Nonhydrolyzable Derivatives Research Articles

Nonhydrolyzable d‑phenylalanine-benzoxazole derivatives retain antitubercular activity

The emergence of drug resistant Mycobacterium tuberculosis, the causative agent of tuberculosis, demands the development of new drugs and new drug targets. We have recently reported that the d-phenylalanine benzoxazole Q112 has potent antibacterial activity against this pathogen with a distinct mechanism of action from other antimycobacterial agents. Q112 and previously reported derivatives were unstable in plasma and no free compound could be observed. Here we expand the structure-activity relationship for antimycobacterial activity and find nonhydrolyzable derivatives with decreased plasma binding. We also show that there is no correlation between antibacterial activity and inhibition of PanG, a putative target for these compounds.

Visualization of Endocannabinoids in the Cell.

A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular membrane compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cells. Herein, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.

Trifluoroacetyl Lysine as a Bromodomain Binding Mimic of Lysine Acetylation.

Genetic code expansion has proven invaluable to the elucidation of functions of defined protein modifications through the site-specific incorporation of noncanonical amino acids. The use of nonhydrolyzable derivatives of post-translational modifications can greatly increase site stoichiometry and half-life. Investigating acetyllysine reader domain (bromodomain) interactions with acetylated nonhistone proteins is challenging due to the limited tools available and dynamic nature of this post-translational modification. Here, we demonstrate that bromodomains bind acetyllysine peptides and those substituted with an acetyllysine derivative, trifluoroacetyllysine, with similar affinity and selectivity. Importantly, both trifluoroacetyllysine and acetyllysine can be site-specifically incorporated into proteins expressed in bacterial and mammalian cells, and the strong electron-withdrawing trifluoro substituent makes the latter resistant to deacetylation by sirtuins (SIRTs). The controlled expression of SIRT-resistant, site-specifically acetylated transcription factors expands the set of available tools for determining the function of acetylation, and it serves as a template for investigating bromodomain interactions with acetylated transcription factors.

Visualization of Endocannabinoids in the Cell.

A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic compound anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cell. Here, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.

Design and NMR studies of cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH.

We report on the design and evaluation of novel cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH from Yersinia. Cyclic peptides have been designed based on a short sequence from the protein SKAP-HOM [DE(pY)DDPF (pY=phosphotyrosine)], and they all contain the motif DEZXDPfK (where Z is a phosphotyrosine or a non-hydrolyzable phosphotyrosine mimetic, X is an aspartic acid or a leucine and f is a d-phenylalanine). These peptides present a 'head to tail' architecture, enabling cyclization through formation of an amide bond in between the side chains of the first aspartic acid and the lysine residues. Chemical shift perturbation studies have been carried out to monitor the binding of these peptides to the N-terminal domain of YopH. Peptides containing a phosphotyrosine moiety exhibit binding affinities in the low micromolar range; substitution of the phosphotyrosine with one of its non-hydrolyzable derivatives dramatically reduces the binding affinities. These preliminary studies may pave the way for the discovery of more potent and selective peptide-based ligands of the YopH N-terminal domain which could be further investigated for their ability to inhibit Yersiniae infections.

Characterization of the DNA Binding Activity of the ZFY Zinc Finger Domain

The ZFY protein is a member of one of the most interesting classes of polydactyl zinc finger proteins. It has a domain of 13 tandem zinc fingers that is organized with an internal repeat of odd-even finger pairs. It has been proposed that each finger pair interacts with six base pairs within a turn of the double helix, the downstream linker crossing the minor groove to place the next finger pair on the following turn of the DNA. Yet putative binding sites for the full-length ZFY protein appear to consist of a six-base AGGCCY consensus sequence that is present in one or two copies. In this study the equilibrium binding of two ZFY-derived zinc finger peptides to 4R DNA with tandem copies of the consensus sequence was investigated. The ZFY5 peptide contains fingers 5-13, including four odd-even finger pairs, and the ZFY11 peptide contains fingers 11-13 and has one odd-even finger pair. Both peptides bound to 4R DNA with equal affinities, forming a bimolecular complex that is mediated by the downstream AGGCCY motif. The additional odd-even finger pairs in ZFY5 made no measurable difference in the mechanism of DNA binding compared to ZFY11. The effects on the DNA-protein interaction of mutations in the 4R DNA and in the key alpha-helical residues of fingers 11-13 indicate that the binding of ZFY to DNA is mediated by the interaction of the GGCC core base pairs with fingers 12 and 13. These results demonstrate that the even-odd repeats in the ZFY zinc finger domain do not make significant contributions to DNA binding.

