Amy M. Barrios

A high throughput drug screen for Entamoeba histolytica identifies a new lead and target

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide, resulting in ∼70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole, which has adverse effects, and potential resistance of E. histolytica to the drug is an increasing concern. To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

Gold(I)-Mediated Inhibition of Protein Tyrosine Phosphatases: A Detailed in Vitro and Cellular Study

Gold(I) complexes containing N-heterocyclic carbene ligands were synthesized, characterized, and along with the antiarthritic drug, auranofin, tested as inhibitors of the cysteine-dependent protein tyrosine phosphatases, which are implicated in several disease states. These compounds exhibit potencies in the low micromolar range against the enzymes in vitro. At therapeutically relevant concentrations, all compounds inhibit PTP activity in Jurkat T leukemia cells with some selectivity. In addition, the gold-carbene compounds inhibit phosphatase activity in primary mouse thymocytes.

Identification of Cystathionine β‐Synthase Inhibitors Using a Hydrogen Sulfide Selective Probe

Buzzing with activity: A hydrogen sulfide selective fluorogenic probe, 7-azido-4-methylcoumarin (AzMC), serves as a highly sensitive assay for cystathionine β-synthase activity, and is suitable for the high-throughput discovery of novel enzyme inhibitors.

Efficient Delivery of Cyclic Peptides into Mammalian Cells with Short Sequence Motifs

Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is l-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7-13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2-5-fold higher than that of nonaarginine (R(9)). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities.

Identifying Potent, Selective Protein Tyrosine Phosphatase Inhibitors from a Library of Au(I) Complexes

Therapeutic inhibition of protein tyrosine phosphatase activity is a compelling yet challenging approach to the treatment of human disease. Toward this end, a library of 40 gold complexes with the general formula R(3)P-Au-Cl was screened to identify novel inhibitors of PTP activity. The most promising inhibitor obtained for the lymphoid tyrosine phosphatase LYP, (2-pyridine)(Ph(2))P-Au-Cl, is one of the most potent and selective LYP inhibitors identified to date with an IC(50) of 1.5 +/- 0.3 microM, 10-fold selectivity for LYP over PTP-PEST, HePTP, and CD45 in vitro, and activity in cellular studies as well.

Early Endosomal Escape of a Cyclic Cell-Penetrating Peptide Allows Effective Cytosolic Cargo Delivery

Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

Spectroscopic Evidence for the Formation of Goldfingers

Gold(I) has long been used in the treatment of rheumatoid arthritis, but the therapeutically relevant biological targets of gold(I) are not well understood. Here, we report the results of a spectroscopic investigation into the formation of goldfingers. By exploiting a thiolate to gold charge-transfer band in the UV, we observed that gold(I) interacts with zinc finger peptides with a stoichiometry of one gold ion for each two cysteine residues, forming 1:1, 1.5:1, and 2:1 adducts with zinc finger peptides containing CCHH, CCHC, and CCCC donor sets, respectively. In addition, circular dichroism experiments provided evidence that goldfingers are more ordered than the corresponding metal-free peptides but do not exhibit the canonical zinc finger structure.

Auranofin is an apoptosis-simulating agent with in vitro and in vivo anti-leishmanial activity.

Cutaneous leishmaniasis remains ignored in therapeutic drug discovery programs worldwide. This is mainly because cutaneous leishmaniasis is frequently a disease of impoverished populations in countries where funds are limited for research and patient care. However, the health burden of individuals in endemic areas mandates readily available, effective, and safe treatments. Of the existing cutaneous leishmaniasis therapeutics, many are growth inhibitory to Leishmania parasites, potentially creating dormant parasite reservoirs that can be activated when host immunity is compromised, enabling the reemergence of cutaneous leishmaniasis lesions or worse spread of Leishmania parasites to other body sites. To accelerate the identification and development of novel cutaneous leishmaniasis therapeutics, we designed an integrated in vitro and in vivo screening platform that incorporated multiple Leishmania life cycles and species and probed a focused library of pharmaceutically active compounds. The objective of this phenotypic drug discovery platform was the identification and prioritization of bona fide cytotoxic chemotypes toward Leishmania parasites. We identified the Food and Drug Administration-approved drug auranofin, a known inhibitor of Leishmania promastigote growth, as a potent cytotoxic anti-leishmanial agent and inducer of apoptotic-like death in promastigotes. Significantly, the anti-leishmanial activity of auranofin transferred to cell-based amastigote assays as well as in vivo murine models. With appropriate future investigation, these data may provide the foundation for potential exploitation of gold(I)-based complexes as chemical tools or the basis of therapeutics for leishmaniasis. Thus, auranofin may represent a prototype drug that can be used to identify signaling pathways within the parasite and host cell critical for parasite growth and survival.

A potent and selective small molecule inhibitor for the lymphoid-specific tyrosine phosphatase (LYP), a target associated with autoimmune diseases

Lymphoid-specific tyrosine phosphatase (LYP), a member of the protein tyrosine phosphatase (PTP) family of signaling enzymes, is associated with a broad spectrum of autoimmune diseases. Herein we describe our structure-based lead optimization efforts within a 6-hydroxy-benzofuran-5-carboxylic acid series culminating in the identification of compound 8b, a potent and selective inhibitor of LYP with a K(i) value of 110 nM and more than 9-fold selectivity over a large panel of PTPs. The structure of LYP in complex with 8b was obtained by X-ray crystallography, providing detailed information about the molecular recognition of small-molecule ligands binding LYP. Importantly, compound 8b possesses highly efficacious cellular activity in both T- and mast cells and is capable of blocking anaphylaxis in mice. Discovery of 8b establishes a starting point for the development of clinically useful LYP inhibitors for treating a wide range of autoimmune disorders.

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157–177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107–137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.