The design of drug delivery systems capable of minimal endolysosomal trapping, controlled drug release, and real-time monitoring of drug effect is highly desirable for personalized medicine. Herein, by using mesoporous silica nanoparticles (MSNs) coated with cell-penetrating poly(disulfide)s and a fluorogenic apoptosis-detecting peptide (DEVD-AAN), we have developed a platform that could be uptaken rapidly by mammalian cells via endocytosis-independent pathways. Subsequent loading of these MSNs with small molecule inhibitors and antisense oligonucleotides resulted in intracellular release of these drugs, leading to combination inhibition of endogenous miR-21 activities which was immediately detectable by the MSN surface-coated peptide using two-photon fluorescence microscopy.

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Two-photon fluorescence microscopy (TPFM) provides key advantages over conventional fluorescence imaging techniques, namely, increased penetration depth, lower tissue autofluorescence and self-absorption, and reduced photodamage and photobleaching and therefore is particularly useful for imaging deep tissues and animals. Enzyme-detecting, small molecule probes provide powerful alternatives over conventional fluorescent protein (FP)-based methods in bioimaging, primarily due to their favorable photophysical properties, cell permeability, and chemical tractability. In this article, we report the first fluorogenic, small molecule reporter system (Y2/Y1) capable of imaging endogenous phosphatase activities in both live mammalian cells and Drosophila brains. The one- and two-photon excited photophysical properties of the system were thoroughly investigated, thus confirming the system was indeed a suitable Turn-ON fluorescence pair for TPFM. To our knowledge, this is the first enzyme reporting two-photon fluorescence bioimaging system which was designed exclusively from a centrosymmetric dye possessing desirable two-photon properties. By conjugation of our reporter system to different cell-penetrating peptides (CPPs), we were able to achieve organelle- and tumor cell-specific imaging of phosphatase activities with good spatial and temporal resolution. The diffusion problem typically associated with most small molecule imaging probes was effectively abrogated. We further demonstrated this novel two-photon system could be used for imaging endogenous phosphatase activities in Drosophila brains with a detection depth of >100 μm.

The design of the first dual-purpose activity-based probe of monoamine oxidase B (MAO-B) is reported. This probe is highly selective towards MAO-B, even at high MAO-A expression levels, and could sensitively report endogenous MAO-B activities by both in situ proteome profiling and live-cell bioimaging. With a built-in imaging module as part of the probe design, the probe was able to accomplish what all previously reported MAO-B imaging probes failed to do thus far: the live-cell imaging of MAO-B activities without encountering diffusion problems.

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Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

Proper folding of cellular proteins is assisted by protein disulfide isomerases (PDIs) in the endoplasmic reticulum of mammalian cells. Of the at least 21 PDI family members known in humans, the 57-kDa PDI has been found to be a potential therapeutic target for a variety of human diseases including cancer and neurodegenerative diseases. Consequently, small molecule PDI-targeting inhibitors have been actively pursued in recent years, and thus far, compounds possessing moderate inhibitory activities (IC50 between 0.1 and 100 μM against recombinant PDI) have been discovered. In this article, by using in situ proteome profiling experiments in combination with in vitro PDI enzymatic inhibition assays, we have discovered a phenyl vinyl sulfonate-containing small molecule (P1; shown) as a relatively potent and specific inhibitor of endogenous human PDI in several mammalian cancer cells (e.g., GI50 ∼ 4 μM). It also possesses an IC50 value of 1.7 ± 0.4 μM in an in vitro insulin aggregation assay. Our results indicate P1 is indeed a novel, cell-permeable small molecule PDI inhibitor, and the electrophilic vinyl sulfonate scaffold might serve as a starting point for future development of next-generation PDI inhibitors and probes.

The protein-lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large-scale mapping studies of the protein-lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne-containing choline head group and a diazirine-modified fatty acid simultaneously into choline-containing phospholipids synthesized from live mammalian cells, protein-phospholipid interactions have been successfully imaged in live cells. Subsequent in situ profiling of the modified Cho phospholipid-crosslinked proteins followed by quantitative proteomics allowed identification of several hundred putative phospholipid-interacting proteins, some of which were further validated.