Novel series of tunable µOR modulators with enhanced brain penetration for the treatment of opioid use disorder, pain and neuropsychiatric indications.

Structural optimization of a previously reported agonist of µOR, PZM21 is described resulting in the discovery of a novel series of amides with at least 4-folds enhanced CNS penetration in rat. Furthermore, these efforts yielded compounds with varying levels of efficacy on the receptor ranging from high efficacy agonists such as compound 20 to antagonists, such as 24. The correlation between in vitro activation of µOR and relative activity in models of analgesia for these compounds is discussed. The compelling results obtained in these studies demonstrate the potential utility of these newly discovered compounds in the treatment of pain and opioid use disorder.

Metabolism and Bioactivation: It's Time to Expect the Unexpected.

Improvements in in vitro ADME tools and pharmacokinetic prediction models have helped to shift attrition rates in early clinical trials from poor exposure to drug safety concerns, such as drug-induced liver injury (DILI). Assessing a new chemical entity's potential for liver toxicity is an important consideration for the likely success of new drug candidates. Reactive intermediates produced during drug metabolism have been implicated as a cause of DILI, and their formation has been correlated to the addition of a black box warning on a drug label. In this work, we will present contemporary examples of the bioactivation of atypical structures usually regarded as benign and often used by medicinal chemists when attempting to avoid bioactivation. Medicinal chemistry strategies used to derisk bioactivation will be discussed, and an emphasis will be placed on the necessity of a multidisciplinary approach.

A traceless linker for aliphatic amines that rapidly and quantitatively fragments after reduction.

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pK a values. Here a traceless self-immolative linker composed of a dithiol-ethyl carbonate connected to a benzyl carbamate (DEC) is presented, which can modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. DEC was able to reversibly modify the lysine residues on CRISPR-Cas9 with either PEG, the cell penetrating peptide Arg10, or donor DNA, and generated Cas9 conjugates with significantly improved biological properties. In particular, Cas9-DEC-PEG was able to diffuse through brain tissue significantly better than unmodified Cas9, making it a more suitable candidate for genome editing in animals. Furthermore, conjugation of Arg10 to Cas9 with DEC was able to generate a self-delivering Cas9 RNP that could edit cells without transfection reagents. Finally, conjugation of donor DNA to Cas9 with DEC increased the homology directed DNA repair (HDR) rate of the Cas9 RNP by 50% in HEK 293T cell line. We anticipate that DEC will have numerous applications in biotechnology, given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

Graphene-based biosensor for on-chip detection of bio-orthogonally labeled proteins to identify the circulating biomarkers of aging during heterochronic parabiosis.

Studies of heterochronic parabiosis, where two animals of different ages are joined surgically, provided proof-of-principle results that systemic proteins have broad age-specific effects on tissue health and repair. In an effort to identify these systemic proteins, we previously developed a method to selectively label the proteome of only one animal joined in parabiosis utilizing bio-orthogonal non-canonical amino acid tagging (BONCAT), which can metabolically label proteins during their de novo synthesis by incorporating a methionine substitute, azido-nor-leucine (ANL), in cells expressing a mutant methionyl-tRNA synthetase (MetRSL274G). Once labeled, we can selectively identify the proteins produced by the MetRSL274G transgenic mouse in the setting of heterochronic parabiosis. This approach enabled the detection of several rejuvenating protein candidates from the young parabiont, which were transferred to the old mammalian tissue through their shared circulation. Although BONCAT is a very powerful technology, the challenges associated with its complexity including large starting material requirements and cost of ANL-labeled protein detection, such as modified antibody arrays and mass spectrometry, limit its application. Herein, we propose a lab-on-a-chip technology, termed Click-A+Chip for facile and rapid digital detection of ANL-labeled proteomes present in minute amount of sample, to replace conventional assays. Click-A+Chip is a graphene-based field effect biosensor (gFEB) which utilizes novel on-chip click-chemistry to specifically bind to ANL-labeled biomolecules. In this study, Click-A+Chip is utilized for the capture of ANL-labeled proteins transferred from young to old parabiotic mouse partners. Moreover, we were able to identify the young-derived ANL-labeled Lif-1 and leptin in parabiotic systemic milieu, confirming previous data as well as providing novel findings on the relative levels of these factors in young versus old parabionts. Summarily, our results demonstrate that Click-A+Chip can be used for rapid detection and identification of ANL-labeled proteins, significantly reducing the sample size, complexity, cost and time associated with BONCAT analysis.

