Protein‐templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein‐templated fragment ligations are chemical reactions ...between small molecules (“fragments”) utilizing a protein's surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. The approach exploits the molecular recognition of reactive small‐molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. In this way, chemical synthesis and bioassay are integrated in one single step. This Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein‐templated ligation products. The chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed.
Can drugs be discovered by using proteins as reactors? Proteins have been found to induce reversible and irreversible ligations of protein‐binding fragments. This Review considers the chemistry and the biophysics of such reactions. The potential of template‐catalyzed reactions in drug discovery and as an alternative mode of drug action is discussed.
The flexible variation of peptidomimetics is of great interest for the identification of optimized protein ligands. Here we present a general concept for introducing side-chain modifications into ...peptides using triarylphosphonium amino acids. Building blocks 4a and 4b are activated for amidation and incorporated into stable peptides. The obtained phosphoranylidene peptides undergo Wittig olefinations and 1,3-dipolar cycloaddition reactions, yielding peptidomimetics with vinyl ketones and 5-substituted 1,2,3-triazoles as non-native peptide side chains.
By using fragments endowed with interesting and complementary properties for the treatment of Alzheimer’s disease (AD), a new family of tacrine–4-oxo-4H-chromene hybrids has been designed, ...synthesized, and evaluated biologically. The tacrine fragment was selected for its inhibition of cholinesterases, and the flavonoid scaffold derived from 4-oxo-4H -chromene was chosen for its radical capture and β-secretase 1 (BACE-1) inhibitory activities. At nano- and picomolar concentrations, the new tacrine–4-oxo-4H-chromene hybrids inhibit human acetyl- and butyrylcholinesterase (h-AChE and h-BuChE), being more potent than the parent inhibitor, tacrine. They are also potent inhibitors of human BACE-1, better than the parent flavonoid, apigenin. They show interesting antioxidant properties and could be able to penetrate into the CNS according to the in vitro PAMPA-BBB assay. Among the hybrids investigated, 6-hydroxy-4-oxo- N-{10-(1,2,3,4-tetrahydroacridin-9-yl)aminodecyl}-4 H-chromene-2-carboxamide (19) shows potent combined inhibition of human BACE-1 and ChEs, as well as good antioxidant and CNS-permeable properties.
During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl⁻) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead ...mediates Cl⁻/proton (H⁺) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl⁻ conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7unc/unc mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl⁻/H⁺ exchange can be taken over by a Cl⁻ conductance. This conductance was even deleterious in Clcn7⁺/unc mice. Clcn7⁻/⁻ and Clcn7unc/unc mice accumulated less Cl⁻ in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes.
Sulfated saccharides are an essential part of extracellular matrices, and they are involved in a large number of interactions. Sulfated saccharide matrices in organisms accumulate heavy metal ions in ...addition to other essential metal ions. Accumulation of heavy metal ions alters the function of the organisms and cells, resulting in severe and irreversible damage. The effect of the sulfation pattern of saccharides on heavy metal binding preferences is enigmatic because the accessibility to structurally defined sulfated saccharides is limited and because standard analytical techniques cannot be used to quantify these interactions. We developed a new strategy that combines enzymatic and chemical synthesis with surface chemistry and label‐free electrochemical sensing to study the interactions between well‐defined sulfated saccharides and heavy metal ions. By using these tools we showed that the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences.
Pattern recognition: The effect of sulfation patterns on the metal binding properties of saccharides is enigmatic because the accessibility to structurally defined sulfated saccharides is limited and because of the absence of suitable analytical techniques. Herein, electrochemical methods are used to show that the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences (see scheme).
Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method ...has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.
Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and ...sulfated GAG, at which the sulfation code—which means the number and positions of sulfate groups along the polysaccharide chain—plays an important role.
GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues.
We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG‐like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode.
tert-Butyl thioesters display an astonishing stability toward secondary amines in basic milieu, in contrast to other alkyl and aryl thioesters. Exploiting this enhanced stability, peptide thioesters ...were synthesized in a direct manner, applying a tert-butyl thiol linker for Fmoc-based solid-phase peptide synthesis.
Rapid and efficient preparation of peptide thioacids from 2-cyanoethyl peptide thioesters has been accomplished. S-2-Cyanoethyl peptide thioesters were obtained cleanly without the need for ...purification from resin-bound tert-butyl peptide thioesters using 3-mercaptopropionitrile as a nucleophile. Elimination of the 2-cyanoethyl group proceeded rapidly (t 1/2 < 8 min) under mild conditions and furnished peptide thioacids up to the size of a 16-mer. Peptide thioacids could be isolated or formed in situ and reacted smoothly with electron-deficient azides yielding an amide as the ligation product.
Carbapenem resistance mediated by metallo-β-lactamases (MBL) such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential β-lactam antibiotics. ...Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing bla NDM‑1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.
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