index

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

-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.