Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.

The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.

Development of anticancer drugs targeting the MAP kinase pathway.

Since the discovery of the role of ras oncogenes in tumorigenesis, we have witnessed an explosion of research in the signal transduction area. In the quest to understand how Ras transmits extracellular growth signals, the MAP kinase (MAPK) pathway has emerged as the crucial route between membrane-bound Ras and the nucleus. The MAPK pathway encompasses a cascade of phosphorylation events involving three key kinases, namely Raf, MEK (MAP kinase kinase) and ERK (MAP kinase). This kinase cascade presents novel opportunities for the development of new cancer therapies designed to be less toxic than conventional chemotherapeutic drugs. Furthermore, as a signal transduction-based approach to cancer treatment, inhibition of any one of these targets has the potential for translational pharmacodynamic evaluation of target suppression. The rationale for targeting the MAP kinase pathway will be reviewed here along with a discussion of various pharmacological approaches and the promise they hold for a new generation of anticancer drugs.

Protein prenylation: from discovery to prospects for cancer treatment.

A specific set of proteins in eukaryotic cells contain covalently attached carboxy-terminal prenyl groups (15-carbon farnesyl and 20-carbon geranylgeranyl). Many of them are signaling proteins including Ras, heterotrimeric G proteins and Rab proteins. The protein prenyltransferases which attach prenyl groups to proteins have been well characterized, and an X-ray structure is available for protein farnesyltransferase. Inhibitors of protein farnesyltransferase are showing sufficient promise in preclinical trials as anti-cancer drugs to warrant widespread interest in the pharmaceutical industry.

Non-peptidic prenyltransferase inhibitors: diverse structural classes and surprising anti-cancer mechanisms.

The development of farnesyltransferase inhibitors (FTIs) has been one of the most active areas of anticancer drug development for the past ten years. This review presents a general overview of the developments in this area, along with a critical appraisal of the anticancer activity of FTIs. A historical survey of the protein prenylation field is given, in particular to emphasize the key role played by the Ras oncoprotein in driving the discovery of prenyltransferase enzymes. The different classes of prenylated proteins will be described along with the biochemical characteristics of the key drug target--farnesyltransferase (FTase). Numerous potent farnesyltransferase inhibitors have been developed. The FTIs developed can be separated into three different categories, based on their origin and/or mechanism of action: a) natural products; b) peptidomimetics and other CAAX-competitive inhibitors; c) farnesyl pyrophosphate (FPP) mimetics or analogs and other FPP-competitive inhibitors. Along with a survey of newer FTIs in each class, the development of several representative, potent compounds will be discussed in depth as we discuss the potential advantages and liabilities of each class. Particular emphasis is given to the discovery of new, more potent FPP-competitive FTIs of several diverse structural classes. Testing of different FTIs for their ability to block the growth of various cancer cell types in animal models will be discussed. There are a number of key differences between these compounds and traditional cytotoxic cancer chemotherapeutic agents, with surprising exceptions to their expected modes of action. As some FTIs have entered human clinical trials, answers may soon become available to key mechanistic questions concerning the extent and nature of their antitumor growth properties.

Enhancement of alkylating agent activity in vitro by PD 128763, a potent poly(ADP-ribose) synthetase inhibitor.

The ability of DNA repair inhibitors to potentiate alkylating agent cytotoxicity was explored with PD 128763, a dihydroisoquinolinone known to effectively inhibit poly(ADP-ribose) synthetase. The cytotoxic activity of streptozotocin in L1210 leukemia cells was maximally potentiated (7-fold decrease in IC50) under conditions of 24 hr exposure to PD 128763 following treatment with the alkylating agent for 1 hr. Similar treatment conditions resulted in a much greater effect (36-fold enhancement in activity) for the 2-nitroimidazole RSU 1069. In contrast, 3-aminobenzamide was only weakly effective at enhancing activity of either streptozotocin or RSU 1069 (2-3 fold potentiation). However, PD 128763 was ineffective at potentiating the cytotoxicity of the bifunctional alkylating agents carmustine (BCNU) and lomustine (CCNU). Our results are consistent with a role for (poly-ADP) ribosylation in the repair of monofunctional alkylating agent damage. This study supports further exploration of the combination of PD 128763 and RSU 1069 as a potentially useful chemotherapeutic regimen.

Pharmacologic/pharmacokinetic evaluation of emesis induced by analogs of RSU 1069 and its control by antiemetic agents.

RB 6145, the ring-opened analog of RSU 1069, and PD 130908, the desoxy ring-opened analog of RSU 1069, were compared to RSU 1069 for their emetic potential in dogs. When RB 6145 and PD 130908 were administered intravenously at doses ranging from 20% to 50% of the mouse equivalent maximum tolerated dose (MTD), both analogs were less emetic than RSU 1069 on a molar basis. Furthermore, the 5HT3 antagonist ondansetron prevented emesis at doses as high as 75% of the MTD. The reactivity, and hence the emetic liability of these compounds, is thought to be mediated by formation of the corresponding aziridine intermediate. In mouse plasma, both analogs rapidly converted to two products, the reactive aziridine and a stable oxiazolidinone species formed upon reaction with bicarbonate in the blood. A positive correlation exists between the amounts of aziridine formed by these analogs and their emetic potential.

