Synthesis and structure-activity relationships of pyrimidine derivatives as potent and orally active FGFR3 inhibitors with both increased systemic exposure and enhanced in vitro potency.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of patients with bladder cancer harboring genetic alterations in FGFR3. We identified pyrimidine derivative 20b, which induced tumor regression following oral administration to a bladder cancer xenograft mouse model. Compound 20b was discovered by optimizing lead compound 1, which we reported previously. Specifically, reducing the molecular size of the substituent at the 4-position and replacing the linker of the 5-position in the pyrimidine scaffold resulted in an increase in systemic exposure. Furthermore, introduction of two fluorine atoms into the 3,5-dimethoxyphenyl ring enhanced FGFR3 inhibitory activity. Molecular dynamics (MD) simulation of 20b suggested that the fluorine atom interacts with the main chain NH moiety of Asp635 via a hydrogen bond.

Structure-based drug design of 1,3,5-triazine and pyrimidine derivatives as novel FGFR3 inhibitors with high selectivity over VEGFR2.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of bladder cancer. We identified 1,3,5-triazine derivative 18b and pyrimidine derivative 40a as novel structures with potent and highly selective FGFR3 inhibitory activity over vascular endothelial growth factor receptor 2 (VEGFR2) using a structure-based drug design (SBDD) approach. X-ray crystal structure analysis suggests that interactions between 18b and amino acid residues located in the solvent region (Lys476 and Met488), and between 40a and Met529 located in the back pocket of FGFR3 may underlie the potent FGFR3 inhibitory activity and high kinase selectivity over VEGFR2.

Crystallization and preliminary X-ray diffraction of the first periplasmic domain of SecDF, a translocon-associated membrane protein, from Thermus thermophilus.

A membrane-integrated Sec component, SecDF, associates with the SecYEG protein-conducting channel and facilitates protein secretion and membrane-protein integration. SecDF contains 12 transmembrane helices and two periplasmic domains. The first periplasmic domain (P1) plays an important role in protein translocation. Here, the overexpression, purification and crystallization of the P1 domain of Thermus thermophilus SecDF are reported. The crystals diffracted X-rays to 2.3 Å resolution and belonged to space group C2, with unit-cell parameters a = 161.1, b = 35.8, c = 181.6 Å, suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by the multiple-wavelength anomalous dispersion method using selenomethionine-labelled crystals.

Structure and function of a membrane component SecDF that enhances protein export.

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.