Structural characterization of the interaction of mTOR with phosphatidic acid and a novel class of inhibitor: compelling evidence for a central role of the FRB domain in small molecule-mediated regulation of mTOR.

The mammalian target of rapamycin (mTOR) is a large, multidomain protein kinase, which plays a central role in the regulation of cell growth and has recently emerged as an essential target of survival signals in many types of human cancer cells. Here, we report the solution structures of complexes formed between the FKBP12-rapamycin binding (FRB) domain of mTOR and phosphatidic acid, an important cellular activator of the kinase, and between the FRB domain and a novel inhibitor (HTS-1). The overall structure of the FRB domain is very similar to that seen in the ternary complex formed with FKBP12 and the immunosuppressive drug rapamycin; however, there are significant changes within the rapamycin-binding site with important consequences for rational drug design. The surface of the FRB domain contains a number of distinctive features that have previously escaped attention, including a potential new regulatory site on the opposite face to that involved in the binding of rapamycin, which displays the features expected for a specific binding site for a small molecule. The interaction sites for phosphatidic acid and HTS-1 were found to closely match the site responsible for rapamycin binding. In addition, the structures determined for the FRB-phosphatidic acid and FRB-HTS-1 complexes revealed a striking similarity between the conformations of buried portions of the ligands and that seen for the rapamycin backbone in contact with the domain. Our findings further highlight the importance of the FRB domain in small molecule-mediated regulation of mTOR, demonstrate the ability to identify novel inhibitors of mTOR that bind tightly to the rapamycin-binding site in the absence of FKBP12, and identify a potential new regulatory site that may be exploited in the design of new anticancer drugs.

Exploring the potential of PI3K inhibitors for inflammation and cancer.

Considerable biological evidence has accumulated in support of nominating the Class I PI3Ks (phosphoinositide 3-kinases) as excellent targets for the development of novel pharmaceuticals to treat cancer and inflammatory disease. Although it remains a goal to deliver compounds with precise PI3K isoform selectivity in order to minimize safety risks, it is not yet certain that this approach will deliver suitable benefit against disease when tested in the clinic. The UCB strategy, therefore, has been to generate a range of compounds covering a broad spectrum of PI3K isoform inhibition. Scaffold diversity has been accomplished by identifying hits using both pharmacophore search and high-throughput screening campaigns, while modulation of potency and isoform selectivity has been achieved through exploratory medicinal chemistry. Simple, high-throughput cell assays relevant to either inflammation or cancer have then been employed to establish a blueprint for defining how isoform selectivity affects biological potency. I will focus on two compounds from our collection: a pan-PI3K inhibitor and UCB1311236, a compound with significant potency against only the PI3Kgamma isoform. These examples will be used to illustrate the extent to which isoform selectivity informs on compound potency against other kinases and to highlight the risks and benefits of developing compounds with limited isoform selectivity.

HVR1 quasispecies analysis from a long-term culture of hepatitis C virus in Hep G2 derived cells grown in a haemodialysis cartridge.

Studies on the in vitro hepatitis C virus (HCV) infection are hampered by the lack of an appropriate system to culture permissive cells to be continuously infected with HCV. Trypsinization required for cell passage can lead to possible temporary loss of permissiveness for infection, whereas refreshment of the medium can result in loss of infectious particles necessary for perpetuation of the infection; it is therefore very difficult to maintain a continuous HCV infection in cell cultures. A new infection method was designed and evaluated in order to prevent these unfavourable circumstances. A cell line derived from the human hepatoblastoma cell line Hep G2 was grown in the extracapillary space of a haemodialysis cartridge, in the presence of a HCV-positive inoculum, while the culture medium was recirculated through the intracapillary space, supplying the cells with nutrients and oxygen. HCV RNA could continuously be detected in the cells up to 77 days of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) revealed that 56% and 75%, respectively, of the clones obtained from the cells at day 20 and 40 after start of the infection were different from the clones obtained from the original inoculum and that certain nucleotide positions in this region were more susceptible to mutations, leading to an alteration in amino acid sequence. As none of these sequences were present in the clones from the inoculum, it is suggested that new HCV quasispecies have emerged as a result of viral replication in the hepatocytes in vitro. This system seems a valuable tool for the in vitro evaluation of antiviral drugs.

Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV.

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.

CDP840: a novel inhibitor of PDE-4.

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor's activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

Matrix metalloproteinases and metastatic cancer.

