkers/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Lactones/chemistry , Lactones/pharmacology , Naphthoquinones/chemical synthesis , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Pyrans/chemistry , Pyrans/pharmacology , Stereoisomerism , Structure-Activity Relationship
12.
Bioorg Med Chem ; 17(5): 2091-100, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19200741

ABSTRACT

Checkpoint deficiency of malignant cells can be exploited in cancer drug discovery. Compounds that selectively kill checkpoint-deficient cells versus checkpoint-proficient cells can be utilized to preferentially target tumor cells, while sparing normal cells. The protein p21(Wafl/Cipl/Sdi1) (hereafter referred to as p21) inhibits progression of the cell cycle by inhibiting the activity of G1 kinases (cyclin D/cdk4 and cyclin E-cdk2) and the G2 kinase (cyclin B/cdkl) in response to DNA damage or abnormal DNA content. The expression of p21 is often low in human cancer cells due to frequent loss of the upstream activator, p53, and is associated with poor prognosis in some cancer patients. Using an isogenic pair of cell lines, HCT116 (p21+/+) and 80S14 (p21-/-), we have disclosed previously a novel series of pyrazolo[1,5-a]pyrimidines that preferentially kill the p21-deficient cells. We will present the synthesis, biological activities and SAR study of a series of pyrazolo[1,5-a]pyrimidines with an optimized phenyl amide moiety at the C-7 position. The mechanism of action of these compounds will also be discussed.


Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Pyrazoles/chemistry , Pyrimidines/chemistry , Amides/chemistry , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/metabolism , Humans , Mice , Mice, Nude , Structure-Activity Relationship , Transplantation, Heterologous
13.
J Med Chem ; 52(1): 105-16, 2009 Jan 08.
Article in English | MEDLINE | ID: mdl-19072540

ABSTRACT

The diphenylsulfonyl sulfonamide scaffold represented by 1 (WAY-316606) are small molecule inhibitors of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators of the Wnt pathway have been proposed as anabolic agents for the treatment of osteoporosis or other bone-related disorders. Details of the structure-activity relationships and biological activity from the first structural class of this scaffold will be discussed.


Subject(s)
Piperidines/chemistry , Proteins/antagonists & inhibitors , Proteins/metabolism , Signal Transduction/drug effects , Sulfanilamides/chemical synthesis , Sulfanilamides/pharmacology , Wnt Proteins/metabolism , Animals , Cell Line, Tumor , Genes, Reporter/genetics , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins , Isomerism , Mice , Molecular Structure , Protein Binding , Skull/drug effects , Structure-Activity Relationship , Sulfanilamides/chemistry , Wnt Proteins/genetics
14.
Bioorg Med Chem Lett ; 19(1): 62-6, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19041240

ABSTRACT

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.


Subject(s)
Antineoplastic Agents/chemical synthesis , Nitriles/chemical synthesis , Quinolines/chemical synthesis , Receptor, IGF Type 1/antagonists & inhibitors , Humans , Nitriles/pharmacology , Quinolines/pharmacology , Structure-Activity Relationship
15.
J Med Chem ; 51(24): 7670-2, 2008 Dec 25.
Article in English | MEDLINE | ID: mdl-19053830

ABSTRACT

Inhibitor of secreted frizzled related protein-1 (sFRP-1) would be a novel potential osteogenic agent, since loss of sFRP-1 affects osteoblast proliferation, differentiation, and activity, resulting in improved bone mineral density, quality, and strength. We have identified small molecule diarylsulfone sulfonamide derivatives as sFRP-1 inhibitors. Structure-activity relationship generated for various regions of the scaffold was utilized to improve the biochemical profile, resulting in the identification of potent selective analogues, such as 16 with desirable pharmaceutical profile.


Subject(s)
Osteoblasts/metabolism , Proteins/antagonists & inhibitors , Sulfonamides/chemistry , Sulfones/chemistry , Animals , Biochemistry/methods , Bone Density , Cells, Cultured , Drug Design , Humans , Intracellular Signaling Peptides and Proteins , Mice , Models, Chemical , Signal Transduction , Structure-Activity Relationship
16.
Antimicrob Agents Chemother ; 52(9): 3327-38, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18559648

ABSTRACT

HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.


Subject(s)
Antiviral Agents/pharmacology , Benzofurans/antagonists & inhibitors , Drug Resistance, Viral , Enzyme Inhibitors/pharmacology , Genetic Variation , Hepacivirus/drug effects , Replicon/drug effects , Antiviral Agents/metabolism , Cell Line, Tumor , Cloning, Molecular , Enzyme Inhibitors/metabolism , Genotype , Hepacivirus/genetics , Hepacivirus/physiology , Humans , Models, Molecular , Mutation , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Replicon/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
17.
Bioorg Med Chem Lett ; 18(12): 3641-5, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18501599

ABSTRACT

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.


Subject(s)
Antineoplastic Agents/pharmacology , Isoquinolines/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Antineoplastic Agents/chemistry , Dose-Response Relationship, Drug , Drug Design , Drug Screening Assays, Antitumor , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Small Molecule Libraries , Structure-Activity Relationship
18.
Mol Cancer Ther ; 6(11): 3028-38, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17989320

ABSTRACT

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 Factors
19.
J Med Chem ; 50(23): 5547-9, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17941624

ABSTRACT

With high-throughput screening, substituted dibenzo[c,f][2,7]naphthyridine 1 was identified as a novel potent and selective phosphoinositide-dependent kinase-1 (PDK-1) inhibitor. Various regions of the lead molecule were explored to understand the SAR requirement for this scaffold. The crystal structure of 1 with kinase domain of PDK-1 confirmed the binding in the active site. The key interaction of the molecule with the active site residues, observed SAR, and the biological profile are discussed in detail.


Subject(s)
Naphthyridines/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , 3-Phosphoinositide-Dependent Protein Kinases , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Naphthyridines/chemistry , Protein Serine-Threonine Kinases/chemistry , Structure-Activity Relationship
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