Solution structures of thiopeptide antibiotics

A detailed NMR study of the thiopeptide amythiamicin D establishes its solution conformation and the presence of a single intramolecular hydrogen bond involving NH13 and O28, and also provides the first evidence for self-association of thiopeptides in solution.     

A proximity-induced covalent fluorescent probe for selective detection of bromodomain 4

Through proximity-induced conjugation reaction, a peptide-based fluorescent probe was designed and synthesized for selective detection of bromodomain 4.     

Differential effects of thiopeptide and orthosomycin antibiotics on translational GTPases

The ribosome is a major target in the bacterial cell for antibiotics. Here, we dissect the effects that the thiopeptide antibiotics thiostrepton (ThS) and micrococcin (MiC) as well as the orthosomycin antibiotic evernimicin (Evn) have on translational GTPases. We demonstrate that, like ThS, MiC is a translocation inhibitor, and that the activation by MiC of the ribosome-dependent GTPase activity of EF-G is dependent on the presence of the ribosomal proteins L7/L12 as well as the G' subdomain of EF-G. In contrast, Evn does not inhibit translocation but is a potent inhibitor of back-translocation as well as IF2-dependent 70S-initiation complex formation. Collectively, these results shed insight not only into fundamental aspects of translation but also into the unappreciated specificities of these classes of translational inhibitors.     

Mapping of the primary mannose binding site of pradimicin A

Pradimicin A (PRM-A) is an actinomycete-derived antibiotic with the lectin-like property of being able to recognize D-mannopyranoside (Man) in the presence of Ca(2+) ion. PRM-A and its derivatives have been attracting a great deal of attention as the only family of natural carbohydrate receptors with nonpeptidic skeleton and, more recently, as conceptually novel drug candidates for human immunodeficiency virus (HIV). Despite its scientific interest and potential therapeutic importance, understanding how PRM-A recognizes Man has been severely limited. Conventional interaction analysis of PRM-A with Man in solution has been frustrated by aggregation of PRM-A and the three-component equilibrium consisting of the [PRM-A(2)/Ca(2+)], [PRM-A(2)/Ca(2+)/Man(2)], [PRM-A(2)/Ca(2+)/Man(4)] complexes, and their mixed oligomers. In this Article, we demonstrate the interaction analysis of PRM-A with methyl α-D-mannopyranoside (Man-OMe) in the solid state, which benefits from aggregate-forming propensity of PRM-A and eliminates the problem associated with the complicated equilibrium in solution. Isothermal titration calorimetry (ITC) analysis and coprecipitation experiments revealed that the primary Man binding of PRM-A is markedly tighter than the secondary one, leading to preparation of the solid aggregate solely composed of the [PRM-A(2)/Ca(2+)/Man-OMe(2)] complex. The simple 1:1 complexes of biosynthetically (13)C-enriched PRM-As and [(13)C(6)]Man-OMe facilitated the analysis of the primary Man binding of PRM-A by two-dimensional dipolar-assisted rotational resonance (2D-DARR), which clearly identified that the cavity consisted of D-alanine moiety and ABC rings of PRM-A is the Man binding site. Interestingly, the proposed Man binding site of PRM-A seems to resemble the typical architecture of artificial carbohydrate receptors.     

Mapping the binding site topology of amyloid protein aggregates using multivalent ligands

A key process in the development of neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases is the aggregation of proteins to produce fibrillary aggregates with a cross β-sheet structure, amyloid. The development of reagents that can bind these aggregates with high affinity and selectivity has potential for early disease diagnosis. By linking two benzothiazole aniline (BTA) head groups with different length polyethylene glycol (PEG) spacers, fluorescent probes that bind amyloid fibrils with low nanomolar affinity have been obtained. Dissociation constants measured for interaction with Aβ, α-synuclein and tau fibrils show that the length of the linker determines binding affinity and selectivity. These compounds were successfully used to image α-synuclein aggregates in vitro and in the post-mortem brain tissue of patients with Parkinson''s disease. The results demonstrate that multivalent ligands offer a powerful approach to obtain high affinity, selective reagents to bind the fibrillary aggregates that form in neurodegenerative disease.

Multivalent ligands offer a powerful approach to obtain high affinity reagents to bind the aggregates that form in neurodegenerative disease. Selectivity for different proteins was achieved by using different linkers to connect the head groups.     

Biosynthesis inspired Diels-Alder route to pyridines: synthesis of the 2,3-dithiazolylpyridine core of the thiopeptide antibiotics

Reaction of serine derived 1-alkoxy-2-azadienes with dehydroalanine derived dienophiles results in Diels-Alder reaction and aromatisation to give 2,3,6-trisubstituted pyridines, thereby establishing the viability of the proposed biosynthetic route to the pyridine ring of the thiopeptide antibiotics originally proposed by Bycroft and Gowland.     

Optimization of protease immobilization by covalent binding using glutaraldehyde

Immobilization of a protease, Flavourzyme, by covalent binding on various carriers was investigated. Lewatit R258-K, activated with glutaraldehyde, was selected among the tested carriers, because of the highest immobilized enzyme activity. The optimization of activation and immobilization conditions was performed to obtain high recovery yield. The activity recovery decreased with increasing carrier loading over an optimal value, indicating the inactivation of enzymes by their reaction with uncoupled aldehyde groups of carriers. The buffer concentrations for carrier activation and enzyme immobilization were optimally selected as 500 and 50 mM, respectively. With increasing enzyme loading, the immobilized enzyme activity increased, but activity recovery decreased. Immobilization with a highly concentrated enzyme solution was advantageous for both the immobilized enzyme activity and activity recovery. Consequently, the optimum enzyme and carrier loadings for the immobilization of Flavourzyme were determined as 1.8 mg enzyme/mL and 0.6 g resin/mL, respectively.     

Synthesis of methyl sulfomycinate, sulfomycinic amide and sulfomycinine, degradation products of the sulfomycin thiopeptide antibiotics

The convergent synthesis of methyl sulfomycinate and sulfomycinic amide, two acidic methanolysis products of the sulfomycin thiopeptide antibiotics, is achieved starting from diethoxyacetonitrile. Further confirmation of structure is obtained by heating methyl sulfomycinate at 110 °C in hydrochloric acid to give (±)-sulfomycinine hydrochloride, the acid hydrolysate of sulfomycin I.     

Immobilization of a recombinant cutinase by entrapment and by covalent binding

Fusarium solani pisi recombinant cutinase, immobilized by entrapment in calcium alginate and by covalent binding on porous silica, was used to catalyze the hydrolysis of tricaprylin. The influence of relevant parameters on the catalytic activity such as pH, temperature, and the substrate concentration were studied. Cutinase immobilized by entrapment presented a Michaelis-Menten kinetics for tricaprylin concentrations up to 200 mM. At higher concentrations of substrate, inhibition was observed. For covalent binding immobilization, diffusional limitations were observed at low substrate concentrations and substrate inhibition occurred for concentrations higher than 150 mM. The stability of immobilized cutinase was also evaluated. The enzyme immobilized by entrapment showed a high stability, in contrast to the immobilization on porous silica.     

Surface modification of proteins by covalent binding of acrylic polymers

Surface modification of enzymes for a potential use in therapy was obtained with a new type of tailor-made copolymers ofNacryloylmorpholine andN-acryloxysuccinimide. The first monomer was designed to confer solubility on the polymer, whereas the second was used to give it reactivity toward protein amino groups. The reactivity of polymers of different composition towards amino acid derivatives and model proteins, such as catalase and ribonuclease-A, is described. Water soluble and catalytically active enzyme derivatives were obained using copolymers prepared with a mixture of N-acryloxysuccinimide andn-acryloylmorpholine in a 1:99 molar ratio. At increasing molar ratio (3:97, 10:90) extensive crosslinking between polymer and enzymes takes place, yielding insoluble adducts.     

Mapping of the binding site on pseudoazurin in the transient 152 kDa complex with nitrite reductase

Nitrite reductase (NiR) catalyzes the reduction of nitrite to nitrite oxide as a part of the denitrification process. In Alcaligenes faecalis S-6, the copper protein pseudoazurin acts as electron donor to NiR. The binding surface of pseudoazurin involved in the formation of the 152 kDa complex with NiR has been determined by NMR using cross saturation from NiR to perdeuterated pseudoazurin. Due to the transient nature of the complex, saturation effects can be observed on the resonances of the unbound protein. The binding site comprises the hydrophobic area surrounding the exposed copper ligand His81, suggesting that this residue is important for efficient electron transfer.     

Total synthesis of the thiopeptide amythiamicin D

The first total synthesis of the thiopeptide antibiotic amythiamicin D is described.     

Investigation on the binding site in heparin by spectrophotometry

Heparin has a variety of biological activities, most of them due to heparin’s high sulfate groups. To gain insight into the mechanism of activation of the spectroscopic probe with sulfate groups of heparin in vitro, we have used a cationic dye by a spectrophotometric method. It is considered that the combination of heparin with methylene blue is due to noncovalent binding forces. Dye binding requires an organic chain structure form with sulfate groups. The solution equilibria of the reaction system are discussed. A new linear regression equation has been proposed, in which the maximum binding number N expresses the binding ability of methylene blue (MB) with sulfate groups of heparin. The linear regression equation can estimate this parameter.     

Elucidating the site of protein-ATP binding by top-down mass spectrometry

A Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for determining the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase is described. Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), but the ability to detect protein-ligand complexes depends on their stability in the gas phase. Previously, we showed that collisionally activated dissociation (CAD) of protein-nucleotide triphosphate complexes yield products from the dissociation of a covalent phosphate bond of the nucleotide with subsequent release of the nucleotide monophosphate (Yin, S. et al., J. Am. Soc. Mass Spectrom. 2008, 19, 1199–1208). The intrinsic stability of electrostatic interactions in the gas phase allows the diphosphate group to remain noncovalently bound to the protein. This feature is exploited to yield positional information on the site of ATP-binding on adenylate kinase. CAD and electron capture dissociation (ECD) of the adenylate kinase-ATP complex generate product ions bearing monoand diphosphate groups from regions previously suggested as the ATP-binding pocket by NMR and crystallographic techniques. Top-down MS may be a viable tool to determine the ATP-binding sites on protein kinases and identify previously unknown protein kinases in a functional proteomics study.     

Direct C-2 arylation of alkyl 4-thiazolecarboxylates: new insights in synthesis of heterocyclic core of thiopeptide antibiotics

The Pd(0)-catalyzed regioselective C-2 (hetero)arylation of tert-butyl 4-thiazolecarboxylate with a broad (hetero)aryl halide is reported, including the direct coupling of pyridinyl halides. The process has allowed the preparation of valuable 2-pyridynyl-4-thiazolecarboxylates which are components of the complex heterocyclic core of thiopeptides antibiotics. As a first application, a synthesis of a tert-butyl sulfomycinamate thio-analogue from tert-butyl 4-thiazolecarboxylate is here described through a three-step direct pyridinylation, halogenation, and Stille cross-coupling sequence.     

Efficient gaseous iodine capture enhanced by charge-induced effect of covalent organic frameworks with dense tertiary-amine nodes

Based on the outstanding application advantages of nitrogen-rich materials with regular porous frameworks in the capture of gaseous radioactive iodine, a series of covalent organic frameworks (COFs) with dual channels and abundant tertiary-amine active sites were constructed herein via a unique multi-nitrogen node design. The high density of up-to-six nitrogen adsorption sites in a single structural unit of the products effectively improved the adsorption capacities of the materials for iodine. Moreover, the adsorption affinity of the active sites can be further regulated by charge-induced effect of different electron-donating groups introduced into the COFs. Adsorption experiments combined with DFT theoretical calculations confirmed that the introduction of electron-donating groups can effectively increase the electron density around the active sites and enhance the binding energy between the materials and iodine, and thus improve the iodine adsorption capacity to 5.54 g/g. The construction strategy of multi-nitrogen node and charge-induced effect proposed in this study provides an important guidance for the study of the structure-activity relationship of functional materials and the design and preparation of high-performance iodine adsorption materials.     

