Synthesis of 2′-Deoxyformycin B and 2′-Deoxyoxoformycin B

Abstract

3-β-D-Ribofuranosylpyazolo[4,3-d]pyrimidines (formycins)¹ modified in the heteroaromatic moiety are of biological interest as analogues of adenosine and guanosine, and have been the objects of intensive synthetic chemical effort by several groups.^2-9 2′-Deoxynucleosides^2c,2d,7b,13 and other analogties of the formycins modified in the sugar moiety^10-12 are also of potential interest, but have been less extensively studied. Examples of the 2′-deoxyribonucleoside type known to date include the 2′-deoxy-6-thioguanosine analogue 1, the 2′-deoxyadenosine (dAdo) analogue 2 (2′-deoxyformycin A),^10,13 and the 2-chloro-2′-deoxyadenosine analogue 3.^7b Compound 2 was found to be 10-15 times more potent than 2′-deoxyadenosine as an inhibitor of the growth of S49 cells, a murine lymphoma line of T-cell origin.¹³ Activity depended on 5′- phosphorylation, since mutants lacking the enzymes adenosine kinase (AK) and deoxycytidine kinase (dCK) were insensitive to the drug. Furthermore, activity was comparable in the presence and absence of an AK inhibitor, suggesting that 2, unlike dAdo, may be a poor substrate for adenosine deaminase. That 5′-phosphorylation of 2 was mediated by AK rather than dCK was indicated by the fact that miitants lacking only dCK retained sensitivity. This contrasted with the behavior of dAdo, which is known to be n substrate for both AK and dCK.¹⁴     

Cytogenetic analysis of the 2B3-4 — 2B11 region of the X chromosome of Drosophila melanogaster

Puffing patterns have been studied both in homozygotes t10/t10, a gene located in the area of the early ecdysone puff 2B5, and in a yellow (y) control stock, at the end of the third instar and during prepupal development. In mutants t10 at the end of the third instar puffing develops normally in general, however, 21 puffs (5 early and 16 late ones) underdevelop or do not develop at all, some larval intermoult puffs regressing slower. The next cycle of puffs (mid prepupal) in mutants t10 proceeds normally, but in the late prepupal cycle 21 puffs underdevelop again or are not formed at all. A model for the induction of early ecdysone puffs is proposed, assigning a key role to the 2B5 puff product in stimulating other early puffs. It is suggested that defects in the activity of early puffs in the mutant t10 may cause underdevelopment of late puffs.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Exploration of 2-benzylbenzimidazole scaffold as novel inhibitor of NF-κB

For finding the novel inhibitor of nuclear factor κB activity, a series of benzimidazole derivatives were rationally designed, synthesized and systematically studied for their in vitro activities against LPS induced NF-κB inhibition in RAW 264.7 cells using the SEAP assay based on the flexible chalcone JSH ((E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxy phenyl)prop-2-en-1-one) which was previously reported. Although most of the benzimidazole derivatives showed strong inhibitory activity in low micromolar potency, 2-(4-methoxybenzyl)-1H-benzo[d]imidazole (3m; IC₅₀ = 1.7 μM) and 2-(2-methoxybenzyl)-1H-benzo[d]imidazole (3n; IC₅₀ = 2.4 μM) showed the best inhibition. The structure activity relationship revealed that 2-benzylbenzimidazole scaffold with hydrogen bonding acceptor on phenyl ring appears as a pharmacophore.     

Crystal Structure of the α Subunit of Human Translation Initiation Factor 2B

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the α subunit of human eIF2B at 2.65 Å resolution. The eIF2Bα structure consists of the N-terminal α-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 Å in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 Å in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Bα. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Bα. Interestingly, the yeast eIF2Bα missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Bα missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The β and δ subunits of eIF2B are homologous to eIF2Bα and may have tertiary structures similar to the present eIF2Bα structure. The sulfate-ion-binding residues of eIF2Bα are well conserved in eIF2Bβ/δ. The abovementioned yeast and human missense mutations of eIF2Bβ/δ were also mapped on the eIF2Bα structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.     

Role of ßarrestins in bradykinin B2 receptor-mediated signalling

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5 min) followed by sustained activation for at least 60 min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.     

Structure–function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure–function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Independent Origins of Subgroup Bl+B2 and Subgroup B3Metallo-β-Lactamases

The metallo--lactamases constitute Class B in the Ambler classification of -lactamases and are divided into three subclasses: Bl, B2, and B3. Bayesian phylogenies of the Subclass B1+B2 and Subclass B3 metallo--lactamases and their homologs show that the -lactam-hydrolyzing function evolved independently within each group. In Subclass B1+B2 that function evolved about 1 billion years ago, and in Subclass B3 it evolved before the divergence of the Gram-positive and Gram-negative eubacteria, about 2 billion years ago. These results lend additional support to the proposal that the metallo--lactamases should be divided into two distinct classes.     

The molecular weight of the basic polypetide chain αB2 of α-crystallin

The molecular weights calculated from the amino acid sequences of the A and B chains of the lens protein -crystallin differ only slightly (19830 and 20070, respectively). SDS gel electrophoresis of these chains and comparison with marker proteins yield apparent molecular weights of 19500 for A and 22500 for B. The discrepancy between the value of 22500 and the real molecular weight of 20070 for B vanishes by the combined use of SDS and 6 M urea in the polyacrylamide gels.     

The Bα and Bδ regulatory subunits of PP2A are necessary for assembly of the CaMKIV·PP2A signaling complex

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that is important in synaptic plasticity and T cell maturation. Activation of CaMKIV requires calcium/calmodulin binding and phosphorylation at T200 by CaMK kinase. Our previous work has shown that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV and negatively regulates its activity. Here we demonstrate that PP2A tightly regulates T200 phosphorylation of endogenous CaMKIV, but has little effect on the phosphorylation of the ectopically-expressed kinase. This differential regulation of endogenous versus exogenous CaMKIV is due to differences in their ability to associate with PP2A, as exogenous CaMKIV associates poorly with PP2A in comparison to endogenous CaMKIV. The inability of exogenous CaMKIV to associate with PP2A appears to be due to limiting amounts of endogenous PP2A regulatory B subunits, since coexpression of Bα or Bδ causes the recruitment of PP2Ac to ectopic CaMKIV, leading to formation of a CaMKIV·PP2A complex. Together, these data indicate that the B subunits are essential for the interaction of PP2A with CaMKIV.     

Phosphatases Involved in Modulation of Gap Junctional Intercellular Communication and Dephosphorylation of Connexin43 in Hamster Fibroblasts: 2B or Not 2B?

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.     

Localization of Two Potassium Channel β Subunit Genes, KCNA1B and KCNA2B

The gating properties and current amplitudes of mammalian voltage-activatedShakerpotassium channels are modulated by at least two associated β subunits (Kvβ1.1 and Kvβ1.2). The human Kvβ1.1 gene (KCNA1B) resides on chromosome 3, as indicated by somatic cell hybrid mapping. More precise localization of KCNA1B to 3q26.1 was obtained with fluorescencein situhybridization (FISH) and was corroborated by PCR screening of the CEPH YAC library. The human Kvβ1.2 gene (KCNA2B) resides on chromosome 1, as indicated by somatic cell hybrid mapping, and has been localized by FISH to 1p36.3.