Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.

Binding and retention of polycationic peptides and dendrimers in the vascular wall.

Extracellular matrix (ECM) of tissues, vascular tissue in particular, contains a high concentration of negatively charged glycosaminoglycans (GAGs), which are involved in the regulation of cell motility, cell proliferation and the regulation of enzyme activities. Previously, we have shown that the vascular ECM is capable of binding an extremely high concentration of positively charged molecules, such as polylysine. Vascular ECM can be used therefore as a substrate for binding and retention of drugs delivered intravascularly, if these drugs are endowed with an ability to bind to the vascular ECM. In this study, we evaluated a number of positively charged molecules as potential affinity vehicles for delivery of drugs to the vascular ECM. We labelled the molecules of interest with fluorescence and compared them ex vivo in terms of binding and retention in the de-endothelialised rat carotid artery after intravascular delivery under pressure. High molecular weight polylysine (84 kDa) and polyamidoamine (PAMAM) dendrimers accumulated in the wall of the artery up to a concentration of 10 mg/ml and were not washed away significantly after 4 h of perfusion of the artery. A 24-mer peptide containing a consensus sequence for binding to GAGs (ARRRAARA)(3), 2.7 kDa, was comparable to high molecular weight polylysine and dendrimers in terms of binding and retention. A 14-mer GAG-binding peptide from vitronectin and low molecular weight polylysine, 3 kDa, accumulated in the vascular wall up to about 3 mg/ml and was washed away after 30 min of perfusion. A 10-mer consensus GAG-binding peptide did not bind significantly to the vascular tissue. We conclude that the consensus 24-mer GAG-binding peptide is by far superior to polylysine of a similar molecular weight in terms of binding to vascular tissue, and can provide high accumulation and long-term retention of a low molecular weight compound (fluorescein, as a model molecule) in the vascular wall. Rationally designed GAG-binding peptides can be useful as affinity vehicles for targeting drugs to the vascular ECM.

One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-D-ribose-5-phosphate. A new strategy in the synthesis of stable isotope labeled deoxynucleosides.

The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP). In the first step the sugar-5-phosphate is enzymatically rearranged to alpha-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12,000 units/84 mg protein) and is used without further purification. In the second step thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl(2) to precipitate inorganic phosphate. In this way the overall yield of the beta-anomeric pure nucleoside increases from 14 to 60%. In contrast to uracil, cytosine is not accepted by TP as a substrate. Therefore, 2'-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2'-deoxy-uridine. The method has been demonstrated by the synthesis of [2',5'-(13)C(2)]- and [1',2',5'-(13)C(3)]thymidine as well as [1',2',5'-(13)C(3)]2'-deoxyuridine and [3',4'-(13)C(2)]2'-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-atomic positions. All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%). In combination with the methods that have been developed earlier it is concluded that each of the (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.

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.

Revised interpretation of the immunological results obtained with pneumococcal polysaccharide 17F derived synthetic di-, tri- and tetrasaccharide conjugates in mice and rabbits.

Recently, the structure for pneumococcal polysaccharide (PS) 17F has been revised. Based on the former PS structure, immunogenicities of PS 17F derived synthetic di-, tri- and tetrasaccharide conjugates have been reported in mice. Here, we present additional data on the immunogenicities of these conjugates in rabbits and re-evaluate the immunogenicity results in the light of the revised PS 17F structure.

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.

Stereoselective transformations on D-glucose-derived eight-membered ring carbocycles.

[structure: see text]. The cyclooctenol derivative 1 can be transformed into the nine-membered ring lactone 3, as well as the amino-containing carbocycles 4 and 5. The corresponding ketone 2 gives access to the conformationally locked azasugar 6.

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.

Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding.

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.

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.

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.

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.

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.