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Erkang Fan

Publications and source records attributed to Erkang Fan.

17 recordsLinked to original sources
PubMed2006

Using fragment cocktail crystallography to assist inhibitor design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase.

The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.

Amino Acid Sequence↗
PubMed2005

Solid-phase and solution-phase syntheses of oligomeric guanidines bearing peptide side chains.

[reaction: see text] Synthetic strategies for preparing N,N'-bridged oligomeric guanidines bearing peptide side chains both on solid support and in solution are presented. Monomers are prepared from common alpha-amino acids and therefore contain conventionally protected peptide side chains. The side chains include alkyl, aromatic, hydroxyl, amino, carboxylic acid, and amide functional groups. Oligomer elongation utilizes acid-sensitive sulfonyl activated thiourea through the formation of carbodiimide intermediate. With proper preparation of monomers, synthesis of oligomer can be performed in two directions (equivalent to N to C terminal or C to N terminal in a peptide sequence) with excellent efficiency.

Chromatography, Liquid↗
PubMed2005

Protein heterodimerization through ligand-bridged multivalent pre-organization: enhancing ligand binding toward both protein targets.

Structure-based design of a bifunctional ligand for two protein pentamers, cholera toxin B pentamer (CTB) and human serum amyloid P component (SAP), leads to multivalent dimerization of CTB and SAP in solution. This multivalent heterodimerization of proteins significantly enhances the affinity of the bifunctional ligand toward both target proteins.

Cholera Toxin↗
PubMed2004

Large cyclic peptides as cores of multivalent ligands: application to inhibitors of receptor binding by cholera toxin.

Large cyclic decapeptides (up to 50-atom ring) were synthesized efficiently on the solid phase with allyl-ester protection of the carboxyl terminus during elongation. Pentavalent ligands, in a "core-linker-finger" modular setup, were assembled by using these cyclic peptide cores to demonstrate large affinity gains for inhibition of surface receptor binding by the cholera toxin B pentamer. The results suggest that the peptide cores retain expanded conformation in solution so that shorter flexible linkers are needed for larger peptide cores to achieve the best inhibitory results.

Amino Acid Sequence↗
PubMed2004

Automated affinity chromatography measurements of compound mixtures using a lab-on-valve apparatus coupled to electrospray ionization mass spectrometry.

We report a fully automated affinity chromatography system using a lab-on-valve (LOV) apparatus coupled to an electrospray ionization ion-trap mass spectrometer (ESI-MS). The system allows simultaneous measurements of multiple ligand affinities to proteins immobilized on beads. Bead regeneration, column repacking, and repetitive measurements are achieved on the time scale of several minutes. In this study, the system was used to screen the binding of a peptide mixture to human and Trypanosoma brucei (T. brucei) truncated Pex5 (tPex5) proteins. Equilibrium dissociation constants (K(d)) were measured for T. brucei tPex5 and compared to the values obtained by a fluorescence-based competition assay. The three peptides that showed affinity toward tPex5 had K(d) values that were comparable in magnitude (within a factor of 5) and showed the same ranking order as those from manual fluorescence measurements. With 12 min of sample infusion, the entire sample-to-sample cycle takes about 15 min and can be repeated without any preparation between runs. For T. brucei tPex5 affinity measurements, 1 mg of protein was sufficient for 35 repetitive analyses in the automated LOV-ESI-MS apparatus. The system allows rapid determination of K(d) in the range of 10(-5)-10(-7) M for sample mixtures and is suitable for screening a large number of compounds against multiple proteins.

Animals↗
PubMed2004

Solution- and solid-phase syntheses of guanidine-bridged, water-soluble linkers for multivalent ligand design.

[reaction: see text] Efficient syntheses of guanidine-bridged poly(ethylene glycol) linkers of various lengths in fully protected form are reported for both solution- and solid-phase protocols. The application of such linkers in the construction of water-soluble and high-affinity multivalent ligands against cholera toxin is demonstrated. Synthetic intermediates for multivalent ligands as large as 20 kDa in molecular weight have been assembled using presynthesized linkers. The final ligands are highly water-soluble, thus enabling proper biophysical characterization.

