Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC.

The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI.

Size matters: DNA binding site kinetics as a function of polyamide size.

Hairpin polyamides (PAs) are remarkable minor groove-binding DNA ligands that demonstrate high affinity and sequence selectivity. Following extensive studies of 6-8 ring hairpin PAs have been more recent descriptions of larger PAs (14 rings or more) and their distinguishing properties and biological activities. However, there are no comparative kinetic studies of PA DNA binding behaviors over a range of PA sizes, making it difficult to understand important structure-activity relationships related to PA size.

Group 14 Metallafluorenes as Sensitive Luminescent Probes of Surfactants in Aqueous Solution.

Sila- and germafluorenes containing alkynyl(aryl) substituents at the 2,7- position are strongly emissive with high quantum yields in organic solvents. Provided they are sufficiently soluble in water, their hydrophobic structures have the potential for many biological and industrial applications in the detection and characterization of lipophilic structures. To that end, the emission behaviors of previously synthesized 2,7- bis[alkynyl(biphenyl)]-9,9-diphenylsilafluorene (1), 2,7- bis[alkynyl(methoxynaphthyl)]-9,9-diphenylgermafluorene (2), 2,7- bis[alkynyl(p-tolyl)]-9,9-diphenylsilafluorene (3), and 2,7- bis[alkynyl(m-fluorophenyl)]-9,9-diphenylsilafluorene (4) were characterized in aqueous solution and in the presence of various surfactants.

DNA binding site kinetics of a large antiviral polyamide.

Polyamides (PAs) are powerful DNA ligands that can bind the minor groove of DNA with high affinity and specificity. While the characterization of PA-DNA behavior has focused principally on hairpin PAs 6-8 rings in size, there is increasing evidence that their behavior does not necessarily reflect the complexities that are emerging from studies of larger hairpin PAs, particularly concerning sequence mismatch tolerance and observed but unaddressed high PA-target site binding stoichiometries. To explore these complexities in more detail, kinetics studies of binding a large anti-HPV hairpin polyamide to an isolated DNA recognition site are described.

DNA binding thermodynamics and site stoichiometry as a function of polyamide size.

The interactions of 6-8 ring hairpin polyamides (PAs) with the minor groove of DNA have been investigated extensively. More recent studies of large antiviral PAs (14-20 rings) active against small DNA tumor viruses lead to questions regarding the extent to which the DNA binding behaviors of the well studied, smaller PAs can be reliably extrapolated to the larger ones. Described here is the first reported study of hairpin PA-DNA binding thermodynamics as a function of PA size (6-20 rings).

Thermodynamics and site stoichiometry of DNA binding by a large antiviral hairpin polyamide.

PA1 (dIm-PyPyβPyPyPy-γ-PyPyβPyPyPyPyβ-Ta) is a large (14-ring) hairpin polyamide that was designed to recognize the DNA sequence 5'-WGW-3', where W is either A or T. As is common among the smaller 6-8-ring hairpin polyamides (PAs), it binds its target recognition sequence with low nM affinity. However, in addition to its large size, it is distinct from these more extensively characterized PAs in its high tolerance for mismatches and antiviral properties.

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from (.

Stereospecific cholinesterase inhibition by O,S-diethylphenylphosphonothioate.

The inhibition kinetics and stereospecificity of the chiral nerve agent derivative O,S-diethylphenylphosphonothioate (DEPP) were examined for two forms of acetylcholinesterase (human and eel) and equine butyrylcholinesterase. Both S- and R-DEPP are poor inhibitors of eel AChE (IC 150μM), consistent with a large, nondiscriminatory binding interaction in the active site of this enzyme. However, S-DEPP is active against human and equine AChE with low μM ICs.

Spectroscopic investigation and direct comparison of the reactivities of iron pyridyl oxidation catalysts.

The growing interest in green chemistry has fueled attention to the development and characterization of effective iron complex oxidation catalysts. A number of iron complexes are known to catalyze the oxidation of organic substrates utilizing peroxides as the oxidant. Their development is complicated by a lack of direct comparison of the reactivities of the iron complexes.

