EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells.

Deficiency for the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) leads to chromosomal instability and diseases such as cancer. Yet, defective HR also results in vulnerabilities that can be exploited for targeted therapy. Here, we identify such a vulnerability and show that BRCA1-deficient cells are dependent on the long-range end-resection factor EXO1 for survival. EXO1 loss results in DNA replication-induced lesions decorated by poly(ADP-ribose)-chains. In cells that lack both BRCA1 and EXO1, this is accompanied by unresolved DSBs due to impaired single-strand annealing (SSA), a DSB repair process that requires the activity of both proteins. In contrast, BRCA2-deficient cells have increased SSA, also in the absence of EXO1, and hence are not dependent on EXO1 for survival. In agreement with our mechanistic data, BRCA1-mutated tumours have elevated EXO1 expression and contain more genomic signatures of SSA compared to BRCA1-proficient tumours. Collectively, our data indicate that EXO1 is a promising novel target for treatment of BRCA1-deficient tumours.

Taspase1: a 'misunderstood' protease with translational cancer relevance.

Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a 'non-oncogene addiction' protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1's pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1's structure-function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field.

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle psi previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

Conformational analysis of cyclophostin and designed analogs in comparison with the potent IP3 receptor agonist adenophostin A.

A conformational analysis of 5'-6"-tethered cyclophostin was carried out in comparison with the mother compound, adenophostin A, which has a potent IP3 receptor agonistic activity. The global minimum 3'-endo/anti conformation of cyclophostin elucidated by a molecular dynamics simulation was in accord with NMR spectroscopic data. In contrast, the 2'-endo/syn conformation was dominant with respect to adenophostin A. Despite the constraint introduced by the tether, the spatial arrangement of the three phosphate groups and the adenine moiety, which are essential for the extremely high potency, was changed only moderately in comparison with adenophostin A. The observed high potency of cyclophostin (EC50 = 38 nM) also indicates that it closely resembles the bioactive conformation of adenophostin A (EC50 = 7 nM). These results led us to estimate the probable active conformation of adenophostin A by comparison with the stable conformations of cyclophostin. Finally, two other tethered analogs were designed and are expected to exhibit high potencies comparable to adenophostin A.

Design and synthesis of a multivalent homing device for targeting to murine CD22.

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail. and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and Crohn's disease. In this situation, synthetic high-affinity ligands for CD22 may be of therapeutic value as inhibitors of B-cell activation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAc alpha-2,6-Lac as the basic binding motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic binding motif (cluster effect). In this paper. the stepwise development of a novel multivalent high-affinity ligand for CD22 is described. In the first stage, a series of monovalent NeuAc alpha-2,6-Glc(Y)X type binding motifs was prepared, and their affinity for murine CD22 was monitored, to obtain more insight into the effect of separate structure elements on ligand recognition. In the second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest affinity for CD22, NeuAc alpha-2,6-GlcNBzNO2OMe (7). This cluster, TRIS(NeuAc alpha-2,6-GlcNBzNO2)3 (52), displayed a more than 58-fold higher affinity for CD22 than the reference structure NeuAc alpha-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antagonists for CD22 yet synthesised.

Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg.

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation.

In addition to highly conserved stem-loop structures located in the 5'- and 3'-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941-1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to the cre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3D(pol) in a reaction that is greatly stimulated by 3CD(pro) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359-10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conserved AAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.

Inhibitors of prenylation of Ras and other G-proteins and their application as therapeutics.

