Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments.

Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

Distribution of fluorescently labeled actin in living sea urchin eggs during early development.

Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.

Hydrodynamic size of DNA/cationic gemini surfactant complex as a function of surfactant structure.

The present study deals with the determination of hydrodynamic size of DNA/cationic gemini surfactant complex in sodium bromide solution using the dynamic light scattering method. Cationic gemini surfactants with polymethylene spacer of variable length were used for the interaction with DNA. The scattering experiments were performed at constant DNA and sodium bromide concentrations and variable surfactant concentration in the premicellar and micellar regions as a function of surfactant spacer length. It was found that the DNA conformation strongly depends on the polymethylene spacer length as well as on the surfactant concentration relative to the surfactant critical micelle concentration. Gemini surfactant molecules with 4 methylene groups in the spacer were found to be the least efficient DNA compacting agent in the region above the surfactant cmc. Gemini molecules with the shortest spacer length (2 methylene groups) and the longest spacer length (8 methylene groups) investigated showed the most efficient DNA compaction ability.

[A new bromine-containing reagent for cysteine modification].

5-Bromo-2[(2-iodoacetyl)amino]benzenesulfonic acid (AIBSA), a reagent for modification of free of cysteine thiol groups in proteins and peptides, was synthesized. Rate constants of its interaction with thiol groups were determined. The presence of a bromine atom allows an easy identification of the AIBSA-labeled peptides in mass spectra due to the characteristic isotope distribution. The compound is stable in solution and under exposure to light.

Double blockade of cell cycle at g(1)-s transition and m phase by 3-iodoacetamido benzoyl ethyl ester, a new type of tubulin ligand.

3-Iodoacetamido benzoyl ethyl ester (3-IAABE) is a new compound synthesized in our laboratory. The primary action of 3-IAABE is to inhibit microtubule assembly by interacting with -SH groups on tubulin. In contrast to other known microtubule disrupters, 3-IAABE caused a double blockade in the cell cycle at G(1)-S transition and in M phase. The blockade was determined by cell cycle analysis and chromosome distribution. Kinase activities of cyclin E and cyclin-dependent kinase 2 responsible for the G(1)-S transition were increased, as were the activities of mitotic cyclin B and cdc2. 3-IAABE treatment also increased p53 expression and dephosphorylated (or activated) retinoblastoma protein. Investigation of the signal transduction pathway showed that 3-IAABE induced bcl-2 phosphorylation, followed by activation of caspase-9, -3, and -6, but not caspase-8. DNA fragmentation factor and poly(ADP-ribose) polymerase, the downstream substrates of caspase-3 and -6, were cleaved after 3 h of exposure to 3-IAABE, followed by DNA fragmentation. Pretreatment of the cells with inhibitors of caspase-9, -3, or -6, respectively, inhibited the cleavage of DNA fragmentation factor and poly(ADP-ribose) polymerase and thus inhibited the onset of apoptosis. 3-IAABE showed antitumor activities in the panel of 60 National Cancer Institute human tumor cell lines with total growth inhibition in the range of 0.22-4.3 micro M for solid tumor lines and 0.025-0.22 micro M for leukemia/lymphoma cell lines. The 3-IAABU total growth inhibition of phytohemagglutinin-stimulated healthy human lymphocytes was 450-fold greater than that of leukemic cells. 3-IAABE significantly inhibited the growth of human hepatocarcinoma (BEL-7402) in nude mice by 72% in tumor volume, more strongly than did vincristine (43 percent inhibition). Besides being a novel lead for the design of new anticancer tubulin ligands, the activity of 3-IAABE in the cell cycle may also help us to understand the molecular pharmacology of microtubule-active drugs.

Effect of carbamidomethylation of cysteine residues G11(104)alpha on the properties of hemoglobin A.

A modified hemoglobin tetramer has been prepared containing carbamidomethylated G11(104)alpha cysteine residues. The molecule is electrophoretically identical to hemoglobin A, at pH 8.6, contains 2 titratable sulfhydryl groups per tetramer, and shows a normal oxygen affinity at half-saturation. However, the cooperative oxygen binding is significantly decreased. As the G11(104)alpha cysteine residues are located at the alpha1beta1 contact point in the hemoglobin tetramer, the results of this study indicate that modification within this portion of the molecule does not interfere with the assembly of subunits to form a tetramer or the resultant p50 but can cause a significant alteration of cooperative oxygen binding. In addition, spin-labels attached to this cysteine residue are not sensitive to changes in conformation which may take place at this contact point during oxygen binding. It is therefore possible that modification of the G11(104)alpha cysteine residue abolishes the contribution of the alpha1beta1 contact point to the cooperative oxygen binding phenomenon.

Identification of reactive vicinal cysteines in Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases.

Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1-pyrenyl)-iodoacetamide. Reagent incorporation was completely prevented by the presence of the respective nucleoside diphosphate plus MnCl2. Under appropriate conditions, 2 mol of reagent per mol of enzyme subunit were incorporated. The fluorescence spectra of the labeled proteins showed the pyrene excimer emission band. The pyrenyl-derivatized enzymes were digested with trypsin after carboxymethylation, and two labeled peptides were isolated for each carboxykinase upon reverse-phase high-performance liquid chromatography. Automated Edman degradation of the labeled peptides indicated that cysteines 364 and 457 (yeast enzyme), and cysteines 288 and 413 (rat enzyme) were labeled with the fluorescence SH-specific reagent. The relative reactivity of these residues was characterized. Labeling experiments utilizing the 5,5'-dithiobis(2-nitrobenzoate)-oxidized enzymes suggested that the reactive SH-groups occupy a vicinal position in the tertiary structure of the proteins, probably in the nucleotide-binding region.

Spin-labeling probe on conformational change at the active sites of creatine kinase during denaturation by guanidine hydrochloride.

The conformational change at the active sites of creatine kinase and its protection by substrates during guanidine denaturation were investigated by monitoring the ESR spectra of the nitroxide radical covalently bound to the reactive thiols of the enzyme. For the enzyme undenatured (pH 9.0) and in the presence of low concentrations of guanidine, i.e. less than 1 M, there are two kinds of enzyme molecule, one of which is bearing a compact structure at the active site and the other is of a looser structure. The content of the latter increases with increasing denaturant concentration. At concentrations of guanidine hydrochloride higher than 1 M, the structure of the enzyme molecule is monomorphic and becomes looser and looser with an increase of guanidine hydrochloride concentration. The existence of a nucleotide substrate complex protects the structure at the active sites of the enzyme from being changed, up to a concentration of denaturant of 0.2 M, while creatine has no protective effect.

Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase.

Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.

Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase.

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 . 10(5) or 6.0 . 10(5) molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4-diisothiocyano-1,2-diphenyl-ethane-2,2'disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.

A novel peptide designed for sensitization of terbium (III) luminescence.

Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.

Selective chemical modification of Cys264 with diiodofluorescein iodacetamide as a tool to study the membrane topology of cytochrome P450scc (CYP11A1).

Cys264 of cytochrome P450scc (CYP11A1) was selectively labelled with diiodofluorescein iodacetamide in solution and in proteoliposomes. The labelling affected the interaction of P450scc with adrenodoxin and significantly inhibited the side-chain cleavage activity of the soluble and membrane-bound hemeprotein in the reconstituted system. In proteoliposomes both the labelled and unlabelled hemeproteins were susceptible to trypsin and split into F1 and F2, two fragments corresponding to the two main domains of P450scc. These results suggest that the hinge connecting the two domains in the region Arg250-Asn257 is exposed to the surface of the membrane and involved in the interaction of P450scc with adrenodoxin.

The excimer fluorescence of N-(1-pyrenyl)iodoacetamide labeled to myosin and its subfragment 1.

