Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors.

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 synthetic inhibitors, we have produced and purified the catalytic domain of ST3, matrilysin, stromelysin-2, and membrane type-1 MMP from inclusion bodies in a bacterial system. Our strategy allowed the purification of MMPs directly in the active form, thereby avoiding in vitro activation. A total of 140,000 synthetic compounds from the Bristol-Myers Pharmaceutical Research Institute chemical deck were tested, using a substrate-based colorimetric enzymatic assay, in which ST3 activity was evaluated through its ability to cleave and inactivate alpha-1 proteinase inhibitor. One ST3 inhibitor belonging to the cephalosporin family of antibiotics was thereby identified.

Glucolipsin A and B, two new glucokinase activators produced by Streptomyces purpurogeniscleroticus and Nocardia vaccinii.

During the screening of the natural products for their ability to increase the activity of glucokinase by relieving inhibition by long chain fatty acyl CoA esters (FAC), two novel compounds, glucolipsin A (1) and B (2) were isolated from the butanol extracts of Streptomyces purpurogeniscleroticus WC71634 and Nocardia vaccinii WC65712, respectively. The structures of these two compounds were established by spectroscopic methods and chemical degradation. Glucolipsin A (1) and B (2) relieved the inhibition of glucokinase by FAC with RC50 values of 5.4 and 4.6 microM.

Quanolirones I and II, two new human cytomegalovirus protease inhibitors produced by Streptomyces sp. WC76535.

Two new naphthacenequinone glycosides, quanolirones I (1) and II (2) were isolated, together with the known compound galtamycin from the fermentation broth of Streptomyces sp. WC76535. The structures 1 and 2 were established by analysis of their spectroscopic data and by comparison of their data to those of galtamycin. Compounds 1, 2, and galtamycin showed inhibitory activity against HCMV protease with IC50 values of 14, 35, and 52 microM, respectively.

A beta-lactam inhibitor of cytosolic phospholipase A2 which acts in a competitive, reversible manner at the lipid/water interface.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid (1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 72 microM). This beta-lactam did not inhibit other phospholipases, including the human nonpancreatic secreted phospholipase A2. The inhibition of cPLA2 was found not to be time-dependent. This, along with the observation that the degradation of the inhibitor was not catalyzed by the enzyme, demonstrates that the inhibition does not result from the formation of an acyl-enzyme intermediate with the active site serine residue. Moreover, the ring-opened form of 1 is also able to inhibit cPLA2 with near-equal potency. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 6-10 mole percent of 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition was defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.5 +/- 0.1 mole% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.4 +/- 0.1 mole%. Thus, 1 represents a novel structural class of inhibitors of cPLA2 which partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site.

1-Methoxy-agroclavine from Penicillium sp. WC75209, a novel inhibitor of the Lck tyrosine kinase.

A high-throughput screen was developed and implemented to identify inhibitors of the Lck tyrosine kinase. This report describes the identification of a specific inhibitor of this enzyme from the solid fermentation culture of the Penicillium sp., WC75209. The active compound was isolated and structurally characterized as 1-methoxy-5R, 10S-agroclavine, a new member of the ergot alkaloid family.

Bripiodionen, a new inhibitor of human cytomegalovirus protease from Streptomyces sp. WC76599.

Bripiodionen (1), a new natural product, was isolated from Streptomyces sp. WC76599 during the screening of microbial fermentation extracts for their ability to inhibit human cytomegalovirus protease. The structure of 1 was elucidated by spectroscopic methods. Compound 1 displayed inhibitory activity against human cytomegalovirus protease with an IC50 value of 30 microM.

The neurotoxic carboxy-terminal fragment of the Alzheimer amyloid precursor binds specifically to a neuronal cell surface molecule: pH dependence of the neurotoxicity and the binding.

One of the hallmarks of Alzheimer's disease neurodegeneration is the accumulation of deposits of amyloid in neuritic plaques and in the cerebral vasculature. Recent studies have implicated carboxy-terminal fragments of the Alzheimer amyloid precursor protein (beta APP) in the processes of amyloidogenesis and neurodegeneration. In particular, the carboxy-terminal 104 amino acids of beta APP (beta APP-C104) have been shown to cause amyloid-like fibrils when expressed in non-neuronal cells and to cause the degeneration of neuronal cells. These data suggest that it may play a role in the development of the progressive neuropathology of Alzheimer's disease. We hypothesized that beta APP-C104 may cause the degeneration of neurons by interacting with a cell surface receptor. In the present report, we show that beta APP-C104 synthesized in vitro binds specifically and with high affinity to the surface of NGF-treated PC12 cells. Both the cell surface binding and the neurotoxicity of beta APP-C104 are pH dependent and are not inhibited by tachykinins. Mutational analysis suggests that both the binding and the neurotoxicity are dependent at least in part on the presence of a tyrosine residue that is a potential site of phosphorylation at the carboxy terminus of the fragment.