The rationale for matrix metalloproteinase (MMP) inhibition as a means to treat disease progression in breast cancer stems from the apparent involvement of MMPs in the hydrolysis of basement membranes during tumour cell invasion and subsequent metastasis. MMP-mediated matrix remodelling also appears to promote the growth of tumour cells, possibly by facilitating the proliferation and migration of endothelial cells and the neovascularization of tumour tissue. We found that transfection of the C127 breast cancer cell line by MMP-2 (gelatinase A), but not by MMP-1 or MMP-3 (collagenase and stromelysin respectively), gave rise to an invasive and metastatic phenotype. We were surprised to find that this phenotype depended not only on the catalytic properties of MMP-2 but also on properties associated with the MMP-2 non-catalytic C-terminal domain. Experiments with a synthetic gelatinase inhibitor revealed that a single dose could prevent the lungs of nude mice being colonized by the MMP-2 transfectants, and that the inhibitor had to be administered during or shortly after injection of the cells, indicating that an early event, such as the extravasation of the cells into the lung, is gelatinase-dependent in this system. In other studies employing long-term treatment with CT1746, an orally active gelatinase inhibitor, we have previously demonstrated a reduction in primary tumour growth rates, localized spread, and spontaneous metastasis, even when the treatment was commenced several days after tumour implantation. Furthermore, additive effects were recorded when gelatinase inhibitor therapy was combined with cytotoxic drug treatment. Since the gelatinase inhibitors can also inhibit bone resorption in vitro, these observations point to their potential for delaying disease recurrence and reducing rates of bone loss following conventional therapeutic strategies, in metastatic breast cancer.

Molecular cloning and expression of a human phosphodiesterase 4C.

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

The secretion of active recombinant human gastric lipase by Saccharomyces cerevisiae.

The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.

An in vivo model for screening peptidomimetic inhibitors of gelatinase A.

Gelatinase A, a matrix metalloproteinase, is frequently associated with human solid tumors, and its secretion and activation in the tumor milieu is considered important in the process of angiogenesis, invasion, and metastasis. Consequently, metalloproteinase inhibitors may be of value in the therapy of cancer as well as other disease states involving tissue remodeling and release of biologically active peptide/protein by proteolytic cleavage. Here we describe the development of a rapid screening assay for in vivo activity of peptidomimetic inhibitors of gelatinase A that involves assessment of inhibition of an enzyme-substrate reaction in a circumscribed body compartment, the mouse pleural cavity. As examples of the utility of this assay, in vivo activity of the aryl sulfonamide, sulfamyl urea, morpholino and carboxylic acid functionality at the P3' position of a series of hydroxamic acid inhibitors was examined after administration both intraperitoneally (ip) (to approximate systemic administration) and orally. For up to 2 h after ip administration, all inhibitors tested showed marked activity (> 90% inhibition) at 17 mumol/kg (approximately 10 mg/kg). This activity declined in a dose-responsive manner to insignificant levels at 0.67 mumol/kg (approximately 0.4 mg/kg). Aryl sulfonamides showed significant inhibition (> 50%) for up to 7 h after administration. A higher dosage (136 mumol/kg, approximately 80 mg/kg) was required to reveal oral activity, which was observed only with morpholino compounds (> 50% inhibition). Thus, the model described may be of value in the identification of orally active gelatinase A inhibitors.

Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A.

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, is secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelatinase A was activated to 38% of maximum by a 6 h incubation at 37 degrees C with equimolar concentrations of trypsin-activated interstitial collagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72,000 progelatinase A to the M(r) 66,000 active enzyme that has Y81 as its N-terminus. At low concentrations, progelatinase A was processed via an inactive intermediate, suggesting that its activation is a biphasic process. This was confirmed by the action of collagenase on proE375-->A (a mutant of progelatinase A that cannot become active) because, in this instance, only an M(r) 68,000 species with L38 as the N-terminus was produced. The remaining propeptide amino acids to Y81 could be readily removed by added active gelatinase A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by the addition of 100 micrograms/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activated by trypsin retains 8 or 14 amino acids of its propeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1.

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Human progelatinase A can be activated by autolysis at a rate that is concentration-dependent and enhanced by heparin bound to the C-terminal domain.

Activation of the latent precursor of human gelatinase A (progelatinase A) was investigated using recombinant proenzyme purified from culture medium conditioned by transfected mouse myeloma cells. A 4.0 microM progelatinase A solution was activated to a maximum of 48% of the activity produced by 4-aminophenylmercuric acetate (APMA) simply by its incubation at 37 degrees C for 12 h, though at lower starting concentrations the rate and extent of activation were reduced. Activation was shown to be the result of a single autolytic cleavage at the Asn80-Tyr81 peptide bond that removes the propeptide and converts the M(r) = 72,000 proenzyme into the M(r) = 66,000 active species also produced by APMA activation. It is proposed that this cleavage is a bimolecular event catalysed by previously activated gelatinase A. The addition of heparin increased by approximately eightfold the initial rate of progelatinase A autolytic activation but did not affect the activation of a deletion mutant that lacked the C-terminal domain [des-(418-631)progelatinase A]. The inference that this increase resulted from an interaction between heparin and the C-terminal domain was supported by the finding that, unlike des-(418-631)gelatinase A, both full-length gelatinase A and the isolated C-terminal domain were able to bind to heparin-Sepharose CL-6B and that, at NaCl concentrations sufficient to abolish this binding, heparin had no effect. We conclude that heparin is able to enhance autolytic activation by acting as a template that approximates active-->latent gelatinase A and suggest that a similar mechanism may account for the cell-surface activation of this enzyme.

The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.