Spotlight pubs.acs.org/acschemicalbiology
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MOLECULAR SWITCHES CONTROL CANCER IMMUNOTHERAPY
In one study, the research team designed CAR-T cells to express an antibody targeting the synthetic dye fluorescein. The corresponding molecular switches consisted of pairs of fluorescein molecules tethered to monoclonal antibodies targeting a specific B cell antigen. Theoretically, treatment would involve inoculating a patient with the fluorescein-targeting CAR-T cells, then dosing the patient with the molecular switch, which would bind to cells bearing the specific B cell antigens and recruit CAR-T cells bearing antifluorescein antibodies. The treatment design described in the other study operated under a similar premise, except the CAR-T cells were engineered to recognize a 14-residue “neo-peptide” that does not occur in the human proteome and thus functions as a bioorthogonal antigen; the corresponding molecular switches were fusion proteins consisting of neo-peptides genetically grafted to specific anti-B cell antibodies. In both studies, the research teams discovered that the effectiveness of the molecular switches depended on the site at which the antigens were tethered to the antibody; each molecular switch had distinct optimal tethering sites that varied between the different anti-B cell antibodies tested. When the switchable CAR-T cell treatments were tested in rodent models of B cell leukemia, they were found to effectively decrease tumor cell burden. Importantly, careful titration of the molecular switches allowed the researchers to induce tumor depletion while mitigating potentially dangerous side effects such as cytokine release syndrome. Furthermore, the researchers demonstrated that the biological target of a single CAR-T cell-type could be alternated simply by changing the antibody component of the corresponding molecular switches. The authors envision that the molecular switch strategy would thus allow CAR-T therapy to be customized to treat tumors with heterogeneous antigen expression. Heidi A. Dahlmann
Image Designer: Travis Young
Improvements to an emerging type of cancer therapy may be on the way, thanks to research recently carried out at the California Institute of Biomedical Research and the Scripps Research Institute. Research teams led by Chan Hyuk Kim, Peter G. Schultz, and Travis S. Young, the corresponding authors on both of two back-to-back reports published in Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (4), E450−E458, DOI: 10.1073/pnas.1524193113 and Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (4), E459−E468, DOI: 10.1073/ pnas.1524155113), anticipate that their methods for controlling the activity of immune cells engineered to seek and destroy certain cancer cells will improve the clinical safety and versatility of this type of therapy, known as CAR-T cell therapy. CAR-T cells are usually made by programming patientderived T lymphocytes to express on their cell surfaces chimeric antibody receptors (CARs) that contain both antibody fragments that specifically recognize cancer-associated antigens as well as domains that trigger the T cells’ inherent cytotoxic activity. Upon reintroduction to the patient, CAR-T cells come into contact with cells displaying their targeted antigen, and subsequent antigen-receptor binding initiates T cell-mediated lysis of the target cells. CAR-T cells designed to target CD19, an antigen found on B lymphocytes, have been shown in clinical studies to induce remission in patients with treatment-resistant B-cell malignancies such as refractory acute lymphoblastic leukemia. However, major drawbacks to this approach are serious side effects resulting from uncontrollable CAR-T cell proliferation and indiscriminate killing of both cancerous and normal B cells. Therefore, Kim and co-workers sought to pair CAR-T cells with molecular switches that would enable external control over the cells’ activation and target specificity. © 2016 American Chemical Society
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ARTEMISININ ACTIVATION PATHWAY CONFIRMED, TARGETS PINPOINTED
Adapted from Nat. Commun, Wang, J. et al., 6, 10111, copyright 2015, DOI: 10.1038/ncomms10111. Copyright 2015 Wang et al. Available under the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). Published: February 19, 2016 293
DOI: 10.1021/acschembio.6b00129 ACS Chem. Biol. 2016, 11, 293−295
ACS Chemical Biology
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drive biological reactions previously reserved for RNA or protein. To date, DNAzymes have been mechanistically dissected by biochemical experiments but have escaped the eyes of structural biologists. Now, Ponce-Salvatierra et al. (Nature 2016, 529, 231−234, DOI: 10.1038/nature16471) present the first three-dimensional look at a DNA catalyst, an X-ray crystal structure of the 9DB1 DNAzyme bound to RNA. This particular enzyme was previously uncovered in a DNA selection for specific 3′−5′ RNA ligase activity. The structure, solved at 2.8 Å, contains a 44-nucleotide DNA bound to a 15-nucleotide RNA, postcatalysis. The enzyme adopts a compact, intricate fold to form an active site despite the lack of 2′-hydroxyl groups. In fact, the less restrictive sugar pucker of the deoxyribose backbone compared to ribose results in a high degree of conformational flexibility. Guided by the structure, follow-up biochemical experiments help explain the mechanism of 3′−5′ specificity and an unforeseen role of DNA position 29 in selecting the RNA substrate’s terminal nucleotide. It was certainly a difficult task to crystallize and solve a DNAzyme after so many have attempted it previously, but the resulting picture is truly impressive. Jason G. Underwood
