Riboflavin synthase from Brucella as target for structure-based drug design

MARÍA L. CERUTTI; SEBASTIÁN KLINKE; LUCÍA M. POTH; MARIELA DEL C. CARRICA; MARÍA I. SERER; FERNANDO A. GOLDBAUM

San Luis

Congreso; XII Reunión Anual de la Asociación Argentina de Cristalografía (AACr); 2016

Asociación Argentina de Cristalografía (AACr)

The enzyme Riboflavin synthase (RS) fromBrucella abortus catalyzes the last step in riboflavin biosynthesis (vitaminB2), being an interesting target for the development of inhibitors against thispathogenic bacterium, since Brucella is unable to take exogenous vitamin B2with efficiency and relies entirely on its own biosynthesis. RS is anintrinsically flexible homotrimer with a molecular weight of 70 kDa. In ourlab, we solved the crystal structure of RS as apo-enzyme and also in thepresence of bound products and product analogue inhibitors. Recently, we havestarted a structure-based drug design project on RS, whose first stepcorresponded to the high-throughput search of inhibitors using a drug-likesmall molecule library. Several hits have been found and we are activelyworking in obtaining the three-dimensional structures of RS in complex withthese inhibitors, with the aim of improving affinities, contacts andcomplementarity by chemical modification of these compounds in a second step. Inthis work we will present our latest results in RS crystal growth andoptimization, exploring different strategies for the introduction of theligands (co-crystallization, soaking) and testing different precipitants, pHvalues and additives to minimize the flexibility of the protein in the crystallattice. Additionally, diffraction data from our Bruker in-house single crystalX-ray diffractometer and from the PROXIMA 1 and 2 beamlines (SOLEILsynchrotron, France) will be presented and analyzed.