DNA looping and unlooping by AraC protein.

Expression of the L-arabinose BAD operon in Escherichia coli is regulated by AraC protein which acts both positively in the presence of arabinose to induce transcription and negatively in the absence of arabinose to repress transcription. The repression of the araBAD promoter is mediated by DNA looping between AraC protein bound at two sites near the promoter separated by 210 base pairs, araI and araO2. In vivo and in vitro experiments presented here show that an AraC dimer, with binding to half of araI and to araO2, maintains the repressed state of the operon. The addition of arabinose, which induces the operon, breaks the loop, and shifts the interactions from the distal araO2 site to the previously unoccupied half of the araI site. The conversion between the two states does not require additional binding of AraC protein and appears to be driven largely by properties of the protein rather than being specified by the slightly different DNA sequences of the binding sites. Slight reorientation of the subunits of AraC could specify looping or unlooping by the protein. Such a mechanism could account for regulation of DNA looping in other systems.

Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.

Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.

Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor.

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.

AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein.

The arabinose operon promoter, pBAD, is negatively regulated in the absence of arabinose by AraC protein, which forms a DNA loop by binding to two sites separated by 210 base-pairs, araO2 and araI1. pBAD is also positively regulated by AraC-arabinose and the cyclic AMP receptor protein, CRP. We provide evidence that CRP breaks the araO2-araI1 repression loop in vitro. The ability of CRP to break the loop in vitro and to activate pBAD in vivo is dependent upon the orientation and distance of the CRP binding site relative to araI1. An insertion of one DNA helical turn, 11 base-pairs, between CRP and araI only partially inhibits CRP loop breaking and activation of pBAD, while an insertion of less than one DNA helical turn, 4 base-pairs, not only abolishes CRP activation and loop breaking, but actually causes CRP to stabilize the loop and increases the araO2-mediated repression of pBAD. Both integral and non-integral insertions of greater than one helical turn completely abolish CRP activation and loop breaking in vitro.

A phase I and pharmacological study of the farnesyl protein transferase inhibitor L-778,123 in patients with solid malignancies.

This Phase I study was performed to assess the feasibility of administering L-778,123, a peptidomimetic farnesyl protein transferase (FPTase) inhibitor, as a continuous i.v. infusion for 7 days every 3 weeks and to determine the recommended dose for subsequent disease-directed trials. This study also sought to characterize the pharmacological behavior of L-778,123 and to determine whether the desired biological effect, inhibition of protein farnesylation, could be detected and assessed during treatment. Patients with advanced solid malignancies were treated with L-778,123 as a continuous i.v. infusion for 7 days every 3 weeks at doses ranging from 35 to 1120 mg/m(2)/day. On the basis of preclinical studies, toxicity assessments included cardiac telemetry, electrocardiograms, and electroretinograms in addition to more routine safety monitoring laboratory tests. Plasma sampling was performed to characterize the pharmacokinetics of L-778,123, and peripheral blood mononuclear cells (PBMCs) were sampled to detect and monitor the inhibitory effects of L-778,123 on the prenylation of HDJ2, a chaperone protein that undergoes farnesylation. Twenty-five patients received 51 complete courses of L-778,123. An unacceptably high incidence of dose-limiting toxicities, consisting of grade 4 thrombocytopenia, significant prolongation of the QT(c) interval, and profound fatigue, was observed at the 1120 mg/m(2)/day dose level. At the next lower L-778,123 dose level, 560 mg/m(2)/day, seven new patients had no unacceptable toxicity. Instead, myelosuppression was mild to moderate and QT(c) prolongation was negligible. Pharmacokinetics were linear, and L-778,123 plasma concentrations at steady-state (mean, 8.09 +/- 3.11 microM at 560 mg/m(2)/day) exceeded IC(50) values (range, 0.07-5.35 microM) required for growth inhibition and cytotoxicity in preclinical studies. The systemic clearance of L-778,123 averaged 106.4 +/- 45.6 ml/min/m(2), and the terminal half-life of elimination was 2.8 +/- 1.0 h. L-778,123 inhibited HDJ2 prenylation for the duration of the drug infusion in a dose-dependent manner, but seemed to plateau above 560 mg/m(2)/day. At the 560 mg/m(2)/day dose level, the mean percentage of HDJ2 protein in its unprenylated form increased from 1.41% +/- 1.71% (pretreatment) to 28.76% +/- 6.10% (day 4) and 30.86 +/- 4.96 (day 8) and declined to 2.28% +/- 2.11% one week after drug discontinuation (day 16). L-778,123 administered as a continuous 7-day i.v. infusion for 7 days every 21 days is well tolerated at doses of 560 mg/m(2)/day and results in biologically relevant concentrations and consistent inhibition of HDJ2 prenylation in PBMCs. Although the relationship between drug-related inhibition of HDJ2 prenylation in PBMCs and both prenylation of relevant proteins and growth inhibition in tumor cells is unknown, serial analyses of HDJ2 prenylation provide a pharmacodynamic marker of protein prenylation that may be useful in optimizing the development of drugs targeting FPTase.

