Discovery of Novel Inhibitors of LpxC Displaying Potent in Vitro Activity against Gram-Negative Bacteria.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-glucosamine deacetylase (LpxC) is as an attractive target for the discovery and development of novel antibacterial drugs to address the critical medical need created by multidrug resistant Gram-negative bacteria. By using a scaffold hopping approach on a known family of methylsulfone hydroxamate LpxC inhibitors, several hit series eliciting potent antibacterial activities against Enterobacteriaceae and Pseudomonas aeruginosa were identified. Subsequent hit-to-lead optimization, using cocrystal structures of inhibitors bound to Pseudomonas aeruginosa LpxC as guides, resulted in the discovery of multiple chemical series based on (i) isoindolin-1-ones, (ii) 4,5-dihydro-6H-thieno[2,3-c]pyrrol-6-ones, and (iii) 1,2-dihydro-3H-pyrrolo[1,2-c]imidazole-3-ones. Synthetic methods, antibacterial activities and relative binding affinities, as well as physicochemical properties that allowed compound prioritization are presented. Finally, in vivo properties of lead molecules which belong to the most promising pyrrolo-imidazolone series, such as 18d, are discussed.

The crystallization additive hexatungstotellurate promotes the crystallization of the HSP70 nucleotide binding domain into two different crystal forms.

The use of the tellurium-centered Anderson-Evans polyoxotungstate [TeW6O24]6- (TEW) as a crystallization additive has been described. Here, we present the use of TEW as an additive in the crystallization screening of the nucleotide binding domain (NBD) of HSP70. Crystallization screening of the HSP70 NBD in the absence of TEW using a standard commercial screen resulted in a single crystal form. An identical crystallization screen of the HSP70 NBD in the presence of TEW resulted in both the "TEW free" crystal form and an additional crystal form with a different crystal packing. TEW binding was observed in both crystal forms, either as a well-defined molecule or in overlapping alternate positions suggesting translational disorder. The structures were solved by both molecular replacement and single wavelength anomalous diffraction (SAD) using the anomalous signal of a single bound molecule of TEW. This study adds one more example of TEW binding to a protein and influencing its crystallization behavior.

Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells.

Rapid expression of recombinant proteins for structure determination is one of the major challenges in pharmaceutical and academic research, since the number of potential drug targets has increased significantly in the last decade. Despite the fact that the baculovirus expression vector system is widely used for this purpose, the system is hampered by three very slow and tedious procedures, namely generation of high titer baculovirus stock, determination of the virus titer and discovery of the best conditions for protein expression. We herein describe the development of the ultraBac system to address and overcome these issues for protein expression in insect cells. We have established a new baculovirus expression technology for insect cells that is based on co-expression of GFP with target genes, a new regime for cell culturing and a highly efficient purification and enrichment procedure for recombinant baculovirus particles. Co-expression of GFP is used to monitor the infection of insect cells, to simplify titer determination and to optimize expression conditions. The new regime for cell culturing with increased viability of non-infected insect cells and its combination with the massive enrichment of virus particles via high-speed centrifugation enables the production of large amounts of recombinant virus in a very short period of time. By combining these techniques and by using the bicistronic vector pUltraBac-1, we have been able to cut the time-lines for protein expression in insect cells by half, approaching those for protein production in Escherichia coli. This new expression system is a significant step forward towards industrialized protein production in both, industry and academia.