Development of a Novel Specific and Sensitive Mass Spectrometry Based Protein Ligand Binding Assay to Discover Novel Beta Lactam Antibiotics

ABSTRACT There is a critical need to discover and develop safe and effective antibiotics for the treatment of multidrug resistant bacterial infections Each year approximately million patients in the US get hospital acquired infections HAIs with a mortality rate of Nearly of the bacteria causing HAIs are resistant to at least one commonly used antibiotic Natural products NPs and their derivatives represent of FDA approved small molecule drugs over the past decades including many important antibiotics The traditional NP activity guided fractionation approach is labor intensive and time consuming and often leads to rediscovery of known compounds or unwanted nuisance compounds Genome mining using state of the art bioinformatic sequence analysis can identify and evaluate the novelty of NP gene clusters early in the discovery process and is quickly replacing the traditional approach Warp Drive Bio has sequenced the genomes of over actinomycete strains from diverse sources worldwide to generate a proprietary genomic database that contains approximately million NP biosynthetic gene clusters Importantly of cluster families identified in our database have yet to be reported in the literature We have identified biosynthetic lactams BLs gene clusters predicted to encode for synthesis of novel BL structures thus providing an unprecedented opportunity to discover BLs possessing new antimicrobial activities We have also developed synthetic biology tools to turn on the biosynthetic gene clusters which otherwise might be expressed at levels below the detection limit or not expressed at all In addition we have initiated development of a novel BL specific and sensitive mass spectrometer MS based ligand binding assay for discovery of novel BLs During our initial experiments we observed a complex formed between a penicillin binding protein Pseudomonas aeruginosa PBP and various BL ligands and between a lactamase E coli TEM and carbapenems However in some experiments the assay sensitivity was decreased significantly due to impurities in the crude extracts and catalytic activity of lactamases In this Phase I application we aim to improve assay sensitivity using various chromatographic separation methods prior the LC MS analysis and by utilization of mutant lactamases defective in deacylation activity Mutant TEM E N CTX M E N and KPC E N can covalently interact with BLs at the catalytic serine residue but will not efficiently hydrolyze the ring opened BLs allowing the acyl protein adduct to be captured yielding increased LC MS detection sensitivity This improved LC MS ligand binding assay using PBPs and lactamase should enable the rapid mass based detection and identification of new BLs in crude extracts thus increasing the likelihood of success for discovery of novel compounds bio activities encoded by specific BL gene clusters The objective of this project is to develop a novel specific and sensitive mass spectrometry MS based ligand binding assay for discovery of novel lactams with improved therapeutic profiles to combat current and emerging multidrug resistant Gram pathogens