Myosin and its subfragment 1 were labeled with the fluorescent probe N-(1-pyrenyl)iodoacetamide. Both of the labeled complexes exhibited the excimer band at 480 nm (pH 8.0, 25 degrees C). SH1 and SH2 are labeled with this probe as judged by Ca2+-ATPase of the labeled complex. Excimers arise both from the interaction of PIAAs in the two different heads within a single myosin molecule and also from the interaction of PIAAs in the same head. ATP affects these excimers depending on the concentration of Ca2+.

Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II.

The excimer fluorescence from two pyrenyl moieties attached to cysteines in human carbonic anhydrase II has been monitored to characterize residual structure retained under strong denaturing conditions. A position in beta-strand 3, N67C, together with the single naturally occurring cysteine 206 in beta-strand 7, were used as attachment sites. The eximer formation by the pyrenyls, requiring proximity of the probes, revealed an unfolding transition at a GuHCl concentration significantly higher than that required to induce unfolding of the molten globule state as monitored by CD. These results indicate that the excimer transition monitors the unfolding of a residual compact structure that spans beta-strands 3-7. This region constitutes the central and the most hydrophobic part of the molecule, emphasizing the importance of hydrophobic interaction in maintaining residual structure under strong unfolding conditions.

Chemical modification of Cys-374 of actin interferes with the formation of the profilactin complex.

Chemical modification of the cysteine residue 374 of actin, both with N-ethylmaleimide and with the fluorescent probe N-(1-pyrenyl)iodoacetamide, is shown to counteract the inhibiting effect of profilin on actin polymerization.

Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin.

The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein (5-IAF). The conjugate is purified by DEAE-cellulose ion exchange chromatography and cycles of polymerization-depolymerization, yielding a relatively homogeneous product with the fluorescein group covalently attached to cystein 373. The fluorescently labeled actin maintains normal polymerizability and activates heavy meromyosin Mg2+ adenosine triphosphatase to the same extent as unlabeled actin. Furthermore, fluoresecent paracrystals are readily detectable in fluroescence microscope upon adding excess Mg2+ or Ni2+ ions. Spectrofluorimetric studies of the bound fluorescein indicate that the peak excitation and emission wavelengths, the shapes of the spectra, and the peak fluorescence intensities are somewhat sensitive to polymerization and heavy meromyosin binding. Possible causes of these spectral changes are analyzed and future applications of this fluorescently labeled actin in vitro as well as in vivo are discussed.

Conformational transitioons of polypeptide chain elongation factor Tu. II. Further studies by electron spin resonance.

The conformational transitions of polypeptide chain elongation factor Tu (EF-Tu) associated with the ligand change from GDP to GTP and also with the displacement of GDP by elongation factor Ts (EF-Ts) have been investigated using the spin-labeling technique. Of the two reactive sulfhydryl groups in EF-Tu, the one essential for interaction with aminoacyl-tRNA was selectively labeled with various kinds of iodoacetamide or maleimide spin-labeling reagents. The electron spin resonance (ESR) spectra of EF-Tu-GDP labeled with these reagents generally consisted of two components, one narrow and one broad, corresponding to labels relatively weakly and strongly immobilized, respectively. The degree of immobilization and the ratio of the narrow to the broad components were different for each kind of label used. The spectra of spin-labeled EF-Tu-GDP changed markedly when its GDP moiety was replaced by GTP through incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], the broad component increasing at the expense of the narrow component. The reversible nature of the conformational change was confirmed with EF-Tu labeled with a maleimide reagent. The GTP-induced spectral change was reversed upon conversion of labeled EF-Tu-GTP to EF-Tu-GDP by addition of excess GDP. A similar type of spectral change was also observed when spin-labeled EF-Tu-GDP was incubated with EF-Ts to form labeled EF-Tu-EF-Ts complex. The extent of the spectral change induced by EF-Ts was even greater than that induced by GTP. These results, together with those obtained by studies with hydrophobic and fluorescent probes (Arai, Arai, Kawakita, & Kaziro (1975) J. Biochem. 77, 1095-1106) indicate that a reversible conformational change is induced in EF-Tu near the sulfhydryl group that is essential for interaction with aminoacyl-tRNA.