Benzyl Mono-P-Fluorophosphonate and Benzyl Penta-P-Fluorophosphate Anions Are Physiologically Stable Phosphotyrosine Mimetics and Inhibitors of Protein Tyrosine Phosphatases

α,α-Difluoro-benzyl phosphonates are currently the most popular class of phosphotyrosine mimetics. Structurally derived from the natural substrate phosphotyrosine, they constitute classical bioisosteres and have enabled the development of potent inhibitors of protein tyrosine phosphatases (PTP) and phosphotyrosine recognition sites such as SH2 domains. Being dianions bearing two negative charges, phosphonates, however, do not permeate membranes and thus are often inactive in cells and have not been a successful starting point toward therapeutics, yet. In this work, benzyl phosphonates were modified by replacing phosphorus-bound oxygen atoms with phosphorus-bound fluorine atoms. Surprisingly, mono-P-fluorophosphonates were fully stable under physiological conditions, thus enabling the investigation of their mode of action toward PTP. Three alternative scenarios were tested and mono-P-fluorophosphonates were identified as stable reversible PTP1B inhibitors, despite of the loss of one negative charge and the replacement of one oxygen atom as an H-bond donor by fluorine. In extending this replacement strategy, α,α-difluorobenzyl penta-P-fluorophosphates were synthesized and found to be novel phosphotyrosine mimetics with improved affinity to the phosphotyrosine binding site of PTP1B.     

Synthesis of 3,5-difluorotyrosine-containing peptides: application in substrate profiling of protein tyrosine phosphatases

Fully protected 3,5-difluorotyrosine (F2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The F2Y-containing peptides display kinetic properties toward protein tyrosine phosphatases (PTPs) similar to their corresponding tyrosine-containing counterparts but are resistant to tyrosinase action. These properties make F2Y a useful tyrosine surrogate during peptide library screening for optimal PTP substrates.     

Identification of a molecular recognition role for the activation loop phosphotyrosine of the SRC tyrosine kinase

A human cDNA phage display library screen, using a phosphopeptide designed to mimic the activation loop phosphotyrosine of the Src tyrosine kinase, has identified the N-terminal SH2 domain of the p85 regulatory subunit of phosphatidyl inositol-3 kinase (PI3K) as an interacting recognition domain. Activation loop phosphorylation is known to play a conformational role in kinase activation, but is largely not thought to play a role in protein/protein recognition. Affinity chromatography and biochemical evaluation in mouse fibroblast cells has confirmed the dependence of this interaction on both the Src activation loop phosphotyrosine and the N-terminal SH2 domain of PI3K.     

基于四氢喹啉酸骈环戊烯骨架的蛋白酪氨酸磷酸酶1B抑制剂的设计、合成及构效关系

基于肿瘤、糖尿病等重大疾病的有限目标,选择蛋白酪氨酸磷酸酶(PTPs)家族这一生物体系中有代表性的PTP家族成员PTP1B,SHP-1,SHP-2,LAR,CDC25B及PRL-3进行研究.通过综合高通量筛选获得的"苗头"化合物结构信息,分析得到四氢喹啉骈环戊烯骨架.初步的构效关系研究表明,四氢喹啉酸骈环戊烯母核结构可...     

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.     

N-->O-Acyl shift in Fmoc-based synthesis of phosphopeptides

Synthetic phosphopeptides are frequently used as chemical probes to explore protein-protein interactions involved in cellular signal transduction. Most commonly, the solid-phase synthesis of phosphotyrosine-containing peptides is performed by applying the Fmoc-strategy and N-Fmoc-protected tyrosine derivatives bearing acid-labile phospho protecting groups. We observed a side-reaction, the isomerisation at threonine, which furnishes depsipeptides. It is shown that the rate of N-->O-acyl migration depends on the sequence context. Depsipeptides were formed most rapidly when the phosphotyrosine was located in the +2 position. Furthermore, different phosphotyrosine building blocks were compared and a suitable method that provides phosphopeptides in enhanced purity and yield is suggested.     

A study of Src SH2 domain protein-phosphopeptide binding interactions by electrospray ionization mass spectrometry

The noncovalent binding of various peptide ligands to pp60^src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries.     

Inhibitors of tyrosine kinase proteins induced Ras signaling pathway as potential anti-tumor agents

Cellular signaling pathways induced by growth-factor receptors with tyrosine kinase activity are frequently deregulated in cancer. Anti-tumor agents that inhibit their enzymatic tyrosine kinase activity have been designed and are now used in human chemotherapy. We review here our data constituting an alternative way to interrupt over-expressed signaling pathway by inhibiting protein-protein interactions. In our approach, the adaptor protein Grb2 over-expressed in connection with HER2/ErbB2/neu in Ras signaling pathway was chosen as a target. Peptides and peptidomimetics with very high affinities for either SH3 or SH2 domains of Grb2 were rationally designed from structural data. We describe their synthesis, their capacity to interrupt the signaling pathway and their anti-proliferative activity. To cite this article: M. Vidal et al., C. R. Chimie 8 (2005).     

Integrating virtual screening and combinatorial chemistry for accelerated drug discovery

Virtual screening is increasingly being used in drug discovery programs with a growing number of successful applications. Experimental methodologies developed to speed up the drug discovery processes include high-throughput screening and combinatorial chemistry. The complementarities between computational and experimental screenings have been recognized and reviewed in the literature. Computational methods have also been used in the combinatorial chemistry field, in particular in library design. However, the integration of computational and combinatorial chemistry screenings has been attempted only recently. Combinatorial libraries (experimental or virtual) represent a notable source of chemically related compounds. Advances in combinatorial chemistry and deconvolution strategies, have enabled the rapid exploration of novel and dense regions in the chemical space. The present review is focused on the integration of virtual and experimental screening of combinatorial libraries. Applications of virtual screening to discover novel anticancer agents and our ongoing efforts towards the integration of virtual screening and combinatorial chemistry are also discussed.     

Optimization of bivalent glutathione S-transferase inhibitors by combinatorial linker design

Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases, including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTP1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120-fold selectivity) and with stoichiometry of one inhibitor: one GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure (Asp-Gly-AMAB-Gly-Asp) and K(D) = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange. A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complementarity between the protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. The results demonstrate the utility of combinatorial, or "irrational", linker design for optimizing bivalent inhibitors.     

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.     

Receptor-assisted combinatorial chemistry: thermodynamics and kinetics in drug discovery

Current drug discovery using combinatorial chemistry involves synthesis followed by screening, but emerging methods involve receptor-assistance to combine these steps. Adding stoichiometric amounts of receptor during library synthesis alters the kinetics or thermodynamics of the synthesis in a way that identifies the best-binding library members. Three main methods have emerged thus far in receptor-assisted combinatorial chemistry: dynamic combinatorial libraries, receptor-accelerated synthesis, and a new method, pseudo-dynamic libraries. Pseudo-dynamic libraries apply both thermodynamics and kinetics to amplify library members to easily observable levels, and attain selectivity heretofore unseen in receptor-assisted systems.     

From protein domains to drug candidates-natural products as guiding principles in the design and synthesis of compound libraries

In the continuing effort to find small molecules that alter protein function and ultimately might lead to new drugs, combinatorial chemistry has emerged as a very powerful tool. Contrary to original expectations that large libraries would result in the discovery of many hit and lead structures, it has been recognized that the biological relevance, design, and diversity of the library are more important. As the universe of conceivable compounds is almost infinite, the question arises: where is a biologically validated starting point from which to build a combinatorial library? Nature itself might provide an answer: natural products have been evolved to bind to proteins. Recent results in structural biology and bioinformatics indicate that the number of distinct protein families and folds is fairly limited. Often the same structural domain is used by many proteins in a more or less modified form created by divergent evolution. Recent progress in solid-phase organic synthesis has enabled the synthesis of combinatorial libraries based on the structure of complex natural products. It can be envisioned that natural-product-based combinatorial synthesis may permit hit or lead compounds to be found with enhanced probability and quality.     

Phosphopeptide selective coordination complexes as promising SRC homology 2 domain mimetics

Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein-protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein-protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (K(a) ～ 10(7) M(-1)) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines.     

2D and 3D spatially addressed arrays for high-throughput automated synthesis of combinatorial libraries

One of the key elements in the drug discovery process is the use of automation to synthesize libraries of compounds for biological screening. The "split-and-mix" approaches in combinatorial chemistry have been recognized as extremely powerful techniques to access large numbers of compounds, while requiring only few reaction steps. However, the need for effective encoding/deconvolution strategies and demands for larger amounts of compounds have somewhat limited the use of these techniques in the pharmaceutical industry. In this paper, we describe a concept of directed sort and combine synthesis with spatially arranged arrays of macroscopic supports. Such a concept attempts to balance the number of reaction steps, the confidence in compound identity, and the quantity of synthesized compounds. Using three-dimensional arrays of frames each containing a two-dimensional array of macroscopic solid supports, we have conceptualized and developed a modular semiautomated system with a capacity of up to 100 000 compounds per batch. Modularity of this system enables flexibility either to produce large diverse combinatorial libraries or to synthesize more focused smaller libraries, both as single compounds in 12-15 micromol quantities. This method using sortable and spatially addressed arrays is exemplified by the synthesis of a 15 360 compound library.     

Library fingerprints: a novel approach to the screening of virtual libraries

We propose a novel method to prioritize libraries for combinatorial synthesis and high-throughput screening that assesses the viability of a particular library on the basis of the aggregate physical-chemical properties of the compounds using a na?ve Bayesian classifier. This approach prioritizes collections of related compounds according to the aggregate values of their physical-chemical parameters in contrast to single-compound screening. The method is also shown to be useful in screening existing noncombinatorial libraries when the compounds in these libraries have been previously clustered according to their molecular graphs. We show that the method used here is comparable or superior to the single-compound virtual screening of combinatorial libraries and noncombinatorial libraries and is superior to the pairwise Tanimoto similarity searching of a collection of combinatorial libraries.     

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site‐specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N‐terminally thiolated peptide hydrazides by hydrazone ligation, b) on‐surface native chemical ligation with self‐purified peptide thioesters, c) radical‐induced desulfurization, and d) a surface‐based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline‐rich ligands.     

Inhibitors of Protein Tyrosine Phosphatases: Next‐Generation Drugs?

The protein tyrosine phosphatases (PTPs) constitute a family of closely related key regulatory enzymes that dephosphorylate phosphotyrosine residues in their protein substrates. Malfunctions in PTP activity are linked to various diseases, ranging from cancer to neurological disorders and diabetes. Consequently, PTPs have emerged as promising targets for therapeutic intervention in recent years. In this review, general aspects of PTPs and the development of small-molecule inhibitors of PTPs by both academic research groups and pharmaceutical companies are discussed. Different strategies have been successfully applied to identify potent and selective inhibitors. These studies constitute the basis for the future development of PTP inhibitors as drugs.     

Library Design Guided by In-house Developed Computer Tools

In recent years, combinatorial library synthesis for drug discovery begins to migrate from library synthesis solely dictated by chemistry availability to design and synthesis of libraries with more drug-like properties. Lipinski's rule of five has been used to evaluate drug-like properties of individual compound; recently LibProTM, a new computation program has been developed at Pharmacopeia to evaluate durg-like properties of libraries. By using LibPrpTM, chemists at Pharmacopeia are able to obtain information of molecular weight and ClogP distribution of a library, and percentage of library members that violate Lipinski's rule after input structures of synthons for each combinatorial step. Currently, a "virtual library design” approach that is to calculate properties of a library at conceptual phase of the library design has been used to predetermine the value of the library. Also a new computer program used to predict "Absorption” of compounds will also be discussed.     

Combinatorial Synthesis and Multidimensional NMR Spectroscopy: An Approach to Understanding Protein–Ligand Interactions

Combinatorial chemistry is a laboratory emulation of natural recombination and selection processes. Strategies in this developing discipline involve the generation of diverse, molecular libraries through combinatorial synthesis and the selection of compounds that possess a desired property. Such approaches can facilitate the identification of ligands that bind to biological receptors, promoting our chemical understanding of cellular processes. This article illustrates that the coupling of combinatorial synthesis, multidimensional NMR spectroscopy, and biochemical methods has enhanced our understanding of a protein receptor used commonly in signal transduction, the Src Homology 3 (SH3) domain. This novel approach to studying molecular recognition has revealed a set of rules that govern SH3–ligand interactions, allowing models of receptor–ligand complexes to be constructed with only a knowledge of the polypeptide sequences. Combining combinatorial synthesis with structural methods provides a powerful new approach to understanding how proteins bind their ligands in general.