Design Principles for Fragment Libraries: Maximizing the Value of Learnings from Pharma Fragment-Based Drug Discovery (FBDD) Programs for Use in Academia

Fragment-based drug discovery (FBDD) is well suited for discovering both drug leads and chemical probes of protein function; it can cover broad swaths of chemical space and allows the use of creative chemistry. FBDD is widely implemented for lead discovery in industry but is sometimes. used less systematically in academia. Design principles and implementation approaches for fragment libraries are continually evolving, and the lack of up-to-date guidance may prevent more effective application of FBDD in academia. This Perspective explores many of the theoretical, practical, and strategic considerations that occur within FBDD programs, including the optimal size, complexity, physicochemical profile, and shape profile of fragments in FBDD libraries, as well as compound storage, evaluation; and screening technologies. This:compilation of industry experience in FBDD will hopefully be useful for those pursuing FBDD in academia

Mutational Studies on 17β-HSD14, Serial Synchrotron X-ray Crystallography, Solubility Enhancement using Cyclodextrins and Fragment-Based Drug Discovery Multiple Blocks to Pave the Road of Drug Design

The first topic of this thesis (Chapter 2) presents a mutational study performed on 17β-hydroxysteroid dehydrogenase type 14 (17β-HSD14) S205 variant. Five different mutations were done with respect to five amino acids which are believed to have an essential role in the enzyme activity and assembly. The five variants are: His93Ala, Gln148Ala, Lys158Ala, Tyr253Ala and Cys255Ala. The mutated amino acids are located in the active site of the enzyme (His93, Gln148 and Lys158) or on a flexible loop of the enzyme, which is located above the active site (Tyr253 and Cys255). X-ray crystallography is the method utilized in this study to obtain a 3D crystal structure of each variant. A non-steroidal potent 17β-HSD14 inhibitor (inhibitor 1) has been crystallized in complex with each variant, that has been used to verify the binding capability of the mutated enzyme. Enzymatic assays have been performed with each variant to compare the activity of each one. Estrogen (estradiol) and androgen (5-diol) have been used as a substrate in the enzyme kinetics assay with NAD⁺ as a cofactor. The second part of this thesis (Chapter 3) is focused on a new crystal sample holder (the Roadrunner I chip) which is used in Serial Synchrotron X-ray Crystallography (SSX). The Roadrunner I chip is a micro-patterned sample holder from single crystalline silicon (waiver technology) with micropores. The aim of using the Roadrunner I chip is to have a sample holder that can present hundreds to thousands of crystals to the high intensity PETRA III beam line P11 (DESY – Hamburg) without interfering with the diffraction pattern. In this study, Thermolysin (TLN) is the protein used to test the limit of this new method. Thermolysin crystals were grown, washed, soaked and frozen at cryogenic temperature without removing them from the chip. Data sets were collected of TLN crystals while they are located on the chip. The experimental part of this study was performed at Deutsches Electronen-Synchrotron (DESY), Hamburg at PETRA III P11 beamline in collaboration with associated laboratories at the facility. The third part of this thesis (Chapter 4) discusses cyclodextrins (CDs) and their ability to enhance hydrophobic compounds solubility in aqueous solutions. The targeted protein in this study is 17β-HSD14. Many compounds were assembled for this study, such as a fluorine-compound library, hydrophobic drugs and sex hormones. The aim of this study is to obtain a compound that binds to the enzyme by introducing it as a compound/CD complex. Most of the compounds used in this study have already been tested with 17β-HSD14 without the use of CDs, but due to their low solubility it was not possible to introduce them in crystallization samples of the enzyme. The data obtained from this study show the effect of the compound/CD complex, as it is introduced to the enzyme via co-crystallization method. The fourth part of this thesis (Chapter 5) focuses on a fragment screening. A 96-fragment library is screened against trypsin using X-ray crystallography. This study focuses on the difference of hits and partial hits obtained from the fragment screening. Fragment screening has been performed on two trypsin crystal form (trigonal and orthorhombic). The data obtained from this study show the different results from each screen and how the crystal form and the fragment delivery method influence the hit ratio. Many aspects were considered in this study, such as the difference in electron density, volume of the binding pocket, anomalous peaks and water channels