An Enzyme-Mediated Amplification Strategy Enables Detection of ß-Lactamase Activity Directly in Unprocessed Clinical Samples for Phenotypic Detection of ß-Lactam Resistance.

Biochemical assays that can identify ß-lactamase activity directly from patient samples have the potential to significantly improve the treatment of bacterial infections. However, current ß-lactamase probes do not have the sensitivity needed to measure ß-lactam resistance directly from patient samples. Here, we report the development of an instrument-free signal amplification technology, DETECT, that connects the activity of two enzymes in series to effectively amplify the activity of ß-lactamase 40 000-fold, compared to the standard ß-lactamase probe nitrocefin.

Nitro Sulfonyl Fluorides are a new pharmacophore for the development of antibiotics.

The development of antibiotics against Gram-negative bacteria is a central problem in drug discovery. In this report, we demonstrate that aromatic sulfonyl fluorides with a nitro group in their ortho position have remarkable antibacterial activity and are active against drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant Acinetobacter baumannii, and Pseudomonas aeruginosa.

Thiophene bridged aldehydes (TBAs) image ALDH activity in cells via modulation of intramolecular charge transfer.

Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of an aldehyde to a carboxylic acid and are implicated in the etiology of numerous diseases. However, despite their importance, imaging ALDH activity in cells is challenging due to a lack of fluorescent imaging probes. In this report, we present a new family of fluorescent probes composed of an oligothiophene flanked by an aldehyde and an electron donor, termed thiophene-bridged aldehydes (TBAs), which can image ALDH activity in cells. The TBAs image ALDH activity via a fluorescence sensing mechanism based on the modulation of intramolecular charge transfer (ICT) and this enables the TBAs and their ALDH-mediated oxidized products, thiophene-bridged carboxylates (TBCs), to have distinguishable fluorescence spectra. Herein, we show that the TBAs can image ALDH activity in cells via fluorescence microscopy, flow cytometry, and in a plate reader. Using TBA we were able to develop a cell-based high throughput assay for ALDH inhibitors, for the first time, and screened a large, 1460-entry electrophile library against A549 cells. We identified α,ß-substituted acrylamides as potent electrophile fragments that can inhibit ALDH activity in cells. These inhibitors sensitized drug-resistant glioblastoma cells to the FDA approved anti-cancer drug, temozolomide. The TBAs have the potential to make the analysis of ALDH activity in cells routinely possible given their ability to spectrally distinguish between an aldehyde and a carboxylic acid.

Thiophene bridged hydrocyanine - a new fluorogenic ROS probe.

Hydrocyanines are a class of commonly used reactive oxygen species (ROS) fluorescent imaging probes, which can image ROS in cell culture, organ culture, and in vivo. However, despite their widespread use, hydrocyanines have several drawbacks that limit their effectiveness, such as a high rate of auto-oxidation, a small Stokes shift, and poor water solubility. In addition, the hydrocyanines oxidize into cyanine dyes, which themselves decompose in the presence of ROS, and this further lowers their sensitivity towards detecting ROS. In this report, we present a new hydrocyanine analog, termed as thiophene-bridged hydrocyanine (TBHC), which has its double bonds replaced with a bisthiophene. TBHC is 8.06-fold more stable to auto-oxidation than the hydrocyanine hydro-Cy5 and is significantly better at imaging ROS in cell culture.