Imbuing Aqueous Solubility to Amphotericin B and Nystatin with a Vitamin

Aqueous solubilities of many drugs in current clinical use are very low, necessitating formulations that often present problems for parenteral administration, including toxicities due to the excipients used. Recognizing that pharmacologically active compounds frequently possess amines, we asked whether pyridoxal phosphate (PLP), an inoccuous, water-soluble vitamin, could be utilized to form prodrug-like complexes via the formation of imine or iminium adducts, and whether the vitamin would impart solubilizing properties to such complexes. Direct spectroscopic and crystallographic data obtained using model primary and secondary amines showed that PLP forms stable imine adducts with primary amines under entirely aqueous conditions and at physiologic pH, while no reaction was observed for secondary amines; the basis of the exceptional stability appears to be a consequence of favorable H-bond interactions of the imine nitrogen with the 5-OH group of PLP. Amphotericin B and nystatin in their native forms display marked aqueous insolubility, and possess lone primary amines. We were able to utilize PLP in achieving excellent solubilization of both these antifungal agents, surpassing aqueous solubilities of 100 mg/mL. In in vitro bioassays, both polyenes in their PLP-adducted form display attenuated antifungal potencies which is attributable to ‘prodrug-like’ complexes. These results point to the utility of excipient-free, entirely aqueous formulations of amphotericin B for parenteral use, and may also be extended to other primary amine-bearing compounds exhibiting poor aqueous solubility

1H, 13C and 15N Backbone Assignment of the EC-1 Domain of Human E-Cadherin

The final publication is available at Springer via http://dx.doi.org/10.1007/s12104-013-9539-6The EC1 domain of E-cadherin has been shown to be important for cadherin-cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell-cell adhesion located at the adherens junction of the biological barriers (i.e., intestinal mucosa and the blood-brain barrier (BBB). Cadherin peptides can modulate cadherin interactions to improve drug delivery through the blood-brain barriers (BBB). However, the mechanism of modulating the E-cadherin interactions by cadherin peptides has not been fully elucidated. To provide a basis for subsequent examination of the structure and peptide-binding properties of the EC1 domain of human E-cadherin using solution NMR spectroscopy, the 1H, 13C and 15N backbone resonance of the uniformly labeled-EC1 were assigned and the secondary structure was determined based on the chemical shift values. These resonance assignments are essential for assessing protein-ligand interactions and are reported here

Natural product (L)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21WAF−1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (−)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (−)-gossypol with the RNA binding pocket of MSI1. We further showed that (−)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (−)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (−)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy

<SUP>1</SUP>H NMR studies on strongly antiferromagnetically coupled dicopper(II) systems

The 1H NMR spectra of three well-characterized &#956; -phenoxo and &#956; -hydroxo spin coupled dicopper(II) complexes 1, 2, and 3 which are strongly antiferromagnetically coupled in the solid state have been studied in solution. The complexes studied were [(Cu2(DAP)2IPA)(OH)(H2O)](ClO4)2&#183;H2O (1) (DAP = 1,3-diaminopropane; IPA = 2-hydroxy-5-methylisophthalaldehyde), [(Cu2(DMDAP)2IPA)(OH)(H2O)](ClO4)2 (2) (DMDAP = N,N-dimethyl-1,3-diaminopropane), and [(Cu2(AEP)2IPA)(OH)(H2O)](ClO4)2 (3) (AEP = 2-(2-aminoethyl)pyridine). All three complexes exhibit relatively sharp hyperfine shifted NMR signals. Signal assignments were based on intensity and T1 values. An analysis of the relaxation data shows that, for these binuclear copper(II) systems, the reorientational correlation time (&#964;c) is dominated probably by a combination of electronic relaxation &#964;s and rotational correlation time (&#964;r) due to an exchange-modulated dipolar mechanism. The temperature dependence of the isotropic shifts has been interpreted in terms of the contact hyperfine interaction constant (A) and exchange coupling constant (-2J). The fitting of these shifts represents a good method for the evaluation of -2J in solution, which is compared to the solid state -2J value obtained by the SQUID method. The results indicate that the structures and magnetic properties of all three complexes (1-3) support a general correlation with the antiferromagnetic coupling constants as evidenced by both solid and solution studies. Our results show that 1H NMR spectroscopy is an excellent tool to probe the solution structures of magnetically coupled binuclear Cu(II) centers in model complexes as well as biological systems. One of these complexes was crystallized from aqueous solution. The crystal and molecular structure of [(Cu2(DMDAP)2IPA)(OH)(H2O)](ClO4)2 (2) has been determined. This crystallizes in the monoclinic system, space group Cc with formula weight = 692.48, a = 12.472(2) &#197;, b = 19.554(2) &#197;, c = 12.185(12) &#197;, &#946; = 107.48 (9)&#176;, Z = 4. The two Cu atoms in this copper(II) complex are bridged by the oxygen atoms of the phenolate and hydroxy groups. The axial position at one Cu atom is occupied by a water molecule, while another Cu has weak interaction with a perchlorate group. The coordination geometries around the two Cu atoms are distorted square pyramidal and square planar

Synthesis, structure, magnetic properties, and <SUP>1</SUP>H NMR studies of a moderately antiferromagnetically coupled binuclear copper(II) complex

A binuclear Cu(II) complex of [(Cu2(HAP)2IPA)(OH)(H2O)](ClO4)2&#183;H2 O (HAP = 3-amino-1-propanol; IPA = 2-hydroxy-5-methylisophthalaldehyde) has been synthesized and characterized by X-ray crystallography, by solid state magnetic susceptibility, and in solution by 1H NMR studies. The binuclear copper(II) complex crystallizes in the orthorhombic system, space group Pbcn, a = 27.9972(9) &#197;, b = 8.8713(3) &#197;, c = 19.5939(6) &#197;, and Z = 8. The two copper(II) atoms in this binuclear Cu(II) complex are bridged by the oxygen atoms of the phenolate and hydroxy groups. The axial position at one Cu atom is occupied by a water molecule while another Cu has weak interaction with a perchlorate group. The coordination geometries around the two Cu atoms are distorted square pyramid and square planar. The solid state magnetic susceptibility measurement reveals a moderate antiferromagnetic exchange interaction between the two Cu atoms with a -2J value of 113 &#177; 9 cm-1. The variable-temperature 1H NMR studies in CD3CN solution show that the observed relatively sharp hyperfine shifted signals follow a Curie behavior. The exchange coupling constant (-2J) obtained in solution by using chemical shift as a function of temperature also reveals a moderate antiferromagnetic exchange interaction between two Cu(II) ions. An analysis of the relaxation data shows that the reorientational correlation time (&#964;c) is dominated probably by a combination of electronic relaxation time &#964;sand rotational correlation time (&#964;r) due to an exchange-modulated dipolar mechanism for this moderately antiferromagnetically coupled binuclear copper(II) system

A novel polymer of a binuclear nickel(II) complex bridged by 1,3-diaminopropane: structure and magnetism

A novel polymer of a binuclear nickel(II) complex of [Ni2L(&#956; -H2N-(CH2)3-NH2)2&#183;(H2O)2]n&#183;(ClO4)n&#183;(H2O)n (where L = [2 + 2] condensation of 2,6-diformyl-4-methylphenol with 1,3-diaminopropane) has been synthesized and characterized. The crystal structure of the compound has been solved. The compound crystallizes in the monoclinic system, space group P21/a, with formula weight = 901.004, a = 13.918(2) &#197;, b = 18.111(3)&#197; , c = 16.079(2) &#197;, &#946; = 103.46(3)&#176;, and Z = 4. The interesting feature of this compound is the bridging behavior of 1,3-diaminopropane in an end-to-end fashion. Nickel atoms are placed in a distorted octahedral environment. The magnetic properties of the compound have been studied by means of susceptibility measurement vs temperature, which reveals a moderately strong intramolecular antiferromagnetic coupling (J) of -30 &#177; 4 cm-1 and a weak intermolecular ferromagnetic coupling (zJ') of 2 &#177; 1 cm-1

Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry.

The lanthanides (Ln3+), or rare earth elements, have proven to be useful tools for biomolecular NMR, X-ray crystallographic, and fluorescence analyses due to their unique 4f orbitals. However, their utility in biological applications has been limited because site-specific incorporation of a chelating element is required to ensure efficient binding of the free Ln3+ ion. Additionally, current Ln3+ chelator syntheses complicate efforts to directly incorporate Ln3+ chelators into proteins as the multi-step processes and a reliance on organic solvents promote protein denaturation and aggregation which are generally incompatible with direct incorporation into the protein of interest. To overcome these limitations, herein we describe a two-step aqueous synthesis of a small molecule lanthanide chelating agent amenable to site-specific incorporation into a protein using copper-free click chemistry with unnatural amino acids. The bioconjugate combines a diethylenetriaminepentaacetic acid (DTPA) chelating moiety with a clickable dibenzylcyclooctyne-amine (DBCO-amine) to facilitate the reaction with an azide containing unnatural amino acid. Incorporating the DBCO-amine avoids the use of the cytotoxic Cu2+ ion as a catalyst. The clickable lanthanide chelator (CLC) reagent reacted readily with p-azidophenylalanine (paF) without the need of a copper catalyst, thereby demonstrating proof-of-concept. Implementation of the orthogonal click chemistry reaction has the added advantage that the chelator can be used directly in a protein labeling reaction, without the need of extensive purification. Given the inherent advantages of Cu2+-free click chemistry, aqueous synthesis, and facile labeling, we believe that the CLC will find abundant use in both structural and biophysical studies of proteins and their complexes

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors

Atypical response regulator ChxR from Chlamydia trachomatis is structurally poised for DNA binding.

ChxR is an atypical two-component signal transduction response regulator (RR) of the OmpR/PhoB subfamily encoded by the obligate intracellular bacterial pathogen Chlamydia trachomatis. Despite structural homology within both receiver and effector domains to prototypical subfamily members, ChxR does not require phosphorylation for dimer formation, DNA binding or transcriptional activation. Thus, we hypothesized that ChxR is in a conformation optimal for DNA binding with limited interdomain interactions. To address this hypothesis, the NMR solution structure of the ChxR effector domain was determined and used in combination with the previously reported ChxR receiver domain structure to generate a full-length dimer model based upon SAXS analysis. Small-angle scattering of ChxR supported a dimer with minimal interdomain interactions and effector domains in a conformation that appears to require only subtle reorientation for optimal major/minor groove DNA interactions. SAXS modeling also supported that the effector domains were in a head-to-tail conformation, consistent with ChxR recognizing tandem DNA repeats. The effector domain structure was leveraged to identify key residues that were critical for maintaining protein - nucleic acid interactions. In combination with prior analysis of the essential location of specific nucleotides for ChxR recognition of DNA, a model of the full-length ChxR dimer bound to its cognate cis-acting element was generated

Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form &alpha;-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects