Sequence-selective peptide detection by small synthetic chemosensors selected from an encoded combinatorial chemosensor library.

Synthetic chemosensors hold great potential in many diagnostic applications. In this study, we describe the design and preparation of the first encoded combinatorial library of chemosensors for tripeptides. Subsequent screening of the library resulted in the discovery of novel chemosensors able to distinguish between random tripeptides.

Inhibition of gene expression in human cells through small molecule-RNA interactions.

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5' end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from D-and L-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC(50) of approximately 50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

Construction of sequence-selective peptide receptors from conformationally restricted eta- and theta- amino acids.

Oligomeric chemoreceptors for tripeptides have been constructed from a set of novel eta- and theta-amino acids. Screening against an encoded combinatorial tripeptide library has revealed two new small molecule chemoreceptor motifs with highly sequence-selective binding properties.

Highly sequence selective nonmacrocyclic two-armed receptors for peptides.

Simple nonmacrocyclic two armed receptors have been synthesized to create a new class of sequence-selective receptors for peptides. Screening several examples of these simple compounds against a 24,389-member library of N-acetyl tripeptides revealed novel binding properties.

Approximate solvent-accessible surface areas from tetrahedrally directed neighbor densities.

A fast analytical formula (TDND) has been derived for the calculation of approximate atomic and molecular solvent-accessible surface areas (SASA), as well as the first and second derivatives of these quantities with respect to atomic coordinates. Extending the work of Stouten et al. (Molecular Simulation, 1993, Vol. 10, pp. 97-120), as well as our own (Journal of Computational Chemistry, 1999, Vol. 20, pp. 586-596), the method makes use of a Gaussian function to calculate the neighbor density in four tetrahedral directions in three-dimensional space, sometimes twice with different orientations. SASA and first derivatives of the 2366 heavy atoms of penicillopepsin are computed in 0.13 s on an SGI R10000/194 MHz processor. When second derivatives are computed as well, the total time is 0.23 s. This is considerably faster than timings reported previously for other algorithms. Based on a parameterization set of nineteen compounds of different size (11-4346 atoms) and class (organics, proteins, DNA, and various complexes) consisting of a total 23,197 atoms, the method exhibits relative errors in the range 0.2-12.6% for total molecular surface areas and average absolute atomic surface area deviations in the range 1.7 to 3.6 A(2).

A new generation of fluorescent chemosensors demonstrate improved analyte detection sensitivity and photobleaching resistance.

Molecular chemosensors have found increased utility in the development of precise and sensitive detection devices. However, chemosensors that report binding via fluorescence through UV excitation are susceptible to destruction via photodegradation of the fluorophore. In the following report, the dansyl fluorophore in a previously reported chemosensor for peptides is replaced with an acridone derivative that is highly resistant to photobleaching. Its spectral properties are closely matched to those of the original dansyl fluorophore, and although quite structurally dissimilar, the new more photostable acridone chemosensor analogue exhibits only minor differences in binding/detection characteristics.

Fluorescent, sequence-selective peptide detection by synthetic small molecules.

Small organic sensor molecules were prepared that bind and signal the presence of unlabeled tripeptides in a sequence-selective manner. Sequence-selective peptide binding is a difficult problem because small peptides are highly flexible and there are no clear rules for designing peptide-binding molecules as there are for the nucleic acids. The signaling system involved the application of fluorescence energy transfer and provided large, real-time fluorescence increases (300 to 500 percent) upon peptide binding. With it, these sensors were sensitive enough to detect unlabeled cognate peptides both in organic solution and in the solid state at low micromolar concentrations.

Parallel synthesis and screening of a solid phase carbohydrate library.

A solid phase carbohydrate library was synthesized and screened against Bauhinia purpurea lectin. The library, which contains approximately 1300 di- and trisaccharides, was synthesized with chemical encoding on TentaGel resin so that each bead contained a single carbohydrate. Two ligands that bind more tightly to the lectin than Gal-beta-1,3-GalNAc (the known ligand) have been identified. The strategy outlined can be used to identify carbohydrate-based ligands for any receptor; however, because the derivatized beads mimic the polyvalent presentation of cell surface carbohydrates, the screen may prove especially valuable for discovering new compounds that bind to proteins participating in cell adhesion.

Engineering of a synthetic receptor to alter peptide binding selectivity.

BACKGROUND: Molecular recognition processes are ubiquitous in nature: substrate binding by enzymes, antigen recognition by antibodies, and hormone activation of receptors provide three classic examples. To better understand these large systems it is valuable to study smaller, well defined host molecules. Previously we found sequence-selective peptide binding with a class of C3 symmetric synthetic receptors. In this work we rationally altered that host structure in order to produce a corresponding change in binding selectivity. RESULTS: A novel C3 symmetric receptor was designed and synthesized such that, unlike previous host molecules, it contained hydrogen-bond accepting functionality within the binding cavity. Screening of this host against a combinatorial tripeptide library revealed an exquisite selectivity for sequences with D-Pro-D-Asn carboxyl termini. Computer simulations and NMR studies indicate that hydrogen bonding of the D-Asn side-chain amide to the amine functionality within the cavity is responsible for this selectivity. CONCLUSIONS: Computer-aided design and combinatorial library screening methods combine to provide a powerful approach to induce and evaluate the results of rational changes in the molecular recognition properties of molecules. Using this approach, we modified the binding properties of a class of molecules to select for hydrogen-bonding residues instead of hydrophobic residues and concomitantly increased the overall sequences selectivity. Structural studies indicate that these changes indeed result from the type of binding mode proposed as part of the initial design. This approach can increase our understanding of molecular recognition processes, and should allow the rational design of larger, more selective systems.

Complex synthetic chemical libraries indexed with molecular tags.

Combinatorial methods of chemical synthesis allow the creation of molecular libraries having immense diversity. The utility of such libraries is dependent upon identifying the structures of the molecules so prepared. We describe the construction of a peptide combinatorial library, having 117,649 different members, synthesized on beads and indexed with inert chemical tags. These tags are used as a binary code to record the reaction history of each bead. The code can be read directly from a single bead by electron capture capillary gas chromatography. We demonstrate the correct selection of members of the library on the basis of binding to a monoclonal antibody.

Synthesis and structure of the platelet aggregation factor thromboxane A2.

In 1975, Hamberg et al. reported evidence for the existence of an unstable platelet-aggregating factor which they named thromboxane A2 (TXA2) and for which they proposed a novel bicyclic oxetane structure (1, below) based on the short half-life of the factor (t1/2 (37 degrees C) = 32 s at pH 7.4) and the isolation of degradation products related to thromboxane (TXB2) (2, below). As natural TXA2 has not yet been isolated and characterized as a pure compound, we have synthesized the proposed structure (1) from TXB2 and compared its biological properties with those of authentic, biologically generated material. Here we present evidence that synthetic material having structure (1) is indistinguishable from platelet-derived TXA2 in various biological assays and that the proposed structure (1) for TXA2 is correct.