Sequence-directed recognition peptides: inhibition of endothelin generation via a substrate-depletion mechanism.

Sequence-directed recognition peptides (SDRPs) were constructed on the basis of their hydropathic complementarity for big-endothelin (bigET). These peptides can inhibit in vitro the proteolytic cleavage that generates endothelin (ET) from its bigET precursor. Comparison of dissociation constants of the complexes SDRP:bigET with kinetic constants obtained for the cleavage of bigET by alpha-chymotrypsin (taken as a model proteinase) provides evidence of the potential of SDRPs. This is a novel application of SDRPs used as inhibitors of a proteolytic reaction.

Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin.

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.

The protonated form of 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine is identified at the active site of adenosine deaminase.

A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (epsilon-EHNA), is protonated at the active site of the enzyme. In epsilon-EHNA [K1 = (4.06 +/- 1.00) 10(-6) M] part of the competitive inhibition of EHNA is combined with spectroscopic properties of etheno-adenines. Computer subtraction of the fluorescence excitation spectrum of ADA from that of its equimolar complex with epsilon-EHNA yielded the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-EHNA at pH 5.5 in buffer solution and is suggested to indicate a shift in protonation equilibrium at the active site.

Quantitative structure-activity analysis in dihydropteroate synthase inhibition by sulfones. Comparison with sulfanilamides.

A set of 25 4'-, eight 2',4'-, and five 2',4',6'-substituted 4-aminodiphenyl sulfones were tested for their inhibitory activity on dihydropteroate synthase of Escherichia coli. Linear regression analysis shows that enzymic inhibition indices correlate well with both quantum chemical and spectroscopic descriptors of the electronic structure of the common moiety 4-NH2-C6H4-SO2 of the sulfones (the above descriptors being expressed in relation to the electronic structure of the enzyme substrate, p-aminobenzoate). Therefore, the biological activity of the sulfones can be related to the electronic structural resemblance between these inhibitors and the substrate of the target enzyme. Since a similar result was previously obtained for a wide series of sulfanilamides in their different (amidic, imidic, and anionic) forms, it appears possible to consider the antibacterial sulfones and sulfanilamides as a congeneric chemical series. On the basis of the present results, the classical theory of antimetabolites would appear to take on a quantitative and sound rationale.

Fluorescence polarization assay for endothelin-converting enzymes.

The conversion of big endothelin to endothelin by alpha-chymotrypsin was determined by following its single Trp fluorescence polarization. This provides a novel, simple, fast, and sensitive identifying assay in the search for a native endothelin-converting enzyme.

Polymer-bound camptothecin: initial biodistribution and antitumour activity studies.

Camptothecin (CPT) is a potent, antitumour drug acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle. Despite its impressive antitumour activity, clinical development was halted for unpredictable toxic events. Two soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain CPT (5 wt.% and 10 wt.%). CPT was covalently linked at its alpha-hydroxyl group to the polymers through a Gly-Phe-Leu-Gly- spacer. In-vitro, CPT-conjugates were fairly resistant to hydrolysis in plasma as in buffer at neutral pH (0.2-0. 4% free CPT/h), while elastase and cysteine-proteases were able to release the active drug. Plasma levels in mice after intravenous administration of CPT-conjugates confirmed the modest hydrolysis in plasma. Plasma levels were approximately 5-fold lower than those observed at the highest tolerated dose of CPT administered in classical vehicles. Biodistribution in HT29 human colon carcinoma bearing mice was carried out after i.v. injection of [3H]CPT-conjugate and free [3H]CPT. Radioactivity uptake in tumour was evident only after [3H]CPT-conjugate treatment. Repeated intravenous administration of CPT-conjugates to HT29-bearing mice gave more than 90% tumour inhibition, some complete tumour regressions and no toxic deaths. The improved pharmacological profile on HT29 human colon carcinoma xenografts of the first poly(HPMA)-CPT conjugates might be ascribed to their prolonged intra-tumour retention and sustained release of the active drug.

Probing the active site of adenosine deaminase by a pH responsive fluorescent competitive inhibitor.