Anchoring of small G-proteins to cellular membranes via a covalently bound lipophylic prenyl group is essential for the functioning of these proteins. For example, the farnesylation of Ras by the action of the enzyme protein:farnesyl transferase (PFT) is pivotal for its signalling function in cell growth and differentiation. The development of inhibitors of PFT was triggered by the role of mutated Ras in certain types of cancer and by the observation that non-farnesylated Ras is inactive. Besides the screening of existing compounds for PFT inhibition, rational drug design has also led to new inhibitors. Our research is in the field of atherosclerosis and concerns the development of inhibitors of the growth of vascular smooth muscle cells. The latter process gives rise to reocclusion of the coronary artery (restenosis) after balloon angioplasty. We and others have developed several analogues of the two substrates of PFT, i.e. farnesyl pyrophosphate (FPP) and the so-called CAAX peptide consensus sequence, which were tested in vitro for the inhibition of PFT and of other enzymes involved in protein prenylation, such as protein:geranylgeranyl transferase-1 (PGGT-1). The FPP analogue TR006, a strong inhibitor of PFT (IC(50) of 67 nM), blocked the proliferation of cultured human smooth muscle cells and inhibited platelet-derived growth factor- and basic fibroblast growth factor-induced DNA synthesis. Similar but more highly charged compounds failed in this respect, probably because of an impaired uptake in the cells. Less charged derivatives were designed to circumvent this problem. The effect on the GF-induced activation of intermediates in signal transduction pathways was investigated in order to gain insight into the mechanism of action within the cells. TR006 decreased the bFGF activation of extracellular signal-regulated kinase 1 (ERK1), suggesting its involvement in inhibiting Ras activity. Although other analogues inhibited DNA synthesis, they affected neither ERK1 activation nor p38/stress-activated protein kinase 2 or Jun N-terminal kinase 1 activation. Since some of these compounds were also shown to be inhibitors of in vitro PGGT-1 activity, the geranylgeranylation of other G-proteins may be decreased by these compounds. Rho seems to be a good candidate as a target for inhibitors of PGGT-1. This uncertainty as to the mechanism of action within non-transformed as well as transformed cells applies to all prenylation inhibitors, but is not holding back their further development as drugs. Their current and possible future application as therapeutics in cancer, restenosis, angiogenesis, and osteoporosis is briefly discussed.

Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity.

The device of new hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with specificity for the asialoglycoprotein receptor on parenchymal liver cells is described. PMEA was conjugated to bi- and trivalent cluster glycosides (K(GN)(2) and K(2)(GN)(3), respectively) with nanomolar affinity for the asialoglycoprotein receptor. The liver uptake of the PMEA prodrugs was more than 10-fold higher than that of the parent drug (52+/-6% and 62+/-3% vs. 4.8+/-0.7% of the injected dose for PMEA) and could be attributed for 90% to parenchymal cells. Accumulation of the PMEA prodrugs in extrahepatic tissue (e.g., kidney, skin) was substantially reduced. The ratio of parenchymal liver cell-to-kidney uptake-a measure of the prodrugs therapeutic window-was increased from 0.058 +/- 0.01 for PMEA to 1.86 +/- 0.57 for K(GN)(2)-PMEA and even 2.69 +/- 0.24 for K(2)(GN)(3)-PMEA. Apparently both glycosides have a similar capacity to redirect (antiviral) drugs to the liver. After cellular uptake, both PMEA prodrugs were converted into the parent drug, PMEA, during acidification of the lysosomal milieu (t(1/2) approximately 100 min), and the released PMEA was rapidly translocated into the cytosol. The antiviral activity of the prodrugs in vitro was dramatically enhanced as compared to the parent drug (5- and 52-fold for K(GN)(2)-PMEA and K(2)(GN)(3)-PMEA, respectively). Given the 15-fold enhanced liver uptake of the prodrugs, we anticipate that the potency in vivo will be similarly increased. We conclude that PMEA prodrugs have been developed with greatly improved pharmacokinetics and therapeutic activity against viral infections that implicate the liver parenchyma (e.g., HBV). In addition, the significance of the above prodrug concept also extends to drugs that intervene in other liver disorders such as cholestasis and dyslipidemia.

Spirophostins: conformationally restricted analogues of adenophostin A.

The synthesis, biological evaluation, and molecular modeling of two conformationally restricted analogues of adenophostinA (1), denominated as spirophostin (3R)-10 and (3S)-11, as novel ligands for the D-myo-inositol 1,4,5-trisphosphate receptor (IP3R), is presented. These diastereoisomeric spiroketals are synthesized by spiroketalization of D-glucose derivatives (2S)-15 and (2R)-16, separation of the protected isomers (3R)-19 and (3S)-20, followed by phosphorylation and deprotection. The spirophostins (3R)-10 and (3S)-11 display comparable biological activity, with a 3H-IP3-displacing and Ca2+-releasing potency less than IP3 and adenophostin A.

