The Chemetics® Platform

Chemetics® , Nuevolutions drug discovery technology, combines organic chemistry and molecular biology enabling the synthesis and DNA-tagging of small drug-like compounds. Each compound within a Chemetics® library is linked to a strand of DNA encoding the synthetic history and chemical structure of the small molecule (Figure 1). The use of DNA to store chemical information provide a highly sensitive means of identifying, by amplification and sequencing, even minute quantities of small molecules isolated from affinity screens. Ultimately, this leads to the identification of potent leads in very small amounts: a prerequisite to screening ultra-large mixtures (combinatorial libraries) of compounds.

Figure 1. Constituents of a Chemetics® molecule.

Chemetics® molecule consisting of a small molecule entity attached through a 20 Ångstrom flexible linker to a DNA sequence that specifically identifies the structure of the small molecule.

Molecular Encoding by Ligation (MEL)

Nuevolution has developed MEL (Molecular Encoding by Ligation) for the production of its ultra-large compound libraries. This technique uses the “split-and-mix” synthesis approach, in which MEL offers a means of tracking the synthesis process for each chemical compound, allowing them to be subsequently identified by their encoding DNA-tag. The basics of MEL library generation is outlined schematically in Figure 2 and involves the following steps:

1. To a pool of microtiter plate reaction wells is added a starter oligonucleotide carrying a flexible linker and a reactive group (X).

2. A unique oligonucleotide sequence (DNA-barcode) is then introduced to each well and ligated to the starting oligonucleotide by use of a ligase.

3. A unique drug fragment carrying reactive groups is then introduced into each well and conjugated with the encoding DNA sequence through reaction with group X.

4. Microtiter plate wells are pooled (mixed) and distributed (split) equally among the wells of a new microtiter plate. Each well on the new microtiter plate will thus contain reaction products from all microtiter wells from the previous reaction round.

5. Steps 2-4 are repeated 2 or 3 times to yield trimeric or tetrameric molecules, i.e. molecules generated from 3 or 4 different drug fragments.

Figure 2. Mel Library Generation: Split & Mix DNA-tagged synthesis.

By this sequential reaction process, each unique drug fragment will become linked to a unique DNA-barcode, with the final molecules containing a complete sequence of DNA describing its synthetic history and composition.

At present, MEL facilitates the production of 100-200 million (108) small molecule libraries in 3-4 weeks, depending on the nature of the compounds, with the scope to extend library size to 1012 compounds.

The MEL protocol is highly flexible and can produce libraries containing linear, branched, macrocyclic and/or scaffolded molecules depending on design preferencies

Chemetics® libraries can contain any chemical motif employed in medicinal chemistry. The MEL system affords chemical reactions in aqueous or organic solvents or any desirable mixture thereof. Thus, reaction conditions can be customized allowing for almost any building block and attachment chemistry compatible with DNA

Chemetics® libraries are drug-like, and specifically designed to have high structural and functional diversity for hit generation and a focused design is applied for hit diversification and hit-to-lead optimization without compromise in regards to library size.

Screening of 100 Million Member Compound Libraries

The Chemetics® small molecule selection procedure enables the isolation and identification of compounds with high affinity and specificity for a relevant drug target. The Chemetics® selection procedure and the subsequent analysis of potential leads are shown in Figure 3 below.

Figure 3. Small Molecule Lead Generation Using Chemetics®.

The key Chemetics® selection process steps are:

1. A library of typically 100-200 million Chemetics® molecules is initially generated using the MEL protocol. This process will typically take from 3-4 weeks.

2. The target protein is immobilized and buffer/washing conditions are optimized to establish optimal small molecule screening conditions. This process will typically take Nuevolution from 2-10 weeks depending on the target.

3. The library of Chemetics® molecules is applied to, for example, a column to which target protein has been immobilized. A portion of the Chemetics® molecules bind to the target protein, whilst non-binders are removed by washing.

4. The pool of ligands (small molecules that bind to the target protein) with their associated unique DNA tag are then eluted and isolated, either by denaturing of the target protein (using for example high salt concentration), or by addition of a specific known ligand to displace site specific ligands.

5. The DNA recovered is amplified by standard PCR methods, cloned and sequenced. In principle, a single DNA strand may be detected and sequenced, allowing the identification of its corresponding small molecule. This process takes approximately a week.

The identified ligands are analyzed for their drug-likeness and clustered into different structural families using standard in silico methods. Based on this analysis, a smaller number of compounds are synthesized in milligram scale and tested using appropriate in vitro and in vivo assays. Based on this in silico analysis and assay data, a new multi-million member “focused” library is generated. This second generation library is focused around the chemical motifs identified in the initial screen, and is therefore designed to achieve a much higher number of hits than the first generation library, from which good leads may be selected. This process can then be repeated as many times as necessary to afford a high-quality optimised lead and back-up compounds.

Classical synthetic chemistry is therefore not performed until drug-like compounds of high affinity have been identified, at which time the identified compounds are readily synthesized by Nuevolution using parallel synthesis techniques.

In summary, the screening of 100 million compound libraries is a “one-pot” process, as the entire library is screened in a single application. New ultra-large focused libraries can then be synthesized and screened in as little as 4-6 weeks.

Flexible Design of Highly Diverse Ultra-Large Libraries

It typically takes only one person 3-4 weeks to produce a 100-200 million member Chemetics® library. Figure 4 illustrates a representative subset of 40 compounds from one Chemetics® library. This compound subset was picked in order to illustrate the structural and functional diversity of the Chemetics® libraries. The 40 compounds cover a wide spectrum of extended and globular three-dimensional structures, as well as a diverse representation of functionalities. None of these compounds violate the Lipinski criteria for drug-likeness, and are therefore (according to Lipinski) considered drug-like. Nuevolution applies this same level of selection rigour to the synthesis of its proprietary scaffolds and building blocks, and to those compounds analyzed from its screenings.

Figure 4. Representative Series of Diverse Chemetics® Library Molecules.

Nuevolution may choose to use highly diverse, naive libraries for primary screening of new targets, and use libraries which have been biased towards over-representing certain properties or chemical motifs in subsequent screening rounds. The use of ultra-large Chemetics® libraries in iterative target screening rounds, allows the library designer full flexibility to adjust the properties of compounds to be screened. The designer may thus, for example, increase or decrease lipophilicity or molecular weight in later library generation rounds. The large size of the Chemetics® libraries ensure that the probability of compromising ligand potency or selectivity is low and much smaller in comparison to the fewer compounds produced and tested by classical medicinal chemistry methods.
The design of drug-like libraries, the synthesis of novel and diverse scaffolds, and the sourcing of diverse sets of commercially available building blocks represent important elements of Nuevolution’s skill sets, know-how and technology base.