Anton Simeonov Ph.D. National Institute of Health

A new simple universal detection system could improve detection of a wide range of current drugs.

Facile monitoring of the actual concentration of a drug inside a patient's blood stream (referred to as therapeutic drug monitoring [TDM]) is crucial in situations in which dose adjustment is necessary, but TDM currently relies on complex clinical laboratory instruments and protocols because most analyses involve immunoassays and chromatographic techniques. Thus, simple devices, not unlike the handheld glucose meter, are needed. Here, the authors attempt to provide a simple universal detection system that is potentially applicable to real-time detection of a very wide range of current drugs. The LUCIDs (luciferase-based indicators of drugs) platform provides ratiometric luminescent reporting of the level of an analyte by using a cognate protein that acts as a receptor for the drug analyte, a fluorescent tracer mimetic of the drug, and luciferase reporter enzyme to serve as a donor of bioluminescence (first figure). 

Design and performance of a sensor for methotrexate. (a) The fusion protein SNAP-Pro30-NanoLuc (NLuc)-cpDHFR is linked to a synthetic molecule containing a fluorophore (red star) and a DHFR inhibitor (gray ball). Free analyte (green ball) can shift the sensor to an open conformation, reducing BRET efficiency. (b) Structure of the synthetic molecules used to assemble the sensor. The benzylguanine group (blue) serves as the reactive moiety for SNAP-tag labeling, the fluorophore Cy3 is colored in red, and the tethered DHFR inhibitors trimethoprim (BG-Cy3-TMP; 1) and methotrexate (BG-Cy3-MTX; 2) are shown in gray. (c) Structure of E. coli DHFR bound to methotrexate. The N terminus is shown in blue, the C terminus is shown in red, and the position of the new termini produced by circular permutation (N23 and L24) as well as the 5-glycine linker used to connect the original termini are shown in orange. (d) Emission spectra of SNAP-Pro30-NanoLuc-DHFRcpL24G5 labeled with BG-Cy3-TMP (1) in human serum spiked with defined concentrations of methotrexate. RLU, relative luminescence units. (e) The sensor protein labeled with BG-Cy3-TMP (1) has a ratio change of 1,340 ± 90% and a c50 of 0.75 ± 0.04 μM (obtained from three independent titrations; the graph shows one titration). The sensor protein labeled with BG-Cy3-MTX (2) has a c50 of 85 ± 6 μM (obtained from three titrations; the graph shows one titration). Error represents SD. (f) Chemical structure of methotrexate.

Bioluminescent resonance energy transfer (BRET) between luciferase and the fluorescent drug tracer bound to the receptor protein serves as the signal generator, while the competitive displacement of the bound tracer by the actual free drug contained within the sample acts to modulate BRET and to ultimately report on the drug's concentration. The authors first established the technology by using the anticancer agent methotrexate. Bacterial dihydrofolate reductase (DHFR) served as the cognate receptor; its inhibitor, trimethoprim, was conjugated to Cy3 and served as the fluorescent tracer; and the engineered minimal luciferase variant NanoLuc (fused to DHFR) served as the BRET donor. To eliminate the inner filter effect caused by intensely colored blood components, the authors thinly spotted blood samples on chromatography paper and analyzed the emitted light from this very thin sample layer in which the BRET-emitted photons had a very low probability of encountering blood component molecules. In a further step toward creation of a simple instrument, the team used a consumer digital camera as an imaging device, with the paper substrate containing the detection components and blood sample set in a regular polystyrene icebox outfitted with a hole, and compared the spots' color change as a function of the changing BRET ratio (second figure). Methotrexate levels in 30 patient samples were thus evaluated and found to compare well to those determined via a standard fluorescence polarization immunoassay. Additional drugs adapted for the LUCIDs platform included cyclosporin A, topiramate, and digoxin. While the overall instrumentation and optical detection component of the present platform appear very likely to be successfully developed into a real product, a major challenge inherent to LUCIDs for the time being remains the design of the requisite receptor protein and the tracer ligand. It is likely that protein evolution or alternative modalities, such as aptamers, will be required for the generation of robust receptor molecules for a number of typical drugs. Such systems could also be applied to measure intracellular compound concentrations within in vitro cell-based assays. 

Measuring methotrexate concentrations in patient samples using a point-and-shoot digital camera. (a) Pictures of the box and camera used for detection. The chromatography paper with printed wax circles can be seen in the bottom picture. (b) Picture of spotted SNAP-Pro30-NanoLuc-DHFRcpL24G5 labeled with BG-Cy3-TMP with varying methotrexate concentrations in human serum taken with a digital camera. Scale bar, 5 mm. The histograms show the intensity distributions of pixels in the red and blue channels. (c) Correlation of the results obtained for patient serum samples using LUCIDs and a traditional fluorescence polarization immunoassay. Each LUCID measurement is given as the mean ± SD of three independent measurements. Regression analysis yielded a Pearson correlation coefficient (R) = 0.995. The two samples highest in concentration were diluted tenfold in commercial human serum before the measurement.

*Abstract from Nat Chem Biol 2014, Vol. 10: 598–603

For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patient's blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column that analyzes two papers published in Science: one titled "Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors" authored by Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, and Rubin LL; and the other titled "Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle" authored by Sinha M, Jang YC, Oh J, Khong D, Wu EY, Manohar R, Miller C, Regalado SG, Loffredo FS, Pancoast JR, Hirshman MF, Lebowitz J, Shadrach JL, Cerletti M, Kim MJ, Serwold T, Goodyear LJ, Rosner B, Lee RT, and Wagers AJ.

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