Downregulation of c-Ki-ras promoter activity by triplex-forming oligonucleotides endogenously generated in human 293 cells.

Exogenous triplex-forming oligodeoxynucleotides (TFO) have the capacity to modulate in vivo the expression of individual genes. As the administration of TFO to cells is not without problems, we analyzed the possibility of generating them directly in the cell, using specific expression vectors. We constructed three vectors, mU6-GA, mU6-CA, and mU6-CT, that direct the synthesis in human 293 cells of 76-mer CU, GU, and AG motif TFO (rTFO) potentially capable of binding to a critical poly (R x Y) sequence contained in the promoter of the Ki-ras proto-oncogene. The ability of the CU, GU, and AG motif rTFO to interact with the double helix of the c-Ki-ras target was investigated in vitro by footprinting and band-shift experiments, using both synthetic and endogenously synthesized oligoribonucleotides. The human 293 cells were transfected with DNA mixtures containing a plasmid, which bears the reporter chloramphenicol acetyltransferase (CAT) gene downstream from the c-Ki-ras promoter (pKRS-413), as well as an rTFO-generating vector (mU6-GA, mU6-CA, or mU6-CT). As control, the cells were transfected with DNA mixtures containing vector mU6-C1 or mU6-C2. These generated transcripts unable to form triple helices with the poly (R x Y) sequence of the c-Ki-ras promoter. Intracellular synthesis of the 76-mer CU, GU, and AG rTFO by mU6-GA, mU6-CA, and mU6-CT was checked by Northern blot hybridization. Through beta-gal and CAT ELISA immunoassays, we found that the 293 cells transfected with either mU6-GA, mU6-CA, or mU6-CT showed a significant inhibition of CAT expression compared with cells transfected with control plasmids mU6-C1 or mU6-C2. The results of five separate transient transfection experiments showed that endogenous GU and AG rTFO, generated by mU6-CA and mU6-CT, produce, respectively, 40% (+/- 4% SE) and 47% (+/- 8% SE) CAT inhibition, whereas CU rTFO, generated by mU6-GA, produces 38% (+/- 7% SE) CAT inhibition. In conclusion, this study suggests that it is possible to downregulate the expression of an individual gene through the use of recombinant vectors encoding the information for the intracellular synthesis of short triplex-forming RNA strands.

Synthesis of potent agonists of the D-myo-inositol 1,4, 5-trisphosphate receptor based on clustered disaccharide polyphosphate analogues of adenophostin A.

Clustered disaccharide analogues of adenophostin A (2), i.e. mono-, di-, and tetravalent derivatives 6-8, respectively, were synthesized and evaluated as novel ligands for the tetrameric D-myo-inositol 1,4, 5-trisphosphate receptor (IP(3)R). The synthesis was accomplished via Sonogashira coupling of propargyl 2-O-acetyl-5-O-benzyl-3-O-(3, 4-di-O-acetyl-2, 6-di-O-benzyl-alpha-D-glucopyranosyl)-beta-D-ribofuranoside (16) with iodobenzene 18, 22, or 25, followed by deacetylation, phosphorylation, and deprotection. The abilities of the target compounds 6-8, as well as ribophostin 4, propylphostin 5, and IP(3) (1), to evoke Ca(2+) release from permeabilized hepatocytes or displacement of [(3)H]IP(3) from its receptor in hepatic membranes were compared. Although the binding affinities of 4-8 were similar, there were modest though significant differences in their potencies in Ca(2+) release assays: tetraphostin 8 > IP(3) approximately diphostin 7 > phenylphostin 6 > ribophostin 4 approximately propylphostin 5.

Studies on the attenuation phenotype of polio vaccines: poliovirus RNA polymerase derived from Sabin type 1 sequence is temperature sensitive in the uridylylation of VPg.

Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in the 5' NTR and coding sequences of the capsid proteins and viral RNA polymerase, 3D(pol). Previous studies have implicated at least two mutations in 3D(pol) of Sabin 1 vaccine strain [PV1(S)], including a Y73H change, as contributing to these phenotypes. We have used an in vitro assay to test the first step in RNA synthesis, the uridylylation of the terminal protein VPg with 3D(pol) isolated from PV1(S). Wt and two mutant 3D(pol) proteins (Y73H, D53N/Y73H) were expressed in Escherichia coli and were purified, and their activities were measured in the synthesis of VPgpU(pU) and of VPg-linked poly(U) at 30 and 39.5 degrees C. Our results show that at 39.5 degrees C the Y73H mutation leads to a defect in the synthesis of VPgpUp(U) and of VPg-poly(U) but not in the elongation of a (dT)(15) primer. The double mutant protein had the same activities as Y73H 3D(pol). Using the yeast two-hybrid assay, we detected a reduced interaction between 3D(pol) molecules carrying either the single or double mutations. Tyrosine-73 maps to the finger domain in the three-dimensional structure of 3D(pol). A model will be presented in which a change of Y73 to H73 may interfere with an interaction between two polymerase molecules that, in turn, may interfere with VPg uridylylation. Alternative explanations, however, cannot be excluded at the present time.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue improves the 3'-exonuclease stability of 2'-5'-oligoadenylate-antisense conjugates.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue at the 3'-terminus of DNA or 2-5A-DNA sequences resulted in a significantly enhanced 3'-exonuclease resistance while the affinity for complementary RNA was only slightly decreased. Furthermore, the binding to and activation of human RNase L by thus modified 2-5A-DNA conjugates was not altered as compared to the parent unmodified 2-5A-DNAs.

The effect of the DNA flanking the lesion on formation of the UvrB-DNA preincision complex. Mechanism for the UvrA-mediated loading of UvrB onto a DNA damaged site.

The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli. To study the formation of this complex, derivatives of a DNA substrate containing a cholesterol adduct were constructed. Introduction of a single strand nick into either the top or the bottom strand at the 3' side of the adduct stabilized the UvrB-DNA complex, most likely by the release of local stress in the DNA. Removal of both DNA strands up to the 3' incision site still allowed formation of the preincision complex. Similar modifications at the 5' side of the damage, however, gave different results. The introduction of a single strand nick at the 5' incision site completely abolished the UvrA-mediated formation of the UvrB-DNA complex. Deletion of both DNA strands up to the 5' incision site also prevented the UvrA-mediated loading of UvrB onto the damaged site, but UvrB by itself could bind very efficiently. This demonstrates that the UvrB protein is capable of recognizing damage without the matchmaker function of the UvrA protein. Our results also indicate that the UvrA-mediated loading of the UvrB protein is an asymmetric process, which starts at the 5' side of the damage.

The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair.

We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequent induction of 3' incision does require ATP binding by UvrB but not hydrolysis. This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5' side of the damage. In contrast, the 5' incision is not dependent on ATP binding because it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5' end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G. F., Monaco, V., van der Marel, G. A., van Boom, J. H., Visse, R., and Goosen, N. (2000) J. Biol. Chem. 275, 8038-8043). To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound. Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed.

Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes.

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P3 induces the activation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P3 receptors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concentration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caused near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2+ release. Similar results were obtained using another Ins(1,4,5)P3-receptor agonist, 2-hydroxyethyl-alpha-d-glucopyranoside 2,3',4'-trisphosphate. Anti-type-1 Ins(1,4,5)P3-receptor monoclonal antibody 18A10 inhibited vasopressin-stimulated Ca2+ inflow but had no observable effect on vasopressin-induced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. However, it did not substantially affect Ca2+ inflow and ER Ca2+ release induced by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release induced by ionomycin or Ins(1,4, 5)P3P4(5)-1-(2-nitrophenyl)ethyl ester ['caged' Ins(1,4,5)P3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)homopiperazine, HCl (ML-9), an inhibitor of myosin light-chain kinase, also inhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow and ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-induced Ins(1,4,5)P3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P3 requires the participation of a small region of the ER, which is distinguished from other regions of the ER by a different apparent affinity for Ins(1,4,5)P3 analogues and is associated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation of SOCs.

2,5-oligoadenylate-peptide nucleic acids (2-5A-PNAs) activate RNase L.

