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At least 37 records · Page 2Linked to original sources
PubMed2006

Chemically engineered extracts as an alternative source of bioactive natural product-like compounds.

The access to libraries of molecules with interesting biomolecular properties is a limiting step in the drug discovery process. By virtue of a long molecular evolution process, natural products are recognized as biologically validated starting points in structural space for library development. We introduce here a strategy to generate natural product-like libraries. A semisynthetic mixture of compounds was produced by diversification of a natural product extract through the chemical transformation of common chemical functionalities in natural products into chemical functionalities rarely found in nature. The resulting mixture showed antifungal activity against Candida albicans, whereas the starting extract did not show such activity. Bioguided fractionation led to the isolation of a previously undescribed active semisynthetic pyrazole. The result illustrates how biological activity can be generated by designed chemical diversification of a natural product mixture, and represents the proof of principle of an alternative strategy for producing natural product-like libraries from natural products libraries.

Antifungal Agents↗
PubMed1997

Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis.

Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.

Avidin↗
PubMed2000

Chemical engineering of a three-fingered toxin with anti-alpha7 neuronal acetylcholine receptor activity.

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.

Amino Acid Sequence↗
PubMed1991

Chemical engineering of the monoclonal antibody A7 by polyethylene glycol for targeting cancer chemotherapy.

The murine monoclonal antibody A7 (Mab A7) against human colon cancer was chemically modified with methoxypolyethylene glycol (PEG) (Mr 5000). A high substitution of PEG molecules on Mab A7 produced a progressive reduction in antibody-binding activity. The pharmacokinetic and immunological properties of PEG-modified monoclonal antibody A7 (Mab A7) and the PEG-modified F(ab')2 fragment, which retained their antibody-binding activity, were assessed and compared with the parent Mab A7 and the parent F(ab')2 fragment. Blood clearance of PEG-modified antibodies appeared to be diminished by PEG modification and was fitted by a two-compartment model. Low PEG-substituted Mab A7 showed less organ uptake in the liver and spleen and similar uptake in the lung and kidney, compared with the parent Mab A7. PEG-F(ab')2 showed less uptake in the liver and kidney. Both preparations exhibited less tissue:blood ratios in all resected organs as compared with parent antibodies. Tumor localization was enhanced by PEG modification for the F(ab')2 fragment, but not by PEG modification for the whole Mab A7. Multiple i.v. administration of PEG-modified antibody to rabbit did not appear to elicit a measurable immune response to the antibody portion of the conjugate. In conclusion, PEG-modified antibodies are promising reagents as drug carriers to the target tumor.

Animals↗
PubMed2004

Protease-sensitive signalling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein.

The native cysteine residues of green fluorescent protein (GFP) at positions 48 and 70 were replaced by non-thiolic amino acids, and new cysteine sites were introduced at specific, surface positions. Based on molecular modeling of the GFP structure, the sites chosen for mutagenesis to Cys were glutamic acid at position 6 and isoleucine at position 229. These new, unique cysteine sites provided reactive thiol groups suitable for site-specific chemical modification by eosin-based fluorescence labels. The new constructs were designed to serve as the basis of proof of principle for fluorescence resonance energy transfer (FRET) using an enzyme-activated (trypsin) intervening sequence between native and chemically conjugated fluorophores. These eosin moieties provided chemical FRET partners for the native GFP chromophore. On excitation, these GFP-eosin constructs exhibited strong intramolecular FRET, with quenching of the native GFP (511 nm) fluorophore emission and emission around 540 nm, corresponding to eosin. GFP mutants engineered with trypsin-sensitive sequences close to the eosin site, so that on trypsinolysis FRET was destroyed, the emission wavelength switching from that of the chemical FRET partner back to that of the native GFP fluorophore, providing efficient, ratio-based detection. This protein engineering provides the basis for novel bioprobes for enzymatic triggering using intramolecular FRET between GFP and carefully sited chemical labels.

Binding Sites↗
PubMed2005

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein.

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.

Amino Acid Sequence↗
PubMed2002

Modelling counter-current chromatography: a chemical engineering perspective.

In conventional chromatography, a solute is usually viewed to be longitudinally transported only in the mobile phase, remaining longitudinally motionless in the stationary phase. In counter-current chromatography, both phases undergo intense mixing in the variable force field of a coil planet centrifuge and longitudinal dispersion of matter in the stationary phase is not to be excluded. To take into account longitudinal mixing in both phases, a cell model of chromatographic process is proposed in which the number of perfectly mixed cells n is determined by the rates of mixing in stationary (Ds) and mobile (Dm) phases by the equation n = LF/(2ADc)/(1 + Sf(lambda - 1)) with A = K(D)D(S)/Dm (F, L, Ac and KD are the mobile phase flow-rate, column length, column cross-section and distribution ratio, respectively). This equation has been derived by comparing the discontinuous cell model with continuous diffusion assuming equilibrium conditions. Parameter determination and their relationships are discussed.

Countercurrent Distribution↗
PubMed2001

Chemical engineering of cell penetrating antibodies.

Antibodies, being exquisitely specific tools in biology, are routinely used to detect and identify intra-cellular structures. However, current intra-cellular application of antibodies requires that the membrane be rendered leaky, resulting in the death of cells. Here, we present a novel method to allow antibodies to penetrate the cellular membrane of living cells without affecting cell viability. A peptide (MTS, membrane transport sequence) that facilitates transport across membranes has been site-specifically attached to antibodies. MTS-antibodies enter the living cells in culture and can be detected by immunofluorescence and ELISA after extraction. Cellular structures are visualized in living cells using a specific MTS-antibody. Antibodies with membrane penetrating properties can become an important tool for the study of intra-cellular processes in living cells. Furthermore, such membrane penetrating antibodies can be used to selectively stimulate or suppress functions of the cellular machinery.

3T3 Cells↗