Threonine Hydroxyl Research Articles

Unraveling of the E-helices and Disruption of 4-Fold Pores Are Associated with Iron Mishandling in a Mutant Ferritin Causing Neurodegeneration

Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 A resolution and was very similar to the wild type between residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.

Role of arginine in chemical cross-linking with N-hydroxysuccinimide esters

In order to clarify whether arginine has a promoting effect on the acylation of hydroxyl groups of serine, threonine, or tyrosine by homobifunctional cross-linking agents in aqueous solution, we carried out systematic experiments with model peptides, comparing relative reaction yields with covalently protected and unprotected arginines by MALDI-MS. The guanidinium group could be demonstrated to contribute to the reactivity of hydroxyl groups toward N-hydroxysuccinimide esters and catalyze the nucleophilic substitution, probably via hydrogen bonds.

Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence 601KMSKS605. Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [32P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.

Quantum Calculations on Potassium Channel Selectivity and Gating

Selectivity: We have extended density functional (DFT) and Hartree Fock calculations (1) on sections of the KcsA K+ channel selectivity filter. HF/3-21G calculations provided geometric optimization, starting from X-ray structures; single point DFT calculations (B3LYP/6-31+G∗∗) were used for energy, charge, potential, and bonding, (techniques chosen to satisfy technical requirements). Calculations on K+ and Na+ at the middle and top of the “cavity” in the channel pore, and at the lowest (S4) position in the selectivity filter, show differences in solvation of the two ions: Na+ depends on more water; K+ in the S4 position has solvation mainly from threonine hydroxyls and backbone carbonyl oxygens. In the cavity Na+ is asymmetrically placed, unlike K+; as seen in geometry and electrostatic potential. Differences in weak covalent bonding to the ions seem responsible. Na+ uses primarily 3s, K+ the more extended 4s orbital, and small contributions from higher orbitals.Gating: We also have an early-stage specific model of the voltage sensing domain of the Kv1.2 channel. A particular salt bridge switch, in response to proton movement initiated upon membrane depolarization, allows both S4 motion, and gating, as separate processes; gating appears similar to that in KcsA, not a response to S4 motion. As in KcsA, protons produce channel opening, and a “nanocrystal” composed of water plus the ion holds the channel closed until the protons place charge near one glutamine from each domain, separating them, destroying the nanocrystal, and allowing the channel to open (2). Calculations are in progress on the model.1) Kariev and Green (2008) J. Phys. Chem B, 112, 1293-1298.2) Kariev, Znamenskiy, and Green (2007) BBA (Biomemb), 1768, 1218-1229.

Curvature Dynamics of α-Synuclein Familial Parkinson's Disease Mutants: Molecular Dynamics Simulations of the Micelle- and Membrane-Bound Forms

α-synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. We have performed a series of all-atom molecular dynamics simulations of wild-type α-synuclein and the three Parkinson's disease familial mutants, A30P, A53T, and E46K, bound to an SDS detergent micelle. Our analysis explains how α-synuclein adapts to, and alters, highly curved membrane surfaces through helical bending. We find that α-synuclein binding induces significant deformation in the micelle, flattening the structure and decreasing its surface area. Similar effects on biological membranes would relieve curvature stress, ameliorating the propensity to fuse, and would perhaps explain α-synuclein's role in stabilizing synaptic vesicles. Consistent with the experimentally determined behavior of A30P, the proline dramatically destabilizes the secondary structure, inducing reversible unfolding up- and down-stream of the substitution. The E46K mutation provides an additional electrostatic interaction between the protein and micelle, offering an explanation for the mutants increased affinity. In the case of the A53T mutant, recent NMR data suggested that an enthalpic interaction might be responsible for a slight rigidification of the helical structure. We show that the equilibrium structure of A53T contains a very tight hydrogen-bond between the threonine's hydroxyl and the backbone carbonyl of Val49. We speculate as to the potential effects of these dynamic and structural changes, on α-synuclein's role in neural signaling. In order to assess the applicability of these results to biological membranes, we have performed simulations of the wild-type and mutants on a DOPS bilayer. These simulations reproduce the major conclusions of the micelle-bound simulations, suggesting that the detergent micelle is an adequate model system.

Antifreeze glycopeptide analogues: microwave-enhanced synthesis and functional studies

Antifreeze glycoproteins enable life at temperatures below the freezing point of physiological solutions. They usually consist of the repetitive tripeptide unit (-Ala-Ala-Thr-) with the disaccharide alpha-D-galactosyl-(1-3)-beta-N-acetyl-D-galactosamine attached to each hydroxyl group of threonine. Monoglycosylated analogues have been synthesized from the corresponding monoglycosylated threonine building block by microwave-assisted solid phase peptide synthesis. This method allows the preparation of analogues containing sequence variations which are not accessible by other synthetic methods. As antifreeze glycoproteins consist of numerous isoforms they are difficult to obtain in pure form from natural sources. The synthetic peptides have been structurally analyzed by CD and NMR spectroscopy in proton exchange experiments revealing a structure as flexible as reported for the native peptides. Microphysical recrystallization tests show an ice structuring influence and ice growth inhibition depending on the concentration, chain length and sequence of the peptides.

Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification that, analogous to phosphorylation, cycles on and off serine and/or threonine hydroxyl groups. Cycling of O-GlcNAc is regulated by the concerted actions of O-GlcNAc transferase and O-GlcNAcase. GlcNAcylation is a nutrient/stress-sensitive modification that regulates proteins involved in a wide array of biological processes, including transcription, signaling, and metabolism. GlcNAcylation is involved in the etiology of glucose toxicity and chronic hyperglycemia-induced insulin resistance, a major hallmark of type 2 diabetes. Several reports demonstrate a strong positive correlation between GlcNAcylation and the development of insulin resistance. However, recent studies suggest that inhibiting GlcNAcylation does not prevent hyperglycemia-induced insulin resistance, suggesting that other mechanisms must also be involved. To date, proteomic analyses have identified more than 600 GlcNAcylated proteins in diverse functional classes. However, O-GlcNAc sites have been mapped on only a small percentage (<15%) of these proteins, most of which were isolated from brain or spinal cord tissue and not from other metabolically relevant tissues. Mapping the sites of GlcNAcylation is not only necessary to elucidate the complex cross-talk between GlcNAcylation and phosphorylation but is also key to the design of site-specific mutational studies and necessary for the generation of site-specific antibodies, both of which will help further decipher O-GlcNAc's functional roles. Recent technical advances in O-GlcNAc site-mapping methods should now finally allow for a much-needed increase in site-specific analyses to address the functional significance of O-GlcNAc in insulin resistance and glucose toxicity as well as other major biological processes.

Quantum Mechanical Calculations on Selectivity in the KcsA Channel: The Role of the Aqueous Cavity

We have carried out quantum calculations on selected residues at the intracellular side of the selectivity filter of the KcsA potassium channel, using the published X-ray coordinates as starting points. The calculations involved primarily the side chains of residues lining the aqueous cavity on the intracellular side of the selectivity filter, in addition to water molecules, plus a K+ or Na+ ion. The results showed unambiguously that Na+ significantly distorts the symmetry of the channel at the entrance to the selectivity filter (at the residue T75), while K+ does so to a much smaller extent. In all, three ion positions have been calculated: the S4 (lowest) position at the bottom of the selectivity filter, the top of the cavity, and the midpoint of the cavity; Na+ is trapped at the cavity top, while K+ is cosolvated by the selectivity filter carbonyl groups plus threonine hydroxyl groups so that it can traverse the filter. Only one water molecule remains in the K+ solvation shell at the upper position in the cavity; this solvation shell also contains four threonine (T75) hydroxyl oxygens and two backbone carbonyls, while Na+ is solvated by five molecules of water and one oxygen from threonine hydroxyls. T75 at the entrance to the selectivity filter has a key role in recognition of the alkali ion, and T74 has secondary importance. The energetic basis for the preferential bonding of potassium by these residues is briefly discussed, based on additional calculations. Taken together, the results suggest that Na+ would have difficulty entering the cavity, and if it did, it would not be able to enter the selectivity filter.

Propargyloxycarbonyl as a protecting group for the side chains of serine, threonine and tyrosine

Propargyloxycarbonyl group is used as a protecting group for the hydroxyl groups of serine, threonine and tyrosine. The propargyloxycarbonyl derivatives of these hydroxy amino acids are stable to acidic and basic reagents commonly employed in peptide synthesis. The deprotection of the O-Poc derivatives using tetrathiomolybdate does not affect commonly used protecting groups such as N-Boc, N-Cbz, N-Fmoc, methyl and benzyl esters. The di-and tripeptides synthesized using O-Poc derivatives of serine, threonine and tyrosine are stable, isolable compounds and give the hydroxy peptides in good yields when treated with tetrathiomolybdate.

