A fast exchange algorithm for designing focused libraries in lead optimization

Combinatorial chemistry is widely used in drug discovery. Once a lead compound has been identified, a series of R-groups and reagents can be selected and combined to generate new potential drugs. The combinatorial nature of this problem leads to chemical libraries containing usually a very large number of virtual compounds, far too large to permit their chemical synthesis. Therefore, one often wants to select a subset of "good" reagents for each R-group of reagents and synthesize all their possible combinations. In this research, one encounters some difficulties. First, the selection of reagents has to be done such that the compounds of the resulting sublibrary simultaneously optimize a series of chemical properties. For each compound, a desirability index, a concept proposed by Harrington,(20) is used to summarize those properties in one fitness value. Then a loss function is used as objective criteria to globally quantify the quality of a sublibrary. Second, there are a huge number of possible sublibraries, and the solutions space has to be explored as fast as possible. The WEALD algorithm proposed in this paper starts with a random solution and iterates by applying exchanges, a simple method proposed by Fedorov(13) and often used in the generation of optimal designs. Those exchanges are guided by a weighting of the reagents adapted recursively as the solutions space is explored. The algorithm is applied on a real database and reveals to converge rapidly. It is compared to results given by two other algorithms presented in the combinatorial chemistry literature: the Ultrafast algorithm of D. Agrafiotis and V. Lobanov and the Piccolo algorithm of W. Zheng et al.     

Simulated molecular evolution in a full combinatorial library

BACKGROUND: The Darwinian concept of 'survival of the fittest' has inspired the development of evolutionary optimization methods to find molecules with desired properties in iterative feedback cycles of synthesis and testing. These methods have recently been applied to the computer-guided heuristic selection of molecules that bind with high affinity to a given biological target. We describe the optimization behavior and performance of genetic algorithms (GAs) that select molecules from a combinatorial library of potential thrombin inhibitors in 'artificial molecular evolution' experiments, on the basis of biological screening results. RESULTS: A full combinatorial library of 15,360 members structurally biased towards the serine protease thrombin was synthesized, and all were tested for their ability to inhibit the protease activity of thrombin. Using the resulting large structure-activity landscape, we simulated the evolutionary selection of potent thrombin inhibitors from this library using GAs. Optimal parameter sets were found (encoding strategy, population size, mutation and cross-over rate) for this artificial molecular evolution. CONCLUSIONS: A GA-based evolutionary selection is a valuable combinatorial optimization strategy to discover compounds with desired properties without needing to synthesize and test all possible combinations (i.e. all molecules). GAs are especially powerful when dealing with very large combinatorial libraries for which synthesis and screening of all members is not possible and/or when only a small number of compounds compared with the library size can be synthesized or tested. The optimization gradient or 'learning' per individual increases when using smaller population sizes and decreases for higher mutation rates.     

REALISIS: a medicinal chemistry-oriented reagent selection, library design, and profiling platform

REALISIS is a software system for reagent selection, library design, and profiling, developed to fit the workflow of bench chemists and medicinal chemists. Designed to be portable, the software offers a comprehensive graphical user interface and rapid, integrated functionalities required for reagent retrieval and filtering, product enumeration, and library profiling. REALISIS is component-based, consisting of four main modules: reagent searching; reagent filtering; library enumeration; and library profiling. Each module allows the chemist to access specific functionalities and diverse filtering and profiling mechanisms. By implementing the entire process of reagent selection, library design, and profiling and by integrating all the necessary functionalities for this process, REALISIS cuts the time required to design combinatorial and noncombinatorial libraries from several days to a few hours.     

Reagent Selector: using Synthon Analysis to visualize reagent properties and assist in combinatorial library design

Reagent Selector is an intranet-based tool that aids in the selection of reagents for use in combinatorial library construction. The user selects an appropriate reagent group as a query, for example, primary amines, and further refines it on the basis of various physicochemical properties, resulting in a list of potential reagents. The results of this selection process are, in turn, converted into synthons: the fragments or R-groups that are to be incorporated into the combinatorial library. The Synthon Analysis interface graphically depicts the chemical properties for each synthon as a function of the topological bond distance from the scaffold attachment point. Displayed in this fashion, the user is able to visualize the property space for the universe of synthons as well as that of the synthons selected. Ultimately, the reagent list that embodies the selected synthons is made available to the user for reagent procurement. Application of the approach to a sample reagent list for a G-protein coupled receptor targeted library is described.     

