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1.
Cancer Immunol Res ; 5(11): 942-949, 2017 11.
Article in English | MEDLINE | ID: mdl-29038296

ABSTRACT

Immunotherapy is rapidly becoming a standard of care for many cancers. However, colorectal cancer had been generally resistant to immunotherapy, despite features in common with sensitive tumors. Observations of substantial clinical activity for checkpoint blockade in colorectal cancers with defective mismatch repair (microsatellite instability-high tumors) have reignited interest in the search for immunotherapies that could be extended to the larger microsatellite stable (MSS) population. The Cancer Research Institute and Fight Colorectal Cancer convened a group of scientists, clinicians, advocates, and industry experts in colorectal cancer and immunotherapy to compile ongoing research efforts, identify gaps in translational and clinical research, and provide a blueprint to advance immunotherapy. We identified lack of a T-cell inflamed phenotype (due to inadequate T-cell infiltration, inadequate T-cell activation, or T-cell suppression) as a broad potential explanation for failure of checkpoint blockade in MSS. The specific cellular and molecular underpinnings for these various mechanisms are unclear. Whether biomarkers with prognostic value, such as the immunoscores and IFN signatures, would also predict benefit for immunotherapies in MSS colon cancer is unknown, but if so, these and other biomarkers for measuring the potential for an immune response in patients with colorectal cancer will need to be incorporated into clinical guidelines. We have proposed a framework for research to identify immunologic factors that may be modulated to improve immunotherapy for colorectal cancer patients, with the goal that the biomarkers and treatment strategies identified will become part of the routine management of colorectal cancer. Cancer Immunol Res; 5(11); 942-9. ©2017 AACR.


Subject(s)
Colorectal Neoplasms/therapy , Immunotherapy , Animals , Colorectal Neoplasms/immunology , Humans
2.
J Med Chem ; 59(14): 6671-89, 2016 07 28.
Article in English | MEDLINE | ID: mdl-27433829

ABSTRACT

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.


Subject(s)
Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Discovery , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Nicotine/analogs & derivatives , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Molecular Conformation , Mutation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Nicotine/chemical synthesis , Nicotine/chemistry , Nicotine/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Rats , Rats, Wistar , Structure-Activity Relationship
3.
Nature ; 534(7605): 129-32, 2016 06 02.
Article in English | MEDLINE | ID: mdl-27251290

ABSTRACT

The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.


Subject(s)
Antineoplastic Agents/pharmacology , Benzeneacetamides/pharmacology , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Mutant Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Thiazoles/pharmacology , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab/pharmacology , Disease Models, Animal , Drug Resistance, Multiple/drug effects , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/drug effects , Drug Synergism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation/drug effects , Protein Multimerization/drug effects
4.
Bioorg Med Chem Lett ; 26(8): 2057-64, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26951753

ABSTRACT

Taking the pyrrolopyrimidine derived IGF-1R inhibitor NVP-AEW541 as the starting point, the benzyl ether back-pocket binding moiety was replaced with a series of 2-cyclic ether methyl ethers leading to the identification of novel achiral [2.2.1]-bicyclic ether methyl ether containing analogues with improved IGF-1R activities and kinase selectivities. Further exploration of the series, including a fluorine scan of the 5-phenyl substituent, and optimisation of the sugar-pocket binding moiety identified compound 33 containing (S)-2-tetrahydrofuran methyl ether 6-fluorophenyl ether back-pocket, and cis-N-Ac-Pip sugar-pocket binding groups. Compound 33 showed improved selectivity and pharmacokinetics compared to NVP-AEW541, and produced comparable in vivo efficacy to linsitinib in inhibiting the growth of an IGF-1R dependent tumour xenograft model in the mouse.


Subject(s)
Antineoplastic Agents/pharmacology , Imidazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Mice , Mice, Nude , Molecular Structure , NIH 3T3 Cells , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazines/chemical synthesis , Pyrazines/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Receptor, IGF Type 1/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor Assays
5.
Cancer Res ; 76(6): 1591-602, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26825170

ABSTRACT

Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Mutation/drug effects , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Female , Lung Neoplasms/metabolism , Mice , Mice, Nude , Phosphorylation/drug effects , Rats , Xenograft Model Antitumor Assays/methods
6.
Bioorg Med Chem Lett ; 26(3): 1090-1096, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26750252

ABSTRACT

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described.


Subject(s)
Antineoplastic Agents/chemical synthesis , Drug Design , Protein Kinase Inhibitors/chemical synthesis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , 4-Aminopyridine/analogs & derivatives , 4-Aminopyridine/chemistry , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Half-Life , Humans , Lymphoma, Large-Cell, Anaplastic/drug therapy , Mice , Mice, SCID , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Rats , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , Transplantation, Heterologous
7.
J Med Chem ; 57(8): 3358-68, 2014 Apr 24.
Article in English | MEDLINE | ID: mdl-24678947

ABSTRACT

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) ( Nature 2011 , 475 , 524 ; 519 ). EBI2 is highly expressed in immune cells ( J. Biol. Chem. 2006 , 281 , 13199 ), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes ( Nature 2010 , 467 , 460 ). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (1), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (2) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (4m). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells.


Subject(s)
Herpesvirus 4, Human , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , CHO Cells , Calcium/metabolism , Cricetulus , Humans , Male , Mice , Mice, Inbred C57BL , U937 Cells
8.
J Med Chem ; 56(14): 5675-90, 2013 Jul 25.
Article in English | MEDLINE | ID: mdl-23742252

ABSTRACT

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.


Subject(s)
Neoplasms/drug therapy , Protein Kinase Inhibitors/chemical synthesis , Pyrimidines/chemical synthesis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Sulfones/chemical synthesis , Anaplastic Lymphoma Kinase , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Dogs , Humans , Macaca fascicularis , Male , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats , Structure-Activity Relationship , Sulfones/pharmacokinetics , Sulfones/therapeutic use , Xenograft Model Antitumor Assays
9.
Toxicol Sci ; 118(1): 71-85, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20631060

ABSTRACT

This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility. Results from initial validation compounds (n = 38) set the stage for differentiating overall positive and negative MN inducers. To delve deeper into the compound categorization process, a more extensive validation set, consisting of a larger set (n = 370) of "drug-like but less optimized" early-stage compounds, was used for further refinement of positive and negative compound categories. The predictivity and applicability of the assay for clinical stage compounds was ascertained using (n = 168) clinically developed marketed drugs or well-studied compounds. Upon full validation, a detailed analysis of results established five compound categories--NEG (negative), NEG/xx µM (negative up to the solubility limit of xx µM), WPOS (weak positive), POS (positive), and INCON (inconclusive). Furthermore, examples of lead-finding applications and ongoing investigative HCMN activities are described. A proposal is offered on how the HCMN assay can be positioned in parallel to the overall stage gates (e.g., scaffold selection, lead optimization, late-stage preclinical development) of drug discovery programs. Because of its greater throughput, 1-week turnaround time, and a substantially reduced (1-2 mg) requirement for compound consumption, the HCMN assay is appropriate for developing structure-genotoxicity relationships and for mechanistic genotoxicity studies. The assay does not replace the Organization for Economic Cooperation and Development-compliant, non-good laboratory practice in vitro MN test (e.g., slide-based MN test in TK6 lymphoblastoid cells) that is used for full characterization of lead candidates.


Subject(s)
Drug Evaluation, Preclinical/methods , Drug Industry/methods , Drug-Related Side Effects and Adverse Reactions , Gene Expression Profiling , Animals , CHO Cells , Cricetinae , Cricetulus , Gene Expression , Micronucleus Tests , Reproducibility of Results
11.
Bioorg Med Chem Lett ; 18(19): 5259-62, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18783949

ABSTRACT

The lead optimization of a novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. The chemical instability of the dihydro-benzo[a]carbazole lead 2 was successfully addressed in the design and evaluation of compounds which also demonstrated improved potency compared to 2. Members of the scaffold have been identified which are full agonists that demonstrate cellular functional potency <50 nM. Analog 21 demonstrates equivalent efficacy in the human megakaryocyte differentiation (CFU-mega) assay compared to Eltrombopag.


Subject(s)
Benzene Derivatives/chemical synthesis , Benzene Derivatives/pharmacology , Carbazoles/chemical synthesis , Carbazoles/pharmacology , Receptors, Thrombopoietin/agonists , Thrombopoietin , Benzene Derivatives/chemistry , Carbazoles/chemistry , Combinatorial Chemistry Techniques , Drug Design , Humans , Inhibitory Concentration 50 , Megakaryocytes/metabolism , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Receptors, Thrombopoietin/chemistry , Structure-Activity Relationship , Thrombopoietin/chemistry , Thrombopoietin/metabolism
12.
J Biol Chem ; 282(19): 14253-61, 2007 May 11.
Article in English | MEDLINE | ID: mdl-17369254

ABSTRACT

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.


Subject(s)
Magnetic Resonance Spectroscopy , Receptors, Thrombopoietin/chemistry , Thrombopoietin/chemistry , Amino Acid Sequence , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Membrane , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Models, Molecular , Molecular Sequence Data , Plasmids , Receptors, Thrombopoietin/metabolism , Sequence Homology, Amino Acid , Thrombopoietin/metabolism , Tumor Cells, Cultured
13.
Bioorg Med Chem Lett ; 16(10): 2621-7, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16524729

ABSTRACT

Several potent, functionally active MCHr1 antagonists derived from quinolin-2(1H)-ones and quinazoline-2(1H)-ones have been synthesized and evaluated. Pyridylmethyl substitution at the quinolone 1-position results in derivatives with low-nM binding potency and good selectivity with respect to hERG binding.


Subject(s)
Quinazolines/chemistry , Quinazolines/pharmacology , Quinolones/chemistry , Quinolones/pharmacology , Receptors, Pituitary Hormone/antagonists & inhibitors , Animals , Mice , Quinazolines/pharmacokinetics , Quinolones/pharmacokinetics
15.
Bioorg Med Chem Lett ; 14(14): 3721-5, 2004 Jul 16.
Article in English | MEDLINE | ID: mdl-15203150

ABSTRACT

A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.


Subject(s)
Body Weight/drug effects , Imidazoles/chemical synthesis , Receptor, Melanocortin, Type 4/antagonists & inhibitors , Animals , Binding Sites , Body Weight/physiology , Cells, Cultured , Imidazoles/pharmacology , Rats , Receptor, Melanocortin, Type 4/metabolism , Structure-Activity Relationship
17.
Bioorg Med Chem ; 11(20): 4503-9, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-13129586

ABSTRACT

The design and synthesis of 10-(2-benzoxazolcarbonyl)-DDACTHF (1) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Ketone 1 and the corresponding alcohol 13 were evaluated for inhibition of GAR Tfase and AICAR Tfase and the former was found to be a potent inhibitor of recombinant human (rh) GAR Tfase (Ki=600 nM).


Subject(s)
Hydroxymethyl and Formyl Transferases/antagonists & inhibitors , Purines/biosynthesis , Tetrahydrofolates/chemistry , Binding Sites , Cell Line , Cell Survival/drug effects , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Phosphoribosylaminoimidazolecarboxamide Formyltransferase , Phosphoribosylglycinamide Formyltransferase , Purines/antagonists & inhibitors , Structure-Activity Relationship , Tetrahydrofolates/pharmacology
18.
Biochemistry ; 42(20): 6043-56, 2003 May 27.
Article in English | MEDLINE | ID: mdl-12755606

ABSTRACT

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydroxymethyl and Formyl Transferases/antagonists & inhibitors , Tetrahydrofolates/chemistry , Tetrahydrofolates/pharmacology , Binding Sites , Cell Line , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemical synthesis , Escherichia coli/enzymology , Humans , Hydroxymethyl and Formyl Transferases/chemistry , In Vitro Techniques , Kinetics , Macromolecular Substances , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphoribosylglycinamide Formyltransferase , Protein Conformation , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Static Electricity , Tetrahydrofolates/chemical synthesis
19.
Bioorg Med Chem ; 10(8): 2739-49, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12057663

ABSTRACT

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.


Subject(s)
Antineoplastic Agents/chemical synthesis , Hydroxymethyl and Formyl Transferases/antagonists & inhibitors , Purines/biosynthesis , Receptors, Cell Surface , Tetrahydrofolates/chemical synthesis , Antineoplastic Agents/pharmacology , Carrier Proteins/physiology , Cell Division/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Folate Receptors, GPI-Anchored , Humans , Peptide Synthases/physiology , Phosphoribosylaminoimidazolecarboxamide Formyltransferase , Phosphoribosylglycinamide Formyltransferase , Purines/antagonists & inhibitors , Structure-Activity Relationship , Tumor Cells, Cultured
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