O-Fucosylation Is Required for ADAMTS13 Secretion

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.

Pristimerin induces apoptosis by targeting the proteasome in prostate cancer cells

Pristimerin is a natural product derived from the Celastraceae and Hippocrateaceae families that were used as folk medicines for anti inflammation in ancient times. Although it has been shown that pristimerin induces apoptosis in breast cancer cells, the involved mechanism of action is unknown. The purpose of the current study is to investigate the primary target of pristimerin in human cancer cells, using prostate cancer cells as a working model. Nucleophilic susceptibility and in silico docking studies show that C6 of pristimerin is highly susceptible towards a nucleophilic attack by the hydroxyl group of N-terminal threonine of the proteasomal chymotrypsin subunit. Consistently, pristimerin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50 2.2 micromol/L) and human prostate cancer 26S proteasome (IC50 3.0 micromol/L). The accumulation of ubiquitinated proteins and three proteasome target proteins, Bax, p27 and I kappa B-alpha, in androgen receptor (AR)-negative PC-3 prostate cancer cells supports the conclusion that proteasome inhibition by pristimerin is physiologically functional. This observed proteasome inhibition subsequently led to the induction of apoptotic cell death in a dose- and kinetic-dependent manner. Furthermore, in AR-positive, androgen-dependent LNCaP and AR-positive, androgen-independent C4-2B prostate cancer cells, proteasome inhibition by pristimerin results in suppression of AR protein prior to apoptosis. Our data demonstrate, for the first time, that the proteasome is a primary target of pristimerin in prostate cancer cells and inhibition of the proteasomal chymotrypsin-like activity by pristimerin is responsible for its cancer cell death-inducing property.

Observation of OH…N scalar coupling across a hydrogen bond in nocathiacin I

We report here the observation of O-H...N hydrogen-bond (1h)J(N,OH) scalar coupling in a biologically active natural product. The intramolecular hydrogen bond between the threonine hydroxyl (Thr-OH) group and the thiazolyl nitrogen at the second thiazole ring (Thz-2) in nocathiacin I was directly detected by a 1H-15N HMBC NMR experiment. The magnitude of the scalar coupling constant (1h)J(N,OH) was accurately measured to be 1.8 +/- 0.1 Hz by a J-resolved 1H-15N HMBC experiment. By adding the O-H...N distance restraint, the 3D solution structure of nocathiacin I was refined. The structure refinement indicated that the distance between the Thr-3 hydroxyl hydrogen and the Thz-2 nitrogen is <or=2.50 A in all the refined structures, and there are no NOE restraint violations >or= 0.23 A. The presence of an intramolecular hydrogen bond in nocathiacin I is further supported by a number of NMR parameters and additional NMR experiments. This observation provides valuable information for characterizing molecular conformations, and for studying structure-activity relationships.

The Tumor Proteasome Is a Primary Target for the Natural Anticancer Compound Withaferin A Isolated from “Indian Winter Cherry”

Withaferin A (WA) is a steroidal lactone purified from medicinal plant "Indian Winter Cherry" that is widely researched for its variety of properties, including antitumor effects. However, the primary molecular target of WA is unknown. By chemical structure analysis, we hypothesized that Withaferin A might be a natural proteasome inhibitor. Computational modeling studies consistently predict that C1 and C24 of WA are highly susceptible toward a nucleophilic attack by the hydroxyl group of N-terminal threonine of the proteasomal chymotrypsin subunit beta5. Furthermore, WA potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50=4.5 microM) and 26S proteasome in human prostate cancer cultures (at 5-10 microM) and xenografts (4-8 mg/kg/day). Inhibition of prostate tumor cellular proteasome activity in cultures and in vivo by WA results in accumulation of ubiquitinated proteins and three proteasome target proteins (Bax, p27, and IkappaB-alpha) accompanied by androgen receptor protein suppression (in androgen-dependent LNCaP cells) and apoptosis induction. Treatment of WA under conditions of the aromatic ketone reduction, or reduced form of Celastrol, had significantly decreased the proteasome-inhibitory and apoptosis-inducing activities. Treatment of human prostate PC-3 xenografts with WA for 24 days resulted in 70% inhibition of tumor growth in nude mice, associated with 56% inhibition of the tumor tissue proteasomal chymotrypsinlike activity. Our results demonstrate that the tumor proteasome beta5 subunit is the primary target of WA, and inhibition of the proteasomal chymotrypsin-like activity by WA in vivo is responsible for, or contributes to, the antitumor effect of this ancient medicinal compound.

The Role of a Conserved Serine Residue within Hydrogen Bonding Distance of FAD in Redox Properties and the Modulation of Catalysis by Ca2+/Calmodulin of Constitutive Nitric-oxide Synthases

The crystal structure of the neuronal nitric-oxide synthase (nNOS) NADPH/FAD binding domain indicated that Ser-1176 is within hydrogen bonding distance of Asp-1393 and the O4 atom of FAD and is also near the N5 atom of FAD (3.7 A). This serine residue is conserved in most of the ferredoxin-NADP+ reductase family of proteins and is important in electron transfer. In the present study, the homologous serines of both nNOS (Ser-1176) and endothelial nitric-oxide synthase (eNOS) (Ser-942) were mutated to threonine and alanine. Both substitutions yielded proteins that exhibited decreased rates of electron transfer through the flavin domains, in the presence and absence of Ca2+/CaM, as measured by reduction of potassium ferricyanide and cytochrome c. Rapid kinetics measurements of flavin reduction of all the mutants also showed a decrease in the rate of flavin reduction, in the absence and presence of Ca2+/CaM, as compared with the wild type proteins. The serine to alanine substitution caused both nNOS and eNOS to synthesize NO more slowly; however, the threonine mutants gave equal or slightly higher rates of NO production compared with the wild type enzymes. The midpoint redox potential measurements of all the redox centers revealed that wild type and threonine mutants of both nNOS and eNOS are very similar. However, the redox potentials of the FMN/FMNH* couple for alanine substitutions of both nNOS and eNOS are >100 mV higher than those of wild type proteins and are positive. These data presented here suggest that hydrogen bonding of the hydroxyl group of serine or threonine with the isoalloxazine ring of FAD and with the amino acids in its immediate milieu, particularly nNOS Asp-1393, affects the redox potentials of various flavin states, influencing the rate of electron transfer.

Post-transfer editing mechanism of a D-aminoacyl-tRNA deacylase-like domain in threonyl-tRNA synthetase from archaea

To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD). High-resolution crystal structures of Pab-NTD with pre- and post-transfer substrate analogs and with L-serine show mutually nonoverlapping binding sites for the seryl moiety. Although the pre-transfer editing is excluded, the analysis reveals the importance of main chain atoms in proper positioning of the post-transfer substrate for its hydrolysis. A single residue has been shown to play a pivotal role in the inversion of enantioselectivity both in Pab-NTD and DTD. The study identifies an enantioselectivity checkpoint that filters opposite chiral molecules and thus provides a fascinating example of how nature has subtly engineered this domain for the selection of chiral molecules during translation.

Post‐translational modification by O‐GlcNAc: Another way to change protein function

Modification of intracellular proteins by the beta-linkage of the monosaccharide, N-acetylglucosamine to serine or threonine hydroxyls (O-GlcNAc) is abundant and reversible. Although many proteins bear this post-translational covalent modification, the changes in function of the proteins as a result of this modification are only starting to be understood. In this article, we describe how aspects of the flux from the glucose backbone to this modification are modified and how the cellular activity and content of the GC-box binding transcription factor, Sp1, is altered by O-glycosylation. The association of the enzyme that puts on the O-GlcNAc modification with the bi-functional enzyme that removes this modification is discussed relative to the transition between transcriptional repression and activation.

Crystal Structure of Bordetella pertussis BugD Solute Receptor Unveils the Basis of Ligand Binding in a New Family of Periplasmic Binding Proteins

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.

Identification of N10-Substituted Phenoxazines as Potent and Specific Inhibitors of Akt Signaling

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 microM), whereas "inactive" derivatives were > or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.

Strategies for the NMR‐Based Identification and Optimization of Allosteric Protein Kinase Inhibitors

Protein kinases are important drug targets, but kinase inhibitors ought to be selective and specific in order to avoid side effects in the clinic. Kinase inhibitors that do not target the highly conserved ATP-binding site, but that target an allosteric site, are generally expected to be more selective for the target kinase and thus have a better clinical profile. Here we propose an NMR-based strategy to discover and optimize allosteric kinase inhibitors. The approach uses a spin-labeled adenine analogue to detect allosteric kinase ligands by paramagnetic relaxation enhancement. Protein kinases comprise a large family of enzymes that catalyze the transfer of the terminal phosphate from ATP (adenosine triphosphate) to protein substrates, specifically to the hydroxyl group of serine or threonine (Ser/Thr kinases) or tyrosine (Tyr kinases). Protein kinases play a crucial role in signal transduction and thereby regulate central cellular processes such as cell-cycle control, growth control, apoptosis, and transcriptional activation. Kinase activity is generally tightly regulated, but can get out of control with overactive or constitutionally activated kinases. Several pathological states or diseases, such as cancer, can be a consequence of kinase overactivation. Small molecules that can modulate kinase activity in vivo are therefore of high therapeutic interest, and those kinases with a central and specific role in a particular disease are pharmaceutically highly relevant drug targets. 3] A recent example of successful target selection and inhibitor design is the clinical success of Gleevec;, a low-molecular-weight inhibitor of the constitutionally activated tyrosine kinase, Bcr-Abl. Protein kinases generally consist of a catalytic (SH1) domain and one or several regulatory (e.g. SH2 or SH3) domains. The catalytic domains have a conserved three-dimensional fold with a bilobed structure: an N-terminal lobe consisting mainly of b sheets and a C-terminal helical lobe (Figure 1). The catalytic site is located near a hinge region that connects these two domains. Kinases can adopt multiple conformational states that are associated with the degree of catalytic activity : fully active kinases are generally phosphorylated in their activation loop, which adopts a conformation that allows for optimal binding of ATP/Mg and substrate protein, and for efficient transfer of the phosphate group of ATP. There are several regulatory mechanisms by which a kinase becomes down-regulated or “inactive”. The conformational consequence of kinase downregulation can be movement of the activation loop or other components so that the substrate cannot be efficiently bound to the kinase catalytic domain. Besides the ATP-binding site and the substrate binding site, allosteric binding sites occur in kinases, often at sites with regulatory control function. More than 500 kinases are estimated to be encoded in the human genome. All of them bind ATP/Mg , and the ATP-binding site is highly conserved both in amino acid sequence and in three-dimensional structure. Kinase inhibitors that target the ATP site in an active kinase conformation (type I inhibitors) might therefore have a higher risk of clinical liabilities due to lack of selectivity against other kinases. The ATP-binding site changes shape and becomes structurally less conserved when it is in a down-regulated conformation. Kinase inhibitors that target the ATP-binding site in a down-regulated conformation (type II inhibitors) might therefore have better selectivity and specificity, and hopefully a better clinical profile. Glivec/Gleevec is such a type II kinase inhibitor. While not being perfectly selective, it targets the ATP site of Bcr-Abl in its down-regulated conformation. The best selectivity profile might be possible for inhibitors that bind outside the ATP site, at the substrate site or an allosteric binding site. These sites are not generally conserved, and high selectivity against other kinases can hopefully be achieved. Most known kinase inhibitors are type I or II inhibitors. 14] This is probably due to the fact that most kinase inhibitor screens are performed by using biochemical functional assays with purified and activated recombinant kinase. Allosteric kinase inhibitors are not identified by these assay types if they do not inhibit kinase catalysis per se, although they might inhibit kinase activation or signal transduction. Allosteric kinase Figure 1. Principle of the experiment. Spin-labeled adenine analogue 3 is bound to the ATP-binding site. Any ligand that binds simultaneously but at a different binding site feels the paramagnetic relaxation enhancement mediated by 3. The structure of MEK2 in complex with PD334581 and ATP is shown here solely to illustrate the technique.

Point mutation of (+)-germacrene A synthase from Ixeris dentata

Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine. To probe the carbocation-stabilizing role, the amino acid residues of the 433TSA435 triad in (+)-germacrene A synthase from Ixeris dentata were altered by site-directed mutagenesis. Enzyme kinetic measurements of the mutants and GC/MS analysis of the enzyme reaction products indicate that mutations of the triad decreased enzyme catalysis rather than substrate binding but did not affect its structural rearrangement in the catalytic mechanism. This is the first report that the hydroxyl group of threonine at the first position of the triad is required for the cyclase activity.