COMBINATORIAL CHEMISTRY IN A HISTORICAL PERSPECTIVE

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1 NUE FEATURE T R A N S F O R M I N G C H A L L E N G E S I N T O M E D I C I N E Nuevolution Feature no. 1 October 2015 Technical Information COMBINATORIAL CHEMISTRY IN A HISTORICAL PERSPECTIVE A PROMISING PARADIGM - WITH CHALLENGES I n the discovery of small-molecule drug candidates, a starting point is required for the optimisation process. This is sometimes an endogenous ligand, if this is known (endogenous ligand being the molecule Nature evolved to bind to the biological target of our interest). However, the endogenous ligand often represents a very challenging starting point from a drug-likeness perspective. Most often, the starting point is therefore generated by screening of a collection of compounds, a high-throughput-screening (HTS) deck, with the hope of identifying hits that can form the starting point for the further optimisation process towards the drug candidate. The more compounds screened, the higher the chance of finding hits, and the more compounds screened, the more structure activity relationship (SAR) information may be obtained, which will help the medicinal chemist in directing the optimisation process. For HTS decks to be highly valuable, they therefore have to comprise a high number of diverse compounds to increase the chance of finding hits, and they must have density, i.e. analogues within the different chemical series to generate SAR. Finally, but not least, compounds should have high drug-likeness, i.e. promising properties from a drug development point of view. Classical HTS decks are rarely homogeneous. Some chemical series are likely to be highly populated, whereas other series only have one or a few representatives. Overall, the diversity and density in classical HTS decks are insufficient. This, combined with application of classical medicinal chemistry for the optimisation process, makes discovery of drug candidates a costly and time-consuming endeavor. An increasing need to access highly diverse sets of compounds (libraries) for screening spurred the establishment of combinatorial chemistry in the late 1980s and early 1990s. It was hoped that the screening of many compounds would provide a significant improvement in the speed of and success rate of the identification of small-molecule drug candidates. However, the challenge in realising this potential was and is multifold: 1. How can large collections of compounds be synthesized quickly and at low cost? 2. How can large collections of compounds be screened quickly and at low cost? 3. How can drug-likeness and diversity be achieved with such an approach? SYNTHESIS OF MANY COMPOUNDS PARALLELIZATION OF CHEMICAL SYNTHESIS S ynthesis of high quality organic compounds is in general both costly and time-consuming. Two principles i.) parallel synthesis and ii.) split-and-mix also known as split-pool synthesis have been developed for the purpose of allowing access to more chemical compounds for hit discovery.

2 i). In parallel synthesis, compounds are synthesised one-by-one in separate compartments (wells, vials, flasks etc.), but in a parallel format allowing hundreds to a few thousands of compounds to be synthesised and purified per campaign. This approach leads to single compounds in a purified state, and each compound can be subjected directly to screening for activity in biological screening assays. However, the production of millions of compounds by this approach is costly, and the production of hundreds of millions to billions of compounds by this approach is nearly impossible and extremely costly. ii). In split-and-mix/split-pool synthesis, compounds are synthesised stepwise by use of two, three or more sets of reagents, which are combined with each other in the production of a mixture (a collection of compounds, a library), in which all or most reagents from one set have been reacted with all or most reagents from other sets in the production of all theoretical possible combinations. In 1998, research in Stuart Schreiber s laboratory achieved an impressive 2.18 million member small-molecule library consisting of a mixture of one compound per bead following a six-step reaction sequence (J. Am. Chem Soc., 1998, 120, ). The screening of complex compound mixtures requires that the structure of active compounds can be identified following screening. In order to obtain this structure, all the compounds synthesised are encoded by a tag that holds information about the reagents and conditions used for synthesis of the individual compound, i.e. by reading the tag, the structure of the active compound can be deduced. This approach of producing code-tagged compounds allows the compounds to be synthesised and screened as a mixture, whereby both production cost and screening cost are significantly lower than for the synthesis and screening of compounds one-by-one. Unfortunately and because of the tags used at the time, it was only to a limited extend possible to reliably screen large compound sets. In most cases, compound collection sets in the order of only thousands of compounds were used in affinity based screenings (selections). Of the two core approaches i.) parallel synthesis and ii.) split-and-mix/split-pool approaches, only the split-andmix/split-pool approach allows access to very large compound libraries. PRINCIPLE FOR SPLIT-AND-MIX COMBINATORIAL LIBRARY SYNTHESIS SCREENING OF MANY COMPOUNDS SCALING TO MILLIONS ROBOTIC SYSTEM FOR FOR HTS T oday s state-of-the-art HTS setup in big pharma may allow screening in the order of an impressive 1 million compounds per week by conventional methods, where compounds are tested one-by-one. However, the screening of, for example, 1 billion compounds by the conventional HTS approach would take years, i.e. that is not practical. Therefore, conventional HTS of compounds applying the testing of compounds one-by-one is not easily scaled further, which limits the size of the screening deck (library) to the order of a few million compounds. Also, conventional screening methods make use of a high concentration of test compounds e.g. HTS typically uses 10 μm, and in Fragment Based Drug Discovery (FBDD) an even higher compound concentration is needed e.g. 100 μm - 1 mm. Precipitation and decomposition of test compounds thereby

