Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Veterinary Comparative Pathobiology
Non Technical Summary
The current costly and time-consuming drug discovery process is ill-equipped to combat rapidly emerging multidrug resistant pathogens. There is a pressing need for the identification of novel strategies to develop antibiotics quickly and efficiently to deal with the rapid emergence of multidrug resistance pathogens. One strategy which warrants more attention as a unique method for identifying new antimicrobials is drug repurposing. Repurposing FDA-approved drugs, with well-characterized toxicology and pharmacology, to find new applications outside the scope of the original medical indication is a novel way to reduce both the time and cost associated with antimicrobial innovation. The FDA-approved drugs represent an untapped reservoir for new antibiotic leads that may lead to identification of new targets that will guide the future development of improved antimicrobial/anti-infective agents. Thus, in addition to the discovery of potential novel drugs and drug targets, the impact will be multiplied by research laboratories/pharmaceutical companies worldwide, using the resulting data to follow up on "hits" from our screening.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our long term research goal is to discover and develop novel antimicrobials with novel mechanism of action from approved human and veterinary non-antimicrobial drugs (drug repurposing) that can be repurposed as therapeutics agents in the treatment of human and animal infections caused by bacterial and fungal pathogens. Recently, drug repurposing is gaining momentum as it has resulted in successes in a number of disease areas and has accounted for approximately 30% of newly approved FDA drugs and vaccines (Ashburn and Thor, 2004; Chong and Sullivan, 2007b; Jin and Wong, 2013). Repurposing existing approved drugs permits companies to bypass much of the preclinical work and early stage clinical trials required for new compounds (particularly toxicological and pharmacological analysis of drugs) thus cutting into the cost associated with bringing a drug to the marketplace (DiMasi et al., 2003). In addition to lower drug discovery-associated costs, repurposing approved drugs (particularly for identification of new antimicrobials) has several additional benefits. Given these drugs have already been tested in animals and human, valuable information pertaining to, toxicity, pharmacokinetic and pharmacodynamic parameters are known. This permits a better understanding of the overall pharmacology of the drug, potential routes of administration, tissue distribution and establishing an appropriate dosing regimen.Our objectives in this proposal are:To assemble library of all approved drugs/clinical molecules (~ 4,000)Screen the library against bacterial and fungal pathogens and Identifying drugs/molecules with promising activityIdentifying the mechanism of action (MOA) and target(s) of the non-antimicrobial drugs
Project Methods
1-Assembling the library of clinical drugs: A major challenge to repurposing approved drugs pertains to the lack of accessibility to libraries containing clinical drug collections. No single collection of the nearly 10,000 known drugs/clinical safe compounds currently exists (Chong and Sullivan, 2007b). Only 40% (~ 4,000 drugs) of the total known approved drugs/clinical safe compounds are available for screening and these drugs are dispersed throughout several different collections. We have already assembled 50% of the commercially available compounds (~ 2,000 drugs: from: 727-NIH Clinical Collections 1 and 2, 1,600-Pharmakon from Microsource, and few small libraries). We identified the remaining 50% of the drugs (the National Institute of Neurological Disorders and Stroke (NINDS) collection of 1,040 compounds, the Prestwick Chemical Library of 1,280 drugs, and the Johns Hopkins Clinical Compound Library of more than 1,500 compounds) (Chong and Sullivan, 2007b). Combined with other drug collections available in our lab, these collections will represent all drugs/clinical molecules (~ 4,000) available for commercial purchase. However, there is redundancy and overlap between these different libraries, which presents an additional problem as a compound may be present in more than one collection making screening these compounds more difficult. Non-redundant collection containing (~ 4,000 drugs) will be assembled at 1 mM concentration in 384 well plates to facilitate the high throughput screening (HTS). 2 Whole-cell screening against multidrug resistant pathogens: The assembled library above will be screened (Cheng et al., 2010) against multidrug resistant pathogens. We will start with ESKAPE pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and E. coli), which can be later expanded to any other pathogen, in 384-well plates at 8 µM concentration in triplicate in a High Throughput Screening Facility at Purdue University. The identified hits (drugs) will be subjected to a secondary manual screen in 96-well plates and according to guidelines of the Clinical and Laboratory Standards (CLSI) (NCCLS, 2012) to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the drugs against ESKAPE. Drugs with direct antimicrobial activity will be identified and screening against additional pathogens of veterinary important. Our findings in ESKAPE pathogens will be broadly relevant to other pathogens and should significantly impact and inform efforts to repurpose therapies against multidrug resistant pathogens.3-dentifying the mechanism of action (MOA) and target(s) of the non-antimicrobial drugsUnderstanding the mechanism of action of a novel antibiotic is an essential step in drug development research. Connecting an antimicrobial compound to its cellular target is a critical step for improving the affinity, selectivity, and antibacterial properties of the drug.Our approach toward identifying the MOA and target will focus mutation and mapping of the mutants. Bacterial Mutants resistant to the drug will be generated in-vitro by two methods: a) large volume of logarithmic culture of the bacteria will be concentrated to 1011-1012 CFU/ml and added to IsoSensitest agar containing 2X, 4X, 10X and 20X MIC drug (MacLeod et al., 2009; Martinez and Baquero, 2000; O'Neill et al., 2001; Zurenko et al., 1996). 10 plates will be used for each concentration (With 10 agar plates, mutations could potentially be detected at frequencies of 1 in 1011 (O'Neill et al., 2001)).b) Using an alternative approach, drug mutants will be isolated by multiple passage methods through progressively increasing concentration of drug in liquid culture (Lama et al., 2012; Peleg et al., 2012). Bacterial cultures that grew at the highest concentrations of drug will be used as an inoculum for the subsequent culture. Colonies from a&b methods above will be selected and stable mutants to the drug will be confirmed (MacLeod et al., 2009; Martinez and Baquero, 2000).Genomic DNA will be isolated from single colonies using standard methods (Skovgaard et al., 2011). Bar coded indexed sequencing libraries will be constructed using standard kits. The Illumina HiSeq 2500 platform (Purdue University Genome Facility) will be used to sequence the mutants and the parenteral strain on one lane using Rapid Run mode that would result in 9-10 million reads per sample. Mapping and Assembly with Qualities software (Purdue University Bioinformatics Facility) will be utilized to map the reads produced by the Illumina sequencer to the reference genome (parental reference strain). Individual high-confidence SNP will be identified. Genetic variant annotation and effect prediction software (snpEff and snpSift) will be utilized to predict the impact of a specific mutation on protein function (Comas et al., 2012; Ng and Henikoff, 2003). Select SNPs will be confirmed independently by PCR amplifying and sequencing of the PCR product.