Interactions of two large antiviral polyamides with the long control region of HPV16.

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K.

Synthesis and comparison of the biological activity of monocyclic phosphonate, difluorophosphonate and phosphate analogs of the natural AChE inhibitor cyclophostin.

New monocyclic phosphate, phosphonate and difluorophosphonate analogs of the natural AChE inhibitor cyclophostin were synthesized and their activity toward human AChE examined. Surprisingly, the phosphate, phosphonate, and difluorophosphonate analogs all showed diminished activity when compared with the natural product.

Rat hormone sensitive lipase inhibition by cyclipostins and their analogs.

Cyclipostins are bicyclic lipophilic phosphate natural products. We report here that synthesized individual diastereomers of cyclipostins P and R have nanomolar IC50s toward hormone sensitive lipase (HSL). The less potent diastereomers of these compounds have 10-fold weaker IC50s.

Binding studies of a large antiviral polyamide to a natural HPV sequence.

PA1 is a large hairpin polyamide (dImPyPy-β-PyPyPy-γ-PyPy-β-PyPyPyPy-β-Ta; Py = pyrrole, Im = imidazole, β = beta alanine) that targets the sequence 5'-WWGWWWWWWW-3' (W = A or T) and is effective in eliminating HPV16 in cell culture (Edwards, T. G., Koeller, K.

Structural insights into the interaction between a potent anti-inflammatory protein, viral CC chemokine inhibitor (vCCI), and the human CC chemokine, Eotaxin-1.

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation.

Mapping small DNA ligand hydroxyl radical footprinting and affinity cleavage products for capillary electrophoresis.

The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand-DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage, and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam-Gilbert indexing is examined for a number of duplexes of mixed sequence.

Promoter scanning of the human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine.

Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methylpyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.

Fluorescence assay of polyamide-DNA interactions.

Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route to their development as therapeutics. Presented here is a simple and convenient fluorescence assay for PA-DNA binding.

DNA targeting and cleavage by an engineered metalloprotein dimer.

Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered "metallohomeodomain" protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, 2004).

Metal ion and DNA binding by single-chain PvuII endonuclease: lessons from the linker.

Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J.

The first total synthesis of (±)-cyclophostin and (±)-cyclipostin P: inhibitors of the serine hydrolases acetyl cholinesterase and hormone sensitive lipase.

Cyclophostin, a structurally unique and potent naturally occurring acetyl cholinesterase (AChE) inhibitor, and its unnatural diastereomer were prepared in 6 steps and 15% overall yield from hydroxymethyl butyrolactone. The unnatural diastereomer of cyclophostin was converted into cyclipostin P, a potent naturally occurring hormone sensitive lipase (HSL) inhibitor, using a one pot dealkylation-alkylation process. The inhibition [IC(50)] of human AChE by cyclophostin and its diastereomer are reported, as well as constituent binding (K(I)) and reactivity (k(2)) constants.

One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies.

The mechanistic details of metallonuclease reactions, typically supported by Mg(II), have a long and contentious history. Two-metal ion mechanisms have enjoyed much favor, based largely in the multitude of X-ray crystal structures of these enzymes with more than one metal ion per active site. Most recently, this mechanism has come under challenge.

Nucleophile activation in PD...(D/E)xK metallonucleases: an experimental and computational pK(a) study.

Metallonucleases conduct metal-dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pK(a)s for active site moieties very close to this species, and measurement of the pK(a) of a specific water molecule is difficult to access experimentally.

Synthesis and kinetic analysis of some phosphonate analogs of cyclophostin as inhibitors of human acetylcholinesterase.

Two new monocyclic analogs of the natural AChE inhibitor cyclophostin and two exocyclic enol phosphates were synthesized. The potencies and mechanisms of inhibition of the bicyclic and monocyclic enol phosphonates and the exocyclic enol phosphates toward human AChE are examined. One diastereoisomer of the bicyclic phosphonate exhibits an IC(50) of 3 microM.

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis.

To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation.