While chemistry is closely related to the identification of new active principle, diagnostic and therapeutic strategies in Life Sciences, it is both a pure and an applied science, whose drivers have a significant impact on drug discovery and development.

These drivers include:

• Molecular understanding of three-dimensional structures of chemical entities involved in biological interactions (target and drug) using computational chemistry/molecular modeling and chemoinformatics;
• The synthesis of small organic molecules as active lead candidates, using medicinal chemistry expertise and experimental protocols aiming to individual compounds (iterative medicinal chemistry) or libraries (parallel/combinatorial chemistry);
• Analytical/ bioanalytical chemistry techniques determining the quality of pharma R&D, and the fate of hits, leads and candidates in any in vitro, ex vivo and in vivo assay.

Queste tecnologie sono presenti in Piattaforme Tecnologiche gestite da CISI scrl, e saranno brevemente discusse qui di seguito. Saranno menzionate anche altre tecnologie reperibili attraverso il network scientifico-tecnico di CISI scrl, ed ubicate nel, o in prossimità del Polo Scientifico Dulbecco.

These technologies are described in detail in a specific section, while a few others available through CNR and/or UniMi and located at or near the Scientific Center Dulbecco are also briefly mentioned.

1. Computational Chemistry/Molecular Modeling

The term molecular modeling is used to denote the set of theoretical approaches and computational techniques that allow to reproduce the behavior of molecules. Molecular modeling is used in pharmaceutical chemistry, bioorganic chemistry, and molecular biology for studying molecular systems ranging from small molecules to large biomolecules and polymeric materials.

The modeling platform at CISI scrl offers state of the art hardware and software for Life Sciences applications, and may also benefit from resources for high performance computing, available from the Interuniversity Consortium CILEA.

Two are the priority themes within the research activities at CISI scrl:

• design and virtual screening (vHTS) of libraries of bioactive molecules;
• Evaluation of drug-likeness (early ADMET properties) of libraries of bioactive molecules.

In the first area of research, through the characterization of a growing number of molecular targets and the development of parallel/combinatorial chemistry, computational modeling can guide the design and generation of molecular diversity in chemical libraries of compounds. The techniques of high-throughput virtual screening (vHTS) allow the analysis of large in silico virtual libraries by examining the affinity of each library individual toward a target with known 3D structure (structure-based drug design, SBDD), or to assess its similarity with a pharmacophore (ligand-based drug design, LBDD). Molecules that interact better with the in silico target (SBDD) or more are similar to a pharmacophore (LBDD) are the output of a virtual screening, and are then characterized (after synthesis or purchase, if commercially available) in suitable biological assays. The results will be used to plan the synthesis of new and more potent analogues, and to refine the computational model, which will be used during the whole project lifespan to lead experimental efforts.

The most promising molecules (virtual hits) may also be subjected to a screening designed to determine rapidly their drug-likeness. In detail, their in silico solubility, permeability, toxicity, potential for metabolic transformation, aspecific protein binding, or inhibitory activity on essential enzymes (hERG, CYP450, etc.) will be measured. This eADMET virtual screening will be routinely used in every project, to prioritize the hits and leads to progress, as well as to their efficacy and selectivity on the target molecules, also according to their probability of being drug-like, i.e. non toxic, bioavailable and stable.

2. Chemoinformatics

The chemoinformatic platform at CISI scrl enables the management of chemicals acquired (reagents), synthetized (by iterative synthesis and by parallel/combinatorial chemistry), or purchased as screening products (catalogs) to make a CISI scrl database. This system enables the selection of building blocks to create and enumerate large virtual libraries (virtual HTS).

1D/2D searches can be made on this 2D database (consisting of ≈ 10M available molecules), either applying filters based on molecular descriptors and settingparameters for substructure search. A smaller subset of drug-like, diverse compounds (≈ 30K) is available as a 3D database of conformers (20 million) that can be easily integrated with molecular modeling software for virtual screening of large virtual libraries, examining their affinity towards a target with known 3D structure (structure-based drug design) or their similarity with a pharmacophore (ligand-based drug design).

The platform was developed using the appropriate software, both open source and commercial, and is available in form of a complete client/server application. The software implemented on the computer server manages all aspects of integrating web graphics to the backend computing and to database management. It is also interfaced with our molecular modeling software (molecular docking, molecular dynamics, pharmacophore screening, etc..) described in computational chemistry/molecular modeling, and may be connected with other softwares in future. Desktop computers only need a web browser and the necessary libraries to interact with Java applets for handling chemical structures (chemical software based on Microsoft Excel).

This platform allows CISI scrl to have a significant database for chemical compounds, and expands the provision of means of investigation for the molecular modeling platform, especially regarding the enumeration of virtual libraries. Its interface with future public and private partners and collaborators will be made to ensure the real-time access to data generated, and to ensure the right level of protection for sensitive data.

3. Iterative Medicinal Chemistry

The platform for iterative medicinal chemistry is devoted to the synthesis of molecules with potential biological activity for at diagnostic and therapeutic applications. Organic synthesis laboratories are equipped with the most modern equipment for the chemical synthesis both in solution and on solid phase, and for parallel/combinatorial chemistry. The laboratories are equipped with advanced equipment for automated purification of products (SP1 and SP4, Biotage; VacMaster, IST) and with modern synthetic systems such as a microwave reactor (Initiator 60, Biotage) and continuous flow reactors (H-Cube, ThalesNano).

Despite the apparent obviousness of this platform and its contents, it largely influences the success of the work done at CISI scrl. Indeed, synthetic strategies used for high throughput synthesis are invariably developed in an iterative format, and the quality (yield, purity) of all compounds produced depends on the validation/assessment performed at this stage.

In detail, compounds synthesized in the early stages of a project (hit discovery, hit-to-lead) have always a high purity, suitable for in vitro profiling. The minimum standards is set at 90% purity (NMR and HPLC/MS), and the quantities for each product will never be below 15 mg, to allow its complete in vitro profiling and storage in the database as a reference sample. As to lead compounds (lead optimization), they will always have an extreme purity, suitable for in vivo administration. The threshold level of purity will be set at 95% and will rise to 98% (NMR and HPLC / MS) for compounds to be tested in vivo. In such a case, the quantities for each product will never be lower than 250 mg, to allow both complete profiling in vivo (in various efficacy and/or PK animal models), and storage as reference samples.

4. Parallel/Combinatorial Chemistry

The use of so-called high-throughput technologies has become common in many areas. Among them, in medicinal chemistry we talk about parallel chemistry (simultaneous synthesis of discrete compounds), or combinatorial chemistry (simultaneous synthesis of all permutations of different replacements for two or more decorative functional groups in a biologically active molecule), or high throughput synthesis (as HT Screening counterpart) meaning the platform that lets you get quickly libraries of compounds that can then be used for many different applications (pharmaceutical, diagnostics, new materials, catalysis, etc.).

The skills and equipment mentioned above are hardly available in their entirety in a single research group. They are all present at CISI scrl, along with a strong expertise in medicinal chemistry, allowing the use of parallel/combinatorial chemistry approaches within pharmaceutical or diagnostic projects, with no limitations related to therapeutic areas or phases of project.

Our organic synthesis laboratories, hosting medicinal chemistry efforts in both an iterative/classic and a parallel/combinatorial format, are equipped with modern instrumentation for manual (Radley Carousels), semi-or fully automated parallel synthesis (Bohdan Miniblock, Mettler Toledo) both in solution and on solid phase allowing the preparation of dozens (reduced format) to several hundreds (expanded format) of compounds structurally related to each other in a single run. This is ideal for fast acquiring structure-activity relationship (SAR). Advanced instruments for automated purification of products, already mentioned for iterative chemistry, are used to their maximum capacity to meet the same stringent standards of purity discussed for iterative medicinal chemistry (90-95-98%) for each library member.

