Here we present a “smart” xenon-129 NMR biosensor that undergoes a peptide conformational change and labels cancer cells at acidic pH. To a cryptophane host molecule with high Xe affinity, we conjugated a 30mer EALA-repeat peptide that is alpha-helical at pH 5.5 and disordered at pH 7.5. The 129Xe NMR chemical shift at rt was strongly pH-dependent (Δδ = 3.4 ppm): δ = 64.2 ppm at pH 7.5 vs. δ = 67.6 ppm at pH 5.5 where Trp(peptide)-cryptophane interactions were evidenced by Trp fluorescence quenching. Using Hyper-CEST NMR, we probed peptido-cryptophane detection limits at low-picomolar (10−11 M) concentration, which compares favourably to other NMR pH sensors at 10−2−10−3 M. Finally, in biosensor-HeLa cell solutions, peptide-cell membrane insertion at pH 5.5 generated a 13.4 ppm downfield cryptophane-129Xe NMR chemical shift relative to pH 7.5 studies. This highlights new uses for 129Xe as an ultrasensitive probe of peptide structure and function, along with potential applications for pH-dependent cell labeling in cancer diagnosis and treatment.
Folate-conjugated
cryptophane was developed for targeting cryptophane
to membrane-bound folate receptors that are overexpressed in many
human cancers. The cryptophane biosensor was synthesized in 20 nonlinear
steps, which included functionalization with folate recognition moiety,
solubilizing peptide, and Cy3 fluorophore. Hyperpolarized 129Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane.
Cellular internalization of biosensor was monitored by confocal laser
scanning microscopy and quantified by flow cytometry. Competitive
blocking studies confirmed cryptophane endocytosis through a folate
receptor-mediated pathway. Flow cytometry revealed 10-fold higher
cellular internalization in KB cancer cells overexpressing folate
receptors compared to HT-1080 cells with normal folate receptor expression.
The biosensor was determined to be nontoxic in HT-1080 and KB cells
by MTT assay at low micromolar concentrations typically used for hyperpolarized 129Xe NMR experiments.
Malaria is a deadly infectious disease caused by parasites of the Plasmodium spp. that takes an estimated 435,000 lives each year, primarily among young African children. For most children, malaria is a febrile illness that resolves with time, but in ∼1% of cases, for reasons we do not understand, malaria becomes severe and life threatening. Cerebral malaria (CM) is the most common form of severe malaria, accounting for the vast majority of childhood deaths from malaria despite highly effective antiparasite chemotherapy. Thus, CM is one of the most prevalent lethal brain diseases, and one for which we have no effective therapy. CM is, in part, an immune-mediated disease, and to fully understand CM, it is essential to appreciate the complex relationship between the malarial parasite and the human immune system. In this study, we provide a primer on malaria for immunologists and, in this context, review progress identifying targets for therapeutic intervention.
The efficient synthesis of enantiopure, trisubstituted cryptophane-A derivatives, organic host molecules with unusually high xenon affinity is reported. Synthesis and chromatographic separation of (+/−) tri-Mosher’s acid-substituted cryptophane diastereomers gave ready access to the enantiopure cryptophanes, which are critical components in the design of enantiomerically pure 129Xe biosensors. Hyperpolarized 129Xe NMR spectroscopy identified single resonances for both trisubstituted cryptophane diastereomers that were separated by 9.5 ppm. This highlights opportunities for using enantiopure xenon biosensors in the simultaneous detection of 129Xe in different biochemical environments.
The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood–brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.
Cerebral malaria is the deadliest complication of malaria, a febrile infectious disease caused by
Plasmodium parasite. Any of the five human
Plasmodium species can cause disease, but, for unknown reasons, in approximately 2 million cases each year
P. falciparum progresses to severe disease, ultimately resulting in half a million deaths. The majority of these deaths are in children under the age of five. Currently, there is no way to predict which child will progress to severe disease and there are no adjunctive therapies to halt the symptoms after onset. Herein, we discuss what is known about the disease mechanism of one form of severe malaria, cerebral malaria, and how we might exploit this understanding to rescue children in the throes of cerebral disease.
We present the first cryptophane-based “turn-on” 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.