Dan Loewenthal

Abstract:

Mass photometry (MP) has emerged as a powerful approach to study quaternary biomolecular structure, dynamics and interactions. The capabilities of the method ultimately hinge on the ability to accurately measure the tiny optical contrast generated by individual molecules at a glass–water interface, which enables mass-resolved quantification of biomolecular mixtures. Ideally, this capability is limited only by photon shot noise, but in practice depends on additional parameters and details of the assay. Here, we focus on the key factors affecting MP performance and present simple steps that can be taken to achieve optimal MP measurements in terms of mass resolution, quantitative detection limit, reproducibility and analyte concentration range without compromising the speed and simplicity of the technique. Each sample takes <10 min to analyse, with an additionial 2 h if amination of the glass surface is desired.

Abstract:

Analytical chemists are often challenged to screen for bioactive compounds in complex matrices, sometimes without a priori knowledge of the exact compound of interest. Therefore, "flagging" techniques, highlighting common characteristics of bioactive compounds, are highly sought after. In this work, we demonstrate a double flagging method, where unknown organophosphorus acetylcholinesterase inhibitors are "flagged" out of a complex matrix by the presence of organophosphorus-indicative ions as well as their acetylcholinesterase inhibition. This is accomplished by flagging the LC chromatographic retention time of phosphorus-indicative ions using accurate mass high-energy in-source CID products, and the retention time of acetylcholinesterase inhibiting compounds using a parallel microfractionation-based bioassay. We successfully apply this method to screen VX, VM, and RVX nerve agents as well as methomyl, a carbamate pesticide, out of soil and whole blood samples at low μM to sub-μM concentrations. This methodology can be easily extended to diverse chemical families and biological activities of interest.

Abstract:

Cholinesterase enzymes are involved in a wide range of bodily functions, and their disruption is linked to pathologies such as neurodegenerative diseases and cancer. While cholinesterase inhibitors are used as drug treatments for diseases such as Alzheimer and dementia at therapeutic doses, acute exposure to high doses, found in pesticides and nerve agents, can be lethal. Therefore, measuring cholinesterase activity is important for numerous applications ranging from the search for novel treatments for neurodegenerative disorders to the on-site detection of potential health hazards. Here, we present the development of a near-infrared (near-IR) fluorescent single-walled carbon nanotube (SWCNT) optical sensor for cholinesterase activity and demonstrate the detection of both acetylcholinesterase and butyrylcholinesterase, as well as their inhibition. We show sub U L^-1 sensitivity, demonstrate the optical response at the level of individual nanosensors, and showcase an optical signal output in the 900-1400 nm range, which overlaps with the biological transparency window. To the best of our knowledge, this is the longest wavelength cholinesterase activity sensor reported to date. Our near-IR fluorescence-based approach opens new avenues for spatiotemporal-resolved detection of cholinesterase activity, with numerous applications such as advancing the research of the cholinergic system, detecting on-site potential health hazards, and measuring biomarkers in real-time.

Abstract:

Government-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood-brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.