Natural Products in Drug Discovery-Concept Current Organic Chemistry.2006.VoL 10,No.8 90 MICROTITER laser COMPOUND COLLECTION + FLUORESCENTLY. LABELED TARGET Fig(7).Principle ofa CEscreening assay.Target-ligand compe oluble to be and the induced flu 7).By modifying the tated mic ong、m and dete nts befo ddition the ell The fe of EMMA provides for fo ted b 711.B test ex ract and the sub e are has been reported.Fo allo d t with the test extract The ligand voir [66).In ave to th In the ubs AC ed as a primary 5 that contro Screening g of a crude atural imulta ne usly as sed towards elated ta GahsiwepofiAGhenelectiophorC MOLECULAR IMPRINTED POLYMERS (MIP) 74).Molecular imprinte Forertnotolecilefe become almost routine 75. The proc cess cula 24 functional er and a target molecule used as a template orm comp are then electrophoresis can be for screenin na小y. the template rer me a variety of eny hape Assay protoc ional grou reported Both ando formats hav approa d te for the detection e of functional monomers and possible target molecule t-column or on line.In the column format,,the substrate and the compoundsNatural Products in Drug Discovery - Concepts Current Organic Chemistry, 2006, Vol. 10, No.8 909 compound collections. In this technology, a soluble macromolecular target is first incubated with the compounds to be tested. The ligand-target complexes are then electrophoretically separated and detected with laser-induced fluorescence (Fig. 7). By modifying the separation conditions (run length, temperature, buffer composition), it is possible to discriminate between strong, moderate and weak binders. Since nuisance compounds migrate away from the labeled target, the methods allows the detection of specific ligands in the presence or interfering substances typically found in natural product extracts. In addition to measurement of functional activity, this method provides information on the binding affinity of ligands and appears well-suited for the investigation of protein-protein interactions. Automation of ACE to a throughput of approximately 1500 analysis per day by using multiple capillary channels has been reported. For subsequent characterization, ligands bound to the target can be purified using a collection reservoir [66]. In a very recent development, the setup of ACE on microchips has been achieved, enabling further miniaturization [67]. ACE has been in particular reported as a primary screening assay to discover binders to Akt1, a key component of biochemical pathways that control apoptosis. Screening of a crude natural extract library resulted in the detection and isolation of a new phthalide (24) from a strain of the fungus Oidiodendron [68]. Capillary electrophoresis can be used for screening inhibitors in functional assays as well. CE is well suited for measuring enzymatic activity in minute biological samples. Assay protocols for a variety of enzymes including transferases, oxidoreductases, lyases and hydrolases have been reported [69]. Both off-line and on-line formats have been developed. CE enzymatic assays have been recently adapted for the detection of inhibitors in complex mixtures such as natural product extracts. The enzymatic reaction can be performed pre-column, post-column or on-line. In the pre￾column format, the enzyme, the substrate and the compounds to be tested are first incubated. The products of the reaction are then injected into the CE column and the conversion of the substrate monitored using UV absorption or laser￾induced fluorescence detection. In an on-line homogeneous version referred to as electrophoretic mediated microanalysis (EMMA) [69, 70], all the steps of the assay including mixing, separation and detection take place within the capillary. One of the greatest advantages of EMMA is that it provides fractionation of sample components before reaction with the substrate. In addition, the reaction products can be separated as well. The feasibility of EMMA technology as a screening method has been demonstrated by the detection of protein phosphatase inhibitors in natural product extracts. [71]. Briefly, the test extract and the substrate are dissolved in the CE running buffer. The enzyme is injected and allowed to equilibrate with the test extract. The voltage is switched off for a brief incubation time (15 s). When the voltage is switched on, the reaction products migrate to the detection window. In the electropherogram, the substrate is observed as a negative peak due to its partial conversion into the product, while the latter appears as a positive signal. Of particular interest is the fact that inhibition properties can be simultaneously assessed towards a mixture of related targets, since the enzymes have been electrophoretically separated in the initial step of EMMA. MOLECULAR IMPRINTED POLYMERS (MIP) Molecular imprinting, first introduced in 1972 by Wulff and Sarhan [72], produces materials with “antibody-like” selectivity [73, 74]. Molecular imprinted materials have found numerous applications. The technology is well established and imprinting of small, organic molecule has become almost routine [75]. The process of molecular imprinting consists of three successive steps (Fig 8): First, a functional monomer and a target molecule used as a template form complexes or covalently react in solution. The imprint￾monomers complexes are then fixed by cross-linking polymerization. Finally, the template is removed through extraction or hydrolysis. Vacant recognition sites of specific shape decorated with functional groups complementary to the original print molecule remain [73]. While covalent imprinting yields a higher density and more homogeneous population of binding sites than the non covalent approach, the latter technique is more flexible with respect to the choice of functional monomers and possible target molecules [75]. The numerous applications of molecular imprinting for screening compounds of biological origin have been recently Fig. (7). Principle of a CE screening assay. Target-ligand complexes show different CE migration than unbound target. O OH HO HO O 24