Iterative deconvolution process

Figure 3.4. Schematic representation of the iterative deconvolution process.

The iterative deconvolution process is explained here using a simple example of a tetrapeptide library made of four different amino acids (R1 to R4, Figure 3.5), where R1 to R4 represent individual amino acids and X represents a mixture of those four amino acids. In the initial screening of this library (Step 1 of Figure 3.6) in a bioassay, the most active... 

With Deconvolution 1 you have access to a fully automatic and interactive mode. In the automatic mode only the region used for deconvolution and a few optional parameters (type of lineshape. number of peaks,. ..) may be set. Whilst the interactive mode allows you to set the initial values for the parameters controlling the iterative fitting process and to create, edit and delete peaks. 

Although not identical, both the orthogonal and positional scan formatted libraries share the features that all mixtures are made at the start of the library process and only individual compound synthesis is required after the first screening of mixtures. This is an extra initial effort with regard to the synthesis of mixtures when compared to an iterative method. The advantage is that no intermediate mixture syntheses will be required. If prepared in sufficient quantity, the library can be screened over a large number of assays, and the added effort of initial mixture syntheses will be translated into an efficiency in deconvolution relative to the continual resynthesis of mixtures with iterative deconvolution. 

The term iterative hbrary deconvolution describes a process consisting of repetitive synthesis, screening, and selection steps, during which all mixture positions of the hbrary (see also Section 4.3.7.3.2.1) are successively defined, resulting in the identification of individual peptides with the desired bioactivify.P At each step of this iterative process, the number of peptides per mixture is reduced by a factor equal to the number of amino adds in the mixture position that was defined at that step. At the final step of the process, individual peptides are synthesized and tested. For a hexapeptide library with two defined and four mixture positions, the iterative deconvolution consists of four cycles of synthesis, screening, and selection (see Table 2).P 1... 

Iterative deconvolution works best when a single compound of the mixture is much more active than all others. Conversely, deconvolution is difficult when all members of a library have similar activity—as is often the case in the later stages of a drug discovery process. Because of the difficulties associated with mixture deconvolution, split-and-pool synthesis is best suited to the early lead discovery phase of the drug discovery process. Later stages, requiring direct comparison of one compound against another, are best served by other methods described below. 

Illustration of iterative deconvolution. Sublibraries are screened for activity and less complex sublibraries with the identified building block incorporated are synthesised. The best building block for every position is identified stepwise through this process. 

Testing of each component of the library, one at a time, is the desired goal of any screening method but has constraints of time and resources. One way to increase the sample throughput is to perform several parallel analyses. An alternative approach to speed up the screening process is to reduce the number of samples for analysis by selecting only a random number of library members [87]. An improvement over this protocol is iterative deconvolution [88]. In one such approach, called the mimotope approach [89], a pool of compounds of soluble libraries is tested first based on this outcome, a smaller pool of compounds is synthesized and sublibraries are retested. The process is repeated until a compound with the highest activity is identified. 

Ihe software routine presents the distribution using a process of iterative deconvolution without an a priori assunnption of the distribution. This allcws multi-model materials to be analysed. 

Pirrung s group is similar to positional scanning. The advantage of positional scanning is that sublibraries need not be resynthesized, but with iterative deconvolution there is the advantage of activity enrichment as the process goes on. 

This potency does not account for the inhibition observed for the library Ai (34%, 65,341 compounds), and examination of the various iterations clearly shows other families of active compounds. Nevertheless, a relatively short process (6 iterations, 51 reactions, 37 deprotections, 1 chromatography) detected a reasonably active lead compound as a starting point for chemical optimization. The sublibrary populations were reduced from 65341 to 12 in only four iterations (sublibrary E2, where only 12 permutations of the four monomers in the structure were possible), the second being a control of the validity of the first selection. Deconvolution of a few other families of positives (for example sublibraries Bs, E2, E4 and F2) could have produced different lead structures while maintaining a relatively modest number of iterations and reactions. 

When a library is deconvoluted via an iterative method, both time and monomers are used in excess for synthesis during iteration rounds. Moreover, the iterative process ends with a single structure which may not be the most active compound of the library (see previous chapters) and the deconvolution of additional positives, when it is possible, considerably lengthens the structure determination process. 

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