Virtual Screening and Biochemical Testing of Borocycles as Immunoproteasome Inhibitors

Inhibition of the immunoproteasome (iCP) offers new opportunities in the treatment of cancer, autoimmune disorders and neurodegenerative diseases. Inspired by the success of boronic acids as proteasome inhibitors we have complied a virtual library of commercially available 5- and 6-membered borocycles and performed a structure based virtual screening against the chymotrypsin-like (β5i) subunit of the iCP. The top scored docking poses were visually inspected to select compounds for experimental testing. Six compounds with 5-membered ring and another six compounds with 6-membered ring were subjected to biochemical tests. All compounds exhibited detectable inhibitory activity at 100 µM concentration and these are the first reported cyclic boronic acid inhibitors of the iCP. Structural variations including the ring size and the substitution of the borocyles and the substitution pattern of the attached aromatic ring resulted in no major variation of the inhibitory activity. We propose that the evaluation of larger cycling boronic acid libraries is needed to fully elucidate the potential of these structures

Evaluation of Fragment Libraries and Their Use for the Discovery of Promising Hit Compounds on Enzyme Targets

Odkrivanje novih učinkovin je zapleten in dolgotrajen proces, ki je skozi zgodovino doživel pomemben napredek. Razvil se je od naključnih odkritij, pogosto iz naravnih proizvodov, do sodobnih pristopov, ki so bolj sistematični in temeljijo na rešetanjih kemijskih knjižnic ali racionalnem načrtovanju. V zadnjih desetletjih se je načrtovanje novih učinkovin na osnovi fragmentov izkazalo kot uspešen pristop in dobra alternativa rešetanjem visoke zmogljivosti. Začne se z rešetanjem majhnih, strukturno enostavnih spojin, ki predstavljajo dobra izhodišča za optimizacijo delovanja in povečevanje strukture do spojin vodnic. V doktorski disertaciji smo raziskali potencial rešetanja fragmentov za odkrivanje obetavnih spojin zadetkov na tarčah, ki jih preiskujemo na fakulteti. Pripravili smo knjižnico več kot 1.000 fragmentov in ovrednotili njihovo delovanje z različnimi biokemijskimi testi. Razvili smo delovni tok za temeljito vrednotenje zadetkov, s katerim lahko izločimo lažno pozitivne rezultate rešetanj. Zajema potrditev čistosti in identitete, eksperimentalno vrednotenje topnosti, reaktivnosti in redoks aktivnosti. Dodatno smo potrdili vezavo oz. delovanje obetavnih zadetkov z ortogonalno metodo. Do sedaj znani testi za določanje redoks aktivnosti niso imeli zadostne zmogljivosti ali niso bili primerni za rešetanje raznolikih kemijskih knjižnic. Z razširjenim naborom pozitivnih kontrol smo jih optimizirali in pripravili protokole, s katerimi lahko zaznavamo različne mehanizme redoks aktivnosti. Pokazali smo široko uporabnost testov, in sicer za vrednotenje spojin zadetkov, že znanih bioaktivnih spojin in kemijskih sond, za razvoj kovalentnih fragmentov in spojin z elektrofilnimi bojnimi glavami. Najbolj obetavne rezultate rešetanja knjižnice fragmentov smo dosegli na bakterijskem encimu D-alanin:D-alanin ligaza in podenoti ß5i imunoproteasoma. Poleg tega smo raziskali alternativne pristope k rešetanju fragmentov, vključno z virtualnim rešetanjem in uporabo kemijskih knjižnic kodiranih z DNA. V vseh primerih je bilo vrednotenje zadetkov ozko grlo in ključen korak za nadaljnji uspeh. S širšo analizo rezultatov rešetanja fragmentov na različnih tarčah smo ugotovili, da je treba biti pozoren na spojine s tiazolnim skeletom, ki zaradi reaktivnosti pogosto lahko izkazujejo nespecifično delovanje.The discovery of new drugs is a complex and long-term process that has made significant progress over time. It has evolved from serendipitous discoveries, often of natural products, to modern, more systematic approaches based on chemical library screening or rational design. In recent decades, fragment-based drug design has emerged as a successful approach and an alternative to high-throughput screening. It starts with screening small, structurally simple compounds as potential starting points for optimization into lead compounds. In this dissertation, we investigated the potential of fragment-based drug design for the discovery of promising hit compounds for targets under investigation by our faculty. We assembled a library of over 1,000 fragments and evaluated their activity using various biochemical assays. We have developed a workflow for thorough hit evaluation to rule out false positives from screenings. This includes confirmation of purity and identity, as well as experimental evaluation of aqueous solubility, reactivity, and redox activity. For most promising hits, we confirmed activity or target binding using an orthogonal method. Previously known assays for the determination of redox activity did not have sufficient capacity or were not suitable for testing diverse chemical libraries. Using an expanded set of positive controls, we optimized them and established protocols to detect various mechanisms of redox activity. We demonstrated the broad applicability of reactivity and redox activity assays, particularly for the evaluation of hit compounds, known bioactive compounds and chemical probes, in the development of targeted covalent fragments and compounds with electrophilic warheads. The most promising results in fragment library screening were obtained with the bacterial enzyme D-alanine:D-alanine ligase and the β5i subunit of the immunoproteasome. In addition, we investigated alternative approaches to fragment screening, including virtual screening and the use of DNA-encoded chemical libraries. In all cases, hit evaluation was a bottleneck and a critical step for further success. A broader analysis of fragment screening results on different targets revealed that it is important to pay attention to compounds with a thiazole scaffold, which can often exhibit nonspecific activity due to their reactivity

Redox active or thiol reactive?

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action

Tunable heteroaromatic nitriles for selective bioorthogonal click reaction with cysteine

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science

DNA-encoded library screening on two validated enzymes of the peptidoglycan biosynthetic pathway

Screening of DNA-encoded libraries is an emerging technology for discovering hits against protein targets. With the recent launch of the DELopen platform, a facile screening of 4.4 billion compounds is available to accelerate the drug discovery process. Here we report an affinity-based screening of the DELopen library for the first time. The screening was performed against two bacterial enzymes of the peptidoglycan biosynthetic pathway, Nacetylglucosamine- enolpyruvyl transferase (MurA) and D-alanine:D-alanine ligase (DdlB). Several binders were obtained and selected for off-DNA synthesis. Hits with confirmed inhibitory potency were deconstructed into smaller fragments. In this way, two new MurA inhibitors with antibacterial activity were obtained and are available for further optimization

Discovery of compounds with viscosity-reducing effects on biopharmaceutical formulations with monoclonal antibodies

For the development of concentrated monoclonal antibody formulations for subcutaneous administration, the main challenge is the high viscosity of the solutions. To compensate for this, viscosity reducing agents are commonly used as excipients. Here, we applied two computational chemistry approaches to discover new viscosity-reducing agents: fingerprint similarity searching, and physicochemical property filtering. In total, 94 compounds were selected and experimentally evaluated on two model monoclonal antibodies, which led to the discovery of 44 new viscosity-reducing agents. Analysis of the results showed that using a simple filter that selects only compounds with three or more charge groups is a good ‘rule of thumb’ for selecting potential viscosity-reducing agents for two model monoclonal antibody formulations

Discovery of a fragment hit compound targeting D-Ala

Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay. Screening revealed a new fragment-hit inhibitor of DdlB with a Ki value of 20.7 ± 4.5 µM. Binding to the enzyme was confirmed by an orthogonal biophysical method, surface plasmon resonance, making the hit a promising starting point for fragment development

Fragment-sized thiazoles in fragment-based drug discovery campaigns

Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action