5. Analytical/Bioanalytical Chemistry

LC-MS methods are among the most versatile analytical techniques for the qualitative and quantitative characterization of small molecules, allowing very low detection limits even in complex matrices. Their application range is extremely broad. At CISI scrl, the main role of these techniques is to support synthetic chemistry (iterative medicinal chemistry and parallel/combinatorial chemistry), and to provide assistance for the purification of products.

HPLC-MS techniques already established at CISI scrl are particularly suitable for the following applications of industrial interest: characterization of compound collections (chemical libraries), isolation and characterization of reference compounds, determination of impurity profiles and structure identification of unknown impurities, development of rapid analytical methods. The available equipment allows analytical (high throughput, timely analysis of samples from iterative and parallel synthesis) and preparative runs (purification of hundreds of milligrams of pure compounds).

The available HPLC-MS instrumentation includes an ion trap instrument (Bruker Esquire 3000+) connected to an Agilent 1100 chromatograph, and an analytical-preparative Waters Fractionlynx chromatograph connected to a single quadrupole mass detector (Waters ZQ), which allows to automate the purification of synthetic compounds. In addition, a UPLC chromatograph (Waters Acquity) connected to a single quadrupole mass detector raises the forefront of efficiency and speed in performing chromatographic analysis (2-3 minutes per analysis).

Nuclear Magnetic Resonance (NMR) spectroscopy is also successfully applied in various fields related to Life Sciences research. The ability to observe ¹H as well as other nuclei such as ¹³C, ¹⁹F, ¹⁵N and ³¹P makes NMR spectroscopy the most powerful and versatile technique available to chemists and biochemists to obtain information on the structure of compounds with high molecular weight, and to understand in depth the biological processes and mechanisms of reaction (for example, through in vitro and in vivo imaging techniques).

The available equipment includes two NMR spectroscopes (Bruker Avance 400 MHz and Avance 600 MHz), both equipped with a 60 positions autosampler, with modern gradient probes and appropriate software for automatic management of single-or two-dimensional experiments, 7 days a week. Two-and three-dimensional sequences allow the study of very complex biomolecules such as proteins or polysaccharides without neglecting the support to be given to synthetic chemistry. The information obtained by NMR spectroscopy, rationalized through molecular modeling studies, allow both the detailed conformational analysis of these molecules in solution and the determination of their bioactive conformations.

Recently NMR was used by us with excellent results for the structural determination of protein-ligand complexes and to screen of ligands to target proteins, providing key information in the search for pharmacologically active molecules.

The experience of our staff and the available instrumentation allows us also, when necessary, to develop HPLC-MS and/or NMR reliable and reproducible standard methods for the quantitative analysis of mixtures of compounds and biological fluids, to support forthcoming in vitro (eg, eADMET assays) and in vivo experiments (e.g., efficacy experiments or bioavailability/ pharmacokinetic profile experiments in several animal species).

6. Other Accessible Platforms

The location of CISI scrl in an environment hosting several UniMi and CNR laboratories provides access to potentially useful techniques on an "if and when necessary” basis, mostly because of the excellent relationships between CISI scrl and its institutional shareholders.

Perhaps the most important accessible platform for Life Sciences R&D, for which it is planned in future the implementation at CISI scrl, is X-ray Crystallography. It is well-known that such a platform can provide information on the structure of target proteins and their complexes with different molecular partners, selected for Biotech/Life Sciences purposes. This platform deals with cloning, expression, purification (mg scale), biochemical and biophysical characterization of proteins, crystal growth and resolution of 3D structures with crystallographic methods. Similar approaches, now popular and known collectively as fragment-based drug discovery (FBDD), promise to make much more efficient the whole process of discovery and optimization of pharmacologically active principles, and are complementary rather than alternative approaches to computational chemistry/molecular modeling. The existing close cooperation with Prof. Martino Bolognesi at UniMi gives access now to this platform for ongoing or future projects, and provides competent support to create a similar platform internally at CISI scrl.