Novel farnesol and geranylgeraniol analogues: A potential new class of anticancer agents directed against protein prenylation.

Protein farnesyltransferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Herein it is shown that certain novel prenyl diphosphate analogues are potent inhibitors of mammalian FTase. Furthermore, the alcohol precursors of two of these compounds are able to block anchorage-independent growth of ras-transformed cells. While 3-allylfarnesol inhibits protein farnesylation, 3-vinylfarnesol instead leads to abnormal prenylation of proteins with the 3-vinylfarnesyl group. In a similar manner, 3-allylgeranylgeraniol acts as a highly specific inhibitor of protein geranylgeranylation, while 3-vinylgeranylgeraniol restores protein geranylgeranylation in cells. This study indicates that certain prenyl alcohol analogues can act as prenyltransferase inhibitors in situ, via a novel prodrug mechanism. These analogues may prove to be valuable tools for investigating the therapeutic consequences of inhibiting geranylgeranylation relative to farnesylation. Furthermore, the 3-vinyl alcohol analogues can inhibit transformed cell growth through a mechanism not involving prenyltransferase inhibition.

Comparative molecular field analysis of in vitro growth inhibition of L1210 and HCT-8 cells by some pyrazoloacridines.

In vitro screening of a number of 2-(aminoalkyl)-5-nitropyrazolo[3,4,5- kl]acridines has previously indicated (Sebolt, J.S.; et al. Cancer Res. 1987, 47, 4299-4304) that these compounds, in general, exhibit selective cytotoxicity against the human colon adenocarcinoma, HCT-8, cell line, relative to mouse leukemia L1210 cells. Comparative molecular field analysis (CoMFA) was applied to HCT-8 and L1210 growth inhibition assays (IC50s) of a series (44) of the pyrazoloacridine derivatives with the objective of predicting improved solid tumor selectivity. In the absence of crystallographic data, the 9-methoxy derivative (15), which is currently in clinical study, was selected as the template molecular model. Two different structural alignments were tested: an alignment of structures based on root mean square (RMS)-fitting of each structure to 15 was compared with an alternative strategy, steric and electrostatic alignment (SEAL). Somewhat better predictive cross-validation correlations (r2) were obtained with models based on RMS vis-à-vis SEAL alignment for both sets of assays. A large change in lattice spacing, e.g., 2 to 1 A, causes significant variations in the CoMFA results. A shift in the lattice of half of its spacing had a much smaller effect on the CoMFA data for a lattice of 1 A than one of 2 A. The relative contribution of steric and electrostatic fields to both models were about equal, underscoring the importance of both terms. Neither calculated log P nor HOMO and/or LUMO energies contribute to the model. Steric and electrostatic fields of the pyrazoloacridines are the sole relevant descriptors to the structure-activity (cross-validated and conventional) correlations obtained with the cytotoxic data for both the L1210 and HCT-8 cell lines. The cross-validated r2, derived from partial least-squares calculations, indicated considerable predictive capacity for growth inhibition of both the leukemia and solid-tumor data. Evidence for the predictive performance of the CoMFA-derived models is provided in the form of plots of actual vs predicted growth inhibition of L1210 and HCT-8 cells, respectively, by the pyrazoloacridines. The steric and electrostatic features of the QSAR are presented in the form of standard deviation coefficient contour maps of steric and electrostatic fields. The maps indicate that increases or decreases in steric bulk that would enhance growth inhibition of HCT-8 cells would likewise promote growth inhibition of L1210 cells. Contour maps generated to analyze the electrostatic field contributions of the pyrazoloacridines to growth inhibition provide an essentially similar set of results.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and evaluation of a series of 3,5-disubstituted benzisoxazole-4,7-diones. Potent radiosensitizers in vitro.

A series of 3,5-disubstituted-2,1-benzisoxazole-4,7-diones was synthesized and evaluated as radiosensitizers both in vitro and in vivo. These compounds were designed as non-nitro electron-affinic agents in an effort to alleviate some of the toxicities seen with the 2-nitroimidazole radiosensitizers evaluated in the clinic. Several compounds in this series were potent radiosensitizers in vitro, with sensitizer enhancement ratios of 2.0-2.3 at concentrations less than 0.5 mM. Compounds with potent in vitro activity were also evaluated in vivo. However, none of these compounds showed radiosensitizing activity in vivo. The reduction potentials of these compounds were determined by cyclic voltammetry and compared to other electron-affinic radiosensitizers. In general, the reduction potentials of this series of compounds was slightly more positive than the 2-nitroimidazoles, but they fell within the range postulated as acceptable to yield in vivo activity. The results suggest that factors other than reduction potential may be responsible for the lack of in vivo radiosensitizing activity observed for this class of radiosensitizers.

A new class of analogues of the bifunctional radiosensitizer alpha-(1-aziridinylmethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069): the cycloalkylaziridines.

A series of compounds related to alpha-(1-aziridinylmethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069, 1) were synthesized and evaluated as selective hypoxic cell cytotoxic agents and as radiosensitizers. The aziridine moiety was replaced with a number of other potential alkylating groups including cycloalkylaziridines and azetidines. The data indicated that modification of the aziridine of 1 resulted in a substantial decrease in the ability of the compounds to selectively kill hypoxic cells. However, these modifications did not affect the compounds' in vitro radiosensitizing activity since many of the derivatives were as potent as 1. All of the compounds that were evaluated in vivo were less toxic than 1, and several members of this series had significant activity. The best compound was trans-alpha-[[(4-bromotetrahydro-2H-pyran-3-yl) amino]methyl]-2-nitro-1H-imidazole-1-ethanol (18), which, due to its activity and log P value, is a candidate for additional in vivo studies.

Structure-activity relationships of cysteine-lacking pentapeptide derivatives that inhibit ras farnesyltransferase.

Mutational activation of ras has been found in many types of human cancers, including a greater than 50% incidence in colon and about 90% in pancreatic carcinomas. The activity of both native and oncogenic ras proteins requires a series of post-translational processing steps. The first event in this process is the farnesylation of a cysteine residue located in the fourth position from the carboxyl terminus of the ras protein, catalyzed by the enzyme farnesyltransferase (FTase). Inhibitors of FTase are potential candidates for development as antitumor agents. Through a high-volume screening program, the pentapeptide derivative PD083176 (1), Cbz-His-Tyr(OBn)-Ser(OBn)-Trp-DAla-NH2, was identified as an inhibitor of rat brain FTase, with an IC50 of 20 nM. Structure-activity relationships were carried out to determine the importance of the side chain and chirality of each residue. This investigation led to a series of potent FTase inhibitors which lack a cysteine residue as found in the ras peptide substrate. The parent compound (1) inhibited the insulin-induced maturation of Xenopus oocytes (concentration: 5 pmol/oocyte), a process which is dependent on the activation of the ras pathway.

Effect of PD 128763, a new potent inhibitor of poly(ADP-ribose) polymerase, on X-ray-induced cellular recovery processes in Chinese hamster V79 cells.

The modifying effects of PD 128763 (3,4-dihydro-5-methyl-1(2H)-isoquinolinone), a potent inhibitor of poly(adenosine-diphosphate (ADP)-ribose) polymerase, on radiation-induced cell killing were examined in Chinese hamster V79 cells. This compound has an IC50 value against the purified enzyme approximately 50X lower than 3-aminobenzamide (3-AB), a widely used specific inhibitor of the enzyme. Exposure of exponentially growing cells to a noncytotoxic concentration (0.5 mM) of PD 128763 for 2 h immediately following X irradiation increased their radiation sensitivity, modifying both the shoulder and the slope of the survival curve. When recovery from sublethal damage and potentially lethal damage was examined in exponential and plateau-phase cells, respectively, postirradiation incubation with 0.5 mM PD 128763 was found not only to inhibit both these processes fully, but also to enhance further the level of radiation-induced cell killing. This is in contrast to the slight effect seen with the less potent inhibitor, 3-AB. The results presented suggest that the mechanism of radiosensitization by PD 128763 is related to the potent inhibition of poly(ADP-ribose) polymerase by this compound.

Novel limonene phosphonate and farnesyl diphosphate analogues: design, synthesis, and evaluation as potential protein-farnesyl transferase inhibitors.

Limonene and its metabolite perillyl alcohol are naturally-occurring isoprenoids that block the growth of cancer cells both in vitro and in vivo. This cytostatic effect appears to be due, at least in part, to the fact that these compounds are weak yet selective and non-toxic inhibitors of protein prenylation. Protein-farnesyl transferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Therefore, several alpha-hydroxyphosphonate derivatives of limonene were designed and synthesized as potentially more potent FTase inhibitors. A noteworthy feature of the synthesis was the use of trimethylsilyl triflate as a mild, neutral deprotection method for the preparation of sensitive phosphonates from the corresponding tert-butyl phosphonate esters. Evaluation of these compounds demonstrates that they are exceptionally poor FTase inhibitors in vitro (IC50 > or = 3 mM) and they have no effect on protein farnesylation in cells. In contrast, farnesyl phosphonyl(methyl)phosphinate, a diphosphate-modified derivative of the natural substrate farnesyl diphosphate, is a very potent FTase inhibitor in vitro (Ki=23 nM).

Dihydroisoquinolinones: the design and synthesis of a new series of potent inhibitors of poly(ADP-ribose) polymerase.

A series of dihydroisoquinolinones, formally rigid analogs of 3-substituted benzamides, and a series of 2,3-disubstituted benzamides were synthesized and evaluated as inhibitors of poly(ADP-ribose) polymerase. The results indicated that the orientation of the amide with respect to the substituent on the aromatic ring was critical for optimum inhibitory activity. Selected compounds were also evaluated for their ability to modify the radiation response of mammalian cells to ionizing radiation. A number of the 5-substituted dihydroisoquinolinones, which were very potent inhibitors of the enzyme, were able to enhance the lethal effects of ionizing radiation in mammalian cells, as measured by changes in the survival curve parameters Do and/or Dq.