Photosensitized reduction and DNA covalent binding of aziridinylquinones

Photolysis of anaerobic aqueous mixtures (at wavelength maxima above 600 nm and at pH 7.4) containing either aluminum phthalocyanine tetrasulfonate (AlPcS₄), chlorin e6 (CHLORIN), pheophorbide-a (PHEO) or a novel tetracationic phthalocyanine derivative (TETCHLORIN) in the presence of the quinones diaziquone (AZQ), carboquone (CARBOQ) or 2,5-dicloro-diaziridinyl-1,4-benzoquinone (AZDClQ) produces the corresponding semiquinones. Photolysis of these mixtures under the conditions stated above, but in the presence of DNA and at pH 5.5 produces quinone–DNA covalent adducts. Absorption bands seen in irradiated solutions suggest binding of these quinones to DNA through the open aziridine ring. In general, the quinone CARBOQ yielded the largest amounts of adducts photosensitized by the dyes studied here. No quinone–DNA adducts were detected if samples were irradiated at pH 7.4.Thus, both photoreduction of these quinones and an acidic environment are needed for these quinones to bind DNA. These results suggest a potential mode of therapy with special applications to hypoxic regions in solid tumors which are characterized by an acidic environment.     

Photolytic covalent binding of indoleacrylic acid to DNA

E-beta-Indol-3-ylacrylic acid (IA), radiolabelled at the 2 position with 14C, and calf thymus DNA have been irradiated with UV light (lambda greater than 280 nm) in phosphate buffer (pH 7.0). Re-isolation of the DNA indicates covalent binding of IA at levels of up to 295 nmol IA/mg native DNA (0.097 IA/base). Binding is observed for both native and heat denatured DNA, but is more efficient with the latter. Quantum efficiencies of 2.60 X 10(-5) and 2.30 X 10(-4) mol IA bound to native DNA/mol photon absorbed have been measured at 308 and 266 nm, respectively. Studies with the four polyribonucleotides indicate a strong preference for binding to poly[U]. Photolysis of either untreated or enzymatically degraded labeled native DNA with 254 nm light leads to the reformation of IA, and a 2 + 2 photocycloadduct of IA and thymidine has been isolated and characterized and matched by HPLC to a DNA derived adduct. Equilibrium dialysis studies provide no evidence for preassociation of IA to DNA.     

Ligand design by targeting a binding site water

Solvent reorganization is a major driving force of protein–ligand association, but the contribution of binding site waters to ligand affinity is poorly understood. We investigated how altered interactions with a water network can influence ligand binding to a receptor. A series of ligands of the A_2A adenosine receptor, which either interacted with or displaced an ordered binding site water, were studied experimentally and by molecular dynamics simulations. An analog of the endogenous ligand that was unable to hydrogen bond to the ordered water lost affinity and this activity cliff was captured by molecular dynamics simulations. Two compounds designed to displace the ordered water from the binding site were then synthesized and evaluated experimentally, leading to the discovery of an A_2A agonist with nanomolar activity. Calculation of the thermodynamic profiles resulting from introducing substituents that interacted with or displaced the ordered water showed that the gain of binding affinity was enthalpy driven. Detailed analysis of the energetics and binding site hydration networks revealed that the enthalpy change was governed by contributions that are commonly neglected in structure-based drug optimization. In particular, simulations suggested that displacement of water from a binding site to the bulk solvent can lead to large energy contributions. Our findings provide insights into the molecular driving forces of protein–ligand binding and strategies for rational drug design.

Solvent reorganization is a major driving force of protein–ligand association, but the contribution of binding site waters to ligand affinity is poorly understood.