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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. |