The adenine analog erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA, a tight reversible inhibitor (KI = 1.6 x 10(-9) M) of adenosine deaminase (EC 3.5.4.4) (ADase), was modified into the fluorescent etheno derivative epsilon-EHNA. The latter is a competitive inhibitor of adenosine deaminase [KI = (2.80 +/- 0.01)10(-6) M], having the fluorescent properties of epsilon-adenines. Affinity to the active site, monitored by both steady-state and dynamic fluorescence polarization, was confirmed by competition experiments with 2'-deoxycoformycin, the substrate adenosine and EHNA. The epsilon-adenine moiety of epsilon-EHNA librates at the shallow active site of ADase. The low absorptivity of epsilon-EHNA required the measurement of fluorescence excitation spectra. Computer subtraction of fluorescence excitation spectrum of ADase from that of its equimolar complex with epsilon-EHNA revealed the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-EHNA at pH 5.5 in buffer solution, implying its protonation at the active site of the enzyme. These results are in agreement with the presence of acidic amino acids that are essential to the catalytic mechanism.

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses.

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation.

Adenosine deaminase-complexing protein from bovine kidney. Isolation of two distinct subunits.

We have isolated a complex of two proteins from bovine kidney that bind to adenosine deaminase immobilized on Sepharose 4B. One protein, with Mr = 110,000, comigrates on both PAGE and SDS-PAGE gels with complexing protein isolated from rabbit kidney by the method of Schrader et al. (Schrader, W.P., Harder, C. M., and Schrader, D. K. (1983) Comp. Biochem. Physiol. B Comp. Biochem. 75, 119-126). The second protein has a Mr = 70,000. Both proteins bind to the adenosine deaminase-Sepharose but not to a control resin of bovine serum albumin bound to Sepharose. Based on a comparison of partial and complete denaturation on SDS-PAGE the two proteins appear to be bound to each other. At adenosine concentrations of 0.5-1 mM the isolated complexing protein increases small subunit adenosine deaminase catalytic activity by 20-30%. There may be some inhibition of catalytic activity at low adenosine concentrations. We have designated the 110,000 Mr protein CP-I, the 70,000 Mr protein CP-II and the complex of these two CP.

Nature of interaction between basic fibroblast growth factor and the antiangiogenic drug 7,7-(Carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino] )bis-(1, 3-naphthalene disulfonate).

PNU145156E (7,7-(carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]) -bis-(1, 3-naphthalene disulfonate)) is a naphthalene sulfonic distamycin A derivative that interacts with heparin-binding growth factors. Because PNU145156E inhibits tumor angiogenesis, it was selected for clinical development. Picosecond time-resolved fluorescence emission and anisotropy were used to characterize the binding of PNU145156E to the basic fibroblast growth factor (a protein associated with tumor angiogenesis). A decrease in PNU145156E fluorescence lifetime was observed as a function of human basic fibroblast growth factor (bFGF) concentration. Nonlinear least-squares fitting of the binding isotherm yielded Kd = 145 nM for a single class of binding sites. Time-resolved anisotropy gave Kd = 174 nM. Kd = 150 nM was independently verified by quantitative high-performance affinity chromatography. The displaced volume of the complex, calculated from its rotational correlation time, fitted a sphere of 1:1 stoichiometry. These results account for the formation of a tight yet reversible PNU145156E:bFGF complex. An evaluation of PNU145156E fluorescence lifetimes in various solvents has highlighted the forces involved in stabilizing the complex. These are mostly electrostatic-hydrophobic in nature, with a relatively low contribution from hydrogen bonding. Both polar and nonpolar groups are involved on the protein-binding site within a largely hydrophobic cleft. A potential binding trajectory, based on a combination of these results with site-directed chemical modification and known bFGF x-ray structure, is suggested.

Laser photobleaching leads to a fluorescence grade adenosine deaminase.

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

Human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells.

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.

Big endothelin-converting enzyme activities in subcellular fractions of bovine aortic endothelial cells.

A combination of HPLC elution patterns and peptide sequencing has been used to characterize two distinct activities present in subcellular fractions of bovine aortic endothelial cells (BAECs) capable of converting human big endothelin-1 (big ET-1) to mature (ET-1). A pepstatin-inhibitable activity with an acidic pH optimum present in a lysosome-enriched fraction cleaved big ET-1 at positions 18 and 21 at similar rates. A neutral pH activity present in a postlysosomal organelles subfraction was also able to convert big ET-1, and was inhibited by EDTA, but not by 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), an inhibitor of chymotrypsin-like serine proteases.

In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.