Design and Synthesis of Novel Dihydroorotate Dehydrogenase Inhibitors

Medicinal Chemistry;Biological Chemistry;Science;Medicinal Chemistry

Madak, JosephSoellner, Matthew Bryan ;

University of Michigan

关键词: Medicinal Chemistry;    Biological Chemistry;    Science;    Medicinal Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/144184/madakj_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Rapidly growing cells are dependent on sufficient concentrations of nucleotides to sustain proliferation. One enzyme essential for the de novo synthesis of pyrimidine-based nucleotides is dihydroorotate dehydrogenase (DHODH); a known therapeutic target for many diseases. In cancer, inhibition of DHODH depletes intracellular pyrimidine nucleotide concentrations and halts the cell cycle at S-phase. This metabolic enzyme is necessary for rapidly growing cells and DHODH inhibition has been demonstrated to sensitize resistant cells to current chemotherapy options. Therefore, we pursued a drug discovery project towards developing novel inhibitors of DHODH.A phenotypic screen was utilized to identify hit compounds that may be suitable leads for a drug discovery project. From this, a lead compound was discovered to inhibit cell growth (MIA PaCa-2, BxPC-3) but through an unknown enzyme target. The essential pharmacophore of the lead compound was elucidated via structure activity relationship (SAR) studies and biologically found to be remarkably similar to brequinar, a potent DHODH inhibitor. Cells were rescued from both the lead compound and brequinar by the addition of uridine, a mimic of a downstream byproduct, and both inhibitors have submicromolar IC50 values toward DHODH. For continued optimization, we sought to improve affinity for the DHODH enzyme.We pursued a structure-guided approach toward the development of improved DHODH inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two residues, T63 and Y356, suitable for novel H-bonding interactions were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of two potent quinoline based analogues with an IC50 against DHODH of 10.6 ± 1.1 nM for one and 32.9 ± 4.6 nM for the other. A co-crystal structure between a quinoline analogue and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to a third 1,7-naphthyridine analogue with an IC50 = 53.9 ± 1.7 nM, which forms a novel H-bond with Y356. In conclusion, the data from our SAR investigation supports further preclinical studies of our improved compounds toward selection of a candidate for early stage clinical development.Finally, to fully understand brequinar-based DHODH inhibition, we developed novel brequinar-based probes. We disclose a 16-step convergent synthesis of the first brequinar-PROTAC and a four-step synthesis to the first mitochondrial-directed brequinar probe. Both of these probes possess cytotoxicity that is superior to brequinar in a colony formation assay and are useful for further pharmacology studies. The collective work described in this dissertation furthers the understanding of DHODH inhibition in cancer, identifies novel sites for electrostatic interaction between brequinar-class inhibitors and DHODH, and has resulted in the first brequinar-based probes for DHODH study. Additionally, this thesis depicts the limitations and effectiveness of DHODH targeted therapy for cancer and suggests potential solutions that may lead to the clinical efficacy. In general, this work provides an excellent foundation to improve brequinar-class inhibitor design and is of board interest to cancer drug discovery.