The Couch Lab has several research projects under investigation, including: 1. The development of new antibiotics. MEP pathway inhibitor development. The increasing prevalence of antibiotic resistant strains, the ease by which antibiotic resistance can be deliberately engineered for bioterrorism, and the shortage of antibiotics in current phase 2 and phase 3 clinical trials all emphasize the need for continued development of new antibiotics with novel mechanism of action. Isoprenoids are a class of molecules fundamentally involved in a variety of crucial biological functions including electron transport (quinones), cell wall biosynthesis (dolichols), signal transduction (prenylated proteins), and the regulation of membrane fluidity (hopanoids and cholesterol). Because mammals use the mevalonic acid pathway for isoprene biosynthesis, while many human pathogens exclusively use the methylerythritol phosphate (MEP) pathway, the latter pathway makes an excellent target for the development of novel antibiotics with new activity. To facilitate MEP pathway inhibitor development, with funding provided by DTRA and NIH, my lab has cloned, expressed, and enzymatically characterized several MEP pathway enzymes, including MEP synthase (aka Dxr or IspC) and MEP cytidylyltransferase (aka IspD) from Francisella tularensis, Yersinia pestis, and Escherichia coli, and we have been given recombinant constructs for the expression, purification, and assay of Mycobacterium tuberculosis Dxr (from Dr. Cynthia Dowd) and Plasmodium falciparum Dxr (from Dr. Audrey Odom). As a result of our work, we have gained insight into feedback mechanisms that naturally regulate the activity of these enzymes, have discovered enzyme structural variations in substrate binding sites that are important for rational drug design, and have identified Y. pestis and F. tularensis IspD as preeminent enzymes for use in high-throughput library screening. After sequentially partnering with Walter Reed Army Institute of Research (WRAIR) then Prof. Cynthia Dowd at George Washington University, we are iteratively deriving structure-activity relationships and performing mechanism of inhibition assays to guide the development of rationally designed synthetic inhibitors of these enzymes (the top inhibitors have nM activity). We also established a multi-well plate assay to screen random compound libraries, and have completed screening a commercially available molecular library and an in-house, proprietary phytochemical library, identifying hit compounds in each. Through a series of follow-on enzyme assays and bacterial growth inhibition assays, we have used the results of the screening to further refine our inhibitor design. Protein crystallography (at WRAIR) is also contributing to inhibitor development. Select publications are listed below. a. Haymond A, Johny C, Dowdy T, Schweibenz B, Villarroel K, Young R, Mantooth CJ, Patel T, Bases J, Jose GS, Jackson ER, Dowd CS, and Couch RD. Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase). PLoS One. 2014 Aug 29;9(8):e106243. b. Chofor, R., Risseeuw, M.D., Pouyez, J., Johny, C., Wouters, J., Dowd, C.S., Couch, R.D., Van Calenbergh, S. Synthetic Fosmidomycin Analogues with Altered Chelating Moieties Do Not Inhibit 1-Deoxy-D-xylulose 5-phosphate Reductoisomerase or Plasmodium falciparum Growth In Vitro. Molecules. 2014; 19(2):2571-2587. c. Jackson, E.R., San Jose, G., Brothers, R.C., Edelstein, E.K., Sheldon, Z., Haymond, A., Johny, C., Boshoff, H.I., Couch, R.D., and Dowd, C.S., The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs. Bioorganic & Medicinal Chemistry Letters, 2014 Jan 15;24(2):649-53. d. San Jose, G., Jackson, E.R., Uh, E., Johny, C., Haymond, A., Lundberg, L., Pinkham, C., Kehn-Hall, K., Boshoff, H.I., Couch, R.D., and Dowd, C.S., Design of Bisubstrate Inhibitors of 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase (Dxr) from Mycobacterium tuberculosis (Mtb). Med. Chem. Comm. 2013 4:1099-1104.
2. Small molecule metabolomics and gastrointestinal health/disease.
In this research project, we are using state-of-the-art metabolomics techniques to evaluate small molecule metabolites present in biological samples, including feces. While we are currently using both GC-MS and LC-MS in our analyses, my group began our metabolomics-based investigations with an NIH funded project wherein we used GC-MS to examine fecal volatile organic compounds (VOCs) and discern their relationship to healthy and alcoholic study participants. The overarching hypothesis is that chronic alcohol consumption causes changes to the gut microbiome, which in turn causes changes to the gut metabolome, contributing to leaky gut and corresponding liver disease. Headspace solid phase microextraction (hSPME) is an established approach to the isolation and analysis of environmental VOCs, so my lab utilized hSPME to analyze feces. However, before we performed a comparative analysis of healthy and alcoholic feces, since little work had been reported using hSPME to analyze feces, we first established the effects of fundamental elements that can influence such an analysis, such as the optimal sorbent composition/combination to use, the effect of extraction duration, the extraction conditions, the collection method for the feces, the storage of the feces, etc. We discovered that the current technologies were inadequate to facilitate a proper hSPME-based metabolomics analysis of fecal VOCs (or other biological samples for that matter), so we developed and patented a device that enables these analyses, and coined the term simulti-hSPME to describe our optimal process of using multiple sorbent types to simultaneously extract VOCs of diverse chemistries from a sample. Concomitantly, I also developed and wrote my own computer algorithms and used them to establish a cheminformatic data analysis pipeline to perform comparative metabolomics analyses in my lab. We then analyzed healthy and alcoholic fecal VOCs and discovered that we could clearly differentiate the two cohorts, and highlighted their distinguishing metabolites. We are currently investigating other human clinical samples in similar types of analyses. Select publications are listed below. a. Dixon, E., Clubb, C., Pittman, S., Ammann, L., Rasheed, Z., Kazmi, N., Keshavarzian, A., Gillevet, P., Rangwala, H., and Couch, R.D., Solid-phase microextraction and the human fecal VOC metabolome.   PLoS ONE 2011 , 6(4): e18471. doi:10.1371/journal.pone.0018471.  PMC3072975 b. Li RW, Wu S, Li W, Navarro K, Couch R.D.,   Hill D, and Urban JF Jr., Alterations in the porcine colon microbiota induced by the gastrointestinal nematode Trichuris suis .   Infect Immun.  2012 80(6):2150-7. c. Couch, R.D., Navarro, K., Sikaroodi, M., Gillevet, P., Forsyth, C.B., Mutlu, E., Engen, P.A., and Keshavarzian, A. The Approach to Sample Acquisition and Its Impact on the Derived Human Fecal Microbiome and VOC Metabolome. PLoS One. 2013 Nov 18;8(11):e81163. d. Couch RD, Dailey A, Zaidi F, Navarro K, Forsyth CB, Mutlu E, Engen PA, and Keshavarzian A. Alcohol Induced Alterations to the Human Fecal VOC Metabolome.  PLoS One. 2015 Mar 9;10(3):e0119362.
4. Small molecule activation of proteins for the chemoprevention of Alzheimer’s disease.
In this project, funded by a GMU provost seed grant and grants from the Virginia Commonwealth Health Research Board and the Virginia Center on Aging, we are applying our small molecule and protein expertise to determine the signal transduction mechanism underlying the ability of select small molecules to induce nerve growth factor release from glial cells. Nerve growth factor keeps neurons alive, and thus has promise for the chemoprevention of Alzheimer’s Disease. Using cultured human glial cells, we have utilized reverse phase protein microarrays to generate temporal maps of signal transduction protein activation, and we are now validating the involvement of these proteins/pathways using pathway specific agonists and antagonists. Related papers are listed below. a. Dixon, E., Schweibenz, T., Hight, A., Kang, B., Dailey, A., Kim, S., Chen, M, Kim, Y., Neale, S., Groth, A., Ike, T., Khan, S., Schweibenz, B., Lieu, D., Stone, D., Orellana, T., and Couch, R.D., Bacteria-Induced Static Batch Fungal Fermentation of the Diterpenoid Cyathin A3, a Small Molecule Inducer of Nerve Growth Factor. J. Ind. Microbiol Biotechnol. 2010, DOI: 10.1007/s10295-010-0805-7 Online First™. PMID: 20714781  b. Davis J, Couch RD. Strategizing the Development of Alzheimer’s Therapeutics. Advances in Alzheimer’s Disease. 2014;03: 107–127. doi:10.4236/aad.2014.33011
3. Biosensor/Electronic Nose. 
In this project we are using our newly developed and patented metabolomics method known as “simultaneous multifiber headspace solid-phase microextraction (simulti-hSPME)” for the rapid and minimally invasive detection of biothreat-relevant microbes. This DTRA funded research project is performed in both BSL2 and BSL3 settings.
A Complete List of Published Work is Available at NCBI’s MyBibliography: