Senescence marker protein 30 (SMP30) expression in eukaryotic cells: existence of multiple species and membrane localization.

Senescence marker protein (SMP30), also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca(2+) homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA). Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ∼28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of ß and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.

Gene-delivered butyrylcholinesterase is prophylactic against the toxicity of chemical warfare nerve agents and organophosphorus compounds.

Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 × 10(10) infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30× LD(50) dose of echothiophate and 5× LD(50) dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5× LD(50) or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.

Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors.

Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily.

Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.

Discovery of pyrrolopyridine-pyridone based inhibitors of Met kinase: synthesis, X-ray crystallographic analysis, and biological activities.

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors.

A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase.

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.

Pyrrolopyridazine MEK inhibitors.

The synthesis and SAR of a series of pyrrolopyridazine MEK inhibitors are reported. Optimal activity was achieved by incorporation of a 4-phenoxyaniline substituent at C4 and an acylated amine at C6.

Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors.

A chemical genetics approach identified a cellular target of several proapoptotic farnesyl transferase inhibitors (FTIs). Treatment with these FTIs caused p53-independent apoptosis in Caenorhabditis elegans, which was mimicked by knockdown of endosomal trafficking proteins, including Rab5, Rab7, the HOPS complex, and notably the enzyme Rab geranylgeranyl transferase (RabGGT). These FTIs were found to inhibit mammalian RabGGT with potencies that correlated with their proapoptotic activity. Knockdown of RabGGT induced apoptosis in mammalian cancer cell lines, and both RabGGT subunits were overexpressed in several tumor tissues. These findings validate RabGGT, and by extension endosomal function, as a therapeutically relevant target for modulation of apoptosis, and enhance our understanding of the mechanism of action of FTIs.

Design, synthesis, and structure-activity relationships of tetrahydroquinoline-based farnesyltransferase inhibitors.

Tetrahydroquinoline-based small molecule inhibitors of farnesyltransferase (FT) have been identified. Lead compounds were shown to have nanomolar to sub-nanomolar activity in biochemical assays with excellent potency in a Ras-mutated cellular reversion assay. BMS-316810 (9e), a 0.7 nM FT inhibitor, was orally-active in a nude mouse tumor allograft efficacy study.

Phase I pharmacokinetic and pharmacodynamic study of weekly 1-hour and 24-hour infusion BMS-214662, a farnesyltransferase inhibitor, in patients with advanced solid tumors.

PURPOSE: BMS-214662 is a potent, nonpeptide, small molecule inhibitor of human farnesyltransferase (FT). We have conducted a phase I pharmacokinetic (PK) and pharmacodynamic study of BMS-214662 administered intravenously weekly with 1- and 24-hour infusions. The objectives were to determine the dose-limiting toxicities and the recommended dose (RD), to describe PKs, and to evaluate the relationships between BMS-214662 exposure, FT inhibition, downstream signaling, and induction of apoptosis in tumor samples. PATIENTS AND METHODS: Patients with advanced solid tumors and adequate organ function were eligible. The dose was escalated according to a modified Fibonacci schedule. RESULTS: high (> 80%) but short-lived (< or = 6 hours) in the 1-hour infusion and moderate (> 40%) but long-lived (24 hours) in the 24-hour infusion. BMS-214662 induced apoptosis in tumors but did not inhibit MAPK signaling. CONCLUSION: BMS-214662 can be safely delivered in both the 1-hour and 24-hour infusions at biologically active doses, with the preclinical, PK, and pharmacodynamic profiles favoring the 24-hour schedule.

Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models.

BMS-214662 and BMS-225975 are tetrahydrobenzodiazepine-based farnesyltransferase inhibitors (FTIs) that have nearly identical structures and very similar pharmacological profiles associated with farnesyltransferase (FT) inhibition. Despite their similar activity against FT in vitro and in cells, these compounds differ dramatically in their apoptotic potency and tumor-regressing activity in vivo. BMS-214662 is the most potent apoptotic FTI known and exhibits curative responses in mice bearing a variety of staged human tumor xenografts such as HCT-116 human colon tumor. By contrast, BMS-225975 does not cause tumor regression and at best causes partial tumor growth inhibition in staged HCT-116 human colon tumor xenografts. Lack of tumor regression activity in BMS-225975 was attributable to its relatively weak apoptotic potency, not to poor cell permeability or pharmacokinetics. Both compounds were equally effective in inhibiting Ras processing and causing accumulation of a variety of nonfarnesylated substrates of FT in HCT-116 cells. Because BMS-225975 has poor apoptotic activity compared with BMS-214662 but inhibits FT to the same extent as BMS-214662, it is very unlikely that FT inhibition alone can account for the apoptotic potency of BMS-214662. Clearly distinct patterns of sensitivities in a cell line panel were obtained for the apoptotic FTI BMS-214662 and the cytostatic FTI BMS-225975. Activation of the c-Jun-NH(2)-terminal kinase pathway was readily observed with BMS-214662 but not with BMS-225975. We developed a highly sensitive San-1 murine xenograft tumor model that is particularly useful for evaluating the in vivo activity of cytostatic FTIs such as BMS-225975.