Using Support Vector Machine Regression to Model the Retention of Peptides in Immobilized Metal-affinity Chromatography.

Retention of histidine-containing peptides in immobilized metal-affinity chromatography (IMAC) has been studied using several hundred model peptides. Retention in a Nickel column is primarily driven by the number of histidine residues; however, the amino acid composition of the peptide also plays a significant role. A regression model based on support vector machines was used to learn and subsequently predict the relationship between the amino acid composition and the retention time on a Nickel column. The model was predominantly governed by the count of the histidine residues, and the isoelectric point of the peptide.

Age dependence of cellular properties of human septal cartilage: implications for tissue engineering.

BACKGROUND: The persistent need for cartilage replacement material in head and neck surgery has led to novel cell culture methods developed to engineer cartilage. Currently, there is no consensus on an optimal source of cells for these endeavors. OBJECTIVES: To evaluate human nasal cartilage as a potential source of chondrocytes and to determine the effect of donor age on cellular and proliferation characteristics. SUBJECTS: Nasal cartilage specimens were obtained after reconstructive surgery from 46 patients ranging in age from 15 to 60 years. METHODS: Specimens were weighed and chondrocytes were isolated by digestion in 0.2% collagenase type II for 16 hours. Cells were maintained in primary cultures until confluency, then seeded onto polylactic acid-polyglycolic acid scaffolds. Seeding efficiency was determined by quantification of DNA content of seeded constructs by means of Hoechst dye 33258. Specimen weights, cell yields, cell content, and doubling time were also measured and correlated to donor age. RESULTS: Mean (+/-SD) cartilage mass obtained (648 +/- 229 mg) is higher than from typical biopsy specimens of auricular cartilage, and the cellular characteristics show a higher proliferation rate than auricular chondrocytes. Cell yield increased with age, while doubling time decreased with age in samples from patients ranging from 15 to 60 years old. CONCLUSIONS: The use of nasal septal cartilage as a source of cells for tissue engineering may be valid over a wide range of patient ages. The large tissue yield and consequent cell yield make this tissue a potential starting source of chondrocytes for large-volume tissue-engineered implants.

New approach for preparation of 2,3,7-trisubstituted 3,4-dihydroisoquinolinone libraries on solid phase.

In an attempt to prepare 7-substituted 3,4-dihydroisoquinolinone family of compounds, we observed an unexpected decarboxylation. The reaction of 4-nitrohomophthalic anhydride with a Schiff base formed on solid support leads to the formation of core structure. LC-MS and 1H NMR analysis confirmed the structure of unexpected intermediate. A library of 38,400 compounds was produced using this new synthetic approach.

New technique for high-throughput synthesis.

A new technique for high throughput solid phase synthesis using the centrifuge based liquid removal from readily available standard microtiterplates is described. This technique eliminates the filtration step and is therefore applicable to simultaneous processing of an unlimited number of reaction compartments. Its application is illustrated on the synthesis of an array of 380 tetrahydroisoquinolinones.

Combinatorial chemistry reveals a new motif that binds the platelet fibrinogen receptor, gpIIbIIIa.

Among cell adhesion molecules, the classic Arg-Gly-Asp (RGD) motif is the best studied. We used combinatorial chemical and affinity immunochemical methods to find a novel motif of unnatural peptide ligands for the fibrinogen receptor of platelets, gpIIbIIIa (alphaIIbbeta3). The new d-amino acid motif, p(f/y)l, is unique among the ligands that bind the RGD pocket: It lacks the carboxylic acid group that is believed to coordinate with calcium in the MIDAS motif of the receptor. With an IC50 of 14 microM for the most potent compound, these linear p(f/y)l peptides had affinities similar to those of linear peptides containing RGD, and reversed sequences failed to compete with binding up to 1 mM. As the new motif was so different, molecular modeling was employed to suggest a model for molecular recognition. A reversed binding mechanism common for d-amino acid mimics of natural l-amino acid peptides offers an attractive hypothesis that suggests three points of contact similar to those made by the RGD-mimicking monoclonal antibody, OPG2. Interestingly, the model proposes that pi-electrons in the new motif may substitute for the carboxylate group present in all other RGD-types of ligands. Although modeling linear peptides is subjective, the pi-bonding model provides intriguing possibilities for medicinal chemistry after appropriate confirmatory studies.

Evaluation of gaseous hydrogen fluoride as a convenient reagent for parallel cleavage from the solid support.

We have tested the limits of gaseous hydrogen fluoride as an agent for parallel detachment of organic molecules from the solid support. Peptides were chosen as relatively sensitive models for this reaction. Acid-catalyzed amide bond hydrolysis, side chain modification (tryptophan and other unnatural amino acids) by the protecting group residues as well as dehydration of serine and asparagine was followed. The technique of cleavage of side chain protection prior to the resin cleavage has given satisfactory results. Two-step deprotection and cleavage from benzhydrylamine resin by TFA and HF was compared to the deprotection and cleavage by TFA from Knorr resin.

Solid-phase synthesis of combinatorial libraries.

Developments in the solid-phase synthesis of combinatorial libraries during 1998 are reviewed with emphasis on the rapid synthesis of libraries, new techniques for analysis of reaction progress and synthetic results, application of solid-phases as reagents, and synthesis of particular molecular entities. An alternative to solid-phase synthesis - fluorous-phase synthesis - is mentioned as an emerging technique that may compete with solid-phase synthesis in the future.

Discovery of a novel, potent, and specific family of factor Xa inhibitors via combinatorial chemistry.

A series of low molecular weight peptide inhibitors of factor Xa, unrelated to any previously described, was identified by screening a combinatorial peptide library composed of L-amino acids. The minimal inhibitory sequence is a tripeptide, L-tyrosinyl-L-isoleucyl-L-arginyl, which competitively inhibits the hydrolysis of small chromogenic substrates by factor Xa but binds in an orientation which prevents a productive nucleophilic attack by serine 195 of the catalytic triad on the carbonyl carbon of the carboxyterminal arginine. The initial leads identified in an octamer combinatorial peptide library ranged in potency from 4 to 15 microM. These peptides were modified into peptide mimetics with a greater than 1000-fold increase in potency while retaining unusual selectivity for factor Xa over the related serine proteases thrombin, factor VIIa/tissue factor, plasmin, activated protein C, kallikrein, and trypsin. One of the most potent analogues, SEL 2711, with a Ki of 0.003 microM for factor Xa and 40 microM for thrombin, is active in in vitro and ex vivo coagulation assays, suggesting the potential application of these inhibitors in anticoagulant therapy.

New methods in combinatorial chemistry-robotics and parallel synthesis.

Technological advances in the automation of parallel synthesis are following the model set by the semiconductor industry: miniaturization, increasing speed, lower costs. Recent work includes preparation of high-density reaction blocks, development of ink-jet dispensing to polypropylene sheets and synthesis inside customized microchips.

Design considerations and computer modeling related to the development of molecular scaffolds and peptide mimetics for combinatorial chemistry.

A critical issue in drug discovery utilizing combinatorial chemistry as part of the discovery process is the choice of scaffolds to be used for a proper presentation, in a three-dimensional space, of the critical elements of structure necessary for molecular recognition (binding) and information transfer (agonist/ antagonist). In the case of polypeptide ligands, considerations related to the properties of various backbone structures (alpha-helix, beta-sheets, etc.; phi, psi space) and those related to three-dimensional presentation of side-chain moieties (topography; chi (chi) space) must be addressed, although they often present quite different elements in the molecular recognition puzzle. We have addressed aspects of this problem by examining the three-dimensional structures of chemically different scaffolds at various distances from the scaffold to evaluate their putative diversity. We find that chemically diverse scaffolds can readily become topographically similar. We suggest a topographical approach involving design in chi space to deal with these problems.

High-volume cellular screening for anticancer agents with combinatorial chemical libraries: a new methodology.

A single-step cancer cell cytotoxic assay system for anticancer drug discovery has been developed which facilitates rapid screening of large combinatorial chemical libraries synthesized using the 'one-bead-one-compound' (OBOC) methodology. Each OBOC library bead incorporates two orthogonally cleavable linkers that release the bead-bound compound at a different pH. The assay utilizes high concentrations of tumor cells mixed directly with OBOC beads and plated in soft agarose containing tissue culture medium. One of the orthogonal linkers is cleaved at neutral pH in tissue culture releasing an aliquot of compound to diffuse at a relatively high local concentration into the soft agarose immediately surrounding the bead. Active compounds are identified visually from a clear ring of tumor cell lysis which forms within 48 h around just the rare bead releasing a cytotoxic compound. The bead releasing a cytotoxin is then plucked from the agar and the remaining compound still linked to the bead can be released for structural analysis, followed by compound resynthesis and confirmatory testing. This assay system has been successfully applied to identification of lead cytotoxic compounds from model peptidic and non-peptidic combinatorial chemical libraries. Use of this methodology may facilitate anticancer drug discovery.

Library generation through successive substitution of trichlorotriazine.

The decreasing reactivity of tri-, di- and monochlorotriazine was utilized for the solid-phase construction of a combinatorial library with three randomized positions, using 20 amino acids and 50 amines as building blocks. The first chlorine atom was selectively substituted by coupling a large excess of trichlorotriazine to the support-bound amino acid, thus avoiding simultaneous substitution of the second chlorine. The second and third diversity positions were selectively introduced by coupling amines at different temperatures. Mixtures of model compounds were synthesized and analyzed, showing the correct representation of all expected components. A library composed of 12,000 compounds was generated using this method.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Inclusion volume solid-phase synthesis.

Solid-phase synthesis of peptides was carried out using only the volume of the solvent included in the swollen solid-phase resin heads (inclusion volume synthesis). This approach enables (i) the use of higher concentrations of activated amino acids, resulting in increased coupling rates, (ii) drastically decreased consumption of solvents, and (iii) the construction of multiple peptide synthesizers having virtually no reaction vessels.

A One-Bead One-Peptide Combinatorial Library Method for B-Cell Epitope Mapping

The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide-beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-beta-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μkappa) of two murine B-cell lymphoma cell lines (antigen unknown).

Structurally homogeneous and heterogeneous synthetic combinatorial libraries.

We have designed and synthesized structurally homogeneous and heterogeneous nonpeptide libraries. Structurally homogeneous libraries are characterized by the presence of one common structural unit, a scaffold, in all library compounds (e.g. cyclopentane, cyclohexane, diketopiperazine, thiazolidine). In structurally heterogeneous libraries different organic reactions (acylation, etherification, reductive amination, nucleophilic displacement) were applied to connect bifunctional building blocks unrelated in structure (aromatic hydroxy acids, aromatic hydroxy aldehydes, amino alcohols, diamines, and amino acids). The focus of this communication is to document the use of bifunctional building blocks for the design and synthesis of structurally heterogeneous libraries of N-(alkoxy acyl)amino acids, N,N'-bis-(alkoxy acyl)diamino acids, N-acylamino ethers, N-(alkoxy acyl)amino alcohols, N-alkylamino ethers, and N-(alkoxy aryl)diamines.

Bifunctional scaffolds as templates for synthetic combinatorial libraries.

A small-molecule synthetic combinatorial library was designed and synthesized that features potential pharmacophores attached to a variety of small cyclic scaffolds. The synthesis of the library involved randomization of three types of building blocks: 20 amino acids, 10 aromatic hydroxy acids and 21 alcohols, totaling a library complexity of 4200 compounds. Mitsunobu polymer-supported etherification was used in the last randomization. The library compounds were attached to beads via an ester-bond linkage enabling both on-bead as well as in-solution screening. When the library was tested against a model target, streptavidin, specific binders were found. The structures of the most active compounds were determined from the fragmentation pattern in MS/MS experiments.