PLUMS: a program for the rapid optimization of focused libraries

PLUMS is a new method to perform rational monomer selection for combinatorial chemistry libraries. The algorithm has been developed to optimize focused libraries with specific two-dimensional and/or three-dimensional properties. A preliminary step is the identification of those molecules in the initial virtual library which satisfy the imposed property constraints; we define these molecules as the virtual hits. From the virtual hits, PLUMS generates a starting library, which is the true combinatorial library that includes all the virtual hits. Monomers are then removed in an iterative fashion, thus reducing the size of the library. At each iteration, the worst monomer is removed. Each sublibrary is selected using a global scoring function, which balances effectiveness and efficiency. The iterative process continues until one is left with a library that consists entirely of virtual hits. The optimal library, which is the best compromise between effectiveness and efficiency, can then be selected according to the score. During the iterative process, equivalent solutions may well occur and are taken into account by the algorithm, according to a user-defined parameter. The number of monomers for each substitution site and the size of the library are parameters that can be either optimized or used to constrain the selection. The results obtained on two test libraries are presented. PLUMS was compared with genetic algorithms (GA) and monomer frequency analysis (MFA), which are widely used for monomer selection. For the two test libraries, PLUMS and GA gave equivalent results. MFA is the fastest method, but it can give misleading solutions. Possible advantages and disadvantages of the different methods are discussed.     

Polyaromatic Scavenger Reagents (PAHSR): A New Methodology for Rapid Purification in Solution-Phase Combinatorial Synthesis

A new method of purification of solution-phase combinatorial libraries has been developed. Development of a chemically inert polyaromatic anchor with a reactive "scavenger reagent" (PAHSR) allows unreacted reagents and impurities to be removed from a reaction by absorption of the PAHSR to charcoal and simple filtration.     

A novel frequency distribution selection method for efficient plate layout of a diverse combinatorial library.

A deterministic method (frequency distribution method) for selecting compounds from a partitioned virtual combinatorial library for efficient synthesis is presented here. The method is based on reagent frequency analysis and can be applied to any library of molecules distributed in any given partitioned chemical space (cluster, cell-based, etc.). Compound selection by reagent frequency distribution can produce a unique, diverse set of molecules that adequately represents the library while requiring the least amount of compounds to be synthesized and minimizing the number of different reagents that must be used. This method also provides a practical solution to the configuration of plate layout. Because the method essentially identifies "expensive" regions in the chemical space to synthesize for a desired diversity or similarity coverage, decisions concerning the necessity to synthesize these compounds can be addressed. Minimum compound generation and efficient plate layout results in savings both in time of synthesis and cost of materials. This method always results in a discrete solution, which can be used for any given library size as well as any combination of reagents and is also readily adaptable to robotic automation.     

Microfluidic platform for combinatorial synthesis in picolitre droplets

This paper presents a droplet-based microfluidic platform for miniaturized combinatorial synthesis. As a proof of concept, a library of small molecules for early stage drug screening was produced. We present an efficient strategy for producing a 7 × 3 library of potential thrombin inhibitors that can be utilized for other combinatorial synthesis applications. Picolitre droplets containing the first type of reagent (reagents A(1), A(2), …, A(m)) were formed individually in identical microfluidic chips and then stored off chip with the aid of stabilizing surfactants. These droplets were then mixed to form a library of droplets containing reagents A(1-m), each individually compartmentalized, which was reinjected into a second microfluidic chip and combinatorially fused with picolitre droplets containing the second reagent (reagents B(1), B(2), …, B(n)) that were formed on chip. The concept was demonstrated with a three-component Ugi-type reaction involving an amine (reagents A(1-3)), an aldehyde (reagents B(1-7)), and an isocyanide (held constant), to synthesize a library of small molecules with potential thrombin inhibitory activity. Our technique produced 10(6) droplets of each reaction at a rate of 2.3 kHz. Each droplet had a reaction volume of 3.1 pL, at least six orders of magnitude lower than conventional techniques. The droplets can then be divided into aliquots for different downstream screening applications. In addition to medicinal chemistry applications, this combinatorial droplet-based approach holds great potential for other applications that involve sampling large areas of chemical parameter space with minimal reagent consumption; such an approach could be beneficial when optimizing reaction conditions or performing combinatorial reactions aimed at producing novel materials.     

Correlation between host-guest binding and host amplification in simulated dynamic combinatorial libraries

We present a versatile computer model of diverse dynamic combinatorial libraries, and examine how molecular recognition between library members and a template can be used to amplify the best binders. The correlation between host-guest binding and amplification was examined for a set of 50 libraries with >300 components each over a wide range of template and building block concentrations. Depending on these concentrations correlations vary from poor (when using a large excess of template) to good (for very dilute libraries and/or substoichiometric template concentrations), highlighting the need to choose the experimental conditions for dynamic combinatorial libraries thoughtfully.     