Carbohydrate Sequence↗
PubMed2004

3,5-Substituted phenyl galactosides as leads in designing effective cholera toxin antagonists; synthesis and crystallographic studies.

With the aim of developing high-affinity mono and multivalent antagonists of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) we are using the galactose portion of the natural receptor ganglioside GM1 as an anchoring fragment in structure-based inhibitor design efforts. In order to establish a better structure-activity relationship for guiding these studies, we designed and prepared a small focused library of twenty 3,5-substituted phenylgalactosides based on two previous leads. The compounds were tested for their ability to block CTB(5) binding to immobilized ganglioside receptor and compared to the two previous leads. The crystal structures of the most promising compounds bound to either CTB(5) or LTB(5) were then determined in order to understand the basis for affinity differences. The most potent new compound yielded a six-fold improvement over our benchmark lead m-nitrophenyl-alpha-d-galactopyranoside (MNPG), and a two-fold improvement in IC(50) over a newer MNPG derivative. These results support the notion that the m-nitrophenyl moiety of MNPG and its derivatives is an important element to retain in future optimization efforts. Additionally, a consensus binding-pocket for the alkylmorpholine or piperazine moiety present in all of the designed antagonists was established as an important area of the GM1 binding site to target in future work.

Animals↗
PubMed2004

Nonspanning bivalent ligands as improved surface receptor binding inhibitors of the cholera toxin B pentamer.

A series of bivalent ligands of varying length were synthesized to inhibit the receptor-binding process of cholera toxin. Competitive surface receptor binding assays showed that significant potency gains relative to the constituent monovalent ligands were achieved independently from the ability of the extended bivalent ligands to span binding sites within the toxin pentamer. Several models that could account for the unexpected improvement in IC(50) values are examined, taking into account crystallographic analysis of each ligand in complex with the toxin pentamer. Evidence is presented that steric blocking at the receptor binding surface may play a role. The results of our study suggest that the use of relatively short, "nonspanning" bivalent ligands, or monovalent ligands of similar topology and bulk may be an effective way of blocking the interaction of multimeric proteins with their cell surface receptors.

Amides↗
PubMed2004

Biogenesis of peroxisomes and glycosomes: trypanosomatid glycosome assembly is a promising new drug target.

In trypanosomatids (Trypanosoma and Leishmania), protozoa responsible for serious diseases of mankind in tropical and subtropical countries, core carbohydrate metabolism including glycolysis is compartmentalized in peculiar peroxisomes called glycosomes. Proper biogenesis of these organelles and the correct sequestering of glycolytic enzymes are essential to these parasites. Biogenesis of glycosomes in trypanosomatids and that of peroxisomes in other eukaryotes, including the human host, occur via homologous processes involving proteins called peroxins, which exert their function through multiple, transient interactions with each other. Decreased expression of peroxins leads to death of trypanosomes. Peroxins show only a low level of sequence conservation. Therefore, it seems feasible to design compounds that will prevent interactions of proteins involved in biogenesis of trypanosomatid glycosomes without interfering with peroxisome formation in the human host cells. Such compounds would be suitable as lead drugs against trypanosomatid-borne diseases.

Animals↗
PubMed2004

Structural biology and structure-based inhibitor design of cholera toxin and heat-labile enterotoxin.

Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels. Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated. At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.

Bacterial Toxins↗
PubMed2003

Analysis of the sequence motifs responsible for the interactions of peroxins 14 and 5, which are involved in glycosome biogenesis in Trypanosoma brucei.