3 are major causes of the generation of false positives during screening, the number of which would further increase if the size of the classical HTS deck is increased. DEVELOPMENT OF ENCODING TAGS SCALING TO BILLIONS W hen a mixture library produced by the split-and-mix method is subjected to screening, it will lead to an output that is also a mixture, a mixture consisting of a minor background of inactive compounds, compounds with low or moderate activity, and potentially compounds with high activity for the target against which the library was screened. The screening method must therefore allow compounds in the output to be ranked according to activity, and the tag used must allow for detection of even trace amounts of material of the individual compounds present in the output. The requirement is therefore that compounds isolated following screening: i.) Can be unambiguously identified by detection of their tag by a robust and high-fidelity process ii.) Are ranked according to the relative activity of compounds for the target against which the library was screened In order to achieve good ranking of ligands (binders to the target) isolated following, for example, affinity chromatographic (panning) screening methods, this approach requires that library members are represented with an approximately similar number of copies, i.e. similar synthesis yield of individual compounds. Therefore, significant investment in establishing multiple reaction protocols for each type of reaction to be used is needed, and furthermore careful testing of the individual reagents to be used in the library is mandatory. This means much preparative hard work prior to library generation in order to maximise the yield of individual library molecules, and to make the library composition as homogeneous as possible. Different tags were developed in the 1990 ies, where e.g. Pharmacopeia Inc. developed the ECLiPS technology (see for example J. Org. Chem., 1994, 59, ) employing library compounds on polymeric support encoded by polyhalogenated aromatic tags, and Neogenesis Inc. developed its ALIS technology (see for example Int. J. Mass Spec., 2004, 238, 77-83) based on virtual mass tags, i.e. each compound within a mixture had a unique molecular weight. These approaches were developed to a level that allowed the screening of up to about 5 million compounds. In the case of Neogenesis, this was performed by the screening of 2000 separate mixtures, each of about 2500 compounds. Many dedicated pioneers were highly engaged in the competitive field of combinatorial chemistry in the 1990s, applying different approaches (see for example the exciting historical interview-based review by Michael Lebl (J. Comb. Chem., 1999, 1, 3-24), but most encoding tags did not at the same time allow the sensitivity needed for handling of compound mixture libraries in the range of hundreds of millions to billions of compounds with high fidelity, while at the same time allow formation of small molecules. Oligonucleotides such as, for example, DNAbased encoding tags offered extremely high sensitivity in terms of detection because the tag could be amplified by PCR methods. However, although peptidic compound libraries containing DNA tags were contemplated in the late 1980s to early 1990s, attempts pioneered by Affymax (see for example Proc. Natl. Acad. Sci., 1993, 90, ) to develop a feasible method proved challenging. In their approach, the DNA-tag was synthesised using chemical synthesis and the peptide or polymer library molecules also required chemical synthesis. Brenner and Lerner suggested a similar AFFYMAX PRINCIPLE FOR ENCODED LIBRARIES concept about the same time in a theoretical patent and paper (Proc. Natl. Acad. Sci., 1992, 89, ). However, the approach pioneered by Affymax and the theoretical concept by Brenner and Lerner required a substantial need for orthogonal protection groups on both the library molecule and the DNA tag in the synthesis. The prerequisite for protection groups that could be removed selectively made the approach very complex and severely limited it. At the same time, it was the general opinion that oligonucleotides as encoding tags were suboptimal from a chemical reaction point of view, which then because of the library design principles also applied at the time made it difficult to realize truly diverse and drug-like small molecule libraries. At the end of the 1990s, the use of DNA for tagging was not preferred by researchers in the field.