The natural product artemisinin is the best line of defense against malaria, a disease resulting from infection by Plasmodium parasites that kills hundreds of thousands of people each year. The drug’s mechanism of action, which is thought to involve iron-mediated bioactivation to generate radicals that form adducts with Plasmodium proteins, has been intensively studied for decades. Answers to fundamental questions about artemisinin’s bioactivation and targets have now been furnished in a recent report by Jigang Wang and co-workers (Nat. Commun. 2015, 6, 10111, DOI: 10.1038/ncomms10111). The research team generated an activity probe (AP1) in which artemisinin was modified with an alkyne tag that would allow it to participate in bioorthogonal “click” reactions. Once AP1 was activated by an iron species and exposed to parasite proteins in vitro or in vivo, it would form covalent adducts with target proteins. The AP1 probe would then be “clicked” to biotin to enable AP1-labeled proteins to be isolated and identified by mass spectrometry; alternatively, AP1 would be clicked to a fluorescent dye to allow adducted proteins to be quantified following SDS-PAGE. Upon incubating live parasites with AP1, the research team isolated 124 parasite proteins that were directly targeted by the drug probe, 33 of which were previously proposed antimalarial drug targets and many of which were involved in key parasitic metabolic pathways. The research team also exposed isolated proteins to AP1 in the presence of several iron species hypothesized to mediate artemisinin bioactivation and discovered that heme rather than free ferrous iron was responsible for bioactivation, resolving a long-standing controversy over the identity of the activating agent. The authors note that this discovery could explain why artemisinin is so effective at killing late-stage parasites, which harbor very high heme concentrations as a consequence of digesting hemoglobin derived from the host’s blood cells. Heidi A. Dahlmann
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TACKLING IMMUNITY CHALLENGES WITH CELL THERAPIES FOR DIABETES
DNA: ACROBATIC AND CATALYTIC Reprinted with permission from Macmillan Publishers Ltd.: Nature Medicine, advance online publication, 25 January 2016, DOI: 10.1038/nm.4030.
Researchers have sought treatments for treatments for type I diabetes that avoid the challenges of daily glucose monitoring and injections with insulin. Transplants of a whole pancreas and islet cells from donor organs are options but require immunosuppressant drugs to prevent rejection. Transplants with betacells derived from human pluripotent cells are promising but could present the same immunity challenges. Taking a step toward a new cell therapy that avoids these problems, Vegas et al. have shown that lab-derived beta cells encapsulated in alginate polymers can control blood sugar levels for more than 100 days in a mouse model of type I diabetes (Nat. Med. 2016, DOI: 10.1038/nm.4030). Previously, these groups had shown that the particle size of materials such as alginates used to encapsulate cells contributes to the immune response. In addition, they developed a modified alginate, triazole-thiomorpholine dioxide (TMTD) alginate, that resists the foreign-body responsethe buildup of immune proteins, cells, and eventually a fibrous capsule that can isolate transplanted cells or tissues within the body. In the current study, Vegas et al. used a strain of immunocompetent mice that produces a strong foreign body response
Reprinted with permission from Macmillan Publishers Ltd.: Nature, advance online publication, 6 January 2016, DOI: 10.1038/nature16471.
Protein and RNA biopolymers are responsible for catalyzing an impressive range of chemical reactions in all kingdoms of life. After the discovery of natural catalytic RNAs with RNAcleaving characteristics, clever in vitro selection schemes identified ribozymes to catalyze many other chemical reactions. Researchers also wondered whether single stranded DNA could be catalytic or whether the signature 2′-hydroxyl group makes RNA uniquely suited to catalysis. Over two decades ago, the first catalytic DNA, or DNAzyme, was uncovered by in vitro selection, and over time, more DNAs have been isolated to 294
DOI: 10.1021/acschembio.6b00129 ACS Chem. Biol. 2016, 11, 293−295
ACS Chemical Biology
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and treated them with streptozotocin, which destroys beta cells, mimicking type 1 diabetes. They treated those mice with 500 μm alginate microcapsules, 1.5 mm spheres of normal alginate, and 1.5 mm spheres of TMTD alginate with various doses of lab-derived beta cells. Mice treated with TMTD alginate encapsulated cells maintained normal glucose levels for more than 70 days, more than twice as long as for mice treated with the 1.5 mm spheres of normal alginate and more than 4 times longer than for mice dosed with microcapsules. The researchers also showed that TMTD alginate capsules had lower amounts of fibrous deposits from immune cells and protected the encapsulated cells from the body’s immune system. In a separate cohort of diabetic mice, the transplanted cells encapsulated in TMTD alginate maintained normal blood sugar for 174 days. These results provide a foundation for further animal and human testing of encapsulated beta cells and support the potential of this type of cell therapy to treat type I diabetes. Sarah A. Webb
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DOI: 10.1021/acschembio.6b00129 ACS Chem. Biol. 2016, 11, 293−295