To potentiate the 2-5A (2',5'-oligoadenylate)-antisense and peptide nucleic acid (PNA) approaches to regulation of gene expression, composite molecules were generated containing both 2-5A and PNA moieties. 2-5A-PNA adducts were synthesized using solid-phase techniques. Highly cross-linked polystyrene beads were functionalized with glycine tethered through a p-hydroxymethylbenzoic acid linker and the PNA domain of the chimeric oligonucleotide analogue was added by sequential elongation of the amino terminus with the monomethoxytrityl protected N-(2-aminoethyl)-N-(adenin-1-ylacetyl)glycinate. Transition to the 2-5A domain was accomplished by coupling of the PNA chain to dimethoxytrityl protected N-(2-hydroxyethyl)-N-(adenin-1-ylacetyl)glycinate. Finally, (2-cyanoethyl)-N,N-diisopropyl-4-O-(4,4-dimethoxytrityl)butylphosphor amidite and the corresponding (2-cyanoethyl)-N,N-diisopropylphosphoramidite of 5-O-(4,4'-dimethoxytrityl)-3-O-(tert-butyldimethylsilyl)-N6-benzoyladeno sine were the synthons employed to add the 2 butanediol phosphate linkers and the four 2',5'-linked riboadenylates. The 5'-phosphate moiety was introduced with 2-[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]ethyl-(2-cyanoethyl) -N,N-diisopropylphosphoramidite. Deprotection with methanolic NH3 and tetraethylammonium fluoride afforded the desired products, 2-SA-pnaA4, 2-5A-pnaA8 and 2-5A-pnaA12. When evaluated for their ability to cause the degradation of two different RNA substrates by the 2-5A-dependent RNase L, these new 2-5A-PNA conjugates were found to be potent RNase L activators. The union of 2-5A and PNA presents fresh opportunities to explore the biological and therapeutic implications of these unique approaches to antisense.

Acyclophostin: a ribose-modified analog of adenophostin A with high affinity for inositol 1,4,5-trisphosphate receptors and pH-dependent efficacy.

Adenophostin A is the most potent known agonist of D-myo-inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptors. Equilibrium competition binding studies with 3H-Ins(1,4,5)P3 showed that the interaction of a totally synthetic adenophostin A with both hepatic and cerebellar Ins(1,4,5)P3 receptors was indistinguishable from that of the natural product. At pH 8.3, a synthetic analog of adenophostin A (which we named acyclophostin), in which most elements of the ribose ring have been removed, bound with substantially higher affinity (Kd = 2.76 +/- 0.26 nM) than Ins(1,4,5)P3 (Kd = 7.96 +/- 1.02 nM) to the 3H-Ins(1,4,5)P3-binding sites of hepatic membranes. At pH 7, acyclophostin (EC50 = 209 +/- 12 nM) and Ins(1,4,5)P3 (EC50 = 153 +/- 11 nM) stimulated 45Ca++ release to the same maximal extent and from the same intracellular stores of permeabilized hepatocytes. Comparison of the affinities of a range of Ins(1,4,5)P3 and adenophostin analogs with their abilities to stimulate Ca++ release revealed that although all other agonists had similar EC50/Kd ratios, that for acyclophostin was significantly higher. Similar results were obtained with cerebellar membranes, which express almost entirely type 1 InsP3 receptors. When the radioligand binding and functional assays of hepatocytes were performed under identical conditions, the higher EC50/Kd ratio for acyclophostin was retained at pH 8.3, but it was similar to that for Ins(1,4,5)P3 when the assays were performed at pH 7. To directly assess whether acyclophostin was a partial agonist of hepatic Ins(1,4,5)P3 receptors, the kinetics of 45Ca++ efflux from permeabilized hepatocytes was measured with a temporal resolution of 80 ms using rapid superfusion. At pH 7, the kinetics of 45Ca++ release, including the maximal rate of release, evoked by maximal concentrations of acyclophostin or Ins(1,4,5)P3 were indistinguishable. At pH 8.3, however, the maximal rate of 45Ca++ release evoked by a supramaximal concentration of acyclophostin was only 69 +/- 7% of that evoked by Ins(1,4,5)P3. We conclude that acyclophostin is the highest affinity ribose-modified analog of adenophostin so far synthesized, that at high pH it is a partial agonist of inositol trisphosphate receptors, and that it may provide a structure from which to develop high-affinity antagonists of inositol trisphosphate receptors.