ABSTRACT
Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF inhibitors based on a tripartite A-B-C system featuring an imidazo[4,5]pyridin-2-one group hinge binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a different A-B-C system that has been modified by the introduction of a range of novel hinge binders (A ring). The optimization of the hinge binding moiety has enabled the development of compounds with low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display optimal pharmacokinetic properties that warrant further in vivo investigations.
Subject(s)
Antineoplastic Agents/chemical synthesis , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazines/chemical synthesis , Pyridines/chemical synthesis , Administration, Oral , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Benzenesulfonates/chemistry , Biological Availability , Crystallography, X-Ray , Female , Humans , Hydrogen Bonding , Imidazoles/chemistry , Mice , Mice, Inbred BALB C , Models, Molecular , Neoplasm Transplantation , Niacinamide/analogs & derivatives , Phenylurea Compounds , Protein Binding , Proto-Oncogene Proteins B-raf/chemistry , Pyrazines/pharmacokinetics , Pyrazines/pharmacology , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyridines/pharmacology , Sorafenib , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified (V600E)BRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated (V600E)BRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, (V600E)BRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.
Subject(s)
Drug Design , Oncogene Proteins v-raf/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Sarcoma Viruses, Murine/enzymology , Sequence Homology , Animals , Cell Line, Tumor , Female , Humans , Inhibitory Concentration 50 , Mice , Models, Molecular , Molecular Conformation , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity RelationshipABSTRACT
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/metabolismABSTRACT
Sixteen novel polyfluorinated benzoic acid mustards have been synthesized for use in gene-directed enzyme prodrug therapy (GDEPT). Eight of these were benzoic acid L-glutamate mustards for evaluation as prodrugs and the other eight were the active drugs formed by the action of the bacterial enzyme carboxypeptidase G2 (CPG2). All of the di- and trifluorinated prodrugs were efficiently cleaved by the enzyme. In contrast, the tetrafluorinated prodrugs were found to be competitive inhibitors of CPG2, the first such inhibitors to have been described. The di- and trifluorinated prodrugs were differentially cytotoxic to human breast carcinoma cells (MDA MB 361) expressing CPG2, compared to control cells that did not express the enzyme. The difluorinated prodrug {4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid and its iodoethylamino analogue were effective substrates for the enzyme and showed excellent therapeutic activity in CPG2-expressing MDA MB 361 xenografts, either curing or greatly inhibiting tumor growth and extending the life of the animals.
Subject(s)
Antineoplastic Agents/chemical synthesis , Fluorine , Mustard Compounds/chemical synthesis , Prodrugs/chemical synthesis , gamma-Glutamyl Hydrolase/genetics , gamma-Glutamyl Hydrolase/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Humans , Mice , Mice, Nude , Mustard Compounds/chemistry , Mustard Compounds/metabolism , Mustard Compounds/pharmacology , Neoplasm Transplantation , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacology , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
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/metabolismABSTRACT
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.