4 WORK AT NUEVOLUTION PIONEERING NEXT GENERATION DNA ENCODED SMALL MOLECULE LIBRARY I n 2001, Nuevolution commenced its work in the field of combinatorial chemistry at a time when combinatorial chemistry was getting out of fashion and certainly the use of compound libraries encoded by oligonucleotides such as DNA was considered not advisable. We chose to revisit the use of oligonucleotides as the encoding tag because of the unique properties providing for single-molecule detection due to the advent of polymerase chain reaction (PCR) and DNA sequencing. In the early years of , we explored multiple encoding setups, which may be roughly categorised into two principles: i.) Amplifiable template-based methods ii.) Non-Amplifiable non-template based methods (split-and-mix/split-pool) For numerous reasons, the split-and-mix principle provided the best option for the efficient production and screening of high-complexity and drug-like small-molecule libraries. We discovered that use of enzymes for synthesis of the encoding tag allowed us the option to avoid the use of protecting groups on the oligonucleotide tag, whereby we could massively improve synthesis options. This combined with changed library design principles made the overall synthesis of ultra diverse drug-like small molecule libraries practical and useful for lead discovery. Since 2001, Nuevolution has invested significantly in the innovative development and optimisation of chemistries, the acquisition and reactivity testing of an unprecedented number of library building blocks, the development of multiple selection methods including methods for minimisation of false positives and elimination of artifacts, the development of customised cheminformatics software to guide optimal design of libraries, and secure analysis of large data screening outputs to maximise the quality of hit identification. All together these, provide for a highly robust and reliable screening engine capable of identifying drug-like small-molecule hits NUEVOLUTION SETUP FOR SCREENING OLIGONUCLEOTIDE ENCODED LIBRARIES from ultra-diverse libraries. FROM SCREENING TECHNOLOGY TO LEAD DISCOVERY PLATFORM TRANSFORMING THE DRUG DISCOVERY PROCESS N omination of the Clinical Candidate is the ultimate goal for the discovery project. Being conscious about this, we have over the years evolved our approach to realize an integrated and efficient platform technology Chemetics for both syntheis and screening of ultra large libraries (1+ billion small molecules), with forward integration of the optimization process to leads and candidates (hundreds of analogues synthesized and purified per week). This represents a massive improvement compared to conventional HTS (few millions of small molecules for screening) and conventional medicinal chemistry (hundreds to thousands of analogues in years).

5 More features: On our website we offer access to a variety of features addressing our technology and information of relevance in the therapeutic areas and business of ours. In addition, we on a regular basis, publish invited features on aspects of drug discovery and development. CONTACT DETAILS NUEVOLUTION RØNNEGADE 8 DK2100 COPENHAGEN DENMARK NUEVOLUTION.COM FEEDBACK@NUEVOLUTION.COM ABOUT NUEVOLUTION N uevolution is a privately-owned biopharmaceutical company focused on developing treatments for human diseases within oncology and chronic inflammatory diseases. Nuevolution is the inventor of Chemetics, a platform which enables efficient discovery of novel small molecule drug candidates for specific indications. Nuevolution has applied the Chemetics platform to deliver leads for pharmaceutical partners and its own pipeline of programs. For additional information about Nuevolution and its programs, please visit nuevolution.com Nuevolution In referring of this feature please use the following reference: NUEsFEATURE, 2015, 1, nuevolution.com/features DISCLAIMER The views and opinions expressed in this Feature may contain statements relating to Nuevolution technology, programs and strategic objectives. Any forward-looking statements made are based on assumptions and expectations, based on current knowledge and they may not be applicable in future performances of the company. Nuevolution undertake no obligation to revise the content of the present Feature whether because of new information, future events or otherwise. Nuevolution shall not be held liable for any damages related to the content and use of information provided in the Feature.