Catalysis of Hydrogen-Deuterium Exchange Reactions by 4-Substituted Proline Derivatives.

The identification and understanding of structure-activity relationships is vital for rational catalyst design. A kinetic study of the hydrogen-deuterium exchange reaction of cyclohexanone in aqueous solution, as catalyzed by proline derivatives, has revealed valuable structure-activity relationships. In phosphate-buffered solution, cis-4-fluoroproline is more active than the trans isomer, a distinction that appears to originate from a destabilizing interaction between the fluorine atom and phosphate anion during general acid-catalyzed dehydration of the carbinolamine intermediate. trans-4-Ammoniumprolines are exceptionally active catalysts owing to favorable Coulombic interactions involving the ammonium group and the alkoxide moiety formed upon 1,2-addition of the proline derivative to the ketone. These results could be used for the optimization of proline catalysts, especially in transformations where the formation of the putative iminium ion is rate-limiting.

Sub-picomolar Inhibition of HIV-1 Protease with a Boronic Acid.

Boronic acids have been typecast as moieties for covalent complexation and are employed only rarely as agents for non-covalent recognition. By exploiting the profuse ability of a boronic acid group to form hydrogen bonds, we have developed an inhibitor of HIV-1 protease with extraordinary affinity. Specifically, we find that replacing an aniline moiety in darunavir with a phenylboronic acid leads to 20-fold greater affinity for the protease. X-ray crystallography demonstrates that the boronic acid group participates in three hydrogen bonds, more than the amino group of darunavir or any other analog. Importantly, the boronic acid maintains its hydrogen bonds and its affinity for the drug-resistant D30N variant of HIV-1 protease. The BOH···OC hydrogen bonds between the boronic acid hydroxy group and Asp30 (or Asn30) of the protease are short ( rO···O = 2.2 Å), and density functional theory analysis reveals a high degree of covalency. These data highlight the utility of boronic acids as versatile functional groups in the design of small-molecule ligands.

Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Concurrent Measurement of Gabapentin, Lamotrigine, Levetiracetam, Monohydroxy Derivative of Oxcarbazepine, and Zonisamide Concentrations in Serum in a Clinical Setting.

BACKGROUND: Therapeutic drug monitoring of antiepileptic drugs (AEDs) is often necessary to prevent associated destructive toxicities. Tandem mass spectrometry (MS/MS) with stable-isotope-labeled internal standards is considered the gold standard for the measurement of AEDs. This study presents the development and validation of a clinical ultra-performance liquid chromatography-MS/MS method for the concurrent measurement of gabapentin, lamotrigine, levetiracetam, monohydroxy derivative of oxcarbazepine, and zonisamide in human serum. METHODS: To determine the optimal assay analyte range, one year of AED therapeutic drug monitoring results (n = 1825) were evaluated. Simple protein precipitation with acetonitrile containing isotopically labeled internal standards was used. Reverse-phase ultra-performance liquid chromatography chromatographic separation was used, having a total run time of 3 minutes. Quantification of analytes was accomplished using electrospray ionization in positive ion mode and collision-induced dissociation MS. Assay parameters were evaluated per Food and Drug Administration bioanalytical guidelines. RESULTS: After evaluating internal patient data, the analytical measuring range (AMR) of the assay was established as 0.1-100 mcg/mL. All AEDs were linear across the AMR, with R values ranging from 0.9988 to 0.9999. Imprecision (% coefficient of variation) and inaccuracy (% difference) were calculated to be <20% for the lower limit of quantitation and <15% for the low, mid, and high levels of quality controls across the AMR. All AEDs demonstrated acceptable assay parameters for carryover, stability under relevant storage conditions, matrix effects, recovery, and extraction and processing efficiency. In addition, the assay displayed acceptable concordance to results obtained from a national reference laboratory, with Deming regression R of 0.99 and slope values ranging from 0.89 to 1.17. CONCLUSIONS: A simple, cost-effective, and robust ultra-performance liquid chromatography-tandem mass spectrometry method for monitoring multiple AEDs was developed and validated to address the clinical needs of patients at our institution.

Interaction of nucleic acids with the glycocalyx.

