High-Throughput Protein Production (HTPP): a review of enabling technologies to expedite protein production.

Recombinant protein production plays a crucial role in the drug discovery process, contributing to several key stages of the pathway. These include exploratory research, target validation, high-throughput screening (HTS), selectivity screens, and structural biology studies. Therefore the quick and rapid production of high-quality recombinant proteins is a critical component of the successful development of therapeutic small molecule inhibitors. This chapter will therefore attempt to provide an overview of some of the current "best-in-class" cloning, expression, and purification strategies currently available that enhance protein production capabilities and enable greater throughput. As such the chapter should also enable a reader with limited understanding of the high-throughput protein production (HTPP) process with the necessary information to set up and equip a laboratory for multiparallel protein production.

N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity.

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.