Orally active α-tocopheryloxyacetic acid suppresses tumor growth and multiplicity of spontaneous murine breast cancer

We recently demonstrated the antitumor efficacy of orally administered alpha-tocopheryloxyacetic acid (alpha-TEA), a redox silent and nonhydrolyzable derivative of naturally occurring vitamin E. In order to move alpha-TEA closer to the clinic to benefit patients with breast cancer, the present study had two goals. First, to determine the minimal effective treatment dose; and second, to test the efficacy of dietary administration of alpha-TEA in the clinically relevant MMTV-PyMT mouse model of spontaneous breast cancer that more closely resembles human disease. The minimal effective dose of alpha-TEA was evaluated in the transplantable 4T1 tumor model and we show a dose-dependent decrease of primary tumor growth and reduction of metastatic spread to the lung. Six-week-old MMTV-PyMT mice were treated with oral alpha-TEA for 9 weeks, with no apparent signs of drug toxicity. The alpha-TEA treatment delayed tumor development and significantly slowed tumor progression, resulting in a 6-fold reduction of the average cumulative tumor size. In addition, oral alpha-TEA caused an 80% reduction in spontaneous metastases. In situ analysis of tumor tissue identified apoptosis as an important mechanism of alpha-TEA-mediated tumor suppression in addition to inhibition of tumor cell proliferation. This study shows, for the first time, the ability of orally administered alpha-TEA to delay tumor onset and to inhibit the progression and metastatic spread of a clinically relevant model of spontaneous breast cancer. Our finding of the high efficacy in this tumor model highlights the translational potential of oral alpha-TEA therapy.

N-Retinylidene-phosphatidylethanolamine Is the Preferred Retinoid Substrate for the Photoreceptor-specific ABC Transporter ABCA4 (ABCR)

ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.

Molecular Cloning, Expression, and Functional Characterization of a Novel Member of the CD38 Family of ADP-ribosyl Cyclases

We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (Mw ∼39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD+) surrogate, NGD+, to its fluorescent nonhydrolyzable derivative, cGDPr.

Two inward K + channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K+ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K+ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K+ channel and a further K+ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K+, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K+ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K+ (EK) was voltage-independent. The channel closed at potentials more positive than EK. A third, hyperpolarization-activated K+ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K+. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K+ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K+ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels.

Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide beta,gamma hydrolysis nor protein phosphorylation.

With some bacterial RNA polymerases and in eukaryotic RNA polymerase II, DNA melting during initiation requires the coupling of energy derived from beta,gamma hydrolysis of ATP. A detailed analysis of this possible requirement for eukaryotic RNA polymerase I reveals no such requirement. However, in some cases, beta,gamma non-hydrolyzable derivatives (beta,gamma imido or methylene) of nucleotide substrates have been found to significantly inhibit transcription initiation because of their inefficient use as the first nucleotide of the transcript. In addition, the results presented here show that protein kinase activity is not required as an integral part of transcription initiation by RNA polymerase I. Prior phosphorylation of proteins participating in the process is not ruled out.

DNA binding of histone H1 is modulated by nucleotides

Histone H1 acts as a general repressor of transcription in eukaryotes by organizing nucleosomes into inaccessible condensed forms of chromatin. The capability of H1 to bind to DNA with some sequence specificity is likely to be critical in the control of these processes. We show here that ATP and several other nucleotides, including non-hydrolyzable derivatives, can inhibit DNA binding of H1. The results also show that ATP differentially affects binding of H1 to DNA in a fashion enhancing nucleotide sequence specificity of the binding. The study suggests a novel mechanism or modulation of H1 activity that has important implications for the role of H1 as a transcriptional regulator.

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.

The gating of nucleotide-sensitive K+ channels in insulin-secreting cells can be modulated by changes in the ratio ATP4-/ADP3- and by nonhydrolyzable derivatives of both ATP and ADP.

The 31P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K+ channels. Adding ATP to the membrane inside closes these channels. However, in the continued presence of ATP adding ADP invariably leads to the reactivation of ATP-inhibited K+ channels, even at ATP4-/ADP3- concentration ratios greater than 7:1. Interactions between ATP4- and ADP3- seem competitive. An increase in the concentration ratio ATP4-/ADP3- consistently evoked a decrease in the open-state probability of K+ channels; conversely, a decrease in ATP4-/ADP3- increased the frequency of K+ channel opening events. Channel gating was also influenced by changes in the absolute concentrations of ATP4- and ADP3-, at constant free concentration ratios. ADP-evoked stimulation of ATP-inhibited channels did not result from phosphorylation of the channel, as ADP-beta-S, a nonhydrolyzable analog of ADP, not only stimulated but enhanced ADP-induced activation of K+ channels, in the presence of ATP. Similarly, ADP was able to activate K+ channels in the presence of two nonhydrolyzable derivatives of ATP, AMP-PNP and beta gamma methylene ATP.

Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells.

Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides.

The binding characteristics of the oligopeptide chemoattractant receptor on guinea pig macrophages and macrophage membrane preparations were characterized using detailed binding studies and computer analysis. Viable macrophages bound the radiolabeled chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine with single dissociation constant (KD) of 18.4 +/- 4.6 nM with 15,300 +/- 1,800 sites per cell. Binding data from membrane preparations indicated the presence of two classes of binding sites with KD of 1.5 +/- 0.4 nM and 25.5 +/- 11.0 nM. Approximately 23% of the receptors were in the high affinity state. In the presence of added guanine nucleotide di- or triphosphates, the high affinity receptors in the membrane preparations were converted to low affinity states with no change in the total receptor number. Nonhydrolyzable derivatives of GTP were most potent in converting the receptor from its high to low affinity state. These data suggest that the affinity state of the oligopeptide chemoattractant receptor in macrophages is regulated by guanine nucleotides and GTPase, implying that the transduction mechanisms of this receptor may be controlled by a guanine nucleotide regulatory unit.

Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes.

The oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte (PMN) membranes exists in two affinity states. Since guanine nucleotides regulate the binding affinity and transductional activity of several other types of receptors, we examined the effect of nucleotides on the binding of N-formyl-methionyl peptides to their receptors on human PMN membranes. The addition of guanylylimidodiphosphate (0.1 mM), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMN membrane preparations reduced the fraction of high-affinity receptors detected in equilibrium binding studies from 21.3 +/- 0.13 to 11.8 +/- 0.05% (P less than 0.03), without altering the binding affinities. Since the total number of receptors remained unchanged, the effect of guanylylimidodiphosphate was to convert a portion of the receptors from the high-affinity state to the low-affinity state. At the maximal concentration of guanine nucleotide tested, approximately 50% of the high-affinity sites were converted to low-affinity sites. The findings obtained by equilibrium binding were supported by kinetic studies since the dissociation of the radiolabeled oligopeptide chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine from PMN membranes was accelerated in the presence of guanine nucleotide. The effect of guanine nucleotides was reversed upon washing, indicating that affinity conversion is bidirectional. The guanine nucleotide effects were greatest with nonhydrolyzable derivatives of GTP followed by GTP then guanosine diphosphate. Neither guanosine monophosphate nor any adenine nucleotide tested had an effect on receptor binding. These data suggest a role for guanine nucleotides in the regulation of stimulus-receptor coupling of chemoattractant receptors on human PMN.

Factors affecting Sertoli cell function in the testis.

The Sertoli cell is the primary target for FSH action in the mammalian testis. These cells contain the majority of testicular plasma membrane receptors for this hormone. Receptor occupancy is directly correlated with a stimulation of adenylyl cyclase and a decrease in the activity of a cytoplasmic Ca++-sensitive cAMP phosphodiesterase. Regulation of these two enzymes allows increased intracellular accumulation of cAMP, activation of cAMP-dependent protein kinase and phosphorylation of a variety of protein substrates. All of these events occur within the first 30 min following exposure of isolated Sertoli cells to FSH. RNA and protein synthesis are also enhanced by FSH. Previous studies have suggested that this gonadotropin may augment the overall cellular synthesis of proteins. Our results reveal that protein kinase inhibitor (PKI) is selectively elevated by FSH both in vivo and in vitro. PKI thus becomes the initial intracellular protein whose synthesis is under FSH control. In addition to effects on protein synthesis, FSH also positively modulates the secretion of several specific proteins. One of the proteins in this latter category is androgen binding protein (ABP). Again, regulation can be observed both in vivo and in vitro. Elevated synthesis of PKI occurs prior to demonstrable secretion of ABP. Both of these events occur subsequent to the effects of FSH on cAMP metabolism. Indeed cAMP (or any of several nonhydrolyzable derivatives) can substitute for FSH in vitro. The temporal sequence of events subsequent to hormone binding and cAMP production are identical, but occur more rapidly. Together these data support the hypothesis that most of the biochemical steps leading to the synthesis and secretion of proteins by FSH are regulated by elevated levels of cAMP.