Laboratory of Clinical Infectious Diseases

Clifton E. Barry, III, Ph.D.

Senior Investigator
Chief, Tuberculosis Research Section

Tuberculosis Research Section

Dr. Barry received his Ph.D. in organic and bioorganic chemistry in 1989 from Cornell University, studying the biosynthesis of complex natural products. Following postdoctoral research at Johns Hopkins University, Dr. Barry joined NIAID's Rocky Mountain Laboratories. In 1998, he was tenured as chief of the Tuberculosis Research Section (TBRS). He is very involved in international activities to develop new chemotherapies for tuberculosis, serving as an advisor to the World Health Organization and the Global Alliance for TB Drug Development. Dr. Barry is a member of several editorial boards and has authored more than 60 research publications in tuberculosis since entering the field 10 years ago.

Description of Research Program

The TBRS is an integrated group of chemists, clinicians, and microbiologists dedicated to improving the chemotherapy of tuberculosis. Through a detailed understanding of Mycobacterium tuberculosis (Mtb)—the natural history of the disease, the advantages and shortcomings of current antibiotics, models for the evaluation of new antibiotics, and the underlying reasons for the development of drug resistance—section scientists work to identify new strategies to improve therapy. Translating these strategies into clinically useful drugs engages the chemistry portion of the group in areas such as chemical modification of promising new synthetic antitubercular compounds, identification and optimization of products derived from natural sources with antitubercular activity, and development of novel delivery methods and formulations.

Molecular determinants of the pathogenesis of tuberculosis
Clinical isolates of Mtb often have discrete virulence traits in vivo that can be linked directly to specific molecules. TBRS scientists, in collaboration with scientists at the Public Health Research Institute in New Jersey, are attempting to understand the molecular basis for these differences in virulence and evaluate the impact on disease outcome in TB patients. Two areas in which we are particularly interested are polyketide metabolites and small molecule secretory systems.

Polyketides are complex natural products with diverse biological activities synthesized by plants, fungi, and bacteria. Several polyketide systems have been ascribed an important role in mycobacterial cell wall structure and virulence of Mtb. We are currently utilizing a broad range of genetic, biochemical, and immunological approaches to understanding the role of this unique family of bioactive lipids in tuberculosis. Secretion of biologically active lipids, glycolipids, and carbohydrates represents a major mechanism of host-pathogen communication. The genome sequence of Mtb revealed the presence of a family of 12 putative transmembrane proteins designated MmpL (major membrane proteins, large). These are predicted to be transport proteins, which may function as efflux pumps for such molecules. TBRS scientists are studying the role of the Mtb MmpL genes in resistance to antibiotics and transport of mycobacterial cell wall components and virulence factors.

The evolution of drug resistance and therapy of multidrug-resistant TB
The emergence of multidrug-resistant tuberculosis (MDRTB) represents a large and growing threat to TB control programs. TBRS scientists tackle important problems in the development of resistance to specific drugs (such as pyrazinamide, isoniazid, and PA-824) and also address the larger issue of whether there exist specific factors that predispose some patients to develop multiple-drug resistance. To study specific drugs, TBRS scientists often utilize DNA microarray analysis of Mtb transcriptional responses to give insight into metabolic networks that are affected, accelerating the process of identifying specific targets. To study the mechanism of emergence of drug resistance, TBRS scientists, in collaboration with scientists from the University of Witswatersrand in Johannesburg, South Africa, have recently shown that the second copy of the major DNA polymerase in Mtb plays a critical, and previously unsuspected, role in error-prone DNA repair that generates the sequence mistakes conferring drug resistance.

TBRS scientists, in collaboration with colleagues from Yonsei University and Masan National Tuberculosis Hospital in Busan, South Korea, are also establishing a center of excellence for the study of multidrug-resistant Mtb.

Masan National TB Hospital Main Administrative Building, Professor Cho (left) and Dr. Park, Chief Surgeon.

The center will address the basic biology underlying the development of drug resistance as well as develop a clinical site for the evaluation of novel antituberculosis agents. Masan Hospital is the national referral center for TB treatment failures in South Korea with the largest population of inpatient MDRTB victims anywhere in the world. Bacterial population dynamics, the frequency of occurrence and phenotype of MDR organisms, the disease history and immune status of the patient, and the innate ability of an individuals' immune system to contain the infection are some of the subjects currently being investigated.

Understanding the evolution of drug-resistance also motivates TBRS studies in South Africa with partners at the University of Cape Town and Stellenbosch University. These studies have previously revealed a major mechanism of resistance to the second-line agent ethionamide. MDRTB patients in South Africa and South Korea will benefit from being pioneers in the development of new cost-effective and accessible therapeutic approaches suitable for the developing world.

Development of new antituberculosis chemotherapies
Using organic synthesis allows TBRS scientists to mount a direct assault on important new targets identified and validated from our biological studies. TBRS scientists have used both combinatorial and structure-based approaches in combination with basic principles of medicinal chemistry to facilitate the identification of promising new agents. Often in partnership with philanthropically motivated pharmaceutical companies, promising new compounds are evaluated in purified protein assays, against whole Mtb cells, and in animal models of human disease. Our goals in such programs have ranged from improving the antitubercular activity of an existing compound to developing a novel antitubercular agent from a previously untapped source.

In collaboration with Sequella, Inc., TBRS launched a project to improve the activity of one of the standard front-line agents, ethambutol. Using solid-phase combinatorial chemistry, a library of over 60,000 analogs was created and screened. Several analogs were discovered with far greater efficacy than the parent compound in vitro, and these have shown promising results in animal models of disease.

Thiolactomycin bound to E. coli  FabB. FabB is a bacterial enzyme responsible for fatty acid elongation. Fatty acids are important components of the bacterial cell wall. Inhibition of FabB and related enzymes leads to inhibition of bacterial growth.

Thiolactomycin (TLM) is a small-molecule natural product that has weak antitubercular activity. Lack of industrial interest in its development left it languishing on the shelves. TLM inhibits an important enzyme in the biosynthesis of the cell wall constituent mycolic acids. Using structure-based design in collaboration with scientists at GlaxoSmithKline and at St. Jude's Children's Research Hospital in Memphis, Tennessee, we are modifying the structure of TLM to improve its activity and increase its antitubercular activity.

Work is also underway to pinpoint critical enzymes involved in cell wall maintenance and turnover as potential cidal targets for nonreplicating organisms. Some key cell wall biosynthetic genes have been identified whose expression is modulated during in vivo infection. These genes may provide valuable clues about the physiology of the pathogen in the host as well as providing important drug targets.

PA-824 is a small molecule antibacterial, related to the structure of metronidazole.

PA-824 was originally synthesized by chemists at the PathoGenesis Corporation (now Chiron) in the late 1990s and characterized in partnership with TBRS. PA-824, in contrast to current antitubercular drugs, exhibits bactericidal activity against both replicating and static Mtb. PA-824 is also a promising lead compound for drug development since it has potent bactericidal activity against multidrug-resistant Mtb strains and shows promising oral activity in animal infection models. Work is underway to identify the target and mode of action of PA-824. Despite its high activity against Mtb, this compound may not advance to the clinic due to its poor physical properties (particularly its poor water solubility). TBRS is engaged in making analogs of PA-824 with improved solubility while maintaining the high activity of the parent compound.

Recently, TBRS has begun a collaborative project involving the screening of ethnobotanically selected natural products for antitubercular activity. Working with scientists at the National Institute for Pharmaceutical and Research Development (NIPRD) in Abuja, we are investigating plant and bacterial products from several areas of Nigeria. This work involves products stemming from the tradition of natural medicine and ethnobotany in that region. It is our hope that novel compounds against TB or other pathogens will be found, and in the process, we hope to build a partnership with the NIPRD scientists that will be productive for years to come. Gene expression signatures developed by microarray analysis of different classes of drugs are used as a tool to classify active components of natural product extracts based on mode of action. Natural compounds with novel transcriptional profiles may prove to be interesting candidates for further development.

Latency and persistence in tuberculosis
Latent tuberculosis in humans is thought to involve the persistence of non-replicating organisms primed to reactivate when the immune response wanes. The relative drug resistance of nonreplicating organisms is also thought to be responsible for the requirement for extremely lengthy antituberculosis therapy. Long-term survival of nonreplicating bacteria is ensured by coordinating the shutdown of active metabolism through a broad transcriptional program called the stringent response. This response is initiated by the enzymatic action of RelMTB in Mtb. We have shown that loss of RelMTB produces an Mtb strain that grows normally but is severely compromised in maintenance of long-term viability in vitro and that the long-term ability of this deficient strain to sustain infection in vivo is severely impaired. We are in the process of defining the relMTb regulon and the contribution of these genes to persistence in vivo.

TBRS scientists are also engaged in developing animal models of disease that more accurately reflect the chronic situation in human tuberculosis patients. These models create bacteria with altered physiological states that show sensitivity to drugs similar to that observed in human clinical trials. The presence of "phenotypically" resistant bacterial populations in human patients on chemotherapy is thought to be one of the principle factors dictating the current 6-8 month chemotherapy required to cure the disease.

Work is also underway to pinpoint critical enzymes involved in cell wall maintenance and turnover as potential cidal targets for nonreplicating organisms. Some key cell wall biosynthetic genes have been identified whose expression is modulated during in vivo infection. These genes may provide valuable clues about the physiology of the pathogen in the host as well as providing important drug targets.

Research Group Members

Memberships

• American Association for the Advancement of Science
• American Chemical Society
• American Society for Biochemistry and Molecular Biology

Editorial Boards:

• Tubercle and Lung Disease (Section Editor)
• Journal of Bacteriology

Selected Publications

(View list in PubMed.)

Barry CE 3rd, Matter A. Next-generation therapeutics: Turning the current 'innovation gap' into an 'innovation leap.' Curr Opin Chem Biol. 2006 Jul 5; [Epub ahead of print].

Barry CE 3rd, Boshoff H. Getting the iron out. Nat Chem Biol. 2005 Aug;1(3):127-8.

Somu RV, Boshoff H, Qiao C, Bennett EM, Barry CE 3rd, Aldrich CC.
Rationally designed nucleoside antibiotics that inhibit siderophore
biosynthesis of Mycobacterium tuberculosis. J Med Chem. 2006 Jan 12;49(1):31-4. 

Manjunatha UH, Boshoff H, Dowd CS, Zhang L, Albert TJ, Norton JE, Daniels L, Dick T, Pang SS, Barry CE 3rd. Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):431-6. 

Domenech P, Reed MB, Barry CE 3rd. Contribution of the  Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance. Infect Immun. 2005 Jun;73(6):3492-501. 

Reed MB, Domenech P, Manca C, Su H, Barczak AK, Kreiswirth BN, Kaplan G, Barry CE 3rd. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature. 2004 Sep 2;431(7004):84-7.

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