Mammalian cells resist the uptake of nucleic acids. The lipid bilayer of the plasma membrane presents one barrier. Here, we report on a second physicochemical barrier for uptake. To create a sensitive probe for nucleic acid-cell interactions, we synthesized fluorescent conjugates in which lipids are linked to DNA oligonucleotides. We found that these conjugates incorporate readily into the plasma membrane but are not retained there. Expulsion of lipid-oligonucleotide conjugates from the plasma membrane increases with oligonucleotide length. Conversely, the incorporation of conjugates increases markedly in cells that lack the major anionic components of the glycocalyx, sialic acid and glycosaminoglycans, and in cells that had incorporated highly cationic lipids into their plasma membrane. We conclude that anionic oligosaccharides provide a formidable barrier to the uptake of nucleic acids by mammalian cells. This conclusion has implications for genomic stability, as well as the delivery of genes and siRNAs into mammalian cells.

A potent, versatile disulfide-reducing agent from aspartic acid.

Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pK(a) values that are ~1 unit lower than those of DTT and forms a disulfide with a similar E°' value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.

Boronate-mediated biologic delivery.

Inefficient cellular delivery limits the landscape of macromolecular drugs. Boronic acids readily form boronate esters with the 1,2- and 1,3-diols of saccharides, such as those that coat the surface of mammalian cells. Here pendant boronic acids are shown to enhance the cytosolic delivery of a protein toxin. Thus, boronates are a noncationic carrier that can deliver a polar macromolecule into mammalian cells.

A Rapid Dilute-and-Shoot UPLC-MS/MS Assay to Simultaneously Measure 37 Drugs and Related Metabolites in Human Urine for Use in Clinical Pain Management.

BACKGROUND: Monitoring of medication compliance and drug abuse is used by clinicians to increase patient prescription drug compliance and reduce illicit drug abuse and diversion. Despite available immunoassays, chromatography-mass spectrometry-based methods are considered the gold standard for urine drug monitoring owing to higher sensitivities and specificities. Herein, we report a fast, convenient ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect or quantify 37 clinically relevant prescription drugs, drugs of abuse, and related glucuronides and other metabolites in human urine by single diluted sample injection. METHODS: Analytes consisted of prescription and illicit opioids, benzodiazepines, and drugs of abuse, including parent compounds and glucuronidated and nonglucuronidated metabolites. Urine samples were diluted with water and supplemented with deuterated internal standards without enzymatic hydrolysis, analyte extraction, or sample purification. Analytes were separated by reversed-phase UPLC and quantified by positive-mode electrospray ionization and collision-induced dissociation MS. Assay validation followed Food and Drug Administration bioanalytical guidelines. RESULTS: Total analytical run time was 5.5 min. All analytes demonstrated acceptable inter- and intraassay accuracy, imprecision, and linearity throughout clinically relevant analytical ranges (1-2000 ng/mL, depending on analyte). All analytes demonstrated acceptable selectivity, stability, matrix effects, carryover, and performance compared to national reference laboratory or previously validated in-house methods. A total of 23 and 14 analytes were validated for quantitative and qualitative testing, respectively. CONCLUSIONS: A convenient UPLC-MS/MS assay for simultaneously monitoring 37 analytes in human urine was validated for use in pain management testing. Advantages of this multiplex assay include facile sample preparation and higher-throughput definitive detection including glucuronide metabolite quantification.

Improving the Gastrointestinal Tolerability of Fumaric Acid Esters: Early Findings on Gastrointestinal Events with Diroximel Fumarate in Patients with Relapsing-Remitting Multiple Sclerosis from the Phase 3, Open-Label EVOLVE-MS-1 Study.

INTRODUCTION: Diroximel fumarate (DRF) is a novel oral fumarate in development for patients with relapsing forms of multiple sclerosis (MS). Clinical findings from the DRF development program suggest that rates of gastrointestinal (GI) treatment-emergent adverse events (TEAEs) and discontinuation due to GI TEAEs are low, based on clinical and real-world observations of other fumaric acid esters, including dimethyl fumarate (DMF). The incidence of GI TEAEs varies from 40 to 88% in clinical and real-world studies of DMF. The objective of this study is to present GI tolerability findings from the EVOLVE-MS-1 study and present biologic hypotheses for the improved GI properties of DRF. METHODS: GI TEAEs and treatment discontinuation because of GI TEAEs were assessed in DRF-treated patients with relapsing-remitting MS who were participating in the ongoing, 96-week, open-label, phase 3 EVOLVE-MS-1 study. RESULTS: As of March 30, 2018, a total of 696 patients were enrolled in EVOLVE-MS-1. GI TEAEs were reported in 30.9% (215/696) of patients; the vast majority (96%; 207/215) experienced events that were mild or moderate in severity. When GI AEs did occur, they occurred early in treatment, resolved (88.8%; 191/215), and were of short duration [median 7.5 (range 1-87) days] in most patients. GI TEAEs led to < 1% of patients discontinuing treatment. CONCLUSIONS: We suggest that the distinct chemical structure of DRF contributes to the observed low rates of GI TEAEs and GI-associated treatment discontinuations, possibly due to a combination of several factors. We hypothesize that these factors may include less reactivity with off-target proteins and/or lower production of a methanol leaving group that may contribute to GI irritation. A direct comparison of GI tolerability with DRF versus DMF is being evaluated in the EVOLVE-MS-2 study. TRIAL REGISTRATION: ClinicalTrials.gov number NCT02634307. FUNDING: Alkermes Inc. (Waltham, MA, USA) and Biogen (Cambridge, MA, USA).

