Chemical Exploration of a Highly Selective Scaffold with Activity against Intracellular Mycobacterium tuberculosis.

We previously identified a phenylthiourea series with activity against intracellular Mycobacterium tuberculosis using a high-throughput, high-content assay. We conducted a catalog structure-activity relationship study with a collection of 35 analogs. We identified several thiourea derivatives with excellent potency against intracellular bacteria and good selectivity over eukaryotic cells. Compounds had much lower activity against extracellular bacteria, which was not increased by using cholesterol as the sole carbon source. Compounds were equally active against strains with mutations in QcrB or MmpL3, thereby excluding common, promiscuous targets as the mode of action. The phenylthiourea series represents a good starting point for further exploration to develop novel antitubercular agents. IMPORTANCE Mycobacterium tuberculosis is responsible for the highest number of deaths from a bacterial pathogen, with >1.5 million in 2020. M. tuberculosis is a sophisticated pathogen that can replicate inside immune cells. There is an urgent need for new drugs to combat M. tuberculosis and to shorten therapy from 6 to 24 months. We have identified a series of molecules that inhibit the growth of M. tuberculosis inside macrophages; we tested a number of derivatives to link structural features to biological activity. The compounds are likely to have novel mechanism of action and so could be developed as new agents for drug-resistant tuberculosis.

Subclinical Genital Herpes Shedding in HIV/Herpes Simplex Virus 2-Coinfected Women during Antiretroviral Therapy Is Associated with an Increase in HIV Tissue Reservoirs and Potentially Promotes HIV Evolution.

Antigen (Ag)-specific immune responses to chronic infections, such as herpes simplex virus type 2 (HSV-2) in HIV/HSV-coinfected persons, may sustain HIV tissue reservoirs by promoting T-cell proliferation but are poorly studied in women on antiretroviral therapy (ART). Mixed anogenital swabs and cervical secretions were self-collected by nine HIV/HSV-2-coinfected women during ART for 28 days to establish subclinical HSV DNA shedding rates and detection of HIV RNA by real-time PCR. Typical herpes lesion site biopsy (TLSB) and cervical biopsy specimens were collected at the end of the daily sampling period. Nucleic acids (NA) isolated from biopsy specimens had HIV quantified and HIV envC2-V5 single-genome amplification (SGA) and T-cell receptor (TCR) repertoires assessed. Women had a median CD4 count of 537 cells/µl (IQR: 483 to 741) at enrollment and HIV plasma viral loads of <40 copies/ml. HSV DNA was detected on 12% of days (IQR: 2 to 25%) from anogenital specimens. Frequent subclinical HSV DNA shedding was associated with increased HIV DNA tissue concentrations and increased divergence from the most recent common ancestor (MRCA), an indicator of HIV replication. Distinct predominant TCR clones were detected in cervical and TLSB specimens in a woman with frequent HSV DNA shedding, with mixing of minor variants between her tissues. In contrast, more limited TCR repertoire mixing was observed in two women with less frequent subclinical HSV DNA shedding. Subclinical HSV shedding in HIV/HSV-coinfected women during ART may sustain HIV tissue reservoirs via Ag exposure or HIV replication. This study provides evidence supporting further study of interventions targeting suppression of Ag-specific immune responses as a component of HIV cure strategies.IMPORTANCE Persons with HIV infection are frequently coinfected with chronic herpesviruses, which periodically replicate and produce viable herpes virions, particularly in anogenital and cervical tissues. Persistent protein expression results in proliferation of CD8+ and CD4+ T cells, and the latter could potentially expand and sustain HIV tissue reservoirs. We found HSV genital shedding rates were positively correlated with HIV DNA concentrations and HIV divergence from ancestral sequences in tissues. Our work suggests that immune responses to common coinfections, such as herpesviruses, may sustain HIV tissue reservoirs during suppressive ART, suggesting future cure strategies should study interventions to suppress replication or reactivation of chronic herpes infections.

The relevance of persisters in tuberculosis drug discovery.

Bacterial persisters are a subpopulation of cells that exhibit phenotypic resistance during exposure to a lethal dose of antibiotics. They are difficult to target and thought to contribute to the long treatment duration required for tuberculosis. Understanding the molecular and cellular biology of persisters is critical to finding new tuberculosis drugs that shorten treatment. This review focuses on mycobacterial persisters and describes the challenges they pose in tuberculosis therapy, their characteristics and formation, how persistence leads to resistance, and the current approaches being used to target persisters within mycobacterial drug discovery.

High content, high-throughput screening for small molecule inducers of NF-κB translocation.

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity.

Human newborn bacille Calmette-Guérin vaccination and risk of tuberculosis disease: a case-control study.

BACKGROUND: An incomplete understanding of the immunological mechanisms underlying protection against tuberculosis (TB) hampers the development of new vaccines against TB. We aimed to define host correlates of prospective risk of TB disease following bacille Calmette-Guérin (BCG) vaccination. METHODS: In this study, 5,726 infants vaccinated with BCG at birth were enrolled. Host responses in blood collected at 10 weeks of age were compared between infants who developed pulmonary TB disease during 2 years of follow-up (cases) and those who remained healthy (controls). RESULTS: Comprehensive gene expression and cellular and soluble marker analysis failed to identify a correlate of risk. We showed that distinct host responses after BCG vaccination may be the reason: two major clusters of gene expression, with different myeloid and lymphoid activation and inflammatory patterns, were evident when all infants were examined together. Cases from each cluster demonstrated distinct patterns of gene expression, which were confirmed by cellular assays. CONCLUSIONS: Distinct patterns of host responses to Mycobacterium bovis BCG suggest that novel TB vaccines may also elicit distinct patterns of host responses. This diversity should be considered in future TB vaccine development.

Serine and proline-rich ligands enriched via phage-display technology show preferential binding to BCR/ABL expressing cells.

BACKGROUND AND OBJECTIVES: Despite the use of targeted therapy, chronic myelogenous leukemia (CML) currently remains incurable with drug therapy, with patients requiring life-long treatment. Developing either a vaccine to prevent the disease or another novel drug to specifically target and eradicate the CML cell will require the identification of CML-associated cell-surface markers and molecules that can bind specifically to the cell surface. In an attempt to discover peptides that bind specifically to cells in the early chronic phase of the disease, we used phage-display technology to identify heptapeptides that bind specifically to the surface of BCR/ABL-expressing fibroblasts. METHODS: An in vitro system using NIH3T3 stably transfected with pGD210 (BCR/ABL) was used as a model for the chronic phase of the disease. The cells were panned using a linear heptapeptide phage library (Ph.D 7.0) in a negative/positive panning strategy with NIH3T3 containing only the plasmid vector as the wild type control. RESULTS: We identified four novel peptides that were enriched through this technique. These peptides contained either multiple proline residues or serine/threonine-proline pairs and showed a confirmed binding preference for BCR/ABL+ fibroblasts. The peptide Y-R-A-P-W-P-P also showed a binding affinity for granulocytes from untreated CML patients. CONCLUSION: We have identified several novel peptides that can be used in future studies to identify specific CML cell-surface antigens or provide a novel drug-delivery mechanism.