Direct STD NMR Identification of β‐Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System

Caraballo, Rémi; Dong, Hai; Ribeiro, João P.; Jiménez‐Barbero, Jesús; Ramström, Olof

doi:10.1002/anie.200903920

Cited by 103 publications

(77 citation statements)

References 71 publications

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“…One-dimensional proton NMR spectra of VG16KRKP at 25 C was carried out on a Bruker Avance III 500 MHz NMR spectrometer (equipped with smart probe), followed by a series of 1D titrations using C. neoformans cell suspensions (OD 600 ¼ 1.0) in same buffer amounting to a final range of~10 7 cells. For live cell STD experiments, a 1 mM VG16KRKP solution was prepared in 10 mM deuterated phosphate buffer of pH 7.2 to rule out the possibility of peptide resonance suppression by water (33,34). Similarly, a live C. neoformans cell suspension of OD 600 ¼ 1.0 was prepared in the same buffer.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

See 1 more Smart Citation

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans

Datta¹,

Yadav

Ghosh³

et al. 2016

Biophysical Journal

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There is a significant need for developing compounds that kill Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis in immunocompromised individuals. Here, we report the mode of action of a designed antifungal peptide, VG16KRKP (VARGWKRKCPLFGKGG) against C. neoformans. It is shown that VG16KRKP kills fungal cells mainly through membrane compromise leading to efflux of ions and cell metabolites. Intracellular localization, inhibition of in vitro transcription, and DNA binding suggest a secondary mode of action for the peptide, hinting at possible intracellular targets. Atomistic structure of the peptide determined by NMR experiments on live C. neoformans cells reveals an amphipathic arrangement stabilized by hydrophobic interactions among A2, W5, and F12, a conventional folding pattern also known to play a major role in peptide-mediated Gram-negative bacterial killing, revealing the importance of this motif. These structural details in the context of live cell provide valuable insights into the design of potent peptides for effective treatment of human and plant fungal infections.

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Section: Nmr Spectroscopymentioning

confidence: 99%

“…7 E). STDD experiments can also help to distinguish between the two conformations (conformations 1 and 2) observed in the presence of live cells (33)(34)(35). A difference of the 1D STD spectra of the peptide VG16KRKP in the presence of live cells ( Fig.…”

Section: Structure Of Vg16krkp When Bound To Live C Neoformans Cellsmentioning

confidence: 99%

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans

Datta¹,

Yadav

Ghosh³

et al. 2016

Biophysical Journal

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“…Conceptually, DLS constitutes a specific application of dynamic covalent chemistry, which has been defined as that branch of organic chemistry studying compounds formed reversibly in thermodynamic equilibrium 6,21 . Therefore in principal all dynamic ligation reactions should be applicable to fragment ligation assays, yielding, for example, imines [22][23][24] , hydrazones 25,26 , hemiacetals 27,28 , hemithioacetals 29 , acetals 30,31 , thioesters 32 or Michael addition products 33 .…”

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confidence: 99%

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Burda

Rademann

2014

Nat Commun

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Sensitive detection of small molecule fragments binding to defined sites of biomacromolecules is still a considerable challenge. Here we demonstrate that protein-binding fragments are able to induce enzymatic reactions on the protein surface via dynamic fragment ligation. Fragments binding to the S1 pocket of serine proteases containing a nitrogen, oxygen or sulphur nucleophile are found to activate electrophilic pre-substrates through a reversible, covalent ligation reaction. The dynamic ligation reaction positions the pre-substrate molecule at the active site of the protein thereby inducing its enzymatic cleavage. Catalytic activation of pre-substrates is confirmed by fluorescence spectroscopy and by high-performance liquid chromatography. The approach is investigated with 3 pre-substrates and 14 protein-binding fragments and the specific activation and the templating effect exerted by the enzyme is quantified for each protease-fragment-pre-substrate combination. The described approach enables the site-specific identification of protein-binding fragments, the functional characterization of enzymatic sites and the quantitative analysis of protein template-assisted ligation reactions.

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“…The nitroaldol reaction is one of the first C-C bond-forming reactions used for complex dynamic systems, and its reversibility has been confirmed under basic conditions. 6,19 Hemithioacetal formation has also been utilized in both aqueous and organic solvents, 9,20,21 leading to the rapid formation of virtual dynamic systems in water, whereas base catalyzed real dynamic systems can be produced in organic solvents. Due to the discrete properties of each of the two reaction types, the equilibration features were first addressed.…”

mentioning

confidence: 99%

Double parallel dynamic resolution through lipase-catalyzed asymmetric transformation

Zhang

Ramström

2013

Chem. Commun.

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Dynamic systems based on double parallel reactions have been generated and resolved in situ by secondary lipase-catalyzed asymmetric transformation, resulting in high chemo-and enantioselectivities.Over the last few decades, along with the powerful impact of supramolecular chemistry, the pursuit to understand the complexity of life has gained increasing momentum within the scientific community. Living systems noticeably depend on intricate networks of molecular reactions and interactions under both thermodynamic and kinetic control, operating in response to various signals and factors. Some of these features can be addressed using the concept of dynamic chemistry. Derived from the feature of reversibility, dynamic chemistry has been established as a useful methodology for generating and studying complex networks on a molecular scale. At the constitutional level, this has led to molecular networks that enable selection and identification of for example new substrates for targets, receptors for ligands, and functional materials.1-3 The concept is based on the generation of dynamic systems, in which all system components mutually undergo reversible-covalent or non-covalent interactions under thermodynamic control. The adaptive nature of these systems makes them responsive to internal or external pressures, and any changes of the important parameters in the system can rearrange the constituent compositions, leading to the amplification of optimal constituents. Among the variety of selection pressures that have been applied to dynamic systems, secondary, kinetically controlled, enzyme-catalyzed transformations have proven especially efficient in constituent selection, due to their high substrate specificities. 4-10 Lipases, which belong to the hydrolase enzyme family, were most often used in the dynamic systems owing to advantages such as showing high catalytic efficiency and good selectivities, and being environmentally friendly. to their generation and control. Yet, this represents an important development since higher order systems would lead to an increased understanding of molecular complexity in general (Fig. 1). This challenge was addressed in the present study, where new dynamic systems were generated based on two different reversible reactions operating in parallel under the same conditions, providing two types of structures for the secondary enzymatic resolution process. Several requirements need to be met by the reactions operating in such systems: (1) compatibility under the same catalytic conditions; (2) similar kinetic properties with comparable substrate distributions; and (3) no dominant side reactions taking place in the system during the time course. In the present case, the nitroaldol reaction together with hemithioacetal formation were deemed able to fulfill all prerequisites, and further evaluated in a more complex parallel resolution process. The nitroaldol reaction is one of the first C-C bond-forming reactions used for complex dynamic systems, and its reversibility has been confirmed under bas...

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Direct STD NMR Identification of β‐Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System

Cited by 103 publications

References 71 publications

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Double parallel dynamic resolution through lipase-catalyzed asymmetric transformation

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