Scalable methods for the construction and analysis of virtual combinatorial libraries

One can distinguish between two kinds of virtual combinatorial libraries: viable and accessible . Viable libraries are relatively small in size, are assembled from readily available reagents that have been filtered by the medicinal chemist, and often have a physical counterpart. Conversely, accessible libraries can encompass millions or billions of structures, typically include all possible reagents that are in principle compatible with a particular reaction scheme, and they can never be physically synthesized in their entirety. Although the analysis of viable virtual libraries is relatively straightforward, the handling of large accessible libraries requires methods that scale well with respect to library size. In this work, we present novel, efficient and scalable techniques for the construction, analysis, and in silico screening of massive virtual combinatorial libraries.     

Design and analysis of a combinatorial library of HEPT analogues: comparison of selection methodologies and inspection of the actually covered chemical space

A large virtual library of 125 396 HEPT analogues, built by combining all fragments present in the published 180-compound HEPT family, has been studied in terms of diversity criteria and the goodness of the 11 available standard diversity selection methods analyzed. All the algorithms under study, except Cell-based Density, have rank above a random selection of compounds, with Optimum and Standard Deviation based Binning and Cell-based Fraction algorithms being the best choices. Furthermore, analysis of the actually tested compounds has been performed to compare the traditional drug discovery methodology versus a rational selection of combinatorial libraries approach.     

Sensitivity analysis and other improvements to tailored combinatorial library design

"Tailoring" combinatorial libraries was developed several years ago as a very general and intuitive method to design diverse compound collections while controlling the profile of other pharmaceutically relevant properties. The candidate substituents were assigned to "categorical bins" according to their properties, and successive steps of D-optimal design were performed to generate diverse substituent sets consistent with required membership quotas from each bin. This serial algorithm was expedient to implement from existing D-optimal design codes, but was order-dependent and did not generally locate the very best possible design. A new "parallel" Fedorov search algorithm has now been implemented that can find the most diverse property-tailored design. An ambiguous mass penalty has been added, whereby most duplicate masses can be eliminated with little loss of library diversity. Sensitivity analysis has also been added to quantitatively explore the diversity trade-offs due to increasing or decreasing each specific kind of bias.     

A general method for designing combinatorial peptide libraries decodable by amino acid analysis

Herein we describe an algorithm for designing combinatorial peptide libraries for split-and-mix synthesis on solid support that are decodable by amino acid analysis (AAA) of the beads. AAA is a standard service analysis available in most biochemical laboratories, and it allows one to control the quality of the peptide on each bead, an important feature that is missing from most library decoding protocols. In the algorithm, each AA is assigned to two variable positions in the sequence grouped in a "unique pair". This arrangement limits sequence design because both the number of unique pairs U (setting the maximum number of variable AA) and the maximum number S of different AA per variable position depend on the peptide length N (U=N(N-1)/2), S=N-1). The method is therefore only suitable for focused libraries. An application example is shown for the selection of peptides with N-terminal proline or hydroxyproline catalyzing an aldol reaction from a combinatorial library of 65536 octapeptides. A simple enumeration program is available to help design combinatorial libraries decodable by amino acid analysis. The method applies to linear and cyclic peptides, can be used for nonnatural building blocks, including beta-amino acids, and should help to explore the vast chemistry of linear and cyclic peptide for catalysis and bioactivity.     

Immunoassays: biological tools for high throughput screening and characterisation of combinatorial libraries

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.     

Combinatorial library design using a multiobjective genetic algorithm

Early results from screening combinatorial libraries have been disappointing with libraries either failing to deliver the improved hit rates that were expected or resulting in hits with characteristics that make them undesirable as lead compounds. Consequently, the focus in library design has shifted toward designing libraries that are optimized on multiple properties simultaneously, for example, diversity and "druglike" physicochemical properties. Here we describe the program MoSELECT that is based on a multiobjective genetic algorithm and which is able to suggest a family of solutions to multiobjective library design where all the solutions are equally valid and each represents a different compromise between the objectives. MoSELECT also allows the relationships between the different objectives to be explored with competing objectives easily identified. The library designer can then make an informed choice on which solution(s) to explore. Various performance characteristics of MoSELECT are reported based on a number of different combinatorial libraries.     

Phage display and colony filter screening for high-throughput selection of antibody libraries

During the last 12 years, antibody combinatorial libraries have provided a new approach for the construction and production of reagents and drugs based on the human monoclonal antibodies. Studies employing antibodies or antibody mimics have become an important part of the explosive growth of proteomics. This places tremendous emphasis on the new approaches for faster library screening, improved methods of selection and evaluation of novel applications. The phage display system, together with its variants of ribosome and bacterial display, is the most extensively used method for the rapid screening of large antibody libraries. However, in the last two years the need to improve selection methods together with a complex patent situation regarding the phage display system, has also directed research towards the possibility of performing antibody selection by colony filter screening. Here, we summarise the results obtained by these different methods of selection comparing their efficacy and advantages.