Evidence for leucine zipper motif in lactose repressor protein.

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.

Thermal denaturation of the core protein of lac repressor.

The thermal denaturation of the core protein of lac repressor was studied alone and in the presence of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the antiinducer o-nitrophenyl beta-D-fucoside (ONPF) by means of high-sensitivity differential scanning calorimetry. The denaturation that takes place at about 65 degrees C is apparently irreversible; i.e., a rescan of a previously scanned sample of protein solution shows no denaturational endotherm. Despite this irreversibility, the denaturation appeared to follow quantitatively the dictates of equilibrium thermodynamics as embodied in the van't Hoff equation. The results obtained indicate clearly that the tetrameric protein dissociates to monomers during denaturation and that the ligands are not dissociated until denaturation takes place. The enthalpy of denaturation of the protein is 4.57 +/- 0.25 cal g-1 and is independent of temperature. The enthalpies of dissociation of IPTG and ONPF at the denaturation temperature are very large, 37 and 42 kcal (mol of ligand)-1, respectively.

lac operator DNA modification in the presence of proteolytic fragments of the repressor protein.

Singly end-labeled DNA fragments containing the lactose operator were methylated in the presence of the lactose repressor and homogeneous preparations of its proteolytic fragments. Binding of core protein produced by mild trypsin digestion yielded a methylation perturbation pattern that differed significantly from that elicited by binding to intact repressor, although similarities in the patterns for these related proteins were noted in the central, asymmetric region of the operator. An NH2-terminal peptide (residues 1 to 56) from lac repressor bound operator fragments in a nitrocellulose filter assay, but failed to perturb DNA methylation significantly relative to the pattern in the absence of peptide. Binding of hybrid tetramers of core and intact repressor monomers produced related but unique methylation patterns for the purines on the operator fragment. The general pattern of perturbation observed suggests preferred binding of a single NH2 terminus to the promoter-distal region of the operator and asymmetric interaction of the core region with the operator sequence. Differences in purine methylation patterns produced by the presence of effector complexes of repressor and core protein suggest the possible nature of changes in protein topology that result in the affinity changes accompanying induction.

Enzymatic digestion of operator DNA in the presence of the lac repressor tryptic core.

The trypsin-resistant core protein of the lac repressor was utilized in protecting operator DNA from two types of enzymatic digestion. Core repressor protects and enhances operator DNA digestion by DNase I in the same fashion as intact repressor, though to a lesser degree on the lower strand. DNase I patterns found for the ternary complexes (protein-sugar-operator) were consistent with the expected affinity alterations of the protein species in response to binding these ligands. The 3' boundaries obtained by exonuclease III digestion for the intact repressor-operator complex varied slightly from those reported by Shalloway et al. (1980). Asymmetric binding to operator by the core repressor fragment was suggested by differences in the 3' boundary for the core compared to intact repressor on the promoter-distal side of the complex. A composite picture of repressor structure and function emerges from the protection studies reported here and in the accompanying paper. In light of these and other results, models for repressor binding are examined.

Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells.

Eighteen cDNAs, cloned from interferon-treated T98G neuroblastoma cells, correspond to seven different mRNAs induced up to 40-fold by interferon. One codes for metallothionein II and another for a class I HLA. The others do not code for proteins of known sequence. In the continued presence of interferon, accumulation of the mRNAs continues for about 1 day but ceases whenever interferon is removed. Once induced, the mRNAs are stable. Synthesis of new proteins is not required for induction. The rate of transcription of one of the genes doubles 5 min after treatment with interferon and reaches a maximum by 60 min. This rate begins to fall after 4-6 hr, reaching the uninduced level by 8-12 hr. Since the mRNA continues to accumulate after 8-12 hr, posttranscriptional events must also play a role in increasing its level.

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.

Model for lactose repressor protein and its interaction with ligands.

A model is presented for the structure of the lactose repressor protein and for its interaction with inducer, operator DNA, and nonspecific DNA. The proposed structure is based on experimental evidence from this laboratory and from the literature and is offered as an integration of the available data on this system. Features unique to this model include: (i) interaction of the core region of the protein with the operator, (ii) primary effects of the conformational change in response to inducer on the core-operator interaction, (iii) contacts between all four subunits of the protein and the operator DNA, and (iv) qualitative differences in operator and nonspecific DNA binding.

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.