A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis.

Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCCs are distinguished by aberrant lipid and glycogen accumulation and are refractory to a broad range of anti-cancer therapies. Here we identify an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCCs. This vulnerability transcends lineage and genetic landscape, and can be exploited by inhibiting glutathione peroxidase 4 (GPX4) with small-molecules. Using CRISPR screening and lipidomic profiling, we identify the hypoxia-inducible factor (HIF) pathway as a driver of this vulnerability. In renal CCCs, HIF-2α selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Our study suggests targeting GPX4 as a therapeutic opportunity in CCCs, and highlights that therapeutic approaches can be identified on the basis of cell states manifested by morphological and metabolic features in hard-to-treat cancers.

Development of a Commercial Reference Laboratory Elective Rotation for Residents in Clinical Pathology.

OBJECTIVES: To develop a curriculum for a commercial reference laboratory clinical pathology training elective. METHODS: A 4-day elective at Quest Diagnostics was developed. The elective included 32 sessions composed of interactive didactic sessions and laboratory tours/demonstrations. Ten residents who attended the elective completed a written evaluation and scored each component of the curriculum. RESULTS: Written comments were very positive and demonstrated the goals of the elective were achieved. Laboratory tours and one-on-one sessions with the medical directors were especially well received. Most of the residents stated that the rotation gave them exposure to an area of laboratory medicine that they were not familiar with. CONCLUSIONS: The elective provided a resident training experience that was highly regarded and exposed residents to an area of laboratory medicine not encountered in most pathology training programs. Our curriculum could serve as a model for establishing a similar elective in other training programs.

Organocatalytic Conversion of Cellulose into a Platform Chemical.

The search for a source of fuels and chemicals that is both abundant and renewable has become of paramount importance. The polysaccharide cellulose meets both criteria, and methods have been developed for its transformation into the platform chemical 5-(hydroxymethyl)furfural (HMF). These methods employ harsh reaction conditions or toxic heavy metal catalysts, deterring large-scale implementation. Here, we describe a low-temperature, one-pot route that uses ortho-carboxyl-substituted phenylboronic acids as organocatalysts in conjunction with hydrated magnesium chloride and mineral acids to convert cellulose and cellulose-rich municipal waste to HMF in yields comparable to processes that use toxic heavy metal catalysts. Isotopic labeling studies indicate that the key aldose-to-ketose transformation occurs via an enediol intermediate. The route, which also allows for facile catalyst recovery and recycling, provides a green prototype for cellulose conversion.

Ribonuclease-Activated Cancer Prodrug.

Cancer chemotherapeutic agents often have a narrow therapeutic index that challenges the maintenance of a safe and effective dose. Consistent plasma concentrations of a drug can be obtained by using a timed-release prodrug strategy. We reasoned that a ribonucleoside 3'-phosphate could serve as a pro-moiety that also increases the hydrophilicity of a cancer chemotherapeutic agent. Herein, we report an efficient route for the synthesis of the prodrug uridine 3'-(4-hydroxytamoxifen phosphate) (UpHT). UpHT demonstrates timed-released activation kinetics with a half-life of approximately 4 h at the approximate plasma concentration of human pancreatic ribonuclease (RNase 1). MCF-7 breast cancer cells treated with UpHT showed decreased proliferation upon co-incubation with RNase 1, consistent with the release of the active drug-4-hydroxytamoxifen. These data demonstrate the utility of a human plasma enzyme as a useful activator of a prodrug.