Targeting Trypanosoma brucei FPPS by Fragment-based drug discovery

Trypanosoma brucei (T. brucei) is the causative agent of the Human African Trypanosomiasis (HAT), which is a neglected disease with an endemic occurrence in 36 sub-Saharan African countries. The current standard of care suffers from low efficacy and severe side effects. Therefore, new drugs with better safety and efficacy profiles are urgently needed. Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their particular pharmacokinetic properties they are not well suited for parasitic therapy. Recently, an additional allosteric site was discovered at the surface of human FPPS that, based on sequence analysis, is likely also present in T. brucei FPPS. The high unmet medical need combined with the discovery of a potential new target site prompted a fragment-based drug discovery approach to identify non-bisphosphonate binders on T. brucei FPPS, which is presented in this work. Fragment screening was performed by NMR and X-ray crystallography. To this end, a robust T. brucei FPPS crystallization system was established enabling high-throughput determination of crystal structures up to 1.67 Å resolution. Structural superimpositions revealed that the allosteric site found on human FPPS is in fact present in T. brucei FPPS. This observation enabled subsequent protein-observed NMR and crystal soaking experiments with established human FPPS binders resulting in three protein-ligand complex structures with bound fragments in the previously unknown allosteric site. For most of the tested binders, Kd by SPR was outside of experimental range for T. brucei FPPS and only for one fragment the Kd on T. brucei FPPS was determined three orders of magnitude higher than the IC50 value on human FPPS. Crystal structural analysis revealed a different binding mode on human and T. brucei FPPS with reduced protein-ligand interactions on T. brucei FPPS, which explains the significantly reduced binding affinity. Encouraged by the detection of first allosteric binders on T. brucei FPPS, fragment pools were screened by ligand-observed NMR and identified hits were followed-up by single compound ligand observed NMR and protein-observed NMR resulting in 25 validated fragment hits for T. brucei FPPS. Validated hits were followed-up by crystal soaking and co-crystallization experiments and seven protein-ligand complex structures were solved using PanDDA. Out of the seven fragments, four fragments were bound in the active site, one fragment was detected in the allosteric site that was identified as part of this thesis, and two fragments were bound in surface exposed binding sites. Notably, an active site bound fragment with a four atom long flexible linker adopted an orthogonal binding mode along αD when compared to the other three ligands. Sixteen fragment analogues of the elongated flexible active site fragment were tested by SAR using additional test compounds retrieved from catalogue and archive, and one crystal structure with a fragment analogue was solved and was surprisingly found in the allosteric site. In addition to the NMR fragment screen, an X-ray screen was performed at XChem (Diamond, UK) and at EMBL/ESRF (Grenoble, FR) resulting in seven protein-ligand structures. One fragment was positioned in the active site, three fragments in the allosteric site, two fragments in a cryptic site between helices αI and αH and one fragment at the opposite side of the allosteric site close to αG and αF. Fragment binding was further validated in protein-observed NMR. As fragments identified by such screening approaches typically exhibit low binding affinities usually in µM to mM range, structure-based fragment optimisation based on a fragment merging and growing approach was performed. In total, ten compounds were synthesised and subjected to protein observed NMR and X-ray structural analysis. Strikingly, a fragment merger based on T. brucei and T. cruzi active-site binders bound in a new binding site close to the SARM instead to the active site. Taken together, this work presents high-resolution structures of T. brucei FPPS and identified 19 compounds binding to seven different sites thereby paving the way for future studies aiming to identify high-affinity non-bisphosphonate inhibitors for T. brucei FPPS with pharmacokinetic properties that are suitable for parasitic indications

Fragment based ligand discovery : library design and screening by thermal shift analysis

The central idea in Fragment Based Ligand Discovery (FBLD) is to identify small, low molecular weight compounds (MW < 250) that bind to a particular protein active site. Hits can be used to efficiently design larger compounds with the desired affinity and selectivity. Three approaches to FBLD are described in this thesis. The first topic is the development and assessment of different chemoinformatics procedures to select those fragments that maximally represent the chemical features of a larger compound library. Such a fragment library could be of great value in the so-called “SAR by Catalogue" approach, where the initial stage of fragment growth is by selecting existing compounds that contain sub-structures of the hit fragments. Five schemes implemented in the Pipeline Pilot software are described. The second project was to develop improved approaches to processing Thermal Shift Analysis (TSA) data. The shift in melting temperature can indicate that a ligand binds and thus stabilises a protein. A program, MTSA, has been written which allows more straightforward processing of the experimental data than existing available software. However, detailed analysis of fragment screening data highlighted difficulties in defining the melting temperature and suggest that TSA is not sufficiently reliable for routine screening use. Finally, a number of proteins were assessed experimentally for suitability for FBLD: N-myristoyl transferase (NMT), the bacterial homologue of a GlcNAcase enzyme (BtGH84) and the model system hen egg white lysozyme (HEWL). It was not possible to produce suitable NMT material due to the inherent instability of the protein produced in York. The screening results of HEWL with a new Surface Plasmon Resonance (SPR) assay, a cell based activity assay and TSA were inconsistent and difficult to interpret. However, BtGH84 was suitable for screening by both TSA and SPR. The resulting fragment hits are suitable starting points for further evolution.EThOS - Electronic Theses Online ServiceGBUnited Kingdo