Glycosome biogenesis in trypanosomatids occurs via a process that is homologous to peroxisome biogenesis in other eukaryotes. Glycosomal matrix proteins are synthesized in the cytosol and imported posttranslationally. The import process involves a series of protein-protein interactions starting by recognition of glycosomal matrix proteins by a receptor in the cytosol. Most proteins to be imported contain so-called PTS-1 or PTS-2 targeting sequences recognized by, respectively, the receptor proteins PEX5 and PEX7. PEX14, a protein associated with the peroxisomal membrane, has been identified as a component of the docking complex and a point of convergence of the PEX5- and PEX7-dependent import pathways. In this paper, the strength of the interactions between Trypanosoma brucei PEX14 and PEX5 was studied by a fluorescence assay, using (i) a panel of N-terminal regions of TbPEX14 protein variants and (ii) a series of different peptides derived from TbPEX5, each containing one of the three WXXXF/Y motifs present in this receptor protein. On the PEX14 side, the N-terminal region of TbPEX14 including residues 1-84 appeared to be responsible for TbPEX5 binding. The results from PEX14 mutants identified specific residues in the N-terminal region of TbPEX14 involved in PEX5 binding and showed that in particular hydrophobic residues F35 and F52 are critical. On the PEX5 side, 13-mer peptides incorporating the first or the third WXXXF/Y motif bind to PEX14 with an affinity in the nanomolar range. However, the second WXXXF/Y motif peptide did not show any detectable affinity. Studies using variants of second and third motif peptides suggest that the alpha-helical content of the peptides as well as the charge of a residue at position 9 in the motif may be important for PEX14 binding. Assays with 7-, 10-, 13-, and 16-mer third motif peptides showed that 16-mers and 13-mers have comparable binding affinity for PEX14, whereas 10-mers and 7-mers have about 10- and 100-fold lower affinity than the 16-mers, respectively. The low sequence identities of PEX14 and PEX5 between parasite and its human host, and the vital importance of proper glycosome biogenesis to the parasite, render these peroxins highly promising drug targets.

Amino Acid Motifs↗
PubMed2003

TFA-sensitive arylsulfonylthiourea-assisted synthesis of N,N'-substituted guanidines.

An efficient synthesis of N,N'-substituted guanidine derivatives was developed via an aromatic sulfonyl-activated thiourea intermediate. The use of certain aromatic sulfonamides, such as PbfNH(2), as the key reagent to incorporate a TFA-labile guanidine protection group greatly facilitates solid-phase synthesis of N,N'-substituted guanidine compounds.

Catalysis↗
PubMed2002

Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin.

The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.

Binding Sites↗
PubMed2002

Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin.

Multivalent ligand design constitutes an attractive avenue to the inhibition of receptor recognition and other biological events mediated by oligomeric proteins with multiple binding sites. One example is the design of multivalent receptor blockers targeting members of the AB(5) bacterial toxin family. We report here the synthesis and characterization of a pentavalent inhibitor for cholera toxin and Escherichia coli heat-labile enterotoxin. This inhibitor is an advance over the symmetric pentacyclen-derived inhibitor described in our earlier work in that it presents five copies of m-nitrophenyl-alpha-D-galactoside (MNPG) rather than five copies of beta-D-galactose. The approximately 100-fold higher single-site affinity of MNPG for the toxin receptor binding site relative to galactose is found to yield a proportionate increase in the affinity and IC50 measured for the respective pentavalent constructs. We show by dynamic light scattering that inhibition of receptor binding by the pentavalent inhibitor is due to 1:1 inhibitor:toxin association rather than to inhibitor-mediated aggregation. This 1:1 association is in complete agreement with a 1.46 A resolution crystal structure of the pentavalent inhibitor:toxin complex, which shows that the favorable single-site binding interactions of MNPG are retained by the five arms of the 5256 Da pentavalent MNPG-based inhibitor and that the initial segment of the linking groups interacts with the surface of the toxin B pentamer.

Bacterial Toxins↗
PubMed2002

Anchor-based design of improved cholera toxin and E. coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes.

The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.

Bacterial Toxins↗
PubMed2002

Combating infectious diseases through multivalent design.

Many biological interactions are multivalent, linking two particles via many copies of the same ligand-receptor binding pair. Examples of multivalent binding range from cell-cell adhesion to the assembly of large protein complexes from constituent multimers to the binding of AB5 bacterial toxins at the cell surface. Multivalent interactions can be effectively mimicked, inhibited, or disrupted through the design of suitable multivalent ligands. We review here recent work on multivalent ligand design based on a number of different chemical scaffolds, with a specific emphasis on the use of structure-based ligand design to target multimeric bacterial toxins.

Anti-Bacterial Agents↗