Novel anti-RSV dianionic dendrimer-like compounds: design, synthesis and biological evaluation.

Human Respiratory Syncytial Virus (RSV) is considered to be the leading cause of lower respiratory tract disease in infants and young children. RSV is also a common pathogen in immunocompromised adults and in the elderly. RSV infection can be epidemic and is evident worldwide. Ribavirin, a small molecule agent, and Synagis, a monoclonal neutralizing antibody, are the only approved drugs for treatment and prevention of RSV in high-risk patients. This review is focused on a group of novel and specific inhibitors discovered at Wyeth-Ayerst Research. Some of these inhibitors have IC50 <50 nM and are active against all the tested group A and B viruses. They also have shown good efficacy in cotton rats and primates. Mechanism of action studies indicate that the compounds inhibit the next step in infection after adsorption suggesting that fusion is the target. A strong relationship between the inhibitor structures and their anti-RSV activity was established. This relationship appears to derive from a multivalent interaction between the functional groupings of the inhibitors and the F protein, which seem to be highly complementary and directional.

Crystal structure of the tet repressor in complex with a novel tetracycline, 9-(N,N-dimethylglycylamido)- 6-demethyl-6-deoxy-tetracycline.

The tetracycline analog 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxy-tetracycline (9glyTc) belongs to a new group of tetracyclines called glycylcyclines. They are strong antibiotics showing reduced sensitivity against the major tetracycline resistance mechanisms. We have determined the crystal structure of 9glyTc in complex with Tet repressor class D, TetR(D), at 2.4 A resolution. Sterical hindrance at the entrance of the tetracycline binding tunnel of TetR by the bulky and charged glycyl amido substituent interferes with conformational changes required for the mechanism of induction, and leads to decreased induction efficiency as observed for point mutations of amino acid residues located in the neighbourhood to the glycylamido moiety of bound 9glyTc.

Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme.

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3' pocket merging with the S1' specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNFalpha-induced pathophysiological effects, and might also help to control related shedding processes.

Tetracycline analogs affecting binding to Tn10-Encoded Tet repressor trigger the same mechanism of induction.

We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (atc) on induction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vitro induction assay. The equilibrium association constants of the modified tc to TetR were independently determined to distinguish effects on binding from those on induction. We found a correlation between the binding affinity and induction of TetR for most tc analogs. While a substitution at position 5 revealed only minor effects, changes at position 6 increased binding and induction efficiencies up to 20-fold. A chlorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively. Substituents at position 9 decreased binding up to 5-fold. Epimerization of the dimethylamino function at position 4 in 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-reduced induction. Substitution of this grouping by hydrogen in 4-de(dimethylamino)-tc resulted in no binding and no induction. The respective atc analog failed to induce as well, although binding was still observed. The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction. Substitution of the 2-carboxamido by a nitrilo function did not influence binding and induction efficiencies. Atc showed about 30-fold increased binding and induction, being the most effective inducer tested in this study. The equilibrium association constants of most TetR-[Mg-tc]+ and TetR-([Mg-tc]+)2 analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free TetR. This suggests that these tc analogs share the same molecular mechanism of TetR induction.

Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

Salt dependence of calicheamicin-DNA site-specific interactions.

Calicheamicin gamma 1I site-specifically binds and cleaves three closely spaced tetranucleotide sequences embedded in an AT-rich region of a 142 base pair DNA restriction fragment. Cleavage is observed predominantly at the TCCT, TTGT, and ATCT sequences, of which TCCT is the primary cleavage site. The Gibbs free energies required to bind calicheamicin to these sequences within the DNA restriction fragment have been determined as a function of NaCl concentration at pH 8.1 and 0 degrees C and at pH 7.5 and 23 and 0 degrees C. Between 150 mM and 1 M NaCl, calicheamicin binding to all three sequences is insensitive to salt. The insensitivity of calicheamicin binding to salt continues to 50 mM NaCl for the TTGT and ATCT sequences; the delta G values for calicheamicin binding to these sequences are on the order of -7.8 to -7.9 kcal mol-1 over the entire range of NaCl concentrations studied. However, between 150 and 125 mM NaCl, the TCCT sequence displays a sharp transition in the delta G of calicheamicin binding from -7.6 to -8.9 kcal mol-1. Below 125 mM NaCl, the delta G values for calicheamicin binding to the TCCT sequence again are invariant. An analysis of the data in terms of polyelectrolyte theory suggests that counterion release from DNA does not contribute significantly to the energetics of the association and that the association of calicheamicin with specific DNA sequences is dominated by nonionic rather than electrostatic forces. Our results further suggest that some calicheamicin binding/cleavage sites are dependent on flanking sequences.

Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage.

We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.

Glycylcyclines. 1. A new generation of potent antibacterial agents through modification of 9-aminotetracyclines.

This report describes the discovery of a new generation of tetracycline antibacterial agents, the "glycylcyclines". These agents are notable for their activity against a broad spectrum of tetracycline-susceptible and -resistant Gram-negative and Gram-positive aerobic and anaerobic bacteria possessing various classes of tetracycline-resistant determinants [tet B (efflux), tet M (ribosomal protection)]. The design and synthesis of a number of 7-substituted 9-substituted-amido 6-demethyl-6-deoxytetracyclines are described.

Cleavage behavior of calicheamicin gamma 1 and calicheamicin T.

Calicheamicin gamma 1 is a potent antitumor antibiotic that cleaves DNA with a high degree of specificity; there is much interest in the recognition process. We have investigated the DNA-cleaving properties of calicheamicin T, a truncated derivative of calicheamicin. We show that calicheamicin T cleaves DNA in a double-stranded fashion, indicating that the first two sugars are sufficient to facilitate binding of the aglycone in the minor groove. However, calicheamicin T cleaves DNA nonselectivity. This result suggests that cyclization kinetics do not determine the cleavage specificity of the intact drug. Instead, cleavage specificity probably reflects binding specificity. Because of the recognition sites reported in the original cleavage paper, calicheamicin has been assumed to recognize oligopyrimidine DNA sequences containing G-C base pairs. We show here that calicheamicin also cuts efficiently at A.T tracts, sometimes in preference to G.C-rich homopyrimidine tracts. Crystallographic data and experiments with chemical probes indicate that DNA sequences including G.C base pairs have significantly different local conformations from DNA sequences containing several (four or more) sequential A.T base pairs. This difference makes it unlikely that calicheamicin simply senses inherent groove conformation and suggests that there is some degree of "induced fit." The ability to recognize both A.T- and G.C-rich oligopyrimidine sequences with a high degree of specificity makes calicheamicin an unusual minor-groove binder.

Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome.

Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.

Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor.

We used the Tn10-encoded Tet repressor, which has a highly specific binding capacity for tetracycline, to probe contacts between the drug and protein by chemical interference studies of the antibiotic. For that purpose, the equilibrium association constants of modified tetracyclines with the Tet repressor and Mg2+ cations were determined quantitatively. The results confirm the previous notion that Mg2+ probably binds with the oxygens at positions 11 and 12 and is absolutely required for protein-drug recognition. Modifications were introduced at positions seven, six, five, and four of the drug, and anhydrotetracycline was also studied. Substitutions or eliminations of functions at these positions influenced binding to the Tet repressor up to 35-fold. The introduction of an azido function at position seven in 7-azidotetracycline and epimerization of the substituents at position four in 4-epitetracycline lead to a 2- or 25-fold reduction, respectively, of Tet repressor affinity in those compounds. Anhydrotetracycline bound about 35-fold more strongly than tetracycline did, indicating that the oxygen at position 11 may be involved in Tet repressor recognition. This increased binding is in contrast to the lower antibiotic activity of anhydrotetracycline and indicates that the Tet repressor and ribosomes recognize the drug differently.

Calicheamicin gamma 1I and DNA: molecular recognition process responsible for site-specificity.

Calicheamicin gamma 1I is a recently discovered diyne-ene-containing antitumor antibiotic that cleaves DNA in a double-stranded fashion, a rarity among drugs, at specific sequences. It is proposed that the cutting specificity is due to a combination of the complementarity of the diyne-ene portion of the aglycone with DNA secondary structures and stabilization by association of the thiobenzoate-carbohydrate tail with the minor groove.

Calicheamicin gamma 1I: an antitumor antibiotic that cleaves double-stranded DNA site specifically.

Calicheamicin gamma 1I is a recently discovered diyne-ene--containing antitumor antibiotic with considerable potency against murine tumors. In vitro, this drug interacts with double-helical DNA in the minor groove and causes site-specific double-stranded cleavage. It is proposed that the observed cleavage specificity is a result of a unique fit of the drug and DNA followed by the generation of a nondiffusible 1,4-dehydrobenzene--diradical species that initiates oxidative strand scission by hydrogen abstraction on the deoxyribose ring. The ability of calicheamicin gamma 1I to cause double-strand cuts at very low concentrations may account for its potent antitumor activity.

Chemistry of maduramicin. II. Decarboxylation, abnormal ketalization and dehydration.

The behavior of the free acid and ammonium salt of maduramicin towards heat and alcohols is examined. In refluxing lower alcohols the free acid material is decarboxylated. In addition a bisketal decarboxylated compound as well as an A-ring monoketal decarboxylated derivative are formed. Heating the ammonium salt of the ionophores in suspension in water, or dissolved in inert solvents such as heptane or xylene can cause decarboxylation as well as concomitant dehydration of the F-ring. Reaction of dansyl chloride with the free acid of maduramicin can cause dehydration of the B-ring under very mild conditions.

Chemistry of maduramicin. I. Salt formation and normal ketalization.

The formation of monovalent and divalent salt complexes of maduramicin is described and the 13C NMR chemical shift assignments of these materials are tabulated and discussed. In the spectrum of the thallium salt, 20 of the 47 signals are split due to the thallium-carbon coupling. Similarly the preparation of both the free acid and the sodium salt complexes of the normal methyl and ethyl ketal derivatives of maduramicin are outlined. Their 13C NMR spectra are fully assigned together with discussion of displacements observed due to this derivatization.

Nuclear magnetic resonance studies on the antibiotic avoparcin, Conformational properties in relation to antibacterial activity.

Avoparcin is a commercially important glycopeptide antibiotic which is active against Gram-positive bacteria. Recently, beta-avoparcin, the major component of the avoparcin mixture, and several other analogues have been structurally characterized and tested for their antibacterial activity. Varying degrees of activity were observed for only minor structural differences. For example, beta-avoparcin and epi-beta-avoparcin, which differ only in the stereochemistry at position 1' of the NH2-terminal phenylsarcosine subunit, exhibit a 10- to 100-fold difference in antibacterial activity. Following the supposition that the conformational properties of these molecules may explain the differences in their antibacterial activity, we have analyzed the conformations of beta-avoparcin, epi-beta-avoparcin, and other avoparcin analogues in aqueous solutions using NMR methods. On the basis of an analysis of the 1H chemical shifts, 1H-1H nuclear Overhauser enhancement data, and pH titration experiments, it was concluded that the conformations of all of the analogues are similar at the COOH terminus. However, for beta-avoparcin and epi-beta-avoparcin, conformational differences were observed in the NH2-terminal region of the molecules. In this paper, we present a detailed description of the over-all conformations of these two glycopeptides as deduced from an in-depth analysis of their corresponding 1H NMR spectra and discuss the possible relationships this may have with their markedly different antibacterial activities.