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Structure-assisted design of inhibitors targeting medicinally relevant enzymes
Djukic, Stefan ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee) ; Kolenko, Petr (referee)
Structure-assisted drug discovery is a powerful approach that utilizes detailed knowledge on 3D structure to design and optimize new inhibitors targeting medically relevant enzymes. X-ray crystallography is a widely used structural biology technique since it provides detailed snapshot of protein-inhibitor complex, which is used to analyze protein- inhibitor interactions. PNP plays an important role in salvage pathway of purine metabolism, it is a target in treatment of T-cell malignancies and/or parasitic infections. Our effort focused on human and M. tuberculosis PNP, and our aim was to develop new inhibitors with high selectivity and specificity. Our inhibitors are acyclic nucleoside phosphates with 9- deazahypoxanthine nucleobase that contain three moieties binding to all three regions of the active site: purine, phenyl and phosphonate moieties. The best inhibitors have IC50 values as low as 19 nM (human) and 4 nM (M. tuberculosis). The presence of short substituents at central phenyl moiety, such as methoxy and bromide group, decreases inhibitor's affinity towards human PNP, but does not affect affinity towards mycobacterial PNP. At the same time, bulky substituents, such as fluorinated phenyl ring, decrease inhibitor's affinity towards human PNP but increase affinity towards mycobacterial...
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Preparation and crystallization of a metabolic repressor LutR from Bacillus subtilis
Soldánová, Anna ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee)
Metabolic transcriptional repressors are proteins controlling transcription of specific genes involved in bacterial metabolism. These proteins typically consist of two domains: N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain. When an effector (usually a metabolite molecule) binds to the protein, the conformation of the protein is changed. This causes a change in affinity to its DNA operator and that subsequently modulates the transcription of genes of the specific metabolic pathway. LutR belongs to the GntR family of bacterial transcriptional regulators. In undomesticated strain RO-NN-1 of Bacillus subtilis, LutR regulates transcription of genes required for L-lactate utilization. Interestingly, LutR from laboratory strains PY79 and 168 has a different function. Due to a mutation, it lacks the first 21 amino acids and this alters its DNA recognition specificity. This LutR variant acts as a global regulator and regulates many genes associated with transition from exponential growth to stationary phase of bacterial population. Knowledge of 3D structure of LutR with DNA could elucidate the impact of this short deletion in LutR DBD on the mechanism of DNA recognition. In this work, the DBD of LutR from undomesticated strains of B. subtilis was prepared by heterologous...
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