Design and biological activity of (S)-4-(5-([1-(3-chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl)imidazol-1-ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyltransferase inhibitor with excellent cell potency.

The synthesis, structure-activity relationships, and biological properties of a novel series of imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-aminopyrrolidinone core, a systematic series of modifications provided 5h, a non-thiol, non-peptide farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and showed full efficacy in the K-rasB transgenic mouse model.

Farnesyl: proteintransferase inhibitors as agents to inhibit tumor growth.

Ras, a signal-transducing protein involved in mediating growth factor-stimulated proliferation, is mutationally activated in over 30% of human tumors. To be functional Ras must bind to the inner surface of the plasma membrane, with post-translational lipid modifications being necessary for this localization. The essential, first modification of Ras is farnesylation catalyzed by the enzyme farnesyl: proteintransferase (FPTase). Inhibitors of FPTase (FTIs) are currently being tested to determine if they are capable of tumor growth inhibition. Here we describe our efforts, along with those of other groups, in testing the biological and biochemical effects of FTIs.

Expression and function of Fc gamma R in mouse mast cells.

Activation of mast cells for the release of secretory granule, membrane lipid, and cytokine mediators via Fc gamma R requires cell surface expression of Fc gamma RIII. A soluble factor(s) from fibroblasts up-regulates the surface expression of Fc gamma RIII in interleukin-3-dependent, bone-marrow-derived mast cells, which otherwise degrade Fc gamma RIII intracellularly. Using a receptor-specific rabbit polyclonal antibody made to a peptide from the cytoplasmic domain of Fc gamma RIII alpha chain, we show that steady-state levels of Fc gamma RIII alpha protein increase in bone-marrow-derived mast cells after coculture with fibroblasts. Thus, the posttranslational stability of this functionally relevant receptor in mast cells is probably regulated by a fibro-blast-derived cytokine(s).

Pre-clinical development of farnesyltransferase inhibitors.

ras is the oncogene most frequently found in human cancers, being detected in 30% of most human cancers and at significantly higher rates in certain cancers including pancreatic (90%) and colon (50%) [1]. Almost 10 years ago it was shown that a C-terminal lipid modification of Ras, catalyzed by a specific farnesyl-protein transferase (FPTase), was required for the function of both normal and oncogenic Ras proteins. This finding spurred the development of FPTase inhibitors (FTIs) as a potential cancer therapy directed at the ras oncogene. FTIs have exhibited potent antiproliferative activity in cell culture and animal tumor models with a surprising lack of toxicity to normal tissues. However, while FTIs were originally conceptualized as Ras-specific agents, their mechanism of action is significantly more complicated than originally envisioned.

Fc gamma R-mediated endocytosis and expression of cell surface Fc gamma RIIb1 and Fc gamma RIIb2 by mouse bone marrow culture-derived progenitor mast cells.

Mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC) bind IgG immune complexes through Fc receptors for IgG (Fc gamma R) but express minimal Fc gamma RIII on their surfaces. BMMC do not degranulate appreciably when their Fc gamma R are perturbed with the rat anti-mouse Fc gamma RII/III mAb 2.4G2 and F(ab')2 mouse anti-rat IgG (MAR). In contrast, after their Fc gamma R were cross-linked with mAb 2.4G2 and Na125I-labeled MAR at 37 degrees C, BMMC rapidly internalized the complex. To identify the Fc gamma R species expressed on the surface of BMMC and therefore implicated in the endocytic response, two rabbit antipeptide antisera were raised, one against a sequence common to the cytoplasmic regions of Fc gamma RIIb1 and Fc gamma RIIb2 and the other to a unique cytoplasmic region of Fc gamma RIIb1. When Fc gamma R were immunoprecipitated with mAb 2.4G2 from detergent extracts of BMMC, digested with N-glycosidase F, subjected to SDS-PAGE, and immunoblotted with the Fc gamma RIIb1- and Fc gamma RIIb1/b2-specific antibodies, BMMC were found to express Fc gamma RIIb1 and Fc gamma RIIb2. Selective immunoprecipitation of plasma membrane-localized Fc gamma RIIb1 and Fc gamma RIIb2 from [3H]leucine-labeled BMMC showed that their ratio at the cell surface was similar to their initial biosynthetic ratio. Thus, in contrast to mature serosal mast cells that degranulate on binding of IgG complexes, immature mast cells, of which BMMC are a prototype, may have a role in the clearance of complexes without concomitant release of proinflammatory mediators.

Intracellular degradation of Fc gamma RIII in mouse bone marrow culture-derived progenitor mast cells prevents its surface expression and associated function.

Although mouse interleukin-3-dependent, bone marrow culture-derived progenitor mast cells (BMMC) and a Kirsten sarcoma virus (KiSV)-immortalized mouse mast cell line (MC4w) both express on their surfaces receptors for the Fc portion of IgG (Fc gamma R), only MC4w degranulate upon Fc gamma R perturbation. As shown by surface iodination and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated proteins immunoprecipitated with the Fc gamma R-specific monoclonal antibody 2.4G2, a 26-kDa protein, identified as Fc gamma RIII by immunoblotting with antibody to Fc gamma RIII, was predominantly expressed on the surface of MC4w but minimally on BMMC. However, both BMMC and MC4w expressed mRNA for Fc gamma RIII as determined by RNA blot analysis, and both translated Fc gamma RIII as assessed by intrinsic radiolabeling and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated monoclonal antibody 2.4G2 immunoprecipitates. Pulse-chase analysis showed that intrinsically radiolabeled Fc gamma RIII was stable in MC4w cells but was degraded rapidly in BMMC and that newly synthesized Fc gamma RIII remained sensitive to digestion by endoglycosidase H in BMMC but rapidly became resistant in MC4w. These data suggest that the deficiency in surface Fc gamma RIII expression on BMMC is due to the degradation of Fc gamma RIII in the endoplasmic reticulum. Immunoprecipitation of surface Fc gamma R and Fc receptors for IgE (Fc epsilon RI) from digitonin-extracted cells followed by immunoblotting with antibody to Fc epsilon RI gamma-chain showed that gamma-chain is associated with surface Fc epsilon RI and Fc gamma R in MC4w, but only with Fc epsilon RI in BMMC, which lack surface Fc gamma RIII. Inasmuch as BMMC are progenitors of serosal mast cells, which, like MC4w, express surface Fc gamma RIII and undergo Fc gamma R-mediated activation, the data suggest that maturation of BMMC enables Fc gamma RIII to bypass degradation in the endoplasmic reticulum, resulting in the acquisition of functional Fc gamma RIII/gamma-chain complexes on the cell surface.

Determinants of heat shock-induced chromosome puffing.

Modified Drosophila heat shock genes were introduced into the germ line by P element transformation. The genes were altered such that several factors could be tested for their influence upon chromosome puffing. Deletion of promoter sequences upstream of position -73 of an hsp70-IacZ hybrid gene was sufficient to abolish puffing. Analysis of progressive 5' deletions defines a 16 bp interval that contains sequences required for both heat-induced puffing and gene expression. An internal deletion of the hsp70-IacZ gene that reduces the transcript size from 9 kb to 0.8 kb results in a dramatic reduction in puff size. The chromosomal insertion sites of 26 variant hsp70 or hsp26 genes fail to influence puffing greatly with one marked exception. This transformant possesses an insert that fails to puff and exhibits a tissue-restricted pattern of expression. These results indicate that variation in either promoter strength or transcript length have profound effects on puffing.

Imidazole-containing diarylether and diarylsulfone inhibitors of farnesyl-protein transferase.

The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Optimization of cysteine-substituted diarylethers led to highly potent imidazole-containing diarylethers and diarylsulfones. Polar diaryl linkers dramatically improved potency and gave highly cell active compounds.

2-Arylindole-3-acetamides: FPP-competitive inhibitors of farnesyl protein transferase.

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.

Oxo-piperazine derivatives of N-arylpiperazinones as inhibitors of farnesyltransferase.

The evaluation of SAR associated with the insertion of carbonyl groups at various positions of N-arylpiperazinone farnesyltransferase inhibitors is described herein. 1-Aryl-2,3-diketopiperazine derivatives exhibited the best balance of potency and pharmacokinetic profile relative to the parent 1-aryl-2-piperazinones.

Anions modulate the potency of geranylgeranyl-protein transferase I inhibitors.

We have identified and characterized potent and specific inhibitors of geranylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibitors of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTase I in vitro. Inhibitors with a strong anion dependence were competitive with geranylgeranyl pyrophosphate (GGPP), rather than with the peptide substrate, which had served as the original template for inhibitor design. One of the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate l-778,123, this increase in potency was shown to result from two major interactions: competitive binding of inhibitor and GGPP, and competitive binding of ATP and GGPP. At 5 mm, ATP caused an increase in the apparent K(d) for the GGPP-GGPTase I interaction from 20 pm to 4 nm, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 10(6) m(-)1 s(-)1 and 10(-)3 s(-)1, respectively. Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accounting for anion requirement, slow binding, and mechanism of competition, the structure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.

Diaryl ether inhibitors of farnesyl-protein transferase.

Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.

Aryloxy substituted N-arylpiperazinones as dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I.

A series of aryloxy substituted piperazinones with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to have potent inhibitory activity in vitro and are promising agents for the inhibition of Ki-Ras signaling.