High-Throughput Screening for Novel Inhibitors of Intracellular Pathogens, Including Chlamydia trachomatis.

High-throughput drug screening (HTS) is a powerful tool that can be used rapidly to identify new potential bacterial inhibitors and/or compounds which enhance host cell control of pathogens, which can then go on to be developed as novel therapeutics. Typically screening is commonly done in artificial culture medium; however, obligate intracellular pathogens, such as Chlamydia trachomatis, cannot be tested this way. Intracellular screening methods allow for such pathogens to undergo HTS, while still giving reliable and consistent data.

The Deconstructed Granuloma: A Complex High-Throughput Drug Screening Platform for the Discovery of Host-Directed Therapeutics Against Tuberculosis.

(Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone.

Early innate immunity determines outcome of Mycobacterium tuberculosis pulmonary infection in rabbits.

Background: Pulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB.

Molecular immunologic correlates of spontaneous latency in a rabbit model of pulmonary tuberculosis.

Background: Infection of humans with Mycobacterium tuberculosis (Mtb) results in latent tuberculosis infection (LTBI) in 90-95% of immune competent individuals, with no symptoms of active disease. The World Health Organization estimates that 1.5 billion people have LTBI, which can reactivate in the setting of waning host immunity, posing a threat to global TB control.

Spontaneous latency in a rabbit model of pulmonary tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts.

Primary and secondary drug screening assays for Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC).

Comparative activities of lipid esters of cidofovir and cyclic cidofovir against replication of herpesviruses in vitro.

Cidofovir (CDV) is an effective therapy for certain human cytomegalovirus (HCMV) infections in immunocompromised patients that are resistant to other antiviral drugs, but the compound is not active orally. To improve oral bioavailability, a series of lipid analogs of CDV and cyclic CDV (cCDV), including hexadecyloxypropyl-CDV and -cCDV and octadecyloxyethyl-CDV and -cCDV, were synthesized and found to have multiple-log-unit enhanced activity against HCMV in vitro. On the basis of the activity observed with these analogs, additional lipid esters were synthesized and evaluated for their activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus, murine cytomegalovirus, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and HHV-8.

In vitro activity and mechanism of action of methylenecyclopropane analogs of nucleosides against herpesvirus replication.

We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8.

Inhibition of herpesvirus replication by a series of 4-oxo-dihydroquinolines with viral polymerase activity.

Herpesviruses cause a wide variety of human diseases ranging from cold sores and genital herpes to encephalitis, congenital infections and lymphoproliferative diseases. These opportunistic viruses cause major problems in immunocompromised individuals such as transplant recipients, cancer patients, and HIV-infected persons. The current treatment of these infections is not optimal and there is a need for more active, less toxic compounds that might be used in place of or in addition to current therapies.

Efficacy of methylenecyclopropane analogs of nucleosides against herpesvirus replication in vitro.

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8).

In vitro activities of benzimidazole D- and L-ribonucleosides against herpesviruses.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8) are responsible for a number of clinical manifestations in both normal and immunocompromised individuals. The parent benzimidazole ribonucleosides evaluated in this series, 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) and maribavir (1263W94), are potent and selective inhibitors of human CMV replication. These nucleosides act by two different mechanisms.

Determination of antiviral efficacy against lymphotropic herpesviruses utilizing flow cytometry.

Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), and human herpesvirus type 8 (HHV-8) comprise a group of lymphotropic herpesviruses which are responsible for a wide range of diseases, including lymphoproliferative disorders and tumors. We have developed several flow cytometric assay (FACS) systems to evaluate antiviral efficacy against EBV, HHV-6 and HHV-8. Assays using either EBV or HHV-8, members of the gammaherpesvirus subfamily, have shown that while EBV responds well to acyclovir (ACV), HHV-8 was most sensitive to cidofovir (CDV).