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1.
Cell ; 140(2): 209-21, 2010 Jan 22.
Article in English | MEDLINE | ID: mdl-20141835

ABSTRACT

We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS. We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling. This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS. Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice. Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression. They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.


Subject(s)
Antineoplastic Agents/adverse effects , Melanoma/drug therapy , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-raf/metabolism , ras Proteins/metabolism , Animals , Cell Line, Tumor , Humans , Mice , Mice, Transgenic , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism
2.
N Engl J Med ; 366(3): 207-15, 2012 Jan 19.
Article in English | MEDLINE | ID: mdl-22256804

ABSTRACT

BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).


Subject(s)
Carcinoma, Squamous Cell/genetics , Genes, ras , Indoles/therapeutic use , Mutation , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Skin Neoplasms/genetics , Sulfonamides/therapeutic use , Aged , Aged, 80 and over , Animals , Carcinoma, Squamous Cell/drug therapy , Female , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Indoles/administration & dosage , Male , Mice , Middle Aged , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Kinase Inhibitors/administration & dosage , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Sulfonamides/administration & dosage , Vemurafenib
3.
Bioorg Med Chem ; 21(5): 1284-304, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23376011

ABSTRACT

The RAS-RAF-MEK-ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine-threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC(50)=190 nM and with cellular GI(50)=2100 nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC(50)=9 nM and GI(50)=220 nM.


Subject(s)
Imidazoles/chemistry , Naphthols/chemistry , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Thiophenes/chemistry , Benzofurans/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Melanoma/metabolism , Melanoma/pathology , Naphthols/chemical synthesis , Naphthols/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/pharmacology
4.
Angiogenesis ; 15(4): 623-41, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22843200

ABSTRACT

Sunitinib is a potent and clinically approved tyrosine kinase inhibitor that can suppress tumour growth by inhibiting angiogenesis. However, conflicting data exist regarding the effects of this drug on the growth of metastases in preclinical models. Here we use 4T1 and RENCA tumour cells, which both form lung metastases in Balb/c mice, to re-address the effects of sunitinib on the progression of metastatic disease in mice. We show that treatment of mice with sunitinib prior to intravenous injection of tumour cells can promote the seeding and growth of 4T1 lung metastases, but not RENCA lung metastases, showing that this effect is cell line dependent. However, increased metastasis occurred only upon administration of a very high sunitinib dose, but not when lower, clinically relevant doses were used. Mechanistically, high dose sunitinib led to a pericyte depletion effect in the lung vasculature that correlated with increased seeding of metastasis. By administering sunitinib to mice after intravenous injection of tumour cells, we demonstrate that while sunitinib does not inhibit the growth of 4T1 lung tumour nodules, it does block the growth of RENCA lung tumour nodules. This contrasting response was correlated with increased myeloid cell recruitment and persistent vascularisation in 4T1 tumours, whereas RENCA tumours recruited less myeloid cells and were more profoundly devascularised upon sunitinib treatment. Finally, we show that progression of 4T1 tumours in sunitinib treated mice results in increased hypoxia and increased glucose metabolism in these tumours and that this is associated with a poor outcome. Taken together, these data suggest that the effects of sunitinib on tumour progression are dose-dependent and tumour model-dependent. These findings have relevance for understanding how anti-angiogenic agents may influence disease progression when used in the adjuvant or metastatic setting in cancer patients.


Subject(s)
Antineoplastic Agents/therapeutic use , Indoles/therapeutic use , Neoplasm Metastasis/drug therapy , Pyrroles/therapeutic use , Animals , Mice , Mice, Inbred BALB C , Positron-Emission Tomography , Sunitinib , Tomography, X-Ray Computed
5.
Bioorg Med Chem Lett ; 22(2): 789-92, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-22222036

ABSTRACT

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.


Subject(s)
Clinical Trials as Topic , Melanoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Humans , Melanoma/metabolism , Molecular Weight , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity Relationship
6.
NMR Biomed ; 24(4): 343-50, 2011 May.
Article in English | MEDLINE | ID: mdl-20891022

ABSTRACT

The pseudomonad protein, carboxypeptidase G2 (CPG2), is a prodrug-activating enzyme utilized in the targeted chemotherapy strategies of antibody- and gene-directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2-based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using (19)F MRSI. The high signal-to-noise ratio afforded by the two MR-equivalent (19)F nuclei of 3,5-DFBGlu, and the 1.4 ppm (19)F chemical shift difference on CPG2-mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5-DFBGlu and its CPG2-mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5-difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2-based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5-DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression.


Subject(s)
gamma-Glutamyl Hydrolase/metabolism , Animals , Benzoic Acid/chemistry , Benzoic Acid/metabolism , Cell Line, Tumor , Female , Fluorine/metabolism , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Humans , Mice , Xenograft Model Antitumor Assays
7.
Bioorg Med Chem ; 18(18): 6934-52, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20667740

ABSTRACT

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.


Subject(s)
Furans/chemistry , Imidazoles/chemistry , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazoles/chemistry , Animals , Binding Sites , Computer Simulation , Female , Humans , Mice , Mutation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Structure-Activity Relationship
8.
Magn Reson Med ; 62(5): 1300-4, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19780183

ABSTRACT

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance (13)C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with (13)C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo.


Subject(s)
Algorithms , Magnetic Resonance Spectroscopy/methods , gamma-Glutamyl Hydrolase/analysis , gamma-Glutamyl Hydrolase/chemistry , Carbon Isotopes/analysis , Carbon Isotopes/chemistry , Enzyme Activation
9.
NMR Biomed ; 22(5): 561-6, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19259950

ABSTRACT

Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid, a prodrug for the GDEPT enzyme carboxypeptidase-G2 (CPG2; K(m) = 1.71 microM; k(cat) = 732 s(-1)), was measured with (19)F magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K(m) = 3.5 microM, k(cat) = 747 s(-1)). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well-resolved (19)F MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half-life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent.


Subject(s)
Fluorine/chemistry , Genes, Transgenic, Suicide , Genetic Therapy , Magnetic Resonance Spectroscopy/methods , gamma-Glutamyl Hydrolase/metabolism , Buffers , Cell Line, Tumor , Humans , Kinetics , Prodrugs/chemistry , Solutions
10.
Clin Cancer Res ; 14(13): 4259-66, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18594008

ABSTRACT

PURPOSE: We engineered the oncolytic Salmonella typhimurium-derived bacterium VNP20009 as a vector to target delivery to tumors of the prodrug-activating enzyme carboxypeptidase G2 (CPG2) and to show enhanced antitumor efficacy on administration of different prodrugs. EXPERIMENTAL DESIGN: We characterized CPG2 expression in vectors by immunoblotting, immunofluorescence, and enzyme activity. We assessed prodrug activation by high-performance liquid chromatography. Target human tumor cell and bacterial vector cell cytotoxicity was measured by flow cytometry and colony-forming assays. Therapy was shown in two human tumor xenografts and one mouse allograft with postmortem analysis of bacterial and CPG2 concentration in the tumors. RESULTS: CPG2 is expressed within the bacterial periplasm. It activates prodrugs and induces cytotoxicity in human tumor cells but not in host bacteria. Following systemic administration, bacteria multiply within xenografts reaching 2 x 10(7)/g to 2 x 10(8)/g at 40 days postinoculation. The concentration of CPG2 in these tumors increases steadily to therapeutic levels of 1 to 6 units/g. The bacteria alone reduce the growth of the tumors. Subsequent administration of prodrugs further reduces significantly the growth of the xenografts. CONCLUSIONS: The bacteria multiply within tumors, resulting in a selective expression of CPG2. The CPG2-expressing bacteria alone reduce the growth of tumors. However, in the presence of prodrugs activated by CPG2, this oncolytic effect is greatly increased. We conclude that bacterial oncolytic therapy, combined with CPG2-mediated prodrug activation, has great potential in the treatment of a range of cancers.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/therapy , Genetic Therapy/methods , Melanoma/drug therapy , Melanoma/genetics , Prodrugs/metabolism , Salmonella typhimurium/metabolism , gamma-Glutamyl Hydrolase/genetics , Animals , Cell Line, Tumor , Chromatography, High Pressure Liquid , Flow Cytometry , Genetic Vectors , Humans , Mice , Neoplasm Transplantation
11.
Cancer Res ; 67(10): 4949-55, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17510425

ABSTRACT

We have designed a targeted systemic suicide gene therapy that combines the advantages of tumor-selective gene expression, using the human telomerase promoter (hTERT), with the beneficial effects of an oncolytic adenovirus to deliver the gene for the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors. Following delivery of the vector (AdV.hTERT-CPG2) and expression of CPG2 in cancer cells, the prodrug ZD2767P was administered for conversion by CPG2 to a cytotoxic drug. This system is sometimes termed gene-directed enzyme prodrug therapy (GDEPT). Here, we have shown that it is applicable to 10 human colorectal carcinoma cell lines with a direct correlation between viral toxicity and CPG2 production. SW620 xenografts were selected for analysis and were significantly reduced or eradicated after a single administration of AdV.hTERT-CPG2 followed by a prodrug course. The oncolytic effect of adenovirus alone did not result in DNA cross-links or apoptosis, whereas DNA cross-links and apoptosis occurred following prodrug administration, showing the combined beneficial effects of the GDEPT system. The apoptotic regions extended beyond the areas of CPG2 expression in the tumors, indicative of significant bystander effects in vivo. Higher concentrations of vector particles and CPG2 were found in the AdV.hTERT-CPG2 plus prodrug-treated tumors compared with the virus alone, showing an unexpected beneficial and cooperative effect between the vector and GDEPT. This is the first time that a tumor-selective GDEPT vector has been shown to be effective in colorectal carcinoma and that apoptosis and significant bystander effects have been identified as the mechanisms of cytotoxicity within the tumor.


Subject(s)
Colonic Neoplasms/therapy , Genetic Therapy/methods , gamma-Glutamyl Hydrolase/genetics , Animals , Cell Line, Tumor , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Colonic Neoplasms/virology , Female , Genetic Vectors/genetics , Humans , Mice , Mice, Nude , Nitrogen Mustard Compounds/pharmacokinetics , Nitrogen Mustard Compounds/pharmacology , Oncolytic Virotherapy/methods , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Telomerase/genetics , Telomerase/metabolism , Xenograft Model Antitumor Assays , gamma-Glutamyl Hydrolase/biosynthesis , gamma-Glutamyl Hydrolase/metabolism
12.
Cancer Res ; 79(22): 5874-5883, 2019 11 15.
Article in English | MEDLINE | ID: mdl-31604713

ABSTRACT

Increased stiffness in the extracellular matrix (ECM) contributes to tumor progression and metastasis. Therefore, stromal modulating therapies and accompanying biomarkers are being developed to target ECM stiffness. Magnetic resonance (MR) elastography can noninvasively and quantitatively map the viscoelastic properties of tumors in vivo and thus has clear clinical applications. Herein, we used MR elastography, coupled with computational histopathology, to interrogate the contribution of collagen to the tumor biomechanical phenotype and to evaluate its sensitivity to collagenase-induced stromal modulation. Elasticity (G d) and viscosity (G l) were significantly greater for orthotopic BT-474 (G d = 5.9 ± 0.2 kPa, G l = 4.7 ± 0.2 kPa, n = 7) and luc-MDA-MB-231-LM2-4 (G d = 7.9 ± 0.4 kPa, G l = 6.0 ± 0.2 kPa, n = 6) breast cancer xenografts, and luc-PANC1 (G d = 6.9 ± 0.3 kPa, G l = 6.2 ± 0.2 kPa, n = 7) pancreatic cancer xenografts, compared with tumors associated with the nervous system, including GTML/Trp53KI/KI medulloblastoma (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 7), orthotopic luc-D-212-MG (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 7), luc-RG2 (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 5), and luc-U-87-MG (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 8) glioblastoma xenografts, intracranially propagated luc-MDA-MB-231-LM2-4 (G d = 3.7 ± 0.2 kPa, G l = 2.2 ± 0.1 kPa, n = 7) breast cancer xenografts, and Th-MYCN neuroblastomas (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 5). Positive correlations between both elasticity (r = 0.72, P < 0.0001) and viscosity (r = 0.78, P < 0.0001) were determined with collagen fraction, but not with cellular or vascular density. Treatment with collagenase significantly reduced G d (P = 0.002) and G l (P = 0.0006) in orthotopic breast tumors. Texture analysis of extracted images of picrosirius red staining revealed significant negative correlations of entropy with G d (r = -0.69, P < 0.0001) and G l (r = -0.76, P < 0.0001), and positive correlations of fractal dimension with G d (r = 0.75, P < 0.0001) and G l (r = 0.78, P < 0.0001). MR elastography can thus provide sensitive imaging biomarkers of tumor collagen deposition and its therapeutic modulation. SIGNIFICANCE: MR elastography enables noninvasive detection of tumor stiffness and will aid in the development of ECM-targeting therapies.


Subject(s)
Breast Neoplasms/metabolism , Collagen/metabolism , Animals , Cell Line, Tumor , Elasticity , Elasticity Imaging Techniques/methods , Extracellular Matrix/metabolism , Female , Humans , Magnetic Resonance Imaging/methods , Mice , Phenotype
13.
J Med Chem ; 51(11): 3261-74, 2008 Jun 12.
Article in English | MEDLINE | ID: mdl-18473434

ABSTRACT

BRAF, a serine/threonine kinase, plays a key role in the development of certain types of cancer, particularly melanoma. 2-(3,4,5-Trimethoxyphenylamino)-6-(3-acetamidophenyl)-pyrazine, 1, was identified as a low micromolar (IC 50 = 3.5 microM) BRAF inhibitor from a high-throughput screen of a library of 23000 compounds. This compound was chosen as the starting point of a program aimed at developing inhibitors of mutant (V600E)BRAF. We have already reported on the optimization of the trimethoxyphenylamino moiety of 1. In this paper, we describe the synthesis of a series of compounds derived from 1 with the purpose of optimization of the pyrazine central core and the phenylacetamido moiety in order to increase the potency against (V600E)BRAF compared to CRAF. The biological activity of the new inhibitors was assessed against mutant (V600E)BRAF in vitro. Several compounds were identified with IC 50s of 300-500 nM for (V600E)BRAF, and all compounds that were assessed showed selectivity for (V600E)BRAF compared to CRAF by 5-->86-fold.


Subject(s)
Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazines/chemical synthesis , Databases, Factual , Humans , Mutation , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-raf/chemistry , Pyrazines/chemistry , Structure-Activity Relationship
14.
Bioconjug Chem ; 19(4): 973-81, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18380471

ABSTRACT

A concise synthesis of long-chain poly(ethylene glycol) (PEG) of defined molecular weight up to 29 ethyleneoxy units is described. These PEG diols were converted in a two-step synthesis into Fmoc-protected PEG amino acids, suitable as long linkers and compatible with solid-phase peptide synthesis. Long PEG chains (MW > 1000) can be readily synthesized with this method, which has the advantage of defined single molecular weight products over the comparable commercial polymers. The application of these PEG linkers to the synthesis of peptide-PEG-folate conjugates on a solid support was investigated. A method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was developed, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on solid support resulted in the synthesis of a conjugate of defined molecular weight and structure.


Subject(s)
Polyethylene Glycols/chemistry , Polyethylene Glycols/chemical synthesis , Amino Acids/chemistry , Folic Acid/chemistry , Molecular Weight , Peptides/chemistry
15.
Clin Cancer Res ; 12(21): 6509-16, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17085666

ABSTRACT

PURPOSE: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti-carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination. EXPERIMENTAL DESIGN: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized. RESULTS: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease. CONCLUSIONS: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.


Subject(s)
Aniline Mustard/analogs & derivatives , Carcinoembryonic Antigen/immunology , Colorectal Neoplasms/drug therapy , Prodrugs/therapeutic use , Recombinant Fusion Proteins/therapeutic use , gamma-Glutamyl Hydrolase/therapeutic use , Aged , Aniline Mustard/blood , Aniline Mustard/pharmacokinetics , Aniline Mustard/therapeutic use , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Dose-Response Relationship, Drug , Female , History, 16th Century , History, 17th Century , Humans , Imaging, Three-Dimensional , Immunoconjugates/blood , Immunoconjugates/pharmacokinetics , Immunoconjugates/therapeutic use , Male , Prodrugs/adverse effects , Prodrugs/pharmacokinetics , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/pharmacokinetics , gamma-Glutamyl Hydrolase/blood , gamma-Glutamyl Hydrolase/pharmacokinetics
16.
Cancer Res ; 65(12): 5003-8, 2005 Jun 15.
Article in English | MEDLINE | ID: mdl-15958540

ABSTRACT

Hepatocellular carcinoma is the fifth most common cancer worldwide, and there is no effective therapy for unresectable disease. We have developed a targeted systemic therapy for hepatocellular carcinoma. The gene for a foreign enzyme is selectively expressed in the tumor cells and a nontoxic prodrug is then given, which is activated to a potent cytotoxic drug by the tumor-localized enzyme. This approach is termed gene-directed enzyme prodrug therapy (GDEPT). Adenoviruses have been used to target cancer cells, have an intrinsic tropism for liver, and are efficient gene vectors. Oncolytic adenoviruses produce clinical benefits, particularly in combination with conventional anticancer agents and are well tolerated. We rationalized that such adenoviruses, if their expression were restricted to telomerase-positive cancer cells, would make excellent gene vectors for GDEPT therapy of hepatocellular carcinoma. Here we use an oncolytic adenovirus to deliver the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors in a single systemic administration. The adenovirus replicated and produced high levels of CPG2 in two different hepatocellular carcinoma xenografts (Hep3B and HepG2) but not other tissues. GDEPT enhanced the adenovirus-alone therapy to elicit tumor regressions in the hepatocellular carcinoma models. This is the first time that CPG2 has been targeted and expressed intracellularly to effect significant therapy, showing that the combined approach holds enormous potential as a tumor-selective therapy for the systemic treatment of hepatocellular carcinoma.


Subject(s)
Carcinoma, Hepatocellular/therapy , Genetic Therapy/methods , Liver Neoplasms/therapy , Nitrogen Mustard Compounds/pharmacology , Prodrugs/pharmacokinetics , gamma-Glutamyl Hydrolase/genetics , Adenoviridae/genetics , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , DNA-Binding Proteins , Female , Humans , Liver Neoplasms/genetics , Liver Neoplasms/virology , Mice , Mice, Nude , Nitrogen Mustard Compounds/pharmacokinetics , Prodrugs/pharmacology , Promoter Regions, Genetic , Telomerase/genetics , Xenograft Model Antitumor Assays , gamma-Glutamyl Hydrolase/biosynthesis , gamma-Glutamyl Hydrolase/metabolism
17.
Curr Gene Ther ; 6(6): 647-70, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17168697

ABSTRACT

Conventional cancer treatments are often hampered by a lack of tumour selectivity, resulting in toxicity to healthy tissue. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy approach that aims to improve the selectivity of chemotherapy by enabling cancer cells to convert non-cytotoxic prodrugs to cytotoxic drugs. Many enzyme/prodrug systems have been described, some of which have already been tested in clinical trials. A key component of GDEPT is a foreign enzyme that is expressed selectively at the tumour site where it converts the prodrug into the cytotoxic agent. The gene encoding the prodrug-activating enzyme needs to be expressed selectively and efficiently in tumour cells in order to spare normal tissue from damage. Substantial efforts have been made to develop gene therapy vectors that are capable of targeting cancer cells. A large number of gene delivery systems have been described for GDEPT: Viral vectors are the most advanced. They include replication-deficient and replication-selective (oncolytic) viruses. Recent advances in engineering viruses for GDEPT are reviewed in this article and data from both preclinical studies and clinical trials are discussed.


Subject(s)
Enzymes/genetics , Genetic Therapy/methods , Genetic Vectors , Neoplasms/therapy , Prodrugs/therapeutic use , Viruses/genetics , Animals , Antineoplastic Agents/therapeutic use , Genetic Therapy/trends , Humans , Neoplasms/drug therapy , Neoplasms/metabolism
18.
J Med Chem ; 49(1): 407-16, 2006 Jan 12.
Article in English | MEDLINE | ID: mdl-16392826

ABSTRACT

B-RAF, a serine/threonine kinase, plays an important role in the development of certain classes of cancer, especially melanoma. As a result of high-throughput screening of a 23,000 compound library, 2-(3,4,5-trimethoxyphenylamino)-6-(3-acetamidophenyl)pyrazine, 1, was identified as a low micromolar (IC(50) = 3.5 microM) B-RAF inhibitor. This compound was chosen as the starting point of a program aimed at producing potent inhibitors of B-RAF. We have synthesized a series of 40 novel compounds, which involved extensive modifications to the 2-(3,4,5-trimethoxyphenylamino) moiety (ring A) of 1. Their biological profiles against isolated B-RAF and mutated B-RAF in a cellular assay have been determined. These efforts led to the identification of two compounds exhibiting activities lower than 800 nM against B-RAF.


Subject(s)
Acetanilides/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazines/pharmacology , Acetanilides/chemical synthesis , Acetanilides/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Pyrazines/chemical synthesis , Pyrazines/chemistry , Structure-Activity Relationship
19.
Cancer Res ; 62(6): 1724-9, 2002 Mar 15.
Article in English | MEDLINE | ID: mdl-11912146

ABSTRACT

Three new prodrugs, [prodrug 1: 4-[bis(2-iodoethyl)amino]-phenyloxycarbonyl-L-glutamic acid; prodrug 2: 3-fluoro-4-[bis(2-chlorethyl)amino]benzoyl-L-glutamic acid; and prodrug 3: 3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl-L-glutamic acid] have been assessed for use with a mutant of carboxypeptidase G2 (CPG2, glutamate carboxypeptidase, EC 3.4.17.11,) engineered to be tethered to the outer tumor cell surface (stCPG2(Q)3) as the activating enzyme in suicide gene therapy systems. All three of the prodrugs produce much greater cytotoxicity differentials between stCPG2(Q)3- and control beta-galactosidase (beta-gal)-expressing breast carcinoma MDA MB 361 and colon carcinoma WiDr cells (70- to 450-fold) than was previously observed (19- to 27-fold) with 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). Prodrug 1 is the most effective antitumor agent in xenografts in mice inoculated with 100% stCPG2(Q)3-expressing MDA MB 361 cells, whereas prodrugs 2 and 3 are most effective when the percentage of stCPG2(Q)3-expressing cells is 50% or 10%. In nude mice bearing xenografts arising from inocula of 100% stCPG2(Q)3-expressing WiDr cells, prodrug 2 is the most effective antitumor agent. All three of the prodrugs produced histological evidence of substantial bystander cell killing in WiDr xenografts in which only 10% or 50% of the cells inoculated were expressing stCPG2(Q)3. We conclude that all three of the prodrugs are more effective therapeutically with stCPG2(Q)3 than is the previously described prodrug CMDA and, also, that the optimal choice of prodrug varies among different tumor types and that prodrugs, optimized for their bystander effect, are effective when only low percentages of cells in a tumor express CPG2.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/therapy , Colorectal Neoplasms/therapy , Genetic Therapy/methods , Glutamic Acid/analogs & derivatives , Prodrugs/pharmacology , gamma-Glutamyl Hydrolase/genetics , Animals , Antineoplastic Agents/pharmacokinetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Prodrugs/pharmacokinetics , Transfection , Xenograft Model Antitumor Assays , beta-Galactosidase/genetics , beta-Galactosidase/metabolism , gamma-Glutamyl Hydrolase/metabolism
20.
Cancer Res ; 64(7): 2338-42, 2004 Apr 01.
Article in English | MEDLINE | ID: mdl-15059882

ABSTRACT

The oncogenic version of B-RAF, (V599E)B-RAF, is found in approximately 70% of human melanomas. However, the role that this oncogene plays in melanoma is unclear because (V559E)B-RAF is also found in approximately 80% of benign nevi. We have examined the role of oncogenic B-RAF in the early stages of melanoma by expressing (V599E)B-RAF in cultured melanocytes. In these cells, (V599E)B-RAF induced constitutive mitogen activated ERK-activating kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, 12-O-tetradecanoylphorbol-13-acetate-independent growth, and tumorigenicity in nude mice. Intriguingly, in RAS-transformed melanocytes, B-RAF depletion did not block MEK-ERK signaling or cell cycle progression. Similarly, B-RAF depletion blocked MEK-ERK signaling in human melanoma cells harboring oncogenic B-RAF, but not in melanoma cells harboring oncogenic RAS. Thus, although B-RAF can act as a potent oncogene in the early stages of melanoma by signaling through MEK and ERK, it is not required for this signaling in RAS-transformed melanocytes due to innate redundancy within the pathway. These findings have important implications for future therapeutic strategies.


Subject(s)
Cell Transformation, Neoplastic/genetics , Melanocytes/physiology , Melanoma/genetics , Oncogenes , Proto-Oncogene Proteins c-raf/genetics , Animals , Carcinogens/pharmacology , Cell Line, Tumor , Female , Humans , MAP Kinase Signaling System , Melanocytes/pathology , Melanoma/pathology , Mice , Mice, Nude , Proto-Oncogene Proteins B-raf , Tetradecanoylphorbol